Mesencephalic astrocyte-derived neurotrophic factor (MANF) alleviates cerebral ischemia/reperfusion injury in mice by regulating microglia polarization via A20/NF-κB pathway

Abstract

Microglia, resident brain immune cells, is critical in inflammation, apoptosis, neurogenesis and neurological recovery during cerebral ischemia/reperfusion (I/R) injury. Mesencephalic astrocyte-derived neurotrophic factor (MANF), a novel identified endoplasmic reticulum stress-inducible neurotrophic factor, can alleviate I/R injury by reducing the inflammatory reaction, but its specific regulatory mechanism on microglia after ischemic stroke has not been fully clarified. To mimic the process of ischemia/reperfusion in vivo and in vitro, middle cerebral artery occlusion/reperfusion (MCAO/R) was induced in C57BL/6J mice and oxygen glucose deprivation/reoxygenation (OGD/R) model was established in BV-2 cells. Moreover, MANF small interfering RNA (siRNA) was used to silence the expression of endogenous MANF, while recombination human MANF protein (rhMANF) acted as an exogenous supplement. Seventy-two hours after MCAO/R, 2,3,5-triphenyltetrazolium staining, neurological scores, brain water content, immunohistochemical staining, immunofluorescent staining, flow cytometry, hematoxylin and eosin staining, quantitative real-time PCR and western blot are applied to evaluate the protective effect and possible mechanism of MANF on cerebral I/R injury. In vitro, cell viability, inflammatory cytokines and the expression of MANF, A20, NF-κB and the markers of microglia were analyzed. The results showed that MANF decreased brain infarct volume, neurological scores, and brain water content. In addition, MANF promoted the polarization of microglia to an anti-inflammatory phenotype both in vivo and in vitro, which are related to A20/NF-κB pathway. In summary, MANF may offer novel therapeutic approaches for ischemic stroke in the process of microglia polarization.