| Background:Ischemic stroke,as an acute cerebrovascular disease,is also one of the most common and frequently reported diseases in the Department of Neurology.Cerebral ischemia-reperfusion injury is an important pathological mechanism of ischemic stroke,and neuroinflammatory response is one of the main pathological mechanisms after ischemia-reperfusion injury.Microglia play a vital role in the neuroinflammatory response.They can rapidly activate and polarize into a pro-inflammatory phenotype(M1 type)and an anti-inflammatory phenotype(M2 type)after ischemia,thereby regulating the inflammatory response.Studies have shown that estrogen regulates the M2-type polarization of microglia through G protein-coupled estrogen membrane receptor,and plays an anti-inflammatory role.Icaritin is a phytoestrogen extracted from Epimedium plants,and has strong anti-inflammatory effect.In our previous studies,we found that ICT had a good protective effect on ischemia-reperfusion in rats,but its potential mechanism is still unclear.Therefore,the purpose of this experiment was to explore the effects and mechanisms of ICT on the regulation of microglia activation and polarization in rats with ischemia-reperfusion injury,so as to provide an experimental basis for further revealing the brain protection of ICT.Objective:To observe the neuroprotective effects of ICT on cerebral ischemia-reperfusion injury in rats through in vivo experiments.To clarify the regulatory effects of ICT on microglia activation and polarization in rats with cerebral ischemia-reperfusion injury;It was clarified that the regulation of microglia polarization by ICT was achieved through the GPER-ERK-NF-κB signaling pathway.Method:Male,healthy SD rats,weighing 250-280 g,were used to prepare a temporary middle cerebral artery occlusion(TMCAO)reperfusion model,ICT treatment and GPER and ERK signal-specific inhibitor intervention experiments using suture method.The neurological function of tMCAO rats was assessed by Garcia JH Neurological Function Score and Fluoro-Jade C staining.Cat Walk gait and rotarod test were used to detect the locomotor function of tMCAO rats.The contents of IL-1β,TNF-α and IL-10 in the ischemic penumbra were detected by ELISA.The gene expressions of IL-1β,TNF-α,CD11 b,CD68,CD206 and NF-κB in the ischemic penumbra were detected by q PCR.The expression levels of Iba1,CD40,CD206,and GPER proteins in the ischemic penumbra and the phosphorylation of NF-κB and ERK proteins were detected by Western blot.Tissue immunofluorescence was used to detect the co-labeling of CD68,CD206,and Iba1.Results:1.ICT can increase Garcia JH nerve function score of tMCAO rats and reduce the number of degenerated neurons in tMCAO rats;2.ICT can obviously improve the gait parameters of tMCAO rats,and increase the walking time and rotation speed of tMCAO rats;3.ICT can significantly reduce the water content of brain tissue and the contents of pro-inflammatory factors IL-1β and TNF-α in tMCAO rats,and significantly increase the content of anti-inflammatory factors IL-10;4.ICT down-regulated the expression levels of Iba1 protein and CD11 b mRNA,down-regulated the expression levels of M1 phenotype marker CD40 protein and CD68 mRNA in microglia,and increased the expression levels of M2 phenotype marker CD206 protein /mRNA in tMCAO rats;5.ICT decreased the expression level of phosphorylated NF-κB protein and NF-κB mRNA in ischemic semi-damaged area of tMCAO rats,and increased the expression level of phosphorylated ERK protein;6.The neuroprotective and anti-inflammatory effects of ICT on tMCAO rats were cancelled after stereotactic administration of specific inhibitors G15 and U0126 of GPER and ERK,respectively.7.G15 and U0126 canceled the influence of ICT on the expression levels of Iba1,CD40,CD206,NF-κB and ERK proteins in the ischemic semi-damaged area of tMCAO rats.Conclusion:1.ICT has the effects of inhibiting neuroinflammation,improving motor function and protecting brain of tMCAO rats;2.ICT inhibits neuroinflammation induced by ischemia-reperfusion by regulating microglia activation and polarization;3.ICT may regulate the activation and polarization of microglia through the GPER-ERK-NF-κB signaling pathway. |
