Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Impact of High Intensity Functional Training and MCT Consumption on Brain Neurometabolites through Magnetic Resonance Spectroscopy in Overweight and Obese Healthy Adults The Impact of High Intensity Functional Training and MCT Consumption on Brain Neurometabolites through Magnetic Resonance Spectroscopy in Overweight and Obese Healthy Adults 17 34 4858 10.22089/spj.2025.18236.2382 FA Maryam Nourshahi Department of Biological Sciences in Sport, Faculty of Sport and Health Sciences, Shahid Beheshti University, Tehran, Iran 0000-0002-5064-8599 Kimia Rahimi Pour Department of Biological Sciences in Sport, Faculty of Sport and Health Sciences, Shahid Beheshti University, Tehran, Iran 0009-0007-0787-7672 Sina Sanaei Dpartment of Exercise Physiology, Faculty of Physical Education, Islamic Azad University of Tehran, East Tehran Branch, Tehran, Iran 0009-0004-0142-9295 Atiye Sadat Mirahmadian Baba Ahmadi Dpartment of Exercise Physiology, Faculty of Physical Education, Islamic Azad University of Tehran, East Tehran Branch, Tehran, Iran 0009-0001-0372-646X Journal Article 2025 08 19 Background and Purpose Obesity and overweight, in addition to being associated with metabolic and cardiovascular diseases, can also affect brain health and accelerate neuronal degeneration and cognitive impairment. Systemic inflammation and oxidative stress caused by obesity can impair the function of various brain regions (including the hippocampus and prefrontal cortex) and disrupt energy metabolism and neurotransmission. N-acetyl aspartate (NAA) is an indicator of neuronal health and density. Choline (Cho) is also an indicator of membrane metabolism. Myo-inositol (mI) is a sensitive indicator of neuronal health, glial activity, and neuroinflammation, and its increase indicates neuronal damage. Increased NAA, cho, and decreased ml are markers of health. Since the importance of nutrition and exercise in neuronal studies has received much attention in recent years, this study was designed to investigate the simultaneous effect of high-intensity functional training and a ketogenic diet with MCT supplementation on NAA, Cho, and mI levels in overweight or obese individuals. Materials and Methods The present study is a randomized, quasi-experimental clinical trial. Thirty adults (aged 25 to 45 years) with overweight and grade 1 obesity (BMI between 25 and 34.9) volunteered for this study, of which 9 were excluded from the study due to non-compliance with diet and exercise. These subjects were divided into three groups: control group (C), extreme functional training (EX), and extreme functional training with ketogenic diet and MCT supplementation (EX+KD). The training program (except for the control group) consisted of three sessions per week for six weeks, and each session consisted of 30 to 40 minutes of high-intensity multi-joint functional training (HIFT) including a combination of aerobic and resistance exercises (including squats, swimming, lunges, chest press, butterfly, plank). In the first two weeks, 3 sets were performed at an intensity of 65%-75% of maximum heart rate, in the second two weeks 4 sets at an intensity of 85% to 95% of maximum heart rate, and in the third two weeks at the same intensity and 5 sets of exercises. The ketogenic diet was adjusted by a nutritionist with a specific ratio of 20% protein, 10% carbohydrate, and 70% fat, including 15 mL of MCT daily based on each individual's BMR, and was consumed by the EX+KD group for six weeks. After 6 weeks (a study without pretest due to ethical considerations and restrictions), neural metabolites were measured using single-voxel hydrogen magnetic resonance spectroscopy (1H-MRS) from the cerebellar vermis and analyzed with Osprey software. Sample size was estimated using G*Power 3.1 software for a three-group design. Data were described as mean and standard deviation. Normality of distribution was checked with the Shapiro-Wilk test and homogeneity of variance was checked with the Levene test. One-way analysis of variance (ANOVA) was used for between-group comparisons, and a significance level of P≥0.05 was considered in all analyses. Effect sizes were also calculated using the Cohen's d test. All statistical analyses were performed using SPSS version 25 software. Results Statistical analysis showed that high-intensity functional training combined with a ketogenic diet with MCT supplementation produced significant changes in the levels of key brain neurometabolites. All values are reported in ppm. NAA levels were significantly different between groups (F(2,18) = 96.228, P < 0.001). The EX group had a 91% increase in NAA compared to the C group (2.623 ± 0.05 → 1.368 ± 0.04, P < 0.001), while the EX+KD group showed a 38% increase (9.5 ± 0.4 → 10.8 ± 0.5, P < 0.05). However, NAA levels in the EX+KD group were only 27% lower than in the EX group (1.891 ± 0.05 → 2.623 ± 0.05, P < 0.001). These results indicate a positive effect of high-intensity functional training on neuronal health. These findings suggest that high-intensity functional training can improve neuronal health by improving mitochondrial oxidative capacity, promoting myelin synthesis, and enhancing neuron-glia function. Cho levels also differed significantly between groups (F(2,18) = 36.410, P < 0.001). The EX group showed a 97% increase (1.8 ± 0.1 → 2.2 ± 0.1, P < 0.05) and the EX+KD group showed a 193% increase compared to C (2.984 ± 0.05 → 1.010 ± 0.03, P < 0.001). The increase in Cho reflects the promotion of neuronal membrane phospholipid synthesis and remodeling, synaptic plasticity, and neurotrophic activity, and is associated with an increase in brain-derived neurotrophic factor (BDNF) and other neurotrophic factors. These results indicate that the combination of exercise and a ketogenic diet has an enhancing effect on neuronal membrane synthesis. mI levels also showed a significant difference between groups (F(2,18) = 205.112, P < 0.001). Both intervention groups had a significant decrease in mI compared to control: EX 12% (5.2 ± 0.3 → 4.6 ± 0.2, P < 0.05) and EX+KD 57% (5.3 ± 0.3 → 4.2 ± 0.2, P < 0.01). The decrease in mI is indicative of reduced glial activity and reduced neuroinflammation, and is likely related to increased ketone body (BHB) utilization and improved redox balance. Overall, increases in NAA and Cho and decreases in mI indicate improved neuronal health, increased mitochondrial oxidative capacity, and reduced glial inflammation. These changes may lead to improved cognitive and motor functions, and protection of neurons from obesity-induced damage. The findings suggest that high-intensity functional training and a ketogenic diet combined with MCTs have strong synergistic interactive effects on metabolism and neuroprotection in addition to their independent effects. Conclusion The results of this study demonstrated that high intensity functional training combined with a ketogenic diet and MCT supplementation significantly altered key brain neurometabolites. Increases in N-acetylaspartate and choline, along with decreased myoinositol, indicate improved neuronal health, enhanced mitochondrial oxidative capacity, increased myelin synthesis, and reduced glial activity and neuroinflammation. These metabolic changes likely enhance cognitive and motor functions, support neural plasticity, and protect neurons from the adverse effects of overweight and obesity. The combination of exercise and diet produced synergistic effects, with the greatest improvements observed in the group receiving both interventions, although each intervention alone also positively influenced brain metabolism. These findings underscore the importance of combined lifestyle interventions in promoting neural function and reducing the risk of obesity-related neuropsychological disorders, suggesting that vigorous exercise paired with a ketogenic diet can serve as an effective, practical approach to enhance brain health and prevent obesity-associated neurological damage in adults. Article Message Overweight and obesity are associated with decreased N-acetyl aspartate and choline and increased myo-inositol in the brain, which can lead to neurological disorders. MRS findings suggest that functional training, alone or in combination with a ketogenic diet, increases these beneficial neurometabolites and decreases myo-inositol. Combining high-intensity exercise with a ketogenic diet containing MCTs is an effective strategy for promoting brain health in overweight and obese individuals by improving neurometabolism and reducing markers of neurodegeneration. Ethical Considerations This study was approved by the ethics code IR.IUMS.REC.1402.057 at Iran University of Medical Sciences Authors’ Contributions Conceptualization: Kimia Rahimi Pour, Maryam Nourshahi Data Collection: Kimia Rahimi Pour, Sina Sanaei Data Analysis: Kimia Rahimi Pour, Manuscript Writing: Atiye Sadat Mirahmadian Baba Ahmadi Review and Editing: Maryam Nourshahi Responsible For Funding: Maryam Nourshahi Literature Review: Maryam Nourshahi Project Manager: Maryam Nourshahi Conflict of Interest <span dir="RTL" lang="FA" style="font-size: 13.0pt; mso-ansi-font-size: 12.0pt; line-height: 107%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">According to the authors, this article has no conflict of interest. Background and Purpose Obesity and overweight, in addition to being associated with metabolic and cardiovascular diseases, can also affect brain health and accelerate neuronal degeneration and cognitive impairment. Systemic inflammation and oxidative stress caused by obesity can impair the function of various brain regions (including the hippocampus and prefrontal cortex) and disrupt energy metabolism and neurotransmission. N-acetyl aspartate (NAA) is an indicator of neuronal health and density. Choline (Cho) is also an indicator of membrane metabolism. Myo-inositol (mI) is a sensitive indicator of neuronal health, glial activity, and neuroinflammation, and its increase indicates neuronal damage. Increased NAA, cho, and decreased ml are markers of health. Since the importance of nutrition and exercise in neuronal studies has received much attention in recent years, this study was designed to investigate the simultaneous effect of high-intensity functional training and a ketogenic diet with MCT supplementation on NAA, Cho, and mI levels in overweight or obese individuals. Materials and Methods The present study is a randomized, quasi-experimental clinical trial. Thirty adults (aged 25 to 45 years) with overweight and grade 1 obesity (BMI between 25 and 34.9) volunteered for this study, of which 9 were excluded from the study due to non-compliance with diet and exercise. These subjects were divided into three groups: control group (C), extreme functional training (EX), and extreme functional training with ketogenic diet and MCT supplementation (EX+KD). The training program (except for the control group) consisted of three sessions per week for six weeks, and each session consisted of 30 to 40 minutes of high-intensity multi-joint functional training (HIFT) including a combination of aerobic and resistance exercises (including squats, swimming, lunges, chest press, butterfly, plank). In the first two weeks, 3 sets were performed at an intensity of 65%-75% of maximum heart rate, in the second two weeks 4 sets at an intensity of 85% to 95% of maximum heart rate, and in the third two weeks at the same intensity and 5 sets of exercises. The ketogenic diet was adjusted by a nutritionist with a specific ratio of 20% protein, 10% carbohydrate, and 70% fat, including 15 mL of MCT daily based on each individual's BMR, and was consumed by the EX+KD group for six weeks. After 6 weeks (a study without pretest due to ethical considerations and restrictions), neural metabolites were measured using single-voxel hydrogen magnetic resonance spectroscopy (1H-MRS) from the cerebellar vermis and analyzed with Osprey software. Sample size was estimated using G*Power 3.1 software for a three-group design. Data were described as mean and standard deviation. Normality of distribution was checked with the Shapiro-Wilk test and homogeneity of variance was checked with the Levene test. One-way analysis of variance (ANOVA) was used for between-group comparisons, and a significance level of P≥0.05 was considered in all analyses. Effect sizes were also calculated using the Cohen's d test. All statistical analyses were performed using SPSS version 25 software. Results Statistical analysis showed that high-intensity functional training combined with a ketogenic diet with MCT supplementation produced significant changes in the levels of key brain neurometabolites. All values are reported in ppm. NAA levels were significantly different between groups (F(2,18) = 96.228, P < 0.001). The EX group had a 91% increase in NAA compared to the C group (2.623 ± 0.05 → 1.368 ± 0.04, P < 0.001), while the EX+KD group showed a 38% increase (9.5 ± 0.4 → 10.8 ± 0.5, P < 0.05). However, NAA levels in the EX+KD group were only 27% lower than in the EX group (1.891 ± 0.05 → 2.623 ± 0.05, P < 0.001). These results indicate a positive effect of high-intensity functional training on neuronal health. These findings suggest that high-intensity functional training can improve neuronal health by improving mitochondrial oxidative capacity, promoting myelin synthesis, and enhancing neuron-glia function. Cho levels also differed significantly between groups (F(2,18) = 36.410, P < 0.001). The EX group showed a 97% increase (1.8 ± 0.1 → 2.2 ± 0.1, P < 0.05) and the EX+KD group showed a 193% increase compared to C (2.984 ± 0.05 → 1.010 ± 0.03, P < 0.001). The increase in Cho reflects the promotion of neuronal membrane phospholipid synthesis and remodeling, synaptic plasticity, and neurotrophic activity, and is associated with an increase in brain-derived neurotrophic factor (BDNF) and other neurotrophic factors. These results indicate that the combination of exercise and a ketogenic diet has an enhancing effect on neuronal membrane synthesis. mI levels also showed a significant difference between groups (F(2,18) = 205.112, P < 0.001). Both intervention groups had a significant decrease in mI compared to control: EX 12% (5.2 ± 0.3 → 4.6 ± 0.2, P < 0.05) and EX+KD 57% (5.3 ± 0.3 → 4.2 ± 0.2, P < 0.01). The decrease in mI is indicative of reduced glial activity and reduced neuroinflammation, and is likely related to increased ketone body (BHB) utilization and improved redox balance. Overall, increases in NAA and Cho and decreases in mI indicate improved neuronal health, increased mitochondrial oxidative capacity, and reduced glial inflammation. These changes may lead to improved cognitive and motor functions, and protection of neurons from obesity-induced damage. The findings suggest that high-intensity functional training and a ketogenic diet combined with MCTs have strong synergistic interactive effects on metabolism and neuroprotection in addition to their independent effects. Conclusion The results of this study demonstrated that high intensity functional training combined with a ketogenic diet and MCT supplementation significantly altered key brain neurometabolites. Increases in N-acetylaspartate and choline, along with decreased myoinositol, indicate improved neuronal health, enhanced mitochondrial oxidative capacity, increased myelin synthesis, and reduced glial activity and neuroinflammation. These metabolic changes likely enhance cognitive and motor functions, support neural plasticity, and protect neurons from the adverse effects of overweight and obesity. The combination of exercise and diet produced synergistic effects, with the greatest improvements observed in the group receiving both interventions, although each intervention alone also positively influenced brain metabolism. These findings underscore the importance of combined lifestyle interventions in promoting neural function and reducing the risk of obesity-related neuropsychological disorders, suggesting that vigorous exercise paired with a ketogenic diet can serve as an effective, practical approach to enhance brain health and prevent obesity-associated neurological damage in adults. Article Message Overweight and obesity are associated with decreased N-acetyl aspartate and choline and increased myo-inositol in the brain, which can lead to neurological disorders. MRS findings suggest that functional training, alone or in combination with a ketogenic diet, increases these beneficial neurometabolites and decreases myo-inositol. Combining high-intensity exercise with a ketogenic diet containing MCTs is an effective strategy for promoting brain health in overweight and obese individuals by improving neurometabolism and reducing markers of neurodegeneration. Ethical Considerations This study was approved by the ethics code IR.IUMS.REC.1402.057 at Iran University of Medical Sciences Authors’ Contributions Conceptualization: Kimia Rahimi Pour, Maryam Nourshahi Data Collection: Kimia Rahimi Pour, Sina Sanaei Data Analysis: Kimia Rahimi Pour, Manuscript Writing: Atiye Sadat Mirahmadian Baba Ahmadi Review and Editing: Maryam Nourshahi Responsible For Funding: Maryam Nourshahi Literature Review: Maryam Nourshahi Project Manager: Maryam Nourshahi Conflict of Interest <span dir="RTL" lang="FA" style="font-size: 13.0pt; mso-ansi-font-size: 12.0pt; line-height: 107%; font-family: 'B Nazanin'; mso-ascii-font-family: 'Times New Roman'; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-ansi-language: EN-US; mso-fareast-language: EN-US; mso-bidi-language: FA;">According to the authors, this article has no conflict of interest.

https://spj.ssrc.ac.ir/article_4858_aea94dc1e6d1dd330cbc2c4a480934d6.pdf

Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Effect of Combined and Separate Interventions of High-Intensity Interval Training and Eryngium Extract on Apoptosis Biomarkers in the Soleus Muscle of Male Rats with Type 2 Diabetes The Effect of Combined and Separate Interventions of High-Intensity Interval Training and Eryngium Extract on Apoptosis Biomarkers in the Soleus Muscle of Male Rats with Type 2 Diabetes 39 54 4849 10.22089/spj.2025.18090.2376 FA Ghassan Amer Bedno Department of exercise physiology and corrective movements, Faculty of Sport Sciences, Urmia University, Urmia, Iran Asghar Tofighi Department of exercise physiology and corrective movements, Faculty of Sport Sciences, Urmia University, Urmia, Iran 0000-0003-3626-1782 Bahram Jamali Department of Basic Sciences, Faculty of Paramedical Sciences, Tabriz University of Medical Sciences, Tabriz, Iran 0000-0002-3138-273X Mohammad Reza Shiri-Shahsavar Department of Nutrition, Faculty of Health, Qazvin University of Medical Sciences, Qazvin, Iran 0000-0002-6243-8361 Journal Article 2025 07 07 Background and Purpose Type 2 diabetes mellitus (T2DM) is a common metabolic disorder characterized by insulin resistance, impaired insulin secretion, and chronic hyperglycemia that together promote tissue damage. Skeletal muscle, a major site of insulin-mediated glucose uptake, is especially vulnerable and in diabetic conditions shows reduced mass, impaired function, mitochondrial dysfunction, and activation of apoptotic pathways. Among the key regulators of apoptosis are the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, and their balance critically determines cell survival. High-intensity interval training (HIIT) is a time-efficient exercise modality that can improve insulin sensitivity, oxidative capacity, and cellular stress tolerance. Eryngium (Buganagh) is a medicinal plant rich in antioxidant and anti-inflammatory constituents that may help counteract oxidative stress and metabolic disturbances in diabetes. The present study investigated the independent and combined effects of HIIT and Eryngium extract on Bax, Bcl-2, and the Bax/Bcl-2 ratio in the soleus muscle of rats with type 2 diabetes. Materials and Methods This experimental study involved fifty male Wistar rats aged 8–10 weeks and weighing 200 ± 20 g. Animals were housed under standard laboratory conditions, including a temperature of 22 ± 2 °C, a 12-hour light/dark cycle, and humidity of 50–60%, with free access to chow and water. Before interventions, rats completed a two-week acclimatization period to the housing environment and treadmill running to reduce stress. Type 2 diabetes was induced by two weeks of high-fat diet followed by a single intraperitoneal injection of streptozotocin (50 mg/kg in citrate buffer, pH 4.5). Seventy-two hours later, fasting blood glucose was measured, and rats with values above 250 mg/dL were considered diabetic. Animals were randomly assigned to five groups (n = 10): healthy control, diabetic control, diabetic plus Eryngium extract, diabetic plus HIIT, and diabetic plus HIIT with Eryngium extract. The extract was administered orally by gavage at 100 mg/kg/day for six weeks. The HIIT protocol was performed on a motorized treadmill five days per week for six weeks using repeated high-speed bouts with active recovery, with intensity progressively increased. At the end, rats were fasted overnight, anesthetized with ketamine/xylazine, and euthanized according to ethical guidelines. Soleus muscles were excised, cleaned, snap-frozen, and stored at −80 °C. Bax and Bcl-2 protein levels were measured with commercial ELISA kits, and the Bax/Bcl-2 ratio was calculated as an apoptotic index. Body weight and glucose were monitored, treadmill intensity was adjusted individually, and investigators were blinded to group allocation during biochemical analyses. All procedures followed institutional animal care standards, ensuring humane treatment, minimized distress, and consistent handling throughout the study. Sample processing, data recording, and assay validation were performed under controlled laboratory conditions to maintain reliability, reduce variability, and ensure that measured apoptotic markers accurately reflected intervention effects on skeletal muscle tissue in this experiment. Results In this study, the soleus muscle was chosen because its oxidative and slow-twitch profile makes it particularly sensitive to diabetes-induced apoptotic alterations.The induction of diabetes produced marked alterations in apoptosis-related proteins within the soleus muscle. One-way ANOVA revealed that Bax protein levels were significantly higher in the diabetic control group compared to all other groups (p = 0.001). Specifically, streptozotocin (STZ) injection resulted in a profound elevation of Bax concentration in diabetic rats relative to healthy controls, confirming the pro-apoptotic impact of diabetes induction.Intervention analyses showed that both high-intensity interval training (HIIT) and the combined protocol of HIIT with Eryngium extract significantly reduced Bax levels in diabetic rats when compared to the diabetic control group (p = 0.001). Importantly, the reduction in Bax was more pronounced in the combined intervention group, demonstrating a significantly greater improvement than that observed in the extract-only group (p = 0.001). In contrast, administration of Eryngium extract alone did not produce a statistically significant reduction in Bax levels (p > 0.05), indicating that the extract by itself was insufficient to counteract diabetes-induced increases in pro-apoptotic signaling.Similarly, the one-way ANOVA for Bcl-2 revealed that healthy controls exhibited significantly higher Bcl-2 protein levels than diabetic controls (p = 0.001), reflecting the suppression of anti-apoptotic mechanisms under diabetic conditions. Treatment with Eryngium extract, HIIT, or their combination all resulted in significant increases in Bcl-2 levels compared to the diabetic control group (p = 0.001). However, no significant differences were observed among the three intervention groups (p > 0.05), suggesting that although all interventions enhanced anti-apoptotic signaling, none was superior in elevating Bcl-2 expression.Analysis of the Bax/Bcl-2 ratio further supported these findings. The ratio was markedly elevated in the diabetic control group relative to the healthy controls (p = 0.001), indicating a strong shift toward apoptosis. Both the HIIT group and the combined HIIT + extract group demonstrated significant reductions in the Bax/Bcl-2 ratio compared with diabetic controls (p = 0.001). Notably, the combined intervention resulted in a significantly greater reduction in the Bax/Bcl-2 ratio compared to the extract-only group (p = 0.001), highlighting the synergistic effect of combining exercise with Eryngium extract.Collectively, these results show that diabetes promotes a pro-apoptotic environment in skeletal muscle by increasing Bax and the Bax/Bcl-2 ratio while suppressing Bcl-2. HIIT—especially when combined with Eryngium extract—exerts stronger protective effects by attenuating pro-apoptotic markers and enhancing anti-apoptotic signaling more effectively than the extract alone. Conclusion This study demonstrated that the combination of high-intensity interval training and Eryngium extract effectively attenuated apoptosis in the soleus muscle of rats with type 2 diabetes. Diabetes induction markedly increased Bax, decreased Bcl-2, and elevated the Bax/Bcl-2 ratio, while both HIIT and the combined intervention significantly reversed these detrimental changes. The extract alone was less effective in lowering Bax but still enhanced Bcl-2, indicating that exercise provided the primary protective stimulus and the plant extract acted mainly as a complementary support. Overall, the combined protocol produced the most favorable profile of apoptotic markers, suggesting improved cellular survival under diabetic conditions. These findings highlight the potential of integrating structured high-intensity exercise with antioxidant herbal supplementation as a non-pharmacological strategy to limit diabetes-related muscle complications and may provide a rationale for future translational and clinical studies targeting diabetic sarcopenia and functional decline. In practice, such combined interventions may help preserve muscle function. Article Message The growing burden of type 2 diabetes demands practical, low-cost strategies to protect vulnerable tissues such as skeletal muscle. In this experimental model, combining high-intensity interval training with Eryngium extract reduced pro-apoptotic signaling, increased anti-apoptotic defenses, and improved the Bax/Bcl-2 balance in the soleus muscle of diabetic rats. These findings suggest that structured high-intensity exercise, particularly when paired with antioxidant herbal supplementation, may mitigate diabetes-related muscle damage and delay progression toward diabetic sarcopenia. Further clinical research is needed to determine optimal protocols and to clarify the translational relevance of this combined non-pharmacological approach. Future work should refine dose and intensity. Ethical Considerations All experimental procedures involving animals were carried out in accordance with the guidelines of the Laboratory Animal Ethics Committee of the University of Urmia. The study was approved under Ethics Code: IR-UU-AEC-3/53. All stages of work with laboratory animals were performed strictly according to the committee’s instructions to ensure humane care, minimization of pain and distress, and adherence to national and institutional standards for animal research. Further information about the ethical framework can be obtained from the Laboratory Animal Ethics Committee of the University of Urmia. Authors’ Contributions Conceptualization: Ghassan Amer Bedno, Asghar Tofighi Data Collection: Ghassan Amer Bedno, Bahram Jamali, Mohammad Reza Shiri-Shahsavar Data Analysis: Ghassan Amer Bedno, Bahram Jamali, Asghar Tofighi Manuscript Writing: Ghassan Amer Bedno, Asghar Tofighi Review and Editing: Asghar Tofighi, Mohammad Reza Shiri-Shahsavar Responsible for Funding: Asghar Tofighi Literature Review: Ghassan Amer Bedno Project Manager: Asghar Tofighi Any Other Contribution: All authors contributed to the interpretation of findings and approved the final version of the manuscript. Conflict of Interest The Authors Declare no Competing Financial or Professional Interests. Acknowledgments We extend our gratitude to all individuals who supported and facilitated this study. Background and Purpose Type 2 diabetes mellitus (T2DM) is a common metabolic disorder characterized by insulin resistance, impaired insulin secretion, and chronic hyperglycemia that together promote tissue damage. Skeletal muscle, a major site of insulin-mediated glucose uptake, is especially vulnerable and in diabetic conditions shows reduced mass, impaired function, mitochondrial dysfunction, and activation of apoptotic pathways. Among the key regulators of apoptosis are the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2, and their balance critically determines cell survival. High-intensity interval training (HIIT) is a time-efficient exercise modality that can improve insulin sensitivity, oxidative capacity, and cellular stress tolerance. Eryngium (Buganagh) is a medicinal plant rich in antioxidant and anti-inflammatory constituents that may help counteract oxidative stress and metabolic disturbances in diabetes. The present study investigated the independent and combined effects of HIIT and Eryngium extract on Bax, Bcl-2, and the Bax/Bcl-2 ratio in the soleus muscle of rats with type 2 diabetes. Materials and Methods This experimental study involved fifty male Wistar rats aged 8–10 weeks and weighing 200 ± 20 g. Animals were housed under standard laboratory conditions, including a temperature of 22 ± 2 °C, a 12-hour light/dark cycle, and humidity of 50–60%, with free access to chow and water. Before interventions, rats completed a two-week acclimatization period to the housing environment and treadmill running to reduce stress. Type 2 diabetes was induced by two weeks of high-fat diet followed by a single intraperitoneal injection of streptozotocin (50 mg/kg in citrate buffer, pH 4.5). Seventy-two hours later, fasting blood glucose was measured, and rats with values above 250 mg/dL were considered diabetic. Animals were randomly assigned to five groups (n = 10): healthy control, diabetic control, diabetic plus Eryngium extract, diabetic plus HIIT, and diabetic plus HIIT with Eryngium extract. The extract was administered orally by gavage at 100 mg/kg/day for six weeks. The HIIT protocol was performed on a motorized treadmill five days per week for six weeks using repeated high-speed bouts with active recovery, with intensity progressively increased. At the end, rats were fasted overnight, anesthetized with ketamine/xylazine, and euthanized according to ethical guidelines. Soleus muscles were excised, cleaned, snap-frozen, and stored at −80 °C. Bax and Bcl-2 protein levels were measured with commercial ELISA kits, and the Bax/Bcl-2 ratio was calculated as an apoptotic index. Body weight and glucose were monitored, treadmill intensity was adjusted individually, and investigators were blinded to group allocation during biochemical analyses. All procedures followed institutional animal care standards, ensuring humane treatment, minimized distress, and consistent handling throughout the study. Sample processing, data recording, and assay validation were performed under controlled laboratory conditions to maintain reliability, reduce variability, and ensure that measured apoptotic markers accurately reflected intervention effects on skeletal muscle tissue in this experiment. Results In this study, the soleus muscle was chosen because its oxidative and slow-twitch profile makes it particularly sensitive to diabetes-induced apoptotic alterations.The induction of diabetes produced marked alterations in apoptosis-related proteins within the soleus muscle. One-way ANOVA revealed that Bax protein levels were significantly higher in the diabetic control group compared to all other groups (p = 0.001). Specifically, streptozotocin (STZ) injection resulted in a profound elevation of Bax concentration in diabetic rats relative to healthy controls, confirming the pro-apoptotic impact of diabetes induction.Intervention analyses showed that both high-intensity interval training (HIIT) and the combined protocol of HIIT with Eryngium extract significantly reduced Bax levels in diabetic rats when compared to the diabetic control group (p = 0.001). Importantly, the reduction in Bax was more pronounced in the combined intervention group, demonstrating a significantly greater improvement than that observed in the extract-only group (p = 0.001). In contrast, administration of Eryngium extract alone did not produce a statistically significant reduction in Bax levels (p > 0.05), indicating that the extract by itself was insufficient to counteract diabetes-induced increases in pro-apoptotic signaling.Similarly, the one-way ANOVA for Bcl-2 revealed that healthy controls exhibited significantly higher Bcl-2 protein levels than diabetic controls (p = 0.001), reflecting the suppression of anti-apoptotic mechanisms under diabetic conditions. Treatment with Eryngium extract, HIIT, or their combination all resulted in significant increases in Bcl-2 levels compared to the diabetic control group (p = 0.001). However, no significant differences were observed among the three intervention groups (p > 0.05), suggesting that although all interventions enhanced anti-apoptotic signaling, none was superior in elevating Bcl-2 expression.Analysis of the Bax/Bcl-2 ratio further supported these findings. The ratio was markedly elevated in the diabetic control group relative to the healthy controls (p = 0.001), indicating a strong shift toward apoptosis. Both the HIIT group and the combined HIIT + extract group demonstrated significant reductions in the Bax/Bcl-2 ratio compared with diabetic controls (p = 0.001). Notably, the combined intervention resulted in a significantly greater reduction in the Bax/Bcl-2 ratio compared to the extract-only group (p = 0.001), highlighting the synergistic effect of combining exercise with Eryngium extract.Collectively, these results show that diabetes promotes a pro-apoptotic environment in skeletal muscle by increasing Bax and the Bax/Bcl-2 ratio while suppressing Bcl-2. HIIT—especially when combined with Eryngium extract—exerts stronger protective effects by attenuating pro-apoptotic markers and enhancing anti-apoptotic signaling more effectively than the extract alone. Conclusion This study demonstrated that the combination of high-intensity interval training and Eryngium extract effectively attenuated apoptosis in the soleus muscle of rats with type 2 diabetes. Diabetes induction markedly increased Bax, decreased Bcl-2, and elevated the Bax/Bcl-2 ratio, while both HIIT and the combined intervention significantly reversed these detrimental changes. The extract alone was less effective in lowering Bax but still enhanced Bcl-2, indicating that exercise provided the primary protective stimulus and the plant extract acted mainly as a complementary support. Overall, the combined protocol produced the most favorable profile of apoptotic markers, suggesting improved cellular survival under diabetic conditions. These findings highlight the potential of integrating structured high-intensity exercise with antioxidant herbal supplementation as a non-pharmacological strategy to limit diabetes-related muscle complications and may provide a rationale for future translational and clinical studies targeting diabetic sarcopenia and functional decline. In practice, such combined interventions may help preserve muscle function. Article Message The growing burden of type 2 diabetes demands practical, low-cost strategies to protect vulnerable tissues such as skeletal muscle. In this experimental model, combining high-intensity interval training with Eryngium extract reduced pro-apoptotic signaling, increased anti-apoptotic defenses, and improved the Bax/Bcl-2 balance in the soleus muscle of diabetic rats. These findings suggest that structured high-intensity exercise, particularly when paired with antioxidant herbal supplementation, may mitigate diabetes-related muscle damage and delay progression toward diabetic sarcopenia. Further clinical research is needed to determine optimal protocols and to clarify the translational relevance of this combined non-pharmacological approach. Future work should refine dose and intensity. Ethical Considerations All experimental procedures involving animals were carried out in accordance with the guidelines of the Laboratory Animal Ethics Committee of the University of Urmia. The study was approved under Ethics Code: IR-UU-AEC-3/53. All stages of work with laboratory animals were performed strictly according to the committee’s instructions to ensure humane care, minimization of pain and distress, and adherence to national and institutional standards for animal research. Further information about the ethical framework can be obtained from the Laboratory Animal Ethics Committee of the University of Urmia. Authors’ Contributions Conceptualization: Ghassan Amer Bedno, Asghar Tofighi Data Collection: Ghassan Amer Bedno, Bahram Jamali, Mohammad Reza Shiri-Shahsavar Data Analysis: Ghassan Amer Bedno, Bahram Jamali, Asghar Tofighi Manuscript Writing: Ghassan Amer Bedno, Asghar Tofighi Review and Editing: Asghar Tofighi, Mohammad Reza Shiri-Shahsavar Responsible for Funding: Asghar Tofighi Literature Review: Ghassan Amer Bedno Project Manager: Asghar Tofighi Any Other Contribution: All authors contributed to the interpretation of findings and approved the final version of the manuscript. Conflict of Interest The Authors Declare no Competing Financial or Professional Interests. Acknowledgments We extend our gratitude to all individuals who supported and facilitated this study.

https://spj.ssrc.ac.ir/article_4849_ec1f764517b7ffb52057af6df18142b7.pdf

Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Effects of High-Intensity Interval Training on Inflammation and Oxidative Stress in Hippocampal Tissue of Rats with Heart Failure The Effects of High-Intensity Interval Training on Inflammation and Oxidative Stress in Hippocampal Tissue of Rats with Heart Failure 55 73 4841 10.22089/spj.2025.18371.2390 FA Fahimeh Fatahi Department of Exercise Physiology, Faculty of Sport and Health Sciences, University of Tehran, Tehran, Iran 0009-0004-6335-2414 Abasali Gaeini Department of Exercise Physiology, Faculty of Sport and Health Sciences, University of Tehran, Tehran, Iran 0000-0002-8679-0669 Reza Nuri faculty of sports and sciences، university of Tehran ، Iran 0000-0003-1976-3561 Alireza Ghardashi Afoosi Department of Exercise Physiology, Faculty of Sport and Health Sciences, University of Tehran, Tehran, Iran 0000-0001-5075-2225 Soheila Adeli Electrophysiology Research Center, Neuroscience Institute, Tehran University of Medical Science, Tehran, Iran 0009-0004-6335-2414 Journal Article 2025 08 11 Background and Purpose Heart failure (HF) is a progressive condition characterized by systemic inflammation, neuroinflammation, and oxidative stress. Persistent inflammatory responses contribute to cognitive decline and impaired brain function in cardiovascular patients. Contributory factors include cerebral hypoperfusion, neurohumoral activation, systemic inflammation, hypertension, atherosclerosis, aging, and genetic predispositions. Chronic cerebral hypoperfusion leads to neuroinflammation, oxidative stress, and disruption of the blood-brain barrier (BBB). Loss of BBB integrity activates further inflammatory and oxidative stress pathways, exacerbating cognitive impairment. Sustained neuroinflammation is marked by elevated cytokines which impair the BBB and promote infiltration of peripheral macrophages into brain tissue, perpetuating inflammation. In HF animal models, increased expression of pro-inflammatory genes such as TLR-4, TNF-α, IL-1β, and IL-6 in the cerebral cortex and hippocampus compromises BBB structure and facilitates neuroinflammation. While physiological cytokine levels support synaptic plasticity and memory consolidation, excessive TNF-α and IL-1β stimulate microglial activation and apoptosis. Additionally, HF decreases hippocampal and cortical superoxide dismutase (SOD) activity, while elevating malondialdehyde (MDA) levels, indicating oxidative neuronal damage associated with cognitive impairments, depression, and anxiety.Exercise has emerged as a promising non-pharmacological strategy to mitigate cardiovascular and neuroinflammatory complications. Mechanistically, exercise attenuates neuroinflammation through modulation of microglial activation, NF-κB signaling, proinflammatory cytokines, mitochondrial function, and reactive oxygen species (ROS). Treadmill exercise enhances brain function by modifying gene expression, promoting neuroplasticity, increasing metabolic efficiency, and boosting antioxidant capacity. It maintains cerebral homeostasis by regulating microglial activity, proinflammatory cytokines, and neuroinflammation, thereby reducing risk of neurodegeneration. Zhang et al. demonstrated that exercise downregulated pro-inflammatory markers and upregulated anti-inflammatory factors in Alzheimer’s disease models, also reducing oxidative stress markers like MDA and enhancing SOD activity. Similarly, treadmill running in aged mice increased microglial activation while suppressing pro-inflammatory cytokine production, conferring neuroprotection. This study investigated the neuroprotective capacity of high-intensity interval training (HIIT) against neuroinflammation in HF rats. Due to its characteristics—brief, intense energy stimulation, recovery periods, and increased cerebral blood flow—HIIT may simultaneously enhance antioxidant defenses and reduce inflammatory responses. Materials and Methods Twenty-four male Wistar rats (8 weeks old, mean weight 250 ± 50 g) were housed under controlled conditions (22 ± 2°C, 12-hour light/dark cycle) with ad libitum access to food and water at the Faculty of Sport Sciences and Health animal facility. After one week of acclimatization, rats were allocated randomly into three groups: healthy control (H, n=8), sedentary HF (HF, n=8), and HF + HIIT (HFT, n=8). HF was induced by subcutaneous administration of isoproterenol (130 mg/kg) daily for four days. Cardiac function was confirmed by echocardiography performed by a veterinary specialist; rats with ejection fraction <45% and fractional shortening <35% were included. After a 25-day recovery period, HFT rats underwent an 8-week HIIT protocol (three 60-minute sessions weekly). Each session comprised a 5-minute warm-up at 40–50% VO₂max, followed by interval running on a zero-degree incline: four minutes at 85–90% VO₂max alternating with two minutes at 50–60% VO₂max, repeated for 30 minutes, concluding with a 5-minute cool-down at 40–50% VO₂max. Training intensity progressively increased by 0.02 m/s weekly, based on prior literature.Forty-eight hours after completing the last session and following a 12-hour fast, rats were anesthetized (ketamine/xylazine), and hippocampal tissues were harvested, snap-frozen in liquid nitrogen, and stored at −80°C. Levels of IL-1β, IL-10, SOD, and MDA were quantified by ELISA kits following manufacturer protocols. Data analysis employed SPSS 25. Normality was assessed by Shapiro-Wilk test; intergroup differences were examined using one-way ANOVA and Tukey’s post hoc test at P<0.05. Data are presented as mean ± standard error. GraphPad Prism was utilized for figure generation. Results HF significantly elevated hippocampal IL-1β and MDA while decreasing IL-10 and SOD compared to healthy controls (P<0.01). Following eight weeks of HIIT, the HFT group exhibited marked reductions in IL-1β and MDA alongside significant increases in IL-10 and SOD compared to the sedentary HF group (P<0.05). These findings indicate that HIIT effectively restores inflammatory balance and antioxidant capacity in HF-affected hippocampal tissue. Conclusion <span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi; color: black; mso-themecolor: text1; font-weight: normal;">An eight-week HIIT regimen improves hippocampal inflammatory status by decreasing pro-inflammatory markers, enhancing anti-inflammatory cytokines, and augmenting antioxidant enzymes in rats with HF. Modulating neuroinflammation and oxidative stress through HIIT offers a potential non-pharmacological intervention to protect neural integrity in HF. Article Message HIIT represents an effective therapeutic modality to mitigate neuroinflammation and oxidative damage in the brain, particularly the hippocampus, in HF. By regulating cytokine expression and antioxidant defense, HIIT may attenuate HF-induced neural injury and improve cognitive resilience. Ethical Considerations This study was approved by the Research Ethics Committee of the Faculty of Sport and Health Sciences, University of Tehran (ethical code: <span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi;">IR.UT.SPORT.REC.1403.014). All experimental procedures conformed to national guidelines for laboratory animal care, with efforts to minimize pain and distress.<span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-fareast-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-language: AR-SA;"> Authors’ Contributions <span style="font-size: 11.0pt; line-height: 107%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi;">Conceptualization: Abbasali Gaeini, Alireza Ghardashi Afousi Data Collection: Fahime Fatahi, Soheila Adeli Data Analysis: Reza Nouri, Fahimeh Fatahi Manuscript Writing: Fahimeh Fatahi, Alireza Ghardashi Afousi Review and Editing: Fahimeh Fatahi, Abbasali Gaeini, Alireza Ghardashi Afousi Funding Responsibility: Soheila Adeli, Fahimeh Fatahi Literature Review: Reza Nouri, Fahimeh Fatahi Project Management: Abbasali Gaeini, Alireza Ghardashi Afousi Additional Contributions: Soheila Adeli Conflict of Interest The authors declare no conflicts of interest regarding this study. Acknowledgments We gratefully acknowledge Ms. Aida Sabouri for her valuable assistance with animal modeling. Background and Purpose Heart failure (HF) is a progressive condition characterized by systemic inflammation, neuroinflammation, and oxidative stress. Persistent inflammatory responses contribute to cognitive decline and impaired brain function in cardiovascular patients. Contributory factors include cerebral hypoperfusion, neurohumoral activation, systemic inflammation, hypertension, atherosclerosis, aging, and genetic predispositions. Chronic cerebral hypoperfusion leads to neuroinflammation, oxidative stress, and disruption of the blood-brain barrier (BBB). Loss of BBB integrity activates further inflammatory and oxidative stress pathways, exacerbating cognitive impairment. Sustained neuroinflammation is marked by elevated cytokines which impair the BBB and promote infiltration of peripheral macrophages into brain tissue, perpetuating inflammation. In HF animal models, increased expression of pro-inflammatory genes such as TLR-4, TNF-α, IL-1β, and IL-6 in the cerebral cortex and hippocampus compromises BBB structure and facilitates neuroinflammation. While physiological cytokine levels support synaptic plasticity and memory consolidation, excessive TNF-α and IL-1β stimulate microglial activation and apoptosis. Additionally, HF decreases hippocampal and cortical superoxide dismutase (SOD) activity, while elevating malondialdehyde (MDA) levels, indicating oxidative neuronal damage associated with cognitive impairments, depression, and anxiety.Exercise has emerged as a promising non-pharmacological strategy to mitigate cardiovascular and neuroinflammatory complications. Mechanistically, exercise attenuates neuroinflammation through modulation of microglial activation, NF-κB signaling, proinflammatory cytokines, mitochondrial function, and reactive oxygen species (ROS). Treadmill exercise enhances brain function by modifying gene expression, promoting neuroplasticity, increasing metabolic efficiency, and boosting antioxidant capacity. It maintains cerebral homeostasis by regulating microglial activity, proinflammatory cytokines, and neuroinflammation, thereby reducing risk of neurodegeneration. Zhang et al. demonstrated that exercise downregulated pro-inflammatory markers and upregulated anti-inflammatory factors in Alzheimer’s disease models, also reducing oxidative stress markers like MDA and enhancing SOD activity. Similarly, treadmill running in aged mice increased microglial activation while suppressing pro-inflammatory cytokine production, conferring neuroprotection. This study investigated the neuroprotective capacity of high-intensity interval training (HIIT) against neuroinflammation in HF rats. Due to its characteristics—brief, intense energy stimulation, recovery periods, and increased cerebral blood flow—HIIT may simultaneously enhance antioxidant defenses and reduce inflammatory responses. Materials and Methods Twenty-four male Wistar rats (8 weeks old, mean weight 250 ± 50 g) were housed under controlled conditions (22 ± 2°C, 12-hour light/dark cycle) with ad libitum access to food and water at the Faculty of Sport Sciences and Health animal facility. After one week of acclimatization, rats were allocated randomly into three groups: healthy control (H, n=8), sedentary HF (HF, n=8), and HF + HIIT (HFT, n=8). HF was induced by subcutaneous administration of isoproterenol (130 mg/kg) daily for four days. Cardiac function was confirmed by echocardiography performed by a veterinary specialist; rats with ejection fraction <45% and fractional shortening <35% were included. After a 25-day recovery period, HFT rats underwent an 8-week HIIT protocol (three 60-minute sessions weekly). Each session comprised a 5-minute warm-up at 40–50% VO₂max, followed by interval running on a zero-degree incline: four minutes at 85–90% VO₂max alternating with two minutes at 50–60% VO₂max, repeated for 30 minutes, concluding with a 5-minute cool-down at 40–50% VO₂max. Training intensity progressively increased by 0.02 m/s weekly, based on prior literature.Forty-eight hours after completing the last session and following a 12-hour fast, rats were anesthetized (ketamine/xylazine), and hippocampal tissues were harvested, snap-frozen in liquid nitrogen, and stored at −80°C. Levels of IL-1β, IL-10, SOD, and MDA were quantified by ELISA kits following manufacturer protocols. Data analysis employed SPSS 25. Normality was assessed by Shapiro-Wilk test; intergroup differences were examined using one-way ANOVA and Tukey’s post hoc test at P<0.05. Data are presented as mean ± standard error. GraphPad Prism was utilized for figure generation. Results HF significantly elevated hippocampal IL-1β and MDA while decreasing IL-10 and SOD compared to healthy controls (P<0.01). Following eight weeks of HIIT, the HFT group exhibited marked reductions in IL-1β and MDA alongside significant increases in IL-10 and SOD compared to the sedentary HF group (P<0.05). These findings indicate that HIIT effectively restores inflammatory balance and antioxidant capacity in HF-affected hippocampal tissue. Conclusion <span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-fareast-font-family: Calibri; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi; color: black; mso-themecolor: text1; font-weight: normal;">An eight-week HIIT regimen improves hippocampal inflammatory status by decreasing pro-inflammatory markers, enhancing anti-inflammatory cytokines, and augmenting antioxidant enzymes in rats with HF. Modulating neuroinflammation and oxidative stress through HIIT offers a potential non-pharmacological intervention to protect neural integrity in HF. Article Message HIIT represents an effective therapeutic modality to mitigate neuroinflammation and oxidative damage in the brain, particularly the hippocampus, in HF. By regulating cytokine expression and antioxidant defense, HIIT may attenuate HF-induced neural injury and improve cognitive resilience. Ethical Considerations This study was approved by the Research Ethics Committee of the Faculty of Sport and Health Sciences, University of Tehran (ethical code: <span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi;">IR.UT.SPORT.REC.1403.014). All experimental procedures conformed to national guidelines for laboratory animal care, with efforts to minimize pain and distress.<span style="font-size: 11.0pt; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-fareast-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-language: AR-SA;"> Authors’ Contributions <span style="font-size: 11.0pt; line-height: 107%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'Times New Roman'; mso-bidi-theme-font: major-bidi;">Conceptualization: Abbasali Gaeini, Alireza Ghardashi Afousi Data Collection: Fahime Fatahi, Soheila Adeli Data Analysis: Reza Nouri, Fahimeh Fatahi Manuscript Writing: Fahimeh Fatahi, Alireza Ghardashi Afousi Review and Editing: Fahimeh Fatahi, Abbasali Gaeini, Alireza Ghardashi Afousi Funding Responsibility: Soheila Adeli, Fahimeh Fatahi Literature Review: Reza Nouri, Fahimeh Fatahi Project Management: Abbasali Gaeini, Alireza Ghardashi Afousi Additional Contributions: Soheila Adeli Conflict of Interest The authors declare no conflicts of interest regarding this study. Acknowledgments We gratefully acknowledge Ms. Aida Sabouri for her valuable assistance with animal modeling.

https://spj.ssrc.ac.ir/article_4841_05ee45de8d877c3949760a94fa691533.pdf

Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Effect of Combined Exercise Sequence on Cardiovascular Risk Factors, C-Reactive Protein, Homocysteine, and Insulin Resistance in Obese Women with Type 2 Diabetes The Effect of Combined Exercise Sequence on Cardiovascular Risk Factors, C-Reactive Protein, Homocysteine, and Insulin Resistance in Obese Women with Type 2 Diabetes 74 90 4835 10.22089/spj.2025.17879.2369 FA Sheida Naghizadeh Department of Physical Education, Boj.C., Islamic Azad University, Bojnourd, Iran 0009-0001-9701-6281 Ali Golestani Department of Sport Sciences, Faculty of Humanity, Bojnourd University, Bojnourd, Iran 0000-0002-5116-8722 Habibeh Sadat Shakeri Department of Endocrinology and Internal Medicine, Faculty of Medicine, North Khorasan University of Medical Sciences, Bojnurd, Iran 0000-0001-9473-1397 Journal Article 2025 05 01 Background and Purpose Abdominal obesity, in particular, represents a cluster of risk factors that significantly increase the likelihood of developing cardiovascular disease and type 2 diabetes. At present, in addition to the classical cardiovascular risk factors—including obesity, hypertension, hypercholesterolemia, metabolic syndrome, physical inactivity, and smoking—several novel risk markers such as homocysteine and C-reactive protein have been identified, which independently and more strongly predict the risk of cardiovascular diseases compared with traditional risk factors.C-reactive protein (CRP) is a member of the pentraxin family composed of five 23-kDa subunits, derived from the liver and the endothelium of coronary arteries, and it contributes to vascular injury and an increased risk of atherosclerosis through mechanisms such as enhancing the generation of reactive oxygen species, reducing nitric oxide activity, and stimulating macrophage-mediated uptake of low-density lipoprotein (LDL) cholesterol. Homocysteine, a sulfur-containing amino acid produced during methionine metabolism, induces endothelial dysfunction by promoting LDL oxidation, suppressing nitric oxide activity and impairing arterial vasodilation, inhibiting nitric oxide synthase function, activating platelets, and generating oxidative stress; its metabolism is dependent on several nutritional and genetic factors. Elevated levels of homocysteine can lead to a prothrombotic state, oxidative stress, and endothelial dysfunction . Numerous studies have demonstrated that hyperhomocysteinemia is a significant risk factor for the development of cardiovascular diseases . However, limited data exist regarding the impact of elevated plasma homocysteine levels on the increased risk of cardiovascular disease in individuals with diabetes. Several studies have reported higher homocysteine concentrations in diabetic patients compared with non-diabetic individuals. According to a meta-analysis of prospective studies, a 5 μmol/L increase in circulating homocysteine levels is associated with a 33.6% rise in all-cause mortality risk.Among the various factors influencing serum homocysteine and CRP concentrations, exercise and physical activity play a significant role. Physical activity induces several biochemical changes that may affect homocysteine metabolism, with oxidative stress likely serving as a major mediator in this process . Aerobic training, a specific form of exercise, is commonly employed to enhance maximal oxygen consumption. The combination of resistance and aerobic training has been suggested as an effective strategy to improve overall performance. Evidence from research indicates that combined resistance–aerobic training, compared with performing each type of exercise alone, elicits distinct resistance-related adaptations that are closely linked to hormonal responses. Relevant studies addressing these effects individually are summarized below.Findings from existing research highlight the dual nature of the immune response to exercise. Intense and prolonged physical activity, characterized by high mechanical stress and eccentric loading, can cause muscle damage and trigger the release of cytokines, whereas exercise involving lower mechanical stress is associated with reduced CRP levels. Currently, limited evidence exists regarding the effects of resistance training—particularly when combined with endurance training—on cardiovascular and immune markers. Therefore, based on these considerations, the present study was conducted to address the central question of whether the sequence of combined training influences cardiovascular risk factors, including C-reactive protein, homocysteine, and insulin resistance, in obese women with type 2 diabetes. Materials and Methods This study employed a quasi-experimental pre-test and post-test design. A total of 36 women aged 40–60 years from Bojnord voluntarily participated and were recruited based on the required research criteria through announcements and informed instructions. After initial assessments and completion of questionnaires, participants were randomly assigned into three groups of 14, which, after attrition, were reduced to 12 per group: a control group that did not perform any exercise, an experimental group that performed aerobic training before resistance training, and a second experimental group that performed aerobic training after resistance training. Following the completion of medical questionnaires, informed consent forms, and readiness assessments, participants were admitted to the study based on inclusion criteria such as absence of medication or supplement use, no history of cardiovascular disease or infections affecting immune factors, and no engagement in regular or intense exercise in the preceding six months. All participants confirmed their readiness to adhere to the training protocol. Blood samples were collected from all three groups during both pre-test and post-test stages, and participants were instructed to refrain from engaging in strenuous physical activity for at least 48 hours before blood collection. Results In this study, the highest mean age was observed in the aerobic–resistance group at 49.7 years. The lowest mean height also belonged to the aerobic–resistance group at 154.2 cm, while the highest mean weight was recorded in the same group at 71.6 kg. The results indicated that ten weeks of combined aerobic–resistance training produced significant effects on HbA1c, homocysteine (Hcy), C-reactive protein (CRP), and insulin resistance in women with type 2 diabetes. In contrast, ten weeks of combined resistance–aerobic training did not significantly affect Hcy levels, although it had significant effects on insulin resistance, CRP, and HbA1c. No significant changes were observed in the control group. The results also showed that ten weeks of combined aerobic–resistance training had no significant effects on the lipid profile (LDL, HDL, TG, TC) of women with type 2 diabetes, except for HDL and TC. Similarly, combined resistance–aerobic training did not significantly affect the lipid profile (LDL, HDL, TG, TC) except for HDL. No significant changes were observed in the control group. Conclusion The findings of this study demonstrated significant differences in HbA1c, homocysteine (Hcy), CRP, and insulin resistance indices before and after aerobic–resistance training. In contrast, ten weeks of resistance–aerobic training did not significantly affect Hcy but did produce significant improvements in insulin resistance, CRP, and HbA1c. Insulin levels before and after training were not significantly different in either experimental group, and no significant differences were observed between the experimental and control groups. Comparison of HbA1c values showed a significant reduction following aerobic–resistance training, whereas no significant change was observed with resistance–aerobic training. After ten weeks of aerobic–resistance training, total cholesterol decreased significantly while HDL increased significantly, whereas in the resistance–aerobic group, only HDL showed a significant increase. These results suggest that aerobic–resistance training may help improve insulin resistance through reductions in fasting glucose and insulin levels. Article Message The sequence of combined exercises significantly affects metabolic and inflammatory markers in obese women with type 2 diabetes. Performing aerobic exercise before resistance training yields greater improvements in insulin resistance, homocysteine, and CRP levels, offering a practical approach for glycemic control and cardiovascular risk reduction. Ethical Considerations All stages of this study were conducted in accordance with the ethical principles of human research approved by the Islamic Azad University. Before participation, informed written consent was obtained from all subjects, and they were assured that their personal information and collected data would be used solely for research purposes. Participants were free to withdraw at any time, and all training sessions were performed under safe conditions and medical supervision. Authors’ Contributions Conceptualization: Sheida Naghizadeh Data Collection and Exercise Protocol Implementation: Shida Naghizadeh and Habibeh-Sadat Shakeri Laboratory Experiments and Sample Preparation: Habibeh-Sadat Shakeri Data Analysis and Interpretation of Results: Shida Naghizadeh and Habibeh-Sadat Shakeri Manuscript Writing: Shida Naghizadeh Review and Editing: Ali Golestani and Habibeh-Sadat Shakeri Literature Review: Ali Golestani and Habibeh-Sadat Shakeri Project Management: Ali Golestani and Habibeh-Sadat Shakeri Conflict of Interest The authors declare that there is no conflict of interest regarding the authorship and publication of this article. Acknowledgments The authors thanks all participants for their cooperation and contribution to the study. Background and Purpose Abdominal obesity, in particular, represents a cluster of risk factors that significantly increase the likelihood of developing cardiovascular disease and type 2 diabetes. At present, in addition to the classical cardiovascular risk factors—including obesity, hypertension, hypercholesterolemia, metabolic syndrome, physical inactivity, and smoking—several novel risk markers such as homocysteine and C-reactive protein have been identified, which independently and more strongly predict the risk of cardiovascular diseases compared with traditional risk factors.C-reactive protein (CRP) is a member of the pentraxin family composed of five 23-kDa subunits, derived from the liver and the endothelium of coronary arteries, and it contributes to vascular injury and an increased risk of atherosclerosis through mechanisms such as enhancing the generation of reactive oxygen species, reducing nitric oxide activity, and stimulating macrophage-mediated uptake of low-density lipoprotein (LDL) cholesterol. Homocysteine, a sulfur-containing amino acid produced during methionine metabolism, induces endothelial dysfunction by promoting LDL oxidation, suppressing nitric oxide activity and impairing arterial vasodilation, inhibiting nitric oxide synthase function, activating platelets, and generating oxidative stress; its metabolism is dependent on several nutritional and genetic factors. Elevated levels of homocysteine can lead to a prothrombotic state, oxidative stress, and endothelial dysfunction . Numerous studies have demonstrated that hyperhomocysteinemia is a significant risk factor for the development of cardiovascular diseases . However, limited data exist regarding the impact of elevated plasma homocysteine levels on the increased risk of cardiovascular disease in individuals with diabetes. Several studies have reported higher homocysteine concentrations in diabetic patients compared with non-diabetic individuals. According to a meta-analysis of prospective studies, a 5 μmol/L increase in circulating homocysteine levels is associated with a 33.6% rise in all-cause mortality risk.Among the various factors influencing serum homocysteine and CRP concentrations, exercise and physical activity play a significant role. Physical activity induces several biochemical changes that may affect homocysteine metabolism, with oxidative stress likely serving as a major mediator in this process . Aerobic training, a specific form of exercise, is commonly employed to enhance maximal oxygen consumption. The combination of resistance and aerobic training has been suggested as an effective strategy to improve overall performance. Evidence from research indicates that combined resistance–aerobic training, compared with performing each type of exercise alone, elicits distinct resistance-related adaptations that are closely linked to hormonal responses. Relevant studies addressing these effects individually are summarized below.Findings from existing research highlight the dual nature of the immune response to exercise. Intense and prolonged physical activity, characterized by high mechanical stress and eccentric loading, can cause muscle damage and trigger the release of cytokines, whereas exercise involving lower mechanical stress is associated with reduced CRP levels. Currently, limited evidence exists regarding the effects of resistance training—particularly when combined with endurance training—on cardiovascular and immune markers. Therefore, based on these considerations, the present study was conducted to address the central question of whether the sequence of combined training influences cardiovascular risk factors, including C-reactive protein, homocysteine, and insulin resistance, in obese women with type 2 diabetes. Materials and Methods This study employed a quasi-experimental pre-test and post-test design. A total of 36 women aged 40–60 years from Bojnord voluntarily participated and were recruited based on the required research criteria through announcements and informed instructions. After initial assessments and completion of questionnaires, participants were randomly assigned into three groups of 14, which, after attrition, were reduced to 12 per group: a control group that did not perform any exercise, an experimental group that performed aerobic training before resistance training, and a second experimental group that performed aerobic training after resistance training. Following the completion of medical questionnaires, informed consent forms, and readiness assessments, participants were admitted to the study based on inclusion criteria such as absence of medication or supplement use, no history of cardiovascular disease or infections affecting immune factors, and no engagement in regular or intense exercise in the preceding six months. All participants confirmed their readiness to adhere to the training protocol. Blood samples were collected from all three groups during both pre-test and post-test stages, and participants were instructed to refrain from engaging in strenuous physical activity for at least 48 hours before blood collection. Results In this study, the highest mean age was observed in the aerobic–resistance group at 49.7 years. The lowest mean height also belonged to the aerobic–resistance group at 154.2 cm, while the highest mean weight was recorded in the same group at 71.6 kg. The results indicated that ten weeks of combined aerobic–resistance training produced significant effects on HbA1c, homocysteine (Hcy), C-reactive protein (CRP), and insulin resistance in women with type 2 diabetes. In contrast, ten weeks of combined resistance–aerobic training did not significantly affect Hcy levels, although it had significant effects on insulin resistance, CRP, and HbA1c. No significant changes were observed in the control group. The results also showed that ten weeks of combined aerobic–resistance training had no significant effects on the lipid profile (LDL, HDL, TG, TC) of women with type 2 diabetes, except for HDL and TC. Similarly, combined resistance–aerobic training did not significantly affect the lipid profile (LDL, HDL, TG, TC) except for HDL. No significant changes were observed in the control group. Conclusion The findings of this study demonstrated significant differences in HbA1c, homocysteine (Hcy), CRP, and insulin resistance indices before and after aerobic–resistance training. In contrast, ten weeks of resistance–aerobic training did not significantly affect Hcy but did produce significant improvements in insulin resistance, CRP, and HbA1c. Insulin levels before and after training were not significantly different in either experimental group, and no significant differences were observed between the experimental and control groups. Comparison of HbA1c values showed a significant reduction following aerobic–resistance training, whereas no significant change was observed with resistance–aerobic training. After ten weeks of aerobic–resistance training, total cholesterol decreased significantly while HDL increased significantly, whereas in the resistance–aerobic group, only HDL showed a significant increase. These results suggest that aerobic–resistance training may help improve insulin resistance through reductions in fasting glucose and insulin levels. Article Message The sequence of combined exercises significantly affects metabolic and inflammatory markers in obese women with type 2 diabetes. Performing aerobic exercise before resistance training yields greater improvements in insulin resistance, homocysteine, and CRP levels, offering a practical approach for glycemic control and cardiovascular risk reduction. Ethical Considerations All stages of this study were conducted in accordance with the ethical principles of human research approved by the Islamic Azad University. Before participation, informed written consent was obtained from all subjects, and they were assured that their personal information and collected data would be used solely for research purposes. Participants were free to withdraw at any time, and all training sessions were performed under safe conditions and medical supervision. Authors’ Contributions Conceptualization: Sheida Naghizadeh Data Collection and Exercise Protocol Implementation: Shida Naghizadeh and Habibeh-Sadat Shakeri Laboratory Experiments and Sample Preparation: Habibeh-Sadat Shakeri Data Analysis and Interpretation of Results: Shida Naghizadeh and Habibeh-Sadat Shakeri Manuscript Writing: Shida Naghizadeh Review and Editing: Ali Golestani and Habibeh-Sadat Shakeri Literature Review: Ali Golestani and Habibeh-Sadat Shakeri Project Management: Ali Golestani and Habibeh-Sadat Shakeri Conflict of Interest The authors declare that there is no conflict of interest regarding the authorship and publication of this article. Acknowledgments The authors thanks all participants for their cooperation and contribution to the study.

https://spj.ssrc.ac.ir/article_4835_20546457187cf3d52ea86538403e47cc.pdf

Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Effect of Intensive Aerobic Training with Omega-3 Supplementation on Level of Pro-Inflammatory Cytokines in Liver Tissue of Obese Mice The Effect of Intensive Aerobic Training with Omega-3 Supplementation on Level of Pro-Inflammatory Cytokines in Liver Tissue of Obese Mice 91 106 4901 10.22089/spj.2025.18512.2402 FA Kosar Anbari Department of Sport Sciences, Faculty of Humanities, Il.C., Islamic Azad University, Ilam, Iran 0009-0000-3072-4251 Abdolhossein Taheri Kalani Department of Sport Sciences, Faculty of Humanities, Il.C., Islamic Azad University, Ilam, Iran 0000-0003-3668-0266 Mahnaz Omidi Department of Sport Sciences, Faculty of Humanities, Il.C., Islamic Azad University, Ilam, Iran 0000-0002-4631-7308 Journal Article 2025 09 22 Background and Purpose An imbalance between energy intake and expenditure leads to overweight and obesity, which is closely associated with excessive fat accumulation in adipose tissue. Adipocytes, the primary cells in adipose tissue, possess the capability to produce and secrete pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). These cytokines play a pivotal role in the pathogenesis of various liver diseases, including non-alcoholic fatty liver disease (NAFLD), by promoting chronic low-grade inflammation and metabolic dysfunction. Accumulating evidence suggests that structured exercise training can significantly improve liver cell function, potentially through mechanisms involving enhanced mitochondrial biogenesis, improved insulin sensitivity, and reduced oxidative stress, although the precise pathways remain incompletely elucidated. Nutrition emerges as another critical modulator of inflammatory processes, with diets enriched in omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), demonstrating robust positive effects on human health, including prevention of inflammation and cardiometabolic disorders. Chronic inflammation constitutes a central driver in the development of metabolic disorders such as obesity, type 2 diabetes, and NAFLD, underscoring the necessity to explore combined exercise and dietary interventions capable of improving overall health by modulating key inflammatory markers. Despite extensive research on individual interventions, available evidence reveals a notable gap regarding the synergistic or potentially interfering effects of concurrent exercise training and ω-3 supplementation. Therefore, the present study was designed to investigate the effect of intensive aerobic training combined with ω-3 supplementation on the protein levels of pro-inflammatory cytokines IL-6 and TNF-α in the liver tissue of obese mice, providing insights into their combined therapeutic potential for obesity-related liver inflammation. Materials and Methods This experimental study involved 30 male mice aged 12-14 weeks, randomly divided into five groups (n=6 per group): normal diet (ND), high-fat diet (HFD), HFD+ω-3 supplementation, HFD+Training (HFD+T), and HFD+ω-3+Training (HFD+ω-3+T). All animals were housed under strictly controlled environmental conditions, including an average temperature of 22±4°C, relative humidity of 40-50%, and a standardized 12:12-hour light/dark cycle, with unrestricted access to water and group-specific food. The entire protocol spanned 16 weeks, comprising two weeks of familiarization, eight weeks of obesity induction, and six weeks of intensive aerobic training intervention with or without ω-3 supplementation. Mice in the ND group received a standard chow diet throughout the study, formulated with 15% fat, 25% protein, and 60% carbohydrates to maintain normal body composition. Following the initial two-week acclimation period on standard diet, obesity was induced in the intervention groups (HFD, HFD+ω-3, HFD+T, HFD+ω-3+T) using a custom high-fat diet (HFD) sourced from the Royan Institute of Isfahan. This HFD composition included 45% fat, 35% carbohydrates, and 20% protein (in kcal), delivering 4.60 kcal/g of energy, which effectively promoted rapid weight gain and metabolic alterations characteristic of obesity. By the conclusion of the eight-week induction phase, the average body weight of obese mice reached 32.89 g, confirming successful model establishment. Subsequently, the HFD was discontinued across all intervention groups, reverting them to the standard diet to isolate the effects of the subsequent interventions. The intensive aerobic training protocol was administered over six weeks, consisting of five sessions per week. Each session involved progressive treadmill running progressing from 10 to 50 minutes at an intensity of 70-75% of maximal oxygen consumption (VO₂max), with speeds ranging from 25-30 meters per minute and a 15% incline to simulate high-intensity aerobic demand. Groups receiving ω-3 supplementation (HFD+ω-3 and HFD+ω-3+T) were administered 500 mg/kg body mass of EPA and DHA daily via oral gavage, ensuring consistent dosing. At study termination, liver tissues were harvested, and protein levels of IL-6 and TNF-α were quantified using the Bradford assay for total protein normalization and enzyme-linked immunosorbent assay (ELISA) for cytokine detection, following standard laboratory protocols. Results Statistical analysis revealed significant differences in body mass across the experimental groups (p<0.05). Tukey's post hoc test indicated that at the onset of the intervention phase, body mass in the HFD, HFD+T, HFD+ω-3, and HFD+ω-3+T groups was markedly higher than in the ND group (all comparisons p=0.0001), validating the obesity induction. At the intervention's conclusion, body mass remained significantly elevated in the HFD group (p=0.0001), HFD+T group (p=0.029), and HFD+ω-3 group (p=0.031) relative to ND, reflecting persistent obesity effects; however, no significant difference emerged between HFD+ω-3+T and ND (p=0.067), suggesting effective mitigation by the combined intervention. Regarding inflammatory markers, data analysis demonstrated a significant elevation in hepatic IL-6 and TNF-α protein levels in the HFD+T (p=0.0001), HFD+ω-3 (p=0.0001), and HFD+ω-3+T (p=0.012) groups compared to ND, consistent with obesity-induced inflammation. Critically, all intervention groups exhibited substantial reductions versus HFD (p=0.0001). Furthermore, IL-6 and TNF-α levels were significantly lower in HFD+ω-3 (p=0.042) and HFD+ω-3+T (p=0.0001) compared to HFD+T alone, although the difference between HFD+ω-3 and HFD+ω-3+T was not statistically significant (p=0.054), indicating additive rather than fully synergistic suppression in cytokine reduction. Conclusion The findings confirm that HFD markedly elevates IL-6 and TNF-α protein levels in liver tissue relative to ND, exacerbating pro-inflammatory states. The mechanisms underlying HFD-induced increases in these markers remain incompletely understood but are hypothesized to involve adipose tissue expansion and ectopic fat deposition in the liver, leading to hepatocyte damage through inflammatory cytokine release and reactive oxygen species (ROS) generation. In contrast, intensive aerobic training, ω-3 supplementation, and their combination effectively attenuated IL-6 and TNF-α levels. Notably, ω-3 supplementation outperformed intensive aerobic training alone in cytokine reduction, highlighting its potent anti-inflammatory properties. Regular aerobic exercise mitigates liver inflammation in models of nonalcoholic fatty liver disease by decreasing macrophage infiltration, elevating antioxidant enzyme expression (e.g., superoxide dismutase, catalase), and normalizing ROS homeostasis. ω-3 fatty acids exert superior anti-inflammatory effects by serving as precursors for specialized pro-resolving mediators such as resolvins, protectins, and maresins, which actively resolve inflammation at sites of injury. These PUFAs competitively inhibit pro-inflammatory ω-6 fatty acid derivatives—including arachidonic acid metabolites like prostaglandins, leukotrienes, and lipoxins—produced via cyclooxygenase and lipoxygenase pathways. The study's culminating observation—a synergistic interaction between intensive aerobic exercise and ω-3 supplementation—manifested in enhanced suppression of IL-6 and TNF-α protein expression in obese mice liver tissue, suggesting complementary mechanisms that amplify anti-inflammatory outcomes beyond individual therapies. Article Message The combination of intensive aerobic exercise and ω-3 supplementation demonstrates a clear synergistic effect in modulating inflammatory markers within liver tissue of obese models. Considering chronic inflammation's foundational role in fatty liver disease pathogenesis and broader metabolic disorders, these findings advocate exercise and targeted nutritional interventions as efficacious strategies for disease management, extending beyond inflammation control to metabolic restoration. Nevertheless, additional mechanistic studies are warranted to delineate precise signaling pathways and translate these benefits to clinical populations. Ethical Considerations The ethics review board of Islamic Azad University, Ilam branch, approved the present study with the code of IR.IAU.ILAM.REC.1402.063. Authors’ Contributions Conceptualization: Abdolhossein Taheri Kalani, Kosar Anbari Data Collection: Kosar Anbari, Mahnaz Omidi Data Analysis: Abdolhossein Taheri Kalani, Mahnaz Omidi Manuscript Writing: Mahnaz Omidi, Kosar Anbari Review and Editing: Abdolhossein Taheri Kalani Literature Review: Abdolhossein Taheri Kalani, Mahnaz Omidi, Kosar Anbari Project Manager: Abdolhossein Taheri Kalani Any Other Contribution: Avin Stem Gen Bio Health Inc. performed experimental analysis. Conflict of Interest The authors declare no conflicts of interest regarding the publication of this study. Acknowledgments The authors thank Avin Stem Gen Bio Health Inc. for technical support performing. Background and Purpose An imbalance between energy intake and expenditure leads to overweight and obesity, which is closely associated with excessive fat accumulation in adipose tissue. Adipocytes, the primary cells in adipose tissue, possess the capability to produce and secrete pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α). These cytokines play a pivotal role in the pathogenesis of various liver diseases, including non-alcoholic fatty liver disease (NAFLD), by promoting chronic low-grade inflammation and metabolic dysfunction. Accumulating evidence suggests that structured exercise training can significantly improve liver cell function, potentially through mechanisms involving enhanced mitochondrial biogenesis, improved insulin sensitivity, and reduced oxidative stress, although the precise pathways remain incompletely elucidated. Nutrition emerges as another critical modulator of inflammatory processes, with diets enriched in omega-3 (ω-3) polyunsaturated fatty acids (PUFAs), particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), demonstrating robust positive effects on human health, including prevention of inflammation and cardiometabolic disorders. Chronic inflammation constitutes a central driver in the development of metabolic disorders such as obesity, type 2 diabetes, and NAFLD, underscoring the necessity to explore combined exercise and dietary interventions capable of improving overall health by modulating key inflammatory markers. Despite extensive research on individual interventions, available evidence reveals a notable gap regarding the synergistic or potentially interfering effects of concurrent exercise training and ω-3 supplementation. Therefore, the present study was designed to investigate the effect of intensive aerobic training combined with ω-3 supplementation on the protein levels of pro-inflammatory cytokines IL-6 and TNF-α in the liver tissue of obese mice, providing insights into their combined therapeutic potential for obesity-related liver inflammation. Materials and Methods This experimental study involved 30 male mice aged 12-14 weeks, randomly divided into five groups (n=6 per group): normal diet (ND), high-fat diet (HFD), HFD+ω-3 supplementation, HFD+Training (HFD+T), and HFD+ω-3+Training (HFD+ω-3+T). All animals were housed under strictly controlled environmental conditions, including an average temperature of 22±4°C, relative humidity of 40-50%, and a standardized 12:12-hour light/dark cycle, with unrestricted access to water and group-specific food. The entire protocol spanned 16 weeks, comprising two weeks of familiarization, eight weeks of obesity induction, and six weeks of intensive aerobic training intervention with or without ω-3 supplementation. Mice in the ND group received a standard chow diet throughout the study, formulated with 15% fat, 25% protein, and 60% carbohydrates to maintain normal body composition. Following the initial two-week acclimation period on standard diet, obesity was induced in the intervention groups (HFD, HFD+ω-3, HFD+T, HFD+ω-3+T) using a custom high-fat diet (HFD) sourced from the Royan Institute of Isfahan. This HFD composition included 45% fat, 35% carbohydrates, and 20% protein (in kcal), delivering 4.60 kcal/g of energy, which effectively promoted rapid weight gain and metabolic alterations characteristic of obesity. By the conclusion of the eight-week induction phase, the average body weight of obese mice reached 32.89 g, confirming successful model establishment. Subsequently, the HFD was discontinued across all intervention groups, reverting them to the standard diet to isolate the effects of the subsequent interventions. The intensive aerobic training protocol was administered over six weeks, consisting of five sessions per week. Each session involved progressive treadmill running progressing from 10 to 50 minutes at an intensity of 70-75% of maximal oxygen consumption (VO₂max), with speeds ranging from 25-30 meters per minute and a 15% incline to simulate high-intensity aerobic demand. Groups receiving ω-3 supplementation (HFD+ω-3 and HFD+ω-3+T) were administered 500 mg/kg body mass of EPA and DHA daily via oral gavage, ensuring consistent dosing. At study termination, liver tissues were harvested, and protein levels of IL-6 and TNF-α were quantified using the Bradford assay for total protein normalization and enzyme-linked immunosorbent assay (ELISA) for cytokine detection, following standard laboratory protocols. Results Statistical analysis revealed significant differences in body mass across the experimental groups (p<0.05). Tukey's post hoc test indicated that at the onset of the intervention phase, body mass in the HFD, HFD+T, HFD+ω-3, and HFD+ω-3+T groups was markedly higher than in the ND group (all comparisons p=0.0001), validating the obesity induction. At the intervention's conclusion, body mass remained significantly elevated in the HFD group (p=0.0001), HFD+T group (p=0.029), and HFD+ω-3 group (p=0.031) relative to ND, reflecting persistent obesity effects; however, no significant difference emerged between HFD+ω-3+T and ND (p=0.067), suggesting effective mitigation by the combined intervention. Regarding inflammatory markers, data analysis demonstrated a significant elevation in hepatic IL-6 and TNF-α protein levels in the HFD+T (p=0.0001), HFD+ω-3 (p=0.0001), and HFD+ω-3+T (p=0.012) groups compared to ND, consistent with obesity-induced inflammation. Critically, all intervention groups exhibited substantial reductions versus HFD (p=0.0001). Furthermore, IL-6 and TNF-α levels were significantly lower in HFD+ω-3 (p=0.042) and HFD+ω-3+T (p=0.0001) compared to HFD+T alone, although the difference between HFD+ω-3 and HFD+ω-3+T was not statistically significant (p=0.054), indicating additive rather than fully synergistic suppression in cytokine reduction. Conclusion The findings confirm that HFD markedly elevates IL-6 and TNF-α protein levels in liver tissue relative to ND, exacerbating pro-inflammatory states. The mechanisms underlying HFD-induced increases in these markers remain incompletely understood but are hypothesized to involve adipose tissue expansion and ectopic fat deposition in the liver, leading to hepatocyte damage through inflammatory cytokine release and reactive oxygen species (ROS) generation. In contrast, intensive aerobic training, ω-3 supplementation, and their combination effectively attenuated IL-6 and TNF-α levels. Notably, ω-3 supplementation outperformed intensive aerobic training alone in cytokine reduction, highlighting its potent anti-inflammatory properties. Regular aerobic exercise mitigates liver inflammation in models of nonalcoholic fatty liver disease by decreasing macrophage infiltration, elevating antioxidant enzyme expression (e.g., superoxide dismutase, catalase), and normalizing ROS homeostasis. ω-3 fatty acids exert superior anti-inflammatory effects by serving as precursors for specialized pro-resolving mediators such as resolvins, protectins, and maresins, which actively resolve inflammation at sites of injury. These PUFAs competitively inhibit pro-inflammatory ω-6 fatty acid derivatives—including arachidonic acid metabolites like prostaglandins, leukotrienes, and lipoxins—produced via cyclooxygenase and lipoxygenase pathways. The study's culminating observation—a synergistic interaction between intensive aerobic exercise and ω-3 supplementation—manifested in enhanced suppression of IL-6 and TNF-α protein expression in obese mice liver tissue, suggesting complementary mechanisms that amplify anti-inflammatory outcomes beyond individual therapies. Article Message The combination of intensive aerobic exercise and ω-3 supplementation demonstrates a clear synergistic effect in modulating inflammatory markers within liver tissue of obese models. Considering chronic inflammation's foundational role in fatty liver disease pathogenesis and broader metabolic disorders, these findings advocate exercise and targeted nutritional interventions as efficacious strategies for disease management, extending beyond inflammation control to metabolic restoration. Nevertheless, additional mechanistic studies are warranted to delineate precise signaling pathways and translate these benefits to clinical populations. Ethical Considerations The ethics review board of Islamic Azad University, Ilam branch, approved the present study with the code of IR.IAU.ILAM.REC.1402.063. Authors’ Contributions Conceptualization: Abdolhossein Taheri Kalani, Kosar Anbari Data Collection: Kosar Anbari, Mahnaz Omidi Data Analysis: Abdolhossein Taheri Kalani, Mahnaz Omidi Manuscript Writing: Mahnaz Omidi, Kosar Anbari Review and Editing: Abdolhossein Taheri Kalani Literature Review: Abdolhossein Taheri Kalani, Mahnaz Omidi, Kosar Anbari Project Manager: Abdolhossein Taheri Kalani Any Other Contribution: Avin Stem Gen Bio Health Inc. performed experimental analysis. Conflict of Interest The authors declare no conflicts of interest regarding the publication of this study. Acknowledgments The authors thank Avin Stem Gen Bio Health Inc. for technical support performing.

https://spj.ssrc.ac.ir/article_4901_a08e32d2f9a8b78894d964ec7fd4172e.pdf

Sport Sciences Research Institute Sport Physiology 2322-164X 17 68 2025 12 22 The Effect of Aerobic Training and Adenosine Injection on Rat Brain HIF-1α Gene Expression Following Ischemic Stroke The Effect of Aerobic Training and Adenosine Injection on Rat Brain HIF-1α Gene Expression Following Ischemic Stroke 107 122 4819 10.22089/spj.2025.18359.2391 FA Saeed Ramezani Department of Exercise Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran 0000-0003-4876-9858 Mahtab Moazami Department of Exercise Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Nahid Bijeh Department of Exercise Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Amir Rashid Lamir Department of Exercise Physiology, Faculty of Sport Sciences, Ferdowsi University of Mashhad, Mashhad, Iran Journal Article 2023 10 13 <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Background and Purpose <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ischemic stroke, resulting from vascular occlusion, induces hypoxia and neuronal death, leading to widespread functional impairments. In response to oxygen deprivation, hypoxia-inducible factor-1 alpha (HIF-1α) is stabilized and translocated to the nucleus, where it upregulates target genes such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), and erythropoietin (EPO), thereby promoting angiogenesis and neuronal survival. The activity of HIF-1α is further modulated by intracellular signaling cascades including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA pathways. Aerobic exercise, a cost-effective nonpharmacological strategy, enhances cerebral blood flow, activates AMP-activated protein kinase (AMPK), and elevates neurotrophic factors like brain-derived neurotrophic factor (BDNF). These changes augment HIF-1α expression, stimulate angiogenesis and neurogenesis, and attenuate post-ischemic inflammation. Preclinical studies confirm that regular aerobic training improves cognitive function, promotes hippocampal neurogenesis, and offers substantial protection against cerebral ischemia. Adenosine, an endogenous nucleoside produced during ATP hydrolysis under stress, regulates immune responses and exerts neuroprotective effects via its four receptors (A1, A2A, A2B, A3). Activation of A2A receptors enhances HIF-1α expression and repair gene activation through PI3K/Akt and cAMP/PKA signaling, while A1 receptor engagement reduces infarct size and mitigates neurological deficits by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2)/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling and pyroptosis. The ATP–adenosine axis thereby shifts the inflammatory milieu toward neuroprotection. Despite strong evidence supporting the individual benefits of aerobic exercise and adenosine, their combined effects on HIF-1α–mediated neuroprotection remain insufficiently studied. This investigation aims to evaluate the combined impact of aerobic exercise and adenosine administration on hippocampal HIF-1α gene expression in a rat model of ischemic stroke, assessing potential synergistic effects on neuronal survival and cognitive recovery. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Materials and Methods <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">This experimental laboratory study employed a multi-group post-test design involving fifty adult male Wistar rats (8–10 weeks old, 240–270 g). Animals were acclimatized for one week under controlled conditions (22 ± 3 °C; 55 ± 3% humidity; 12-hour light/dark cycle), with ad libitum access to water and standard chow (5 g per 100 g body weight). Ischemic stroke was induced under intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine (4–5 mg/kg) by occluding the right common carotid artery for 45 minutes using a vascular clamp. Neurological deficits were verified by stereomicroscopic assessment, followed by a 5-minute reperfusion period. Rats were randomized into five groups (n=10 each): Sham (surgical procedure without occlusion), Stroke + Control (ischemia + saline), Stroke + Aerobic Exercise (AE), Stroke + Adenosine (Ad), and Stroke + Adenosine + Aerobic Exercise (Ad + AE). Adenosine (0.3 mg/kg) or saline was administered intraperitoneally 3 hours prior to exercise sessions daily. After three treadmill familiarization sessions (15 m/min for 10–15 minutes), rats underwent an eight-week aerobic protocol: five sessions weekly, each comprising a 5-minute warm-up, progressively increasing main bouts (from 20 m/min for 18 minutes in week 1 to 30 m/min for 50 minutes by week 8), and a 3-minute cool-down. Forty-eight hours post-intervention and following a 12-hour fast, hippocampal tissue was harvested. Total RNA was isolated using Qiagen kits; cDNA synthesis was performed with Fermentas reagents and Oligo dT primers. HIF-1α expression levels were quantified via reverse transcription quantitative PCR (RT-qPCR). Statistical analysis employed one-way ANOVA followed by Tukey’s post hoc tests in SPSS v26, with significance at p ≤ 0.05. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Findings <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Aerobic exercise, with or without adenosine, induced the most substantial body weight reductions (2.76% in the Stroke + Ad + AE group and 2.18% in the Stroke + AE group) and significantly elevated hippocampal HIF-1α expression compared to Sham, Stroke + Control, and Stroke + Ad groups (P < 0.01). No statistically significant difference was found between the Stroke + AE and Stroke + Ad + AE groups (P = 0.1), nor did the Stroke + Ad group differ from Sham or Stroke + Control groups. These results indicate that aerobic exercise is the principal stimulus for HIF-1α upregulation in this model. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Conclusion <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ischemic stroke, precipitated by sudden vascular occlusion, triggers hypoxic injury, neuronal death, and functional deficits. HIF-1α stabilization and nuclear translocation activate genes such as VEGF, GLUT1, and EPO, vital for angiogenesis and neuroprotection. This gene expression is intricately regulated by signaling pathways including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA. Aerobic exercise enhances cerebral perfusion, activates AMPK and SIRT1, and increases neurotrophic factors like BDNF, collectively promoting HIF-1α expression and suppressing inflammation. Exercise-induced shear stress stimulates endothelial nitric oxide synthase (eNOS), augmenting nitric oxide synthesis, which improves tissue perfusion and alleviates secondary ischemic damage. Adenosine, released during ATP catabolism, modulates immune response and cell survival through A1 and A2A receptor pathways. Activation of A2A receptors potentiates HIF-1α expression via PI3K/Akt and cAMP/PKA mechanisms, whereas A1 receptor activation diminishes infarct volume by repressing pyroptosis and NLRP3 inflammasome pathways. Although both interventions possess neuroprotective capabilities, this study demonstrates that aerobic training predominantly drives HIF-1α upregulation, with adenosine alone exerting limited effects and no additive benefit when combined with exercise. Future studies should optimize adenosine dosing regimens and administration timing to potentiate synergistic neuroprotection. These findings endorse aerobic exercise as a key nonpharmacological intervention to attenuate ischemic brain injury, promote cognitive recovery, and inform rehabilitation protocols for stroke survivors. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ethical Considerations <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Aerobic exercise constitutes an effective nonpharmacological modality offering neuroprotection in ischemic stroke by modulating molecular pathways centered on HIF-expression, its combined use with aerobic exercise may enhance cellular repair mechanisms. These data support the integration of structured physical training with or without adjunct pharmacological agents to advance innovative stroke rehabilitation strategies and improve neuronal outcomes. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Conflicts of Interest <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">This research was conducted as part of a doctoral dissertation without external funding. The authors report no conflicts of interest related to this work. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Acknowledgement <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">We express our sincere gratitude to all individuals who supported us throughout this research. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Background and Purpose <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ischemic stroke, resulting from vascular occlusion, induces hypoxia and neuronal death, leading to widespread functional impairments. In response to oxygen deprivation, hypoxia-inducible factor-1 alpha (HIF-1α) is stabilized and translocated to the nucleus, where it upregulates target genes such as vascular endothelial growth factor (VEGF), glucose transporter 1 (GLUT1), and erythropoietin (EPO), thereby promoting angiogenesis and neuronal survival. The activity of HIF-1α is further modulated by intracellular signaling cascades including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA pathways. Aerobic exercise, a cost-effective nonpharmacological strategy, enhances cerebral blood flow, activates AMP-activated protein kinase (AMPK), and elevates neurotrophic factors like brain-derived neurotrophic factor (BDNF). These changes augment HIF-1α expression, stimulate angiogenesis and neurogenesis, and attenuate post-ischemic inflammation. Preclinical studies confirm that regular aerobic training improves cognitive function, promotes hippocampal neurogenesis, and offers substantial protection against cerebral ischemia. Adenosine, an endogenous nucleoside produced during ATP hydrolysis under stress, regulates immune responses and exerts neuroprotective effects via its four receptors (A1, A2A, A2B, A3). Activation of A2A receptors enhances HIF-1α expression and repair gene activation through PI3K/Akt and cAMP/PKA signaling, while A1 receptor engagement reduces infarct size and mitigates neurological deficits by inhibiting nuclear factor erythroid 2-related factor 2 (Nrf2)/NLR family pyrin domain containing 3 (NLRP3) inflammasome signaling and pyroptosis. The ATP–adenosine axis thereby shifts the inflammatory milieu toward neuroprotection. Despite strong evidence supporting the individual benefits of aerobic exercise and adenosine, their combined effects on HIF-1α–mediated neuroprotection remain insufficiently studied. This investigation aims to evaluate the combined impact of aerobic exercise and adenosine administration on hippocampal HIF-1α gene expression in a rat model of ischemic stroke, assessing potential synergistic effects on neuronal survival and cognitive recovery. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Materials and Methods <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">This experimental laboratory study employed a multi-group post-test design involving fifty adult male Wistar rats (8–10 weeks old, 240–270 g). Animals were acclimatized for one week under controlled conditions (22 ± 3 °C; 55 ± 3% humidity; 12-hour light/dark cycle), with ad libitum access to water and standard chow (5 g per 100 g body weight). Ischemic stroke was induced under intraperitoneal anesthesia with ketamine (60 mg/kg) and xylazine (4–5 mg/kg) by occluding the right common carotid artery for 45 minutes using a vascular clamp. Neurological deficits were verified by stereomicroscopic assessment, followed by a 5-minute reperfusion period. Rats were randomized into five groups (n=10 each): Sham (surgical procedure without occlusion), Stroke + Control (ischemia + saline), Stroke + Aerobic Exercise (AE), Stroke + Adenosine (Ad), and Stroke + Adenosine + Aerobic Exercise (Ad + AE). Adenosine (0.3 mg/kg) or saline was administered intraperitoneally 3 hours prior to exercise sessions daily. After three treadmill familiarization sessions (15 m/min for 10–15 minutes), rats underwent an eight-week aerobic protocol: five sessions weekly, each comprising a 5-minute warm-up, progressively increasing main bouts (from 20 m/min for 18 minutes in week 1 to 30 m/min for 50 minutes by week 8), and a 3-minute cool-down. Forty-eight hours post-intervention and following a 12-hour fast, hippocampal tissue was harvested. Total RNA was isolated using Qiagen kits; cDNA synthesis was performed with Fermentas reagents and Oligo dT primers. HIF-1α expression levels were quantified via reverse transcription quantitative PCR (RT-qPCR). Statistical analysis employed one-way ANOVA followed by Tukey’s post hoc tests in SPSS v26, with significance at p ≤ 0.05. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Findings <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Aerobic exercise, with or without adenosine, induced the most substantial body weight reductions (2.76% in the Stroke + Ad + AE group and 2.18% in the Stroke + AE group) and significantly elevated hippocampal HIF-1α expression compared to Sham, Stroke + Control, and Stroke + Ad groups (P < 0.01). No statistically significant difference was found between the Stroke + AE and Stroke + Ad + AE groups (P = 0.1), nor did the Stroke + Ad group differ from Sham or Stroke + Control groups. These results indicate that aerobic exercise is the principal stimulus for HIF-1α upregulation in this model. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Conclusion <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ischemic stroke, precipitated by sudden vascular occlusion, triggers hypoxic injury, neuronal death, and functional deficits. HIF-1α stabilization and nuclear translocation activate genes such as VEGF, GLUT1, and EPO, vital for angiogenesis and neuroprotection. This gene expression is intricately regulated by signaling pathways including PI3K/Akt/mTOR, MAPK/ERK, JAK/STAT, and cAMP/PKA. Aerobic exercise enhances cerebral perfusion, activates AMPK and SIRT1, and increases neurotrophic factors like BDNF, collectively promoting HIF-1α expression and suppressing inflammation. Exercise-induced shear stress stimulates endothelial nitric oxide synthase (eNOS), augmenting nitric oxide synthesis, which improves tissue perfusion and alleviates secondary ischemic damage. Adenosine, released during ATP catabolism, modulates immune response and cell survival through A1 and A2A receptor pathways. Activation of A2A receptors potentiates HIF-1α expression via PI3K/Akt and cAMP/PKA mechanisms, whereas A1 receptor activation diminishes infarct volume by repressing pyroptosis and NLRP3 inflammasome pathways. Although both interventions possess neuroprotective capabilities, this study demonstrates that aerobic training predominantly drives HIF-1α upregulation, with adenosine alone exerting limited effects and no additive benefit when combined with exercise. Future studies should optimize adenosine dosing regimens and administration timing to potentiate synergistic neuroprotection. These findings endorse aerobic exercise as a key nonpharmacological intervention to attenuate ischemic brain injury, promote cognitive recovery, and inform rehabilitation protocols for stroke survivors. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Ethical Considerations <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Aerobic exercise constitutes an effective nonpharmacological modality offering neuroprotection in ischemic stroke by modulating molecular pathways centered on HIF-expression, its combined use with aerobic exercise may enhance cellular repair mechanisms. These data support the integration of structured physical training with or without adjunct pharmacological agents to advance innovative stroke rehabilitation strategies and improve neuronal outcomes. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Conflicts of Interest <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">This research was conducted as part of a doctoral dissertation without external funding. The authors report no conflicts of interest related to this work. <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">Acknowledgement <span style="font-size: 11.0pt; line-height: 115%; mso-ascii-font-family: 'Times New Roman'; mso-ascii-theme-font: major-bidi; mso-hansi-font-family: 'Times New Roman'; mso-hansi-theme-font: major-bidi; mso-bidi-font-family: 'B Titr';">We express our sincere gratitude to all individuals who supported us throughout this research.

https://spj.ssrc.ac.ir/article_4819_30d454f09b771b9f65e3eaf6e00fa7bd.pdf