Metformin Plays A Protective Role In Cerebral Ischemia-reperfusion Injury By Regulating Autophagy And Pyroptosis

In recent years,the role of autophagy and pyroptosis in neuronal injury after CIRI has attracted more and more attention.Metformin（Met）,as a classic hypoglycemic drug,has been found to alleviate the neural damage caused by cerebral ischemia and hypoxia.However,the direct mechanism of Met on（nucleotide binding oligomerization domain like receptor protein 3）NLRP3 has not been discovered yet.The classic function of Met is to activate（adenosine monophosphate activated protein kinase）AMPK pathway,which leads to autophagy.Whether Met can inhibit inflammation through this pathway has not been reported.Based on this idea,this paper will investigate the effects of Met on autophagy and pyroptosis in brain tissue of rats with CIRI,so as to better understand the mechanism of Met,clarify its pharmacological effect,and provide theoretical support for its clinical application.In Part 1,the protective effects of Met in different concentrations on rats with CIRI were studied.Objective:To investigate the effect of Met in different concentrations on rats with CIRI.Methods:In this study,the middle cerebral artery of adult male SD rats were embolized by thread embolism for 120 min,and then reperfused to establish CIRI model.90 SD rats were selected and randomly divided into Sham group,Model group,Model+Met 50,100 and 200 mg·kg^-1group,Model+EDA 10 mg·kg^-1group,15 rats in each group,and 10 selected for the experiment.Met was given i.g.21 days before operation,and（edaravone）EDA was given i.p.7 days before operation.The body mass,blood glucose,neurological score,water maze data,infarct volume and pathological section indexes were observed.Results:Pretreatment with different concentrations of Met had no effect on blood glucose.Met can effectively improve neurobehavioral scores,improve cognitive function of rats with CIRI,reduce the volume of cerebral infarct volume after CIRI,and reduce the pathological damage of neurons in the hippocampal CA1 area,with 200mg·kg^-1Met be the best.Conclusion:Pretreatment with different concentrations of Met can play a role in brain protection and reduce CIRI.200 mg·kg^-1Met was selected for the following experiment.In Part 2,the effect of Met on autophagy and pyroptosis of brain tissue in rats with CIRI.Objective:To investigate the effect of Met on autophagy and pyroptosis in brain tissue of rats with CIRI.Methods:AMPK inhibitor Compound C（Cc）,autophagy inhibitor trimethyladenine（3-MA）,and Caspase-1 inhibitor Z-YVAD-FMK（Z-YVAD）were applied to block p-AMPK,LC3-II and Caspase-1 pathway respectively.135 rats were chosen as the research object and randomly divided into nine groups:Sham group,Model group,Model+Met 200 mg·kg^-1group（i.g.）,Model+Cc 20 mg·kg^-1group（i.p.）,Model+Met+Cc group,Model+3-MA 200 mmol·L^-1group（i.c.v 2μL）,Model+Met+3-MA group,Model+Z-YVAD 8μg·kg^-1·d^-1group（i.c.v 2μL）and Model+Met+Z-YVAD group.There were 15 rats in each group,and 10 selected for the experiment.Met was given 21 days before operation,Cc,3-MA and Z-YVAD were given 30 minutes before operation.The effects of nerve function score,cerebral infarction volume,pathological section indexes and the expression of related protein were under observation.Results:The expression of p-AMPK was increased and the brain injury was alleviated after Met was given to rats with CIRI.After blocking AMPK with inhibitor Cc,the brain protective effect of Met was blocked;Met could increase the expression of LC3-II and decrease the expression of p62.After blocking autophagy with 3-MA,the expression of LC3-II was decreased;the expression of p62 was increased;the expression of AMPK was not affected,but the protective effect of Met on brain injury was blocked.Met could reduce the expression of NLRP3 and Caspase-1,suggesting that Met could inhibit the production of inflammasome.After Z-YVAD,which is a Caspase-1 specific inhibitor,was applied to block the production of Caspase-1,in Model+Met group and Model+Met+Z-YVAD group,the expression of Caspase-1was decreased;the expression of LC3-II was increased;the expression of p62 was decreased,and the brain protective effect of Met+Z-YVAD was stronger than that of Met alone.Conclusion:The protective effect of Met was related to the activation of AMPK pathway,which may be able to remove the damaged mitochondria by autophagy and reduce the activation of NLRP3 inflammasome caused by the damaged mitochondria.In Part 3,the specific mechanism of Met regulating autophagy and pyroptosis was explored at the cellular level.Objective:To explore the mechanism of Met on autophagy and pyroptosis pathway at the cellular level,at the same time,to explore the effect of Met on the oxidative stress level of PC12 cells.Methods:AMPK inhibitor Compound C（Cc）,autophagy inhibitor trimethyladenine（3-MA）and Caspase-1 inhibitor Z-YVAD-FMK（Z-YVAD）were used to treat PC12 Cells induced by H₂O₂injury model.The experiment had explored the effect of Met on injured cells,observed cell morphology,and determined the survival rate of MTT,mitochondrial membrane potential,superoxide dismutase（SOD）,malondialdehyde（MDA）and glutathione（GSH）content,and the expression of proteins related to autophagy and pyroptosis.Results:Cells treated with different concentrations of H₂O₂had different degrees of damage,and the mitochondrial transmembrane potential decreased gradually,which indicated that H₂O₂could reduce the mitochondrial transmembrane potential of PC12cells.Met can improve the morphology of injured cells,increase cell survival rate,increase mitochondrial transmembrane potential,reduce MDA content,increase SOD and GSH content.Western blotting results showed that the protein was consistent with those from experiment 2.Conclusion:Met can reduce the oxidative stress injury of PC12 cells induced by H₂O₂,which is related to the activation of AMPK pathway.It can play a role through autophagy,reducing the expression of NLRP3 inflammasome,and inhibiting the role of pyroptosis as showed in Experiment 2.