Naloxone modulates the miR-30a-5p/ follistatin-like 1 axis to attenuate neurological deficits and hippocampal neuronal injury in ischemic stroke.

Abstract

The aim of the study was to investigate the underlying molecular mechanisms by which Naloxone enhances neurological function after ischemic stroke (IS). The permanent middle cerebral artery occlusion (PMCAO) model was utilized to simulate ischemic stroke in mice. Neurological function was assessed through behavioral scoring, and infarct volume as well as brain water content were measured to evaluate the extent of ischemic damage. Histopathological changes in the hippocampus were analyzed using hematoxylin and eosin staining, while neuronal apoptosis was quantified using TUNEL staining. An oxygen-glucose deprivation (OGD) injury model was established in HT22 cells, with cell viability assessed by MTT assay, apoptosis measured by flow cytometry, and lactate dehydrogenase release used to evaluate cellular toxicity. Proinflammatory cytokines were measured by enzyme-linked immunosorbent assay. The miR-30a-5p and Follistatin-like 1 (FSTL1) were quantified by RT-qPCR, and Western blotting was performed to detect FSTL1 protein levels as well as key apoptotic markers. Bioinformatic analysis, luciferase reporter assays, and RNA pulldown assays were conducted to confirm the direct interaction between miR-30a-5p and FSTL1. We found that Naloxone demonstrated a dose-dependent improvement in neurological function in PMCAO mice, as evidenced by reduced infarct volume, diminished cerebral edema, and attenuation of neuronal apoptosis and inflammation. Naloxone treatment significantly enhanced the viability of HT22 cells subjected to OGD, while also reducing apoptosis and inflammatory damage. Furthermore, Naloxone upregulated miR-30a-5p expression, and this upregulation contributed to the amelioration of OGD-induced cellular injury. The protective effects of Naloxone were partially reversed by silencing miR-30a-5p. miR-30a-5p directly targeted FSTL1, and silencing FSTL1 mitigated the reversal effect of miR-30a-5p inhibition on Naloxone's neuroprotective action. We conclude that Naloxone exerts its neuroprotective effects in ischemic stroke by upregulating miR-30a-5p, which inhibits the expression of FSTL1, ultimately improving neurological function and reducing brain injury in ischemic stroke models.