| Objective The protective effect of electroacupuncture at "Neiguan","Zusanli" and "Guanyuan" points on myocardial ischemia-reperfusion injury in rats and its mechanism were observed from the perspective of apoptosis,pyrocytosis and autophagy.The mechanism of FXR/SHP signaling pathway and the elationship between apoptosis,pyrosis and autophagy was explored by using FXR/SHP signaling pathway agonist gw4064 and inhibitor z-guggulsterone,so as to provide a new theoretical basis and therapeutic target for electroacupuncture pretreatment in the prevention and treatment of MIRI.Methods1 Theoretical discussion Based on the theory of traditional Chinese medicine and modern medicine,from the perspective of cardiomyocyte pyrocytosis,autophagy and apoptosis,to explore the protective effect and mechanism of electroacupuncture preconditioning on myocardial ischemia-reperfusion rats;from the persp-ective of FXR/SHP apoptosis signal pathway,to explore the effect and mechanism of FXR/SHP pathway on cardiomyocyte apoptosis,pyrocytosis and autophagy.2.Animal experiment80 Wistar rats(male,SPF grade)were randomly divided into four groups according to the "random number table method",namely:normal group(N Group),sham operation group(S group),Model group(M group)and EA pretreatment group(EA group),with 20 rats in one group.All the experimental animals were fed adaptively for one week before the formal experiment.MIRI rat model was established by ligation of left coronary artery,and corresponding intervention measures were given to each group.Another 40 Wistar rats(male,SPF grade)were randomly divided into two groups with 20 rats in each group:EA+GW4064 agonist group(EA+GW group)and EA+Z-guggulsterone(EA+Z group)inhibitor group.(1)He staining was used to observe the morphological structure of myocardial tissue;TTC staining was used to measure the size and mass of myocardial infarction;TUNEL staining was used to calculate the apoptosis index of myocardial cells;double antibody sandwich enzyme-linked immunosorbent assay(ELISA)was used to determine the myocardial injury markers(LDH,CK and c Tn I);so as to explore the protective effect of electroacupuncture pretreatment on MIRI rats.(2)Western blot was used to detect the expression of NLRP3,ASC,Caspase-1,gasdermin D and other pyrocytosis related proteins,so as to preliminarily explore the regulatory effect of electroa-cupuncture pretreatment on cardiomyocyte pyrocytosis pathway in MIRI rats.(3)Western blot was used to detect the expression of Beclin1,LC3?,LC3?protein and the ratio of LC3 ?/LC3,to study the protectiveeffect of electroacupuncture pretreatment on cardiomyocytes,and to preliminarily explore the regulatory effect of electro-acupuncture pretreatment on autophagy pathway of myocardial cells in MIRI rats.(4)Western blot was used to detect the expression of Bcl-2 and Bax protein,and fluorescent quantitative PCR was used to detect the expression of caspases-9 and caspases-3 m RNA.The protective effect of electroacupuncture pretreatment on MIRI rat model was studied,and the regulatory effect of electroacupuncture pretreatment on cardiomyocyte apoptosis pathway in MIRI rats was preliminarily explored.(5)TUNEL staining was used to calculate the apoptosis index of cardiomyocytes,and Western blot was used to detect pyrocytosis related indicators gasdermin D.IL-?,IL-18 protein expression,autophagy related indicators beclin-1,LC3?,LC3? protein content and LC3?/LC3?ratio,apoptosis related indicators Bcl-2,Bax,Caspase3 protein expression.At the same time,fluorescence quantitative PCR was used to detect apoptosis related indicators FXR,SHP gene expression,to explore the effect of Electroacupuncture pretreatment on FXR/SHP signaling pathway in MIRI model rats,and the effect of FXR/SHP signaling pathway on apoptosis,the effects and mechanisms of apoptosis,pyrocytosis and autophagy.Results Experiment 1:Effects of electroacupuncture pretreatment on myocardial injury markers,apoptosis index and morphology in MIRI rats(1)He staining:the morphological results of myocardial cells of rats in each group were observed and compared under light microscope as follows:in group N and group S,the myocardial fibers were orderly,the transverse stripes were clear,the cell structure was complete,the morphology was normal,there was no inflammatory infiltration,the nuclear position was normal,and there was no vacuolar degeneration;in group M,the myocardial fibers were disordered,the myocardial fibers were broken,and the myocardial cell structure was incomplete,In EA group,the morphology of myocardial tissue was normal,the damage was alleviated,the arrangement of myocardial fibers was slightly disordered,a few fibers were broken,a few inflammatory cells were infiltrated,and a few cells were edematous and necrotic.These results indicate that EA preconditioning can effectively alleviate myocardial ischemia-reperfusion injury in rats.(2)Myocardial infarct area:compared with group N,the infarct size of group S did not change significantly(P>0.05);compared with group N,the infarct area of group M and group EA all increased significantly(all P<0.01);compared with group M,the infarct area of group EA decreased significantly(P<0.01).(3)Myocardial infarct mass:Compared with group N,the infarct mass of group S did not change significantly(P>0.05);compared with group N,the infarct size and infarct mass of group M and group EA all increased significantly(all P<0.01);compared with group M,the infarct mass of group EA decreased significantly(P<0.01).(4)Apoptotic index of myocardial cells:a small number of apoptotic nuclei were found in the myocardial tissues of group N and group S,a large number of apoptotic cells were found in group M,with irregular shape and fragmented nuclei,and a small number of apoptotic nuclei were found in group EA.Compared with group N and group S,the apoptotic index of cardiomyocytes in group M and group EA were all significantly increased(all P<0.01),and compared with group M,the apoptotic index of cardiomyocytes in group EA was significantly decreased(P<0.01).(5)Serum LDH,CK,c Tn I content:compared with group N,serum LDH,CK,c Tn I content of group S had no significant change(P> 0.05);compared with group N,serum LDH,CK,c Tn I content of group M and group EA were all increased(all P<0.01),serum LDH,CK,c Tn I content of group EA were lower than group M(P<0.01).Experiment 2 :The regulation of electroacupuncture preconditioning on the pyroptosis pathway of myocardial ischemia-reperfusion in rats(1)The expression of NLRP3 and ASC protein in myocardial tissue:compared with group N and group s,the expression of NLRP3 and ASC protein in group M and group EA all increased(all P< 0.05),and compared with group M,the expression of NLRP3 and ASC protein in group EA decreased significantly(all P<0.05).(2)Expression of caspase-1 and gasdermin D protein in myocardial tissue: compared with group N and group S,the expression of caspase-1and gasdermin D protein in group M and EA all increased(all P<0.05),and compared with group M,the expression of caspase-1 and gasdermin D protein in group EA all decreased significantly(all P<0.05).Experiment 3: regulation of electroacupuncture preconditioning on autophagy pathway in myocardial ischemia-reperfusion model rats(1)The expression of Beclin-1 protein in myocardial tissue: compared with group N and group s,the expression of Beclin-1 protein in group M and group EA all increased(all P< 0.05),and compared with group M,the expression of Beclin-1 protein in group EA decreased significantly(all P<0.05).(2)Expression of LC3 ?,LC3 ? protein and LC3?/LC3? ratioin myocardial tissue: compared with group N and group S,the expression of LC3?,LC3 ? protein and LC3 ?/LC3 ? ratio in group M and EA all increased(all P<0.05),and compared with group M,the expression of LC3 ?,LC3 ?protein and LC3 ?/LC3 ? ratio in group EA all decreased significantly(all P<0.05).Experiment 4:Effect of electroacupuncture preconditioning on apoptosis of myocardial ischemia-reperfusion model rats(1)The expression of Bcl-2 in myocardial tissue: compared with group N and group S,the expression of Bcl-2 protein in myocardial cells of group M and EA all decreased(all P<0.05).Compared with M group,the expression level of Bcl-2 protein in EA group was significantly higher(P<0.05).(2)Caspase-9 gene,caspase-3 m RNA and Bax protein expression in myocardial tissue: the expression levels of caspase-9 gene,caspase-3 gene and Bax protein in group M and EA were significantly higher than those in group N and group S(all P<0.05),and the expression levels of caspase-9gene,caspase-3 gene and Bax protein in group EA were significantly lower than those in group M(all P<0.05).Experiment 5:Effect of electroacupuncture preconditioning on FXR/SHP signaling pathway in myocardial ischemia-reperfusion model rats(1)Myocardial cell apoptosis index: compared with group N and group S,the myocardial cell apoptosis index of group M,EA,EA+ GW and EA+Z all increased significantly(all P<0.05);compared with group M,the myocardial cell apoptosis index of group EA,EA+GW and EA+Z all decreased significantly(all P<0.05);compared with group EA,the myocardial cell apoptosis index of group EA+Z decreased significantly(P<0.05),and the myocardial cell apoptosis index of group EA+GW decreased significantly(P<0.05).(2)FXR and SHP m RNA expression in myocardial tissue:compared with group N and group S,FXR and SHP gene expression levels in myocardial cells of group M,EA group and EA+GW group were significantly increased(all P<0.05);compared with group M,FXR and SHP gene expression levels in EA group and EA+GW group were significantly decreased(all P<0.05);compared with group EA,FXR and SHP gene expression levels in EA+Z group were significantly decreased(P<0.05),FXR and SHP gene expression levels in EA+Z group were significantly increased(P<0.05).(3)The expression of Bcl-2,Bax protein and Caspase-3 m RNA in myocardial tissue: compared with group N and group S,the expression levels of Bcl-2 in myocardial cells of group M,EA,EA + GM and EA+Z were decreased(all P<0.05),and the expression levels of Bax and caspase-3 were significantly increased(all P<0.05).Compared with group M,the expression levels of Bcl-2 protein in EA group,EA+GW group and EA+Z group were significantly increased(all P<0.05),and the expression levels of Bax and caspase-3 protein were significantly decreased(all P<0.05).Compared with group EA,the expression levels of Bcl-2 protein in EA+Z group were significantly increased(P<0.05),and the expression levels of Bax and caspases-3 protein in EA+GW group were significantly decreased(all P<0.05).(4)Beclin-1,LC3 ?,LC3 ? protein expression and LC3 ?/LC3 ? ratio in myocardial tissue: compared with group N and group S,beclin-1,LC3?,LC3 ? protein expression and LC3 ?/LC3 ? ratio in myocardial cells of group M,EA,EA+GW and EA+Z were significantly increased(all P<0.05).Compared with group M,beclin-1,LC3 ?,LC3 ? protein expression and LC3?/LC3 ? ratio in myocardial cells of group EA,EA+GW and EA+Z were significantly decreased(all P<0.05)Compared with EA group,the expression of beclin-1,LC3 ?,LC3 ? protein and LC3 ?/LC3 ? ratio in EA+Z group were significantly decreased,while those in EA+GW group were significantly increased(P<0.05).(5)The protein expression of gasdermin D,IL-1? and IL-18 in myocardial tissue: compared with group N and group S,the protein expression levels of gasdermin D,IL-1 ? and IL-18 in myocardial cells of group M,EA,EA+GW and EA+Z were significantly increased(all P<0.05);compared with group M,the protein expression levels of gasdermin D,IL-1 ? and IL-18 in myocardial cells of group EA,EA+GW and EA+Z were significantly decreased(all P<0.05).Compared with EA group,the expression levels of gasdermin D,IL-1 ?and IL-18 protein in EA+Z group were significantly lower(P<0.05),while those in EA+GW group were significantly higher(P<0.05).Conclusion1.EA pretreatment can significantly reduce the infarct size and infarct degree of MIRI model rats,reduce the degree of mitochondrial swelling and necrosis,reduce the infiltration of inflammatory factors between myocardial cells,and reduce the apoptosis index.Pathomorphological examination shows that EA can significantly improve the pathological damage degree of damaged myocardial tissue.2.EA pretreatment can reduce the contents of LDH,CK and c Tn I in serum of MIRI model rats and protect myocardial tissue.3.EA pretreatment can inhibit the protein expression of NLRP3,ASC,Caspase-1 and gasdermin D,and protect myocardial tissue by regulating the pyrocytosis pathway.4.EA pretreatment can inhibit the protein expression of beclin-1,LC3?,LC3 ? and the increase of LC3 ?/LC3 ? ratio,and protect myocardial tissue by regulating autophagy pathway.5.EA pretreatment can regulate the expression of Bcl-2,Bax protein,caspases-9,caspases-3 m RNA,and protect myocardial tissue by regulating apoptosis pathway.6.Electroacupuncture pretreatment can regulate apoptosis mediated by FXR/SHP apoptotic signal pathway,and FXR/SHP apoptotic pathway can also indirectly regulate autophagy and pyrocytosis,indicating that the protective mechanism of electroacupuncture pretreatment on MIRI may be a multi-level and multi-channel comprehensive effect. |
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