Molecular mechanism of hydromorphone preconditioning in cerebral ischemia/reperfusion‑induced inflammatory injury

The present study explored the protective mechanism of hydromorphone (HM) preconditioning in cerebral ischemia/reperfusion injury (CIRI). An animal model of CIRI was established by middle cerebral artery occlusion/reperfusion in male C57BL/6J mice. An I/R cell model was induced by oxygen‑glucose deprivation/reperfusion. Brain tissue injury and cell injury were evaluated by hematoxylin and eosin staining, 2,3,5‑triphenyltetrazolium chloride staining, lactate dehydrogenase testing and a Cell Counting Kit‑8 assay. Nod‑like receptor protein 3 (NLRP3)‑positive expression and caspase‑1 activity were assessed by immunostaining and caspase‑1 activity assays, respectively. Reverse transcription‑quantitative PCR or western blotting were used to quantify the expression levels of microRNA (miR)‑195‑5p, RNA‑binding motif protein 15 (RBM15) and upstream stimulatory factor 2 (USF2). RNA immunoprecipitation (RIP) and dual‑luciferase assays verified the binding between miR‑195‑5p and RBM15, followed by RIP analysis of N6‑methyladenosine (m6A) enrichment on USF2 and quantitative analysis of m6A content. The binding of RBM15 and insulin‑like growth factor 2 mRNA‑binding protein 3 (IGF2BP3) to the USF2 m6A site was analyzed by a dual‑luciferase assay. HM preconditioning inhibited NLRP3‑mediated pyroptosis, alleviated neurological deficits, and reduced inflammatory injury in brain tissue. Mechanistically, hydromorphone (HM) preconditioning targeted RBM15 and reduced IGF2BP3‑mediated m6A modification by upregulating miR‑195‑5p expression, thus decreasing USF2 expression, reducing the enrichment of USF2 on the NLRP3 promoter, and inhibiting NLRP3‑mediated pyroptosis. In conclusion, HM preconditioning may repress NLRP3‑mediated pyroptosis and alleviate CIRI via the effects of miR‑195‑5p/RBM15/USF2.