Author(s): JIN, Y., JANG, S., JEONG, H.S., KIM, S.H., KIM, J., AN, H., KIM, S.H., KIM, D., Institution: CHONNAM NATIONAL UNIVERSITY, Country: KOREA, SOUTH, Abstract-ID: 598

INTRODUCTION:
An ischemic stroke, the most common type of stroke, is caused by thrombosis or embolism that reduces blood flow to the brain and induces cell death. Only two treatments, plasminogen activators and mechanical thrombectomy, have been approved for ischemic stroke; however, their overall therapeutic efficacy remains limited. Consequently, this study investigates whether combined exercise training and miR-495-3p inhibitor (miR-495-3p-I) treatment can attenuate ER stress-linked autophagy, reduce apoptosis, and enhance neuronal survival in a transient middle cerebral artery (MCA) occlusion (tMCAo) rat model.
METHODS:
To establish of tMCAo model, male Sprague-Dawley rats (250-270 g, 8 weeks old) were subjected to intraluminal occlusion of the right MCA for 60 min, followed by reperfusion to restore blood flow. Animals were assigned to four groups: sham, tMCAo, tMCAo+Exercise (10 min/day, 12 m/min treadmill running for 2 weeks), and tMCAo+Exercise+miR-495-3p-I (5′-AAGAAGUGCACCAUGUUUGUUU-3’, 10 pmol/5 uL PBS, intracerebroventricular injection). Infarct volume was assessed using TTC staining. Motor function was evaluated using cylinder test, elevated body swing test, rota-rod test, and neurological scoring (Zea-Longa). Gene and protein expressions were assessed using qPCR, western blotting analysis, immunohistochemistry, and RNA sequencing. Statistical analysis was performed using GraphPad Prism 10.6.1.
RESULTS:
Exercise combined with miR-495-3p-I treatment significantly reduced the infarct volume and enhanced the motor function compared to tMCAo. RNA sequencing revealed 18 genes with fold change ≥3.5 and 49 genes with fold change ≥2 were differentially expressed, enriched in MAPK, PI3K-AKT, and autophagy-related pathways. qPCR analysis confirmed altered expression of JNK1, JNK2, p38α, NFH, and GAP-43 in treatment groups. Western blot analysis demonstrated that combined intervention significantly decreased phosphorylation of IRE1α and ASK1, ER-stress markers, and suppressed downstream JNK and p38 MAPK activation compared to tMCAo group. All the autophagy markers including Beclin-1, ATG7, ATG12, LC3B-II, and p62 were markedly reduced in combined treatment group compared with MCAo group. For analysis of apoptosis, immunohistochemistry showed decreased BAX and increased BCL2 expression. Neuronal markers, NeuN and NFH, were significantly elevated in exercise and combined treatment groups, indicating enhanced neuronal preservation and axonal integrity.
CONCLUSION:
Combined exercise training and miR-495-3p-I treatment provides synergistic neuroprotection in tMCAo rats by attenuating IRE1α-dependent ER stress, suppressing excessive autophagy through MAPK–BCL2–ATG–LC3B-II axis, and reducing neuronal apoptosis. These effects were associated with improved motor function recovery and enhanced neuronal survival. This study demonstrates that exercise training with targeted microRNA therapy represents a promising multimodal strategy for ischemic stroke recovery.