_{Extended Abstract
Background and Purpose}
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by severe cognitive decline and neuronal loss, primarily driven by the accumulation of amyloid-beta (Aβ) plaques and neurofibrillary tau tangles. These pathological hallmarks disrupt synaptic plasticity and memory formation, posing significant challenges for current therapeutic approaches. Recent evidence highlights the critical role of epigenetic regulators, particularly long non-coding RNAs (lncRNAs) such as TUG1 and MEG3, in AD pathogenesis. TUG1 is known to mitigate neuronal apoptosis by modulating specific molecular cascades, whereas MEG3 is essential for maintaining neuronal survival and cognitive integrity. Consequently, targeting these lncRNAs offers a promising avenue for neuroprotection. Non-pharmacological interventions, including aerobic interval training (AIT) and Ginkgo biloba (GB) supplementation, have gained attention for their multi-modal benefits. AIT enhances cerebral function by upregulating neurotrophic factors and attenuating oxidative stress, while GB exerts antioxidant and anti-inflammatory effects that counteract Aβ toxicity. However, the synergistic potential of combining these strategies to modulate hippocampal lncRNA expression remains underexplored. This study investigates the combined effects of AIT and GB on TUG1 and MEG3 expression in the hippocampus of a rat model of AD, aiming to elucidate their capacity to alleviate cognitive decline and provide a novel epigenetic perspective for AD management.

Materials and Methods
Forty-nine male Wistar rats, aged 8–10 weeks and weighing 250 ± 20 g, were housed in a controlled environment with a standard 12-hour light-dark cycle and free access to food and water. From an initial cohort of 56 animals, six were lost during the Alzheimer’s disease (AD) induction phase, and one was excluded due to sample preparation issues, resulting in a final sample size distributed across seven groups: Healthy Control (HC, n=7), Alzheimer’s Control (AC, n=8), Ginkgo Biloba (AD+GB, n=8), Ginkgo Biloba + Aerobic Interval Training (AD+GB+AIT, n=8), Aerobic Interval Training (AD+AIT, n=8), Sham (n=5), and Placebo (n=5). AD was induced via bilateral intracerebroventricular injection of Aβ1-42 (5µg/µL, 2 µL/site) using a stereotaxic apparatus. Following a 4-week post-injection period to confirm pathology, interventions commenced. The AD+AIT and AD+GB+AIT groups underwent an 8-week progressive treadmill aerobic interval training program, 5 days per week, at intensities ranging from 40% to 55% of VO₂max. Concurrently, the AD+GB and AD+GB+AIT groups received Ginkgo biloba extract (100 mg/kg/day) via oral gavage. Cognitive performance was evaluated using the shuttle box passive avoidance test to assess memory retention. Upon completion, hippocampal tissues were extracted, and the expression levels of lncRNAs TUG1 and MEG3 were quantified using quantitative real-time PCR (qPCR), normalized to β-actin as a housekeeping gene. Data were analyzed using one-way ANOVA followed by LSD post-hoc tests, with statistical significance set at P ≤ 0.05.

Results
Evaluation of cognitive performance via the shuttle box passive avoidance test demonstrated that the Healthy Control (HC) group achieved the highest step-through latency (STLA), reflecting superior memory retention relative to all other groups (P ≤ 0.05). In contrast, the Placebo group exhibited significantly reduced latency compared to the Ginkgo Biloba (AD+GB), Alzheimer’s Control (AC), and Sham groups (P ≤ 0.05), indicating negligible cognitive effects from the placebo procedure. Regarding hippocampal gene expression, no statistically significant differences were observed in TUG1 levels across the study cohorts (P > 0.05). Although the AC group presented a slight, non-significant increase in TUG1 expression (1.36 ± 0.76) compared to HC (1.27 ± 0.98), AD+AIT (1.08 ± 0.75), AD+GB+AIT (1.22 ± 0.82), AD+GB (1.06 ± 1.00), Sham (1.13 ± 0.54), and Placebo (1.22 ± 0.71), these variations were not meaningful. On the other hand, MEG3 expression was significantly amplified in the AD+GB+AIT group (2.19 ± 1.98) when compared to the AC (0.93 ± 0.12, P<0.0001), HC (P = 0.002), AD+AIT (0.83 ± 0.47, P<0.0001), Sham (1.00 ± 0.08, P<0.0001), and Placebo (0.95 ± 0.17, P = 0.001) groups. While the AD+GB group showed elevated MEG3 levels (1.69 ± 0.27) versus the AC group, statistical significance was not reached (P > 0.05). Collectively, these data imply that the combined regimen of aerobic interval training and Ginkgo biloba selectively upregulates MEG3, suggesting a neuroprotective role, whereas TUG1 expression remains unaffected by these treatments.

Conclusion
In conclusion, this study demonstrates that the synergistic application of aerobic interval training and Ginkgo biloba supplementation exerts selective regulatory effects on hippocampal lncRNAs in a rat model of Alzheimer’s disease. While the interventions did not alter TUG1 expression, they significantly upregulated MEG3 levels in the combined group, surpassing even healthy control levels. This distinct upregulation suggests a robust neuroprotective mechanism, likely mediated by enhanced neurotrophic support, reduced oxidative stress, and the activation of survival pathways such as PI3K/Akt. These findings underscore the therapeutic potential of non-pharmacological strategies in modulating epigenetic markers to counteract neuronal apoptosis and cognitive decline. Consequently, MEG3 represents a promising target for AD management, whereas the role of TUG1 requires further investigation with extended intervention periods to fully elucidate its responsiveness and molecular interactions in AD pathology.

Article Message
This investigation highlights that the concurrent use of aerobic interval training and Ginkgo biloba supplementation induces selective neuroprotective outcomes by markedly enhancing hippocampal MEG3 expression, without modulating TUG1. The results indicate that integrating non-pharmacological approaches can effectively regulate epigenetic mechanisms to mitigate neuronal apoptosis and oxidative stress associated with Alzheimer's disease. Therefore, directing therapeutic strategies toward lncRNA MEG3 via combined lifestyle modifications presents a viable and cost-effective method for managing AD pathology, underscoring the significant role of physical activity and phytochemicals in promoting neuroprotection and cognitive resilience.
 
Ethical Considerations
All experimental procedures were approved by the Ethics Committee of the Sport Sciences Research Institute, Tehran, Iran (Ethical Code: IR.SSRC.REC.1403.001) and adhered to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 2011).  

Authors’ Contributions
Conceptualization: Sepideh Basirat Dehkordi, Mehdi Mogharnasi, Majid Vahidian-Rezazadeh, Mohsen Saravani
Data curation: Sepideh Basirat Dehkordi
Formal analysis: Sepideh Basirat Dehkordi
Funding acquisition: Sepideh Basirat Dehkordi
Investigation: Sepideh Basirat Dehkordi
Methodology: Majid Vahidian-Rezazadeh
Project administration: Mehdi Mogharnasi
Resources: Sepideh Basirat Dehkordi
Software: Mohsen Saravani
Supervision: Majid Vahidian-Rezazadeh
Validation: Mohsen Saravani
Visualization: Mohsen Saravani
Writing–original draft: Sepideh Basirat Dehkordi
Writing–review & editing: Sepideh Basirat Dehkordi, Mohsen Saravani

Conflict of Interest
The authors declare no competing interests

Acknowledgments
This project was supported by Zahedan University of Medical Sciences and Imam Ali Hospital Research Center, Zahedan, Iran. The authors express their sincere gratitude for this support.