RKIP regulates the ERK/MAPK signaling pathway to improve neuroinflammatory injury in male rats with ischemic stroke.

Abstract

BACKGROUND: To explore the mechanism by which RAF kinase inhibitory protein (RKIP) alleviates neuroinflammatory damage in male rats with ischemic stroke (IS). METHODS: Male rats were subjected to middle cerebral artery occlusion (MCAO) to establish an IS model. Two weeks before MCAO, a single tail-vein injection of lentivirus or an equivalent volume of normal saline was administered to rats. The neurological deficit scores, infarct volume fractions, and pathological changes of the ischemic penumbra were evaluated in the rats. Immunohistochemstry, double-labeled immunofluorescence, ELISA, and Western blot were performed to assess microglial polarization, neuroinflammation and ERK/MAPK-related protein expressions. RESULTS: IS rats exhibited elevated neurological deficit scores and enlarged infarct volume fractions, accompanied by aggravated histopathological damage in the ischemic penumbra. Microglial M1 polarization was enhanced, meanwhile, IL-6 and TNF-α were up-regulated, whereas the anti-inflammatory mediator IL-10 was down-regulated, indicating a pronounced neuroinflammatory response. In particular, Western blot results showed that the ischemic penumbra expression of RKIP in IS rats was markedly lower in IS group than that in Control group. After achieving RKIP overexpression via lentivirus mediation, the polarization direction of microglia in the ischemic penumbra of IS rats shifted toward the M2 phenotype. This was specifically manifested by a significant decrease in the proportion of iNOS⁺/Iba1⁺ double-positive microglia, while the proportion of Arg-1⁺/Iba1⁺ double-positive microglia was significantly increased, and the neuroinflammatory response was alleviated. Moreover, its overexpression significantly reduced the expressions of p-ERK1/2 and p-p38 MAPK in ischemic penumbra. Interestingly, rmEGF-activated ERK elevated the protein levels of p-ERK1/2 and p-p38 MAPK in ischemic penumbra without altering RKIP expression itself. Consequently, the proportion of iNOS⁺/Iba1⁺ double-positive microglia rebounded, while that of Arg-1⁺/Iba1⁺ double-positive microglia decreased . Finally, functional experiments demonstrated that ERK partially reversed the neuroinflammatory protection conferred by RKIP overexpression in IS rats. CONCLUSION: Overexpression of RKIP may alleviate the neuroinflammatory damage in IS rats by inhibiting ERK/MAPK pathway, thereby improving neurological function.