| Background:How to reduce cardiomyocyte oxidative stress injury is critical to repair infarcted myocardium.After the occurrence of myocardial infarction(MI),cardiomyocytes are in a state of ischemia and hypoxia,resulting in the decrease of mitochondrial membrane potential(ΔΨm),the inhibition of the function of the mitochondrial respiratory chain complex,the generation of excessive ROS,and the induction of oxidative stress to start mitophagy.Mitophagy is a potential mechanism that promotes cardiomyocyte survival under oxidative stress conditions.SIRT3 is an intracellular protease with deacetylation modification as its main function,and SIRT3 regulates mitophagy by effecting deacetylation and nuclear translocation of FOXO3a during oxidative stress injury.The previous research found that the number of autophagosomes in rat myocardial tissue increased significantly after MI.Autophagy related proteins were highly expressed in the 28th day after MI.Yiqi Huoxue decoction affects autophagy through the m-TOR pathway to exert a protective effect on cardiomyocytes.Objectives:1.To evaluate the oxidative stress injury in cardiomyocytes of infarcted rats,and to study the effects of Yiqi Huoxue decoction on mitophagy levels and SIRT3 pathway in the myocardial infarction border zone.2.To observe the mitochondrial damage,apoptosis,and mitophagy in the H9c2 cell oxidative stress injury model,and to reveal the molecular mechanism of MI by Yiqi Huoxue decoction based on the SIRT3 pathway.Methods:This study is divided into two parts:animal experiments and cell experiments.Animal experiments:Healthy male SD rats underwent ligation of the left anterior descending coronary to replicate the myocardial infarction model.After successful modeling,they were divided into sham Group(Group S),myocardial infarction Group(Group M),Yiqi Huoxue decoction Group(Group Y)and Perindopril Group(Group P).Cardiac function was assessed by echocardiography after 28 days;HE staining,Masson staining,and transmission electron microscopy were used to observe the pathological alterations of heart structure;The LDH,CK,and oxidative stress-related indicators such as SOD,MDA,GSH,GSH-Px,and CAT levels were detected by commercial kits.Detection of LC3B,p62,PINK1,Parkin,SIRT3,FOXO3a mRNA and protein expression levels by RT-qPCR and Western blot.Immunofluorescence was uesd to observe the co-localization of LC3B,PINK1/Parkin and mitochondria;Immunohistochemistry was uesd to observe nuclear translocation changes of FOXO3a.Cell experiments:Oxidative stress in H9c2 cardiomyocytes was induced by H2O2.CCK8 and DPPH were used to screen drugs and H2O2 concentrations.The LDH,CK,and oxidative stress-related indicators such as SOD,MDA,GSH,GSH-Px,and CAT levels were detected by commercial kits.Fluorescent cell labeling and/or flow cytometry were used to detect reactive oxygen species,mitochondrial permeability transition pore(mPTP),ΔΨm,and calcium(Ca2+)concentration to evaluate mitochondrial damage.Annexin V-FITC and TUNEL staining were used to detect apoptosis.MDC staining and Ad-mCherry GFP-LC3B fluorescence labeling were used to observe cellular autophagy levels.Mitophagy Detection Kit was used to stain mitochondria and lysosomes in living cells,and mitophagy levels were observed using fluorescence microscopy and a fluorescent microplate reader.Western blot were used to detect LC3B,p62,PINK1,Parkin,SIRT3 and FOXO3a protein levels.Immunofluorescence staining was used to observe the localization changes of LC3B,p62,PINK1,and Parkin.Results:1.Effect of Yiqi Huoxue decoction on Cardiac Function in Rats with MI:on Day 28 after MI,compared with the Group S,left ventricular ejection fraction(LVEF),short-axis rate(LVFS)decreased,and end-systolic internal diameter and end-diastolic internal diameter(LVIDs/d)increased in Group M(P<0.05),and the Yiqi Huoxue decoction and perindopril increased LVEF and LVFS and decreased LVIDs(P<0.05).2.Effect of Yiqi Huoxue decoction on Heart Structure of Rats with MI:HE staining showed that the cells in Group S were neatly arranged and clearly structured,with the nuclei distributed in the middle and morphologically intact,and no inflammatory cell infiltration was observed.In the Group M,the number of cardiomyocytes in the myocardial infarction border zone decreased,with the irregular arrangement,increased intercellular space,myocardial fiber breakage,and obvious inflammatory cell infiltration.The degree of structural destruction and inflammatory cell infiltration of cardiomyocytes in the Group Y and Group P were lighter than those in the Group M.Masson staining showed that the cell structure in Group M was surrounded with collagen fibers,shown in blue.The area of blue staining in Group Y and Group P was reduced compared with that in Group M.3.Effects of Yiqi Huoxue decoction on LDH and CK and oxidative stress-related factors in MI rats:Compared with Group S,LDH and CK levels were increased in Group M(P<0.05),while Yiqi Huoxue decoction and perindopril reduced LDH and CK(P<0.05).Compared with Group S,SOD,GSH-Px and CAT levels were decreased and MDA levels were increased in Group M(P<0.05),while Yiqi Huoxue decoction increased SOD,GSH-Px and CAT levels and decreased MDA levels(P<0.05).4.Electron microscopy to observe mitochondrial damage and autophagosomes in the myocardial infarction border zone:The myocardial fibers of cardiac myocytes in Group S were well arranged,with clear Z and M lines,intact mitochondrial structure,clear cristae structure and uniform distribution of matrix particles.Myofilaments of cardiac myocytes in Group M were broken and the mitochondrial structure was severely damaged,and autophagosomes were seen wrapped around the phagocytosed damaged mitochondria.The myofilament gap was larger in cardiac myocytes of Groups Y and P.Mitochondria were seen to be phagocytosed by lysosomes,but the mitochondrial morphology was better than that of Group M.Flameng scores were significantly higher in Group M compared with Group S,while Flameng scores decreased in Groups Y and P(P<0.05).5.Effects of Yiqi Huoxue decoction on Mitophagy-related Genes and Proteins in the myocardial infarction border zone:The RT-qPCR results showed that compared with the Group M,the levels of PINK 1 and Parkin mRNA increased in the Group Y and Group P(P<0.05).The Western blot results showed that compared with Group S,the expression level of PINK1 protein in Group M was significantly increased(P<0.05).Compared with the Group M,Yiqi Huoxue decoction and Perindopril significantly increased the expression levels of PINK1 and Parkin protein(P<0.05).Immunofluorescence showed that compared with Group M,Yiqi Huoxue decoction promoted the co localization of LC3B and mitochondria,as well as the co localization of PINK1,Parkin and mitochondria.6.Effect of Yiqi Huoxue decoction on SIRT3 Pathway in the myocardial infarction border zone:RT-qPCR results showed that SIRT3 mRNA was significantly decreased in Group M compared with Group S(P<0.05),while both Yiqi Huoxue decoction upregulated SIRT3 mRNA(P<0.05).FOXO3a mRNA expression was not significantly different in each Group(P>0.05).Western blot results showed that compared with Group S,SIRT3 and FOXO3a protein expression was decreased and acetylated FOXO3a protein expression was increased in the Group M compared with the Group S,while the Yiqi Huoxue decoction could attenuate this change(P<0.05).IHC results showed that in the Group S,FOXO3a was localized to the nucleus;the co-localization of FOXO3a with the nucleus was reduced in the Group M and mostly distributed in the cytoplasm,while the co-localization of FOXO3a with the nucleus was increased in the Group Y and Group P.7.Effects of Yiqi Huoxue decoction on H2O2-induced H9c2 cardiomyocyte viability and oxidative stress:Yiqi Huoxue decoction had an appreciable scavenging capacity on DPPH radicals with a dose-dependent effect(P<0.05).After treatment with 400 μM H2O2 for 4 h,LDH,CK and MDA levels were increased and SOD,GSH-Px and CAT levels were decreased in the model Group compared with the control Group,while Yiqi Huoxue decoction could effectively reduce LDH,CK and MDA levels and increase SOD,GSH-Px and CAT(P<0.05).8.Effects of Yiqi Huoxue decoction on mitochondrial damage and apoptosis in H9c2 cells:Compared with the control Group,ROS and Ca2+concentration levels increased,mPTP remained open,ΔΨm decreased and apoptotic cells increased in the model Group,whereas Yiqi Huoxue decoction reduced ROS and Ca2+concentrations,promoted mPTP closure,increased ΔΨm and reduced apoptosis(P<0.05),and this effect was attenuated by the mitophagy inhibitor Mdivi-1(P<0.05).9.Effect of Yiqi Huoxue decoction on Mitophagy in H9c2 Cells:The results of mitophagy staining showed that cellular autophagy and mitophagy levels were elevated in the model Group compared with the control Group,while Yiqi Huoxue decoction could promote mitophagy levels(P<0.05).Western blot results showed that compared with the control Group,the ratio of LC3B Ⅱ to LC3B Ⅰ was significantly higher in the model Group and the Yiqi Huoxue decoction Group(P<0.05),while the protein levels of LC3B were not significantly altered between the remaining Groups;PINK1 and Parkin protein levels were increased in the model Group,while Yiqi Huoxue decoction could promote the increase of PINK1 and Parkin protein levels,and this alteration was attenuated by 3-TYP(P<0.05).Immunofluorescence results showed that in the control Group,LC3B and p62 exhibited weak diffuse fluorescence,and both LC3B and p62 in the model Group showed punctate aggregation,but Yiqi Huoxue decoction attenuated this change.In addition,compared with the control Group,the fluorescence intensity of Parkin and PINK1 increased in the model Group,and their co-localization increased.Parkin co-localized with Tomm20,a mitochondrial marker protein,which was promoted by Yiqi Huoxue decoction,and this alteration was attenuated by 3-TYP.10.Effect of Yiqi Huoxue decoction on SIRT3 Pathway in H9c2 Cells:Western blot results showed decreased SIRT3 protein and increased Ace-FOXO3a protein levels in the model Group compared with the control Group(P<0.05),whereas SIRT3 protein levels were increased and Ace-FOXO3a protein levels were decreased in the Yiqi Huoxue decoction Group compared with the model Group(P<0.05),but this effect was partially reversed by 3-TYP.FOXO3a protein was not significantly altered in any of the Groups(P>0.05).Immunofluorescence results showed that in normal cells,FOXO3a was almost exclusively localized to the nucleus,and H2O2 stimulation led to a decrease in the co-localization of FOXO3a with the nucleus.However,Yiqi Huoxue decoction increased the co-localization of FOXO3a with the nucleus,and this change was partially reversed by 3-TYP.Conclusions:1.Yiqi Huoxue decoction could improve the structure and function of the myocardium after MI and reduce myocardial oxidative stress damage.It also promotes the occurrence of mitophagy in the myocardial infarction border zone and affects the expression of the SIRT3 pathway.2.Yiqi Huoxue decoction could reduce mitochondrial function damage and apoptosis in H9c2 oxidative stress injury model by oxidative stress,which was related to mitophagy.3.Yiqi Huoxue decoction could upregulate the expression of SIRT3,enhance its deacetylation function,promote the nucleation of FOXO3a,and promote mitophagy in an oxidative stress cell model. |
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