Background and Purpose
Brain aging is a time‑dependent physiological process closely associated with progressive mitochondrial dysfunction, particularly within the hippocampus. Mitochondrial quality control (MQC) is essential for maintaining neuronal integrity and protecting cells against metabolic and oxidative stress. Mitophagy, primarily regulated by the PINK1/Parkin pathway, is a key mechanism that preserves MQC by eliminating dysfunctional mitochondria; however, its efficiency declines with age, enhancing to oxidative stress, metabolic imbalance, and neurodegeneration. Exercise training is recognized as a potent modulator of MQC, with moderate‑intensity continuous training (MICT) consistently shown to enhance mitophagy‑related pathways, whereas the effects of high‑intensity interval training (HIIT) on mitophagy in the aging brain remain unclear inconsistent. Clarifying how different exercise intensities influence PINK1/Parkin-mediated mitophagy is essential for optimizing exercise interventions to protect brain function during aging. Therefore, this study aimed to investigate the effects of eight weeks of MICT and HIIT on some mitophagy markers (PINK1, Parkin) in the hippocampus of D-galactose–induced aging rats.
Materials and Methods
Twenty eight male Wistar rats (3 months old; 220 ± 40 g) were obtained and housed in the Animal Facility of the University of Isfahan under controlled environmental conditions (12-h light/dark cycle, 23 ± 2 °C, 50 ± 5% humidity) with ad libitum access to standard chow and water. After a one-week acclimation period, rats completed five days of treadmill familiarization (5–10 m/min, 0% incline, 10 min/day). Subsequently, the animals were randomly divided into four groups (n = 7 each): control (C), D-galactose- induced aging model (D-gal), moderate-intensity continuous training plus D-galactose (MICT+D), and high-intensity interval training plus D-galactose (HIIT+D). Aging was induced in all groups except the control group via daily intraperitoneal injections of D-galactose (150 mg/kg/day, dissolved in 0.9% saline) for eight weeks. was monitored weekly throughout the intervention. Baseline exercise capacity and maximal running velocity (Vmax) were determined using an incremental treadmill test. The protocol consisted of ten consecutive 3-min stages at 0% incline, beginning at 0.3 km/h and increasing by 0.3 km/h per stage until volitional exhaustion. Vmax was reassessed at weeks 2, 4, 6, and 8 to adjust training intensities. Exercise training was conducted five days per week for eight weeks and  each sessions included a 5 min warm up and cool down at 40% Vmax. The MICT protocol involved continuous running at 60% Vmax for 33.3 -38.5 min, while the HIIT protocol consisted of nine 1.5-min bouts at 85% Vmax interspersed with nine 2-min recovery bouts at 45% Vmax. Forty-eight hours after the final training session, and following a 12-h fast, rats were anesthetized using ketamine (90 mg/kg) and xylazine (10 mg/kg) and subsequently euthanized. Hippocampal tissues were rapidly dissected on an ice-cold surface, immediately snap-frozen in liquid nitrogen, and stored at −70 °C until analysis. To assess expression levels of PINK1 and Parkin by Western blotting, tissues were homogenized in a lysis buffer containing protease inhibitors, followed by centrifugation at 14,000 rpm for 10 minutes at 4 °C. The resulting supernatants were collected, and protein concentrations were determined using the Bradford assay. Samples were separated on 12% SDS–PAGE gels, transferred to PVDF membranes, blocked with 5% skim milk, and incubated with primary antibodies against PINK1, Parkin, and β-actin, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized by enhanced chemiluminescence and quantified relative to β-actin. A one‑way ANOVA was used to analyze PINK1 and Parkin levels, and a two‑way repeated‑measures ANOVA assessed changes in body weight and Vmax over time. The Bonferroni post hoc test was used for pairwise comparisons between groups. A significance level of p < 0.05 was applied.
Results
The results of the repeated-measures ANOVA for body weight revealed a significant main effect of time (F(1,24) = 130.180, p = 0.001، η²p = 0.844), indicating that mean body weight changed significantly over the course of the 8-week intervention. However, the main effect of group was not significant (F(3,24) = 0.154, p = 0.926، η²p = 0.019), demonstrating that mean body weight did not differ across groups. Despite the absence of group differences, the time × group interaction was statistically significant (F(3,24) = 3.935, p = 0.020, η²p = 0.330), suggesting that patterns of weight change over time varied among groups. For Vmax, there was a significant main effect of time (F(1,24) = 7.105, p = 0.014, η²p=0.228), along with a significant main effect of group (F(3,24) = 26.527, p = 0.001, η²p=0.768). Furthermore, a highly significant time × group interaction was detected (F(3,24) = 70.095, p = 0.001, η²p=0.898), indicating that the progression of Vmax over the 8-weeks period differed substantially between groups. Bonferroni post hoc tests indicated no significant difference in Vmax between the control and D-gal groups (p = 1.000). In contrast, both training groups showed significant increases in Vmax compared with the control and D-gal groups (p = 0.001). No significant difference was found between the MICT+D and HIIT+D groups (p = 1.000), indicating that both training modalities comparable improvements in Vmax. To determine the effect of exercise intensity on the expression levels of mitophagy-related proteins, a one-way ANOVA was conducted for PINK1 and Parkin. The analysis revealed significant differences among groups for both proteins (PINK1: F(3,24) = 385.30, p = 0.001, η²p = 0.979; Parkin: F(3,24) = 591.40, p = 0.001, η²p = 0.986). Post hoc Bonferroni comparisons indicated that D-gal administration significantly increased levels of PINK1 and Parkin expression relative to  the control group (both, p = 0.001). in contrast, eight weeks of exercise training markedly reduced the expression of both proteins in the MICT+D and HIIT+D groups compared with the D-gal group (p = 0.001), demonstrating that aerobic training effectively attenuated mitochondrial stress responses. Moreover, the MICT+D group exhibited a significantly greater reduction in PINK1 and Parkin levels than the HIIT+D group (p = 0.001). These findings indicate that both exercise protocols improved functional capacity (Vmax), while MICT exerted a more pronounced regulatory effect on mitophagy-related protein expression in the aging-induced hippocampal tissue.
Conclusion
The present study demonstrates that chronic D-galactose administration the significantly elevation of PINK1 and Parkin expression in the hippocampus. This increase likely reflects a compensatory response to increased oxidative stress and the accumulation of damaged mitochondria, which is consistent with previous evidence indicating that D-galactose promotes ROS over production, accelerates brain aging, and impairs mitochondrial integrity. In contrast, eight weeks of exercise training reversed these alterations, as both MICT and HIIT significantly reduced PINK1 and Parkin levels compared with the D-gal group. These findings are in line with the established role of exercise in modulating mitophagy-related pathways  and  improving mitochondrial function. Notably, MICT induced a more pronounced reduction in these proteins, suggesting that moderate, sustained intensity may more effectively regulate the mitophagy signaling in vulnerable hippocampal tissue, and facilitate a more efficient restoration of mitochondrial homeostasis compared with HIIT. Collectively, these results underscore the therapeutic potential of appropriately prescribed exercise training for mitigating mitochondrial dysfunction during aging and highlight MICT as a particularly promising strategy for maintaining hippocampal cellular integrity.
Article Message
The findings of the present study demonstrate that both continuous and intermittent exercise training (MICT, HIIT), effectively improved hippocampal MQC-related protein expression and attenuated D-galactose–induced neurodegenerative alterations. Notably, MICT induced a more pronounced restoration of MQC balance compared with HIIT, suggesting that moderate, sustained exercise may exert a more favorable regulatory influence on mitochondrial homeostasis in the aging hippocampus. These results highlight the importance of considering exercise intensity and training structure when designing interventions aimed at mitigating or preventing brain aging.
Ethical Considerations
The study was approved ethically by the ethics committee of the University of Isfahan, Iran Code No, IR.UI.REC.1403.027). All methods were carried out in accordance with relevant guidelines and regulations.  
Authors’ Contributions
Conceptualization: Mohammad Faramarzi
Data Collection: Hajar Allahverdi, Siamak Beheshti
Data Analysis: Hajar Allahverdi, Jalil Reisi
Manuscript Writing: Hajar Allahverdi
Review and Editing: Hajar Allahverdi, Mohammad Faramarzi, Jalil Reisi, Siamak Beheshti
Responsible for funding: Hajar Allahverdi
Literature Review: Hajar Allahverdi
Project Manager: Mohammad Faramarzi
Conflict of Interest
The authors declare no competing interests
Acknowledgments
This study was supported by the Iran National Science Foundation (INSF) under project No.4030891. The authors gratefully acknowledge this support.