Therapeutic effects of miR-495–3p inhibition and exercise in a transient middle cerebral artery occlusion rat model

Ischemic stroke is a leading cause of neurological disability, with limited therapeutic options beyond thrombolysis and thrombectomy. This study investigated whether combined exercise training and miR-495–3p inhibitor (miR-495–3p-I) treatment could attenuate endoplasmic reticulum (ER) stress–associated autophagy, reduce apoptosis, and enhance neuronal survival in a transient middle cerebral artery occlusion (tMCAo) rat model. Rats subjected to tMCAo received intracerebroventricular injection of miR-495–3p-I, treadmill exercise training (12 m/min, 10 min/day for 2 weeks), or a combination of both treatments (miR-495–3p-I + Ex). Functional recovery was evaluated using behavioral tests, while molecular and histological analyses were performed using RNA sequencing, quantitative PCR, western blotting analysis, and immunohistochemistry or immunofluorescence. The combined treatment significantly reduced infarct volume and improved motor function compared with tMCAo alone. Transcriptomic analysis identified differentially expressed genes enriched in MAPK, PI3K–AKT, and autophagy-related pathways. Western blotting analysis demonstrated suppression of IRE1α-mediated ER stress signaling and a reduction in autophagy-related markers. Immunofluorescence confirmed decreased LC3B and p62 expression in Ex and miR-495–3p-I + Ex groups. Furthermore, apoptosis was attenuated as evidenced by decreased BAX and increased BCL2, as well as neuronal markers (NeuN and NFH). These findings suggest that combined exercise and miR-495–3p inhibition synergistically promote neuroprotection after tMCAo by suppressing ER stress–mediated signaling, attenuating excessive autophagy, and reducing apoptosis. This multimodal strategy may offer a promising therapeutic and rehabilitation approach for ischemic stroke.