Study On The Role Of Activin A/P38/MEF2D Pathway In Regulating Endoplasmic Reticulum Stress In Isoflurane Against Ischemia-reperfusion Injury

To investigate the effect of isoflurane post-processing on improving the behavioral function of rats against cerebral ischemia-reperfusion injury by regulating endoplasmic reticum stress and reducing cell apoptosis through Activin A/P38/MEF2 D signaling pathway in the middle cerebral artery(MCAO)embolization model and rat hippo hippoal slices hypoxic-sugar free injury group(OGD group).To further explore the effect of MEF2 D transcription factor on endoplasmic reticulum stress and apoptosis in rat hippocampus by regulating the Activin A/P38 signaling pathway.Methods:In the vivo part of the experiment,a rat model of middle cerebral artery embolization(MCAO)was prepared after ischemia and reperfusion was performed.The samples were collected 24 hours after modeling.The isoflurane post treatment group was immediately treated with isoflurane posttreatment during reperfusion.The behavioral indicators of rats were performed 24 hours after modeling.In vitro,hippocampal slices were prepared from 2-week-old newborn SD rats.First,TTC staining was used to observe and analyze the difference in cerebral infarction volume between each group in vivo model.Nissl staining and TUNEL staining were used to observe the apoptosis of cells in hippocampal CA1 region of rats.Transmission electron microscopy was used to observe the changes of organelles in each group in vitro.The expressions of apoptosis-related proteins Bax,Bcl-2 and Caspase3 in the hippocampus of rats in vitro were further verified.The expressions of ER stress-related proteins CHOP,GRP78,ATF6 and Caspase12 were detected by Western blot and immunofluorescence.Then the protein expression of Activin A and MEF2 D in hippocampal CA1 region of rats was observed.Then,microinjection of adenovirus was performed in the hippocampus 21 days before the modeling of the in vivo model,and in vitro injection was performed on P0 of SD rats 2 weeks before the operation to knockdown the expression of MEF2 D.Preparation in vivo in vitro respectively after I/R model,using the TTC staining observation in vivo model rat infarction volume difference and analysis,with TUNEL staining and observation of cell apoptosis of hippocampus CA1 area,using transmission electron microscope from the change of cell organelle between groups of rats,validation in vitro between groups in the rat hippocampus in vivo expression of apoptosis related proteins,The expression of ER stress-related proteins was detected by Western blot and immunofluorescence.Then,the pathway inhibitor SB431542 was used to reduce the expression of Activin A in vitro model,and the protein expressions of Activin A,P38,p-p38 and MEF2 D were observed.The protein expressions of Activin A,P38,p-p38 and MEF2 D were observed while the expression of MEF2 D was inhibited.Thus,the regulatory relationship between pathways can be clarified.Results:In the vivo part of the experiment,a rat model of middle cerebral artery embolization(MCAO)as prepared after ischemia and reperfusion was performed.The samples were collected 24 hours after modeling.The isoflurane posttreatment group was immediately treated with isoflurane posttreatment during reperfusion.The behavioral indicators of rats were performed 24 hours after modeling.In vitro,hippocampal slices were prepared from 2-week-old newborn SD rats.First,TTC staining was used to observe and analyze the difference in cerebral infarction volume between each group in vivo model.Nissl staining and TUNEL staining were used to observe the apoptosis of cells in hippocampal CA1 region of rats.Transmission electron microscopy was used to observe the changes of organelles in each group in vitro.The expressions of apoptosis-related proteins Bax,Bcl-2 and Caspase3 in the hippocampus of rats in vitro were further verified.The expressions of ER stress-related proteins CHOP,GRP78,ATF6 and Caspase12 were detected by Western blot and immunofluorescence.Then the protein expression of Activin A and MEF2 D in hippocampal CA1 region of rats was observed.Then,microinjection of adenovirus was performed in the hippocampus 21 days before the modeling of the in vivo model,and in vitro injection was performed on P0 of SD rats 2 weeks before the operation to knockdown the expression of MEF2 D.Preparation in vivo in vitro respectively after I/R model,using the TTC staining observation in vivo model rat infarction volume difference and analysis,with Nissl staining,TUNEL staining and observation of cell apoptosis of hippocampus CA1 area,using transmission electron microscope from the change of cell organelle between groups of rats,validation in vitro between groups in the rat hippocampus in vivo expression of apoptosis related proteins,The expression of ER stress-related proteins was detected by Western blot and immunofluorescence.Then,the pathway inhibitor SB431542 was used to reduce the expression of Activin A in vitro model,and the protein expressions of Activin A,P38,p-p38 and MEF2 D were observed.The protein expressions of Activin A,P38,p-p38 and MEF2 D were observed while the expression of MEF2 D was inhibited.Thus,the regulatory relationship between pathways can be clarified.Conclusion:(1)MEF2D can regulate the Activin A/P38/MEF2 D signaling pathway to reduce endoplasmic reticulum stress,thereby reducing cell apoptosis and improving the behavioral function of rats.(2)Isoflurane post-processing can further exert A brain protective effect on the Activin A/P38/MEF2 D signaling pathway by regulating MEF2 D transcription factors.