Effect Of Inhibition Of TRPM2 Channel On Cerebral Ischemia-reperfusion Injury In Mice

Objective The present study aimed to explore the Effect of inhibition of TRPM2 channel on cerebral ischemia-reperfusion injury in mice.Methods Eighty C57BL/6 male mice were randomly divided into sham group,MCAO/R group and ACA group.The mouse model of MCAO/R was established by the suture blotting method,and the models with the same degree of injury,which were evaluated by the Bederson score method,for follow-up experiments.The mice were intraperitoneally injected with 25 mg/kg ACA 2 hours after ischemia(ACA group).By Bederson score method,we evaluated the neurological function of the mouse after 24 hours of MCAO/R.Laser speckle blood flow imaging was used for detecting changes in cerebral blood flow in the cerebral cortex of mice in vivo.The volume of cerebral infarction in mice was evaluated by TTC Staining.The morphological changes in the cerebral temporal cortex,hippocampus CA1,CA3 and ischemic penumbra of the mouse were observed by HE staining and Nissl staining.The apoptosis of cells on the brain ischemic side was evaluated by TUNEL staining.The expression of Iba-1,iNOS and LC3 in the ischemic penumbra area was observed by immunofluorescence staining.In this experiment,fluorescence quantitative PCR(qPCR)was used to measure the relative expression level of TRPM2 mRNA in the ischemic brain damage area of mice,and the expression of tumor necrosis factor alpha(TNF-α)in the serum was detected by ELISA.Western Blot was used to evaluate the protein expression of autophagy substrate P62 on the ischemic side of mice.Results Compared with the Sham group,the Bederson score of the MCAO/R group increased(P<0.05),and compared with the MCAO/R group,the Bederson score of the ACA group decreased(P<0.05).The relative reduction of cerebral cortex blood flow on the ischemic side of the MCAO/R group was significantly higher than that of the Sham group(P<0.05).Compared with the MCAO/R group,the relative reduction of cortical blood flow of the cerebral ischemic side of the ACA group was significantly reduced(P<0.05).TTC staining results showed that compared with the Sham group,the cerebral infarction volume of the MCAO/R group increased significantly(P<0.05),but it decreased in ACA group compared with that of in MCAO/R group(P<0.05).HE and Nissl staining showed that the morphology and organization structure were destroyed,and the number of nerve cells decreased in the cerebral temporal lobe cortex,hippocampus CA1,CA3 and ischemic penumbra area of the MCAO/R group,and it significantly ameliorated in ACA group.The number of TUNEL-positive cells in the ischemic penumbra of the cerebral cortex of the MCAO/R group is significantly increased(P<0.05),also it was decreased in ACA group(P<0.05).A large amount of Iba-1,iNOS and LC3 positive cells appeared in the ischemic penumbra of mice in the MCAO/R group(P<0.05),but the positive cells’ number of Iba-1,iNOS and LC3 was decrease in ACA group(P<0.05).The MCAO/R group’s relative TRPM2 mRNA expression level in the ischemic side of brain was significantly up-regulated(P<0.05),and it was down-regulated in ACA group compared with that of in MCAO/R group(P<0.05).ELISA results demonstrated that the production of TNF-α in the MCAO/R group was significantly higher than that in the Sham group(P<0.05),but it dropped in ACA group compared with that of in MCAO/R group(P<0.05).Western Blot results showed that compared with the Sham group,the protein of P62 expression level in the MCAO/R group was remarkably down-regulated(P<0.05),but it up-regulated in ACA group contrast with that of in MCAO/R group(P<0.05).Conclusion It has a protective effect on cerebral ischemia-reperfusion injury in mice by inhibition of TRPM2 channel,which might be related to the activation of microglia,the secretion of inflammatory factors and autophagy.