Optimization And Mechanism Of Ginsenoside Re On Quality Control Of Myocardial Mitochondrial With Ischemic/Hypoxic Injury

Acute myocardial infarction(AMI)refers to myocardial tissue necrosis caused by acute and persistent ischemia and hypoxia of coronary artery,which can lead to progressive cardiac insufficiency and even heart failure(HF).It is the most serious ischemic heart disease with high morbidity and mortality in the world,which is one of the clinical problems to be solved urgently.Adverse ventricular remodeling(AVR)after myocardial infarction is strongly correlated with the occurrence of HF and poor prognosis of patients.The change of mitochondrial energy metabolism is also one of the key mechanisms involved in the occurrence and progression of AVR,while the structure and function dysfunction of mitochondria is the core of myocardial metabolism disorder.Myocardial mitochondrial structural damage and dysfunction can lead to increased apoptosis.Therefore,protecting the integrity of cardiomyocyte mitochondrial structure and function is of great value in reducing apoptosis,alleviating post-infraction AVR and delaying the process of HF after AMI.The imbalance of mitochondrial quality control(MQC)system in cardiomyocytes can directly lead to changes in mitochondrial structure and function,and then induce the dysfunction and even death of cardiomyocytes.Therefore,the optimization of MQC system is very important for maintaining mitochondrial function.In this study,the AMI model of SD rats and the hypoxic injury model of H9c2 cardiomyocytes were established respectively.Ginsenoside Re was used as an intervention to observe its effects on cardiac structure and function,mitochondrial structure and function after ischemia and hypoxia injury,and to further study its regulation of MQC system related signal pathway by regulating SIRT3 deacetylase activity and protein expression.The purpose of this study is to provide scientific basis for TCM to prevent AVR after AMI and improve the clinical prognosis of AMI.Part 1 Effects of ginsenoside Re on cardiac function and ventricular remodeling of SD rats after AMI.Object:To observe the effects of ginsenoside Re intervention on cardiac function and myocardial pathology of SD rats after AMI,and to explore the mechanism of ginsenoside Re reducing myocardial apoptosis,reducing or reversing AVR,and then delaying the development of HF after AMI.Methods:The animal model was established by ligating the left anterior descending branch.After Modeling,the experimental rats were randomly divided into the following 5 groups,the sham group(Sham),the model group(Model),the ginsenoside Re low dose group(G-L),the ginsenoside Re high dose group(G-H)and the trimetazidine hydrochloride group(TMZ).15 rats in each group were treated once a day after surgery for 8 weeks.After the intervention,echocardiography was utilized to assess the cardiac function,with left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)as the main markers.Heart weight index(HWI)and left ventricular weight index(LVWI)were calculated separately to reflect the compensatory hypertrophy of myocardium after AMI.ELISA was used to detect the contents of CK-MB and BNP in serum.Histopathological staining was performed with HE,Masson and TUNEL respectively to observe the myocardial tissue structure,fibrosis and myocardial cell apoptosis after AMI.WST-8 method and colorimetry were adopted to detect the activity of superoxide dismutase(SOD)and the content of malondialdehyde(MDA)in myocardium.Western blot was used to detect the expression of Bax and Bcl2.Results:(1)Compared with Sham,LVEF and LVFS in Model decreased significantly(P<0.01),while LVEF and LVFS increased significantly in G-L,G-H and TMZ compared with Model(P<0.01).(2)Compared with Sham,the levels of CK-MB and BNP in Model significantly increased(P<0.01).While the levels of CK-MB and BNP in G-L,G-H and TMZ significantly decreased compared with Model(P<0.01).(3)Compared with Sham,HWI and LVWI increased in Model,G-L,G-H and TMZ(P<0.01),while HWI and LVWI decreased significantly in G-L,G-H and TMZ compared with Model(P<0.05).(4)Compared with Sham,the structure of myocardial fiber in Model was disordered with abundant inflammatory cell infiltration.And compared with Model,the pathological changes of myocardium in G-L,G-H and TMZ improved significantly.(5)Compared with Sham,the collagen volume fraction(CVF)and the apoptosis index(AI)were higher in Model(P<0.01),while CVF and AI in GL,G-H and TMZ were remarkably lower in Model(P<0.01).(6)Compared with Sham,the SOD activity in Model decreased significantly(P<0.01)and the MDA content increased significantly(P<0.01).Compared with Model,the SOD activity increased in different degrees in G-L,G-H and TMZ(P>0.05,P<0.05,P<0.05),and the MDA content decreased significantly in G-L,G-H and TMZ(P<0.01).(7)The expression of Bax protein significantly increased in Model compared with Sham(P<0.05),while the expression level of Bax protein significantly decreased in G-H and TMZ compared with Model(P<0.05).The expression of Bcl2 protein in Model decreased compared with Sham(P<0.05),while the expression level of Bcl2 protein in G-L,G-H and TMZ increased in different levels compared with Model(P<0.05,P<0.01,P<0.05).Compared with Sham,the ratio of Bax/Bcl2 in Model increased significantly(P<0.01)while that in G-L,G-H and TMZ decreased significantly(P<0.01).Conclusion:Ginsenoside Re can reduce myocardial ischemic injury of SD rats after AMI,inhibit myocardial compensatory hypertrophy and fibrosis,reduce oxidative stress injury and apoptosis,protect cardiac function and delay the process of HF after AMI.Part 2 Effects and mechanism of ginsenoside Re on the structure and function of mitochondria of SD rats after AMI.Objective:To observe the effects of ginsenoside Re on myocardial mitochondrial ultrastructure and mitochondrial function of SD rats after AMI,and to explore the possible mechanism.Method:The AMI model of SD rats was established according to the method in"Part 1",and the intervention was conducted after randomization.The ultrastructure of myocardial mitochondria,mitochondrial membrane potential(MMP),energy efficiency of mitochondrial electron chain and energy supply of mitochondria were analyzed after intervention.The deacetylase activity of SIRT3 was tested by fluorescence,and the expression of SIRT3,Ac-FoxO1 and MQC system related proteins was detected by Western blot.Results:(1)Compared with Sham,the number of myocardial mitochondria in Model was reduced,and the structure was disordered with obvious hyperplasia and hypertrophy of which some were even in a huge spindle shape.In the meantime,mitochondrial crest fractured.Compared with Model,the ultrastructure of myocardial mitochondria in G-L,G-H and TMZ was significantly improved.(2)Compared with Sham,MMP decreased significantly in Model,G-L,G-H and TMZ(P<0.01).Compared with Model,MMP increased in different degrees in G-L,G-H and TMZ(P>0.05,P<0.05,P<0.01).(3)Compared with Sham,the ratio of NAD+/NADH was significantly lower in Model(P<0.01),while the NAD+/NADH ratio in G-L,G-H and TMZ was significantly higher than Model(P<0.01).(4)The ATP content in Model decreased significantly compared with Sham(P<0.01),while that in G-L,G-H and TMZ increased compared with Model(P>0.05,P<0.05,P<0.05).(5)Compared with Sham,SIRT3 deacetylase activity decreased significantly in Model(P<0.01).Compared with Model,that increased in G-L,G-H and TMZ differently(P>0.05,P<0.01,P<0.01).(6)The expression of SIRT3 protein in Model decreased compared with Sham(P<0.05).Compared with Model,the expression of SIRT3 protein increased in different degrees in G-L,G-H and TMZ(P<0.05,P<0.01,P<0.01).(7)Compared with Sham,the degree of FoxO1 protein acetylation in Model was higher(P<0.05),while the degree of FoxO1 acety lation was lower in G-L,G-H and TMZ compared with Model(P>0.05,P<0.05,P<0.05).(8)Compared with Sham,the expression level of Fisl protein in Model increased(P<0.05).And that in G-L,G-H and TMZ decreased compared with Model(P<0.05).Compared with Sham,the expression of SOD2 protein significantly decreased in Model(P<0.01).Compared with Model,G-L,G-H and TMZ increased the relative expression of this protein to varying degrees(P<0.05,P<0.01,P<0.05).Compared with Sham,the expressions of Mfn1 and Mfn2 protein in Model decreased(P<0.05).Compared with Model,G-L,G-H and TMZ increased the expression of Mfn1 and Mfn2 protein in different levels(P>0.05,P<0.01,P<0.05),(P<0.05,P<0.01,/P>0.05).Compared with Sham,the expressions of PGC-lα protein decreased significantly(P<0.01).Compared with Model,G-L,G-H and TMZ increased the relative expression of this protein to varying degrees(P>0.05,P<0.01,P>0.05).Conclusion:Ginsenoside Re can protect the integrity of mitochondrial structure and function from ischemic injury,stabilize MMP,improve mitochondrial productivity efficiency and increase energy supply in AMI rats.Furthermore,it can reduce myocardial oxidative stress and cardiomyocyte apoptosis.The mechanism may be related to the regulation of SIRT3 deacetylase activity and protein expression,so as to optimize the MQC system.Part 3 Study on the mechanism of ginsenoside Re regulating SIRT3 deacetylation to protect H9c2 cardiomyocytes from hypoxia injuryObject:To observe the effects of ginsenoside Re on mitochondrial structure and mitochondrial related functions such as oxidative stress and apoptosis in H9c2 cardiomyocytes injured by hypoxia,and to explore whether the above effects are related to the regulation of MQC system by SIRT3.Methods:The hypoxia injury model of H9c2 myocardium was established by hypoxia intervention.The range of ginsenoside Re intervention concentration and the optimal intervention concentration of trimetazidine hydrochloride were screened by CCK-8 method.The experiment was divided into 6 groups,the control group(Control),the model group(Model),the ginsenoside Re low concentration group(G-L),the ginsenoside Re medium concentration group(G-M),the ginsenoside Re high concentration group(G-H)and the trimetazidine hydrochloride group(TMZ).After intervention,cardiomyocytes or mitochondria was collected for further detection,including the content of lactate dehydrogenase(LDH),the SOD activity,the MDA content,the ROS level,MMP,ATP,Mito TrackerTM Red,mitochondrial SIRT3 deacetylase activity and protein expression,cardiomyocyte apoptosis and the expression of MQC system related proteins.Results:(1)Through CCK-8 detection,24-hour was confirmed as the optimal intervention time for hypoxic injury of H9c2 cardiomyocytes.(2)Through CCK-8 detection,50μmol/L,100μmol/L and 200μmol/L ginsenoside Re were set as low,medium and high concentration of ginsenoside Re in the experiment.And 10 μmol/L trimetazidine hydrochloride was used as the best intervention concentration of the positive control group.(3)Compared with Control,the LDH content in Model significantly increased(P<0.01).Compared with Model,LDH level of cell supernatance in G-L,G-M,G-H and TMZ significantly decreased(P<0.01).(4)Compared with Control,the SOD activity in Model significantly decreased(P<0.01)and the MDA content significantly increased(P<0.01).Compared with Model,the SOD activity in G-L,G-M,G-H and TMZ significantly increased(P<0.01),while the MDA content significantly decreased(P<0.01).(5)Compared with Control,the average fluorescence intensity of ROS in Model significantly increased(P<0.01).Compared with Model,it decreased to varying degrees in G-L,G-M,G-H and TMZ(P>0.05,P<0.01,P<0.05,P>0.05).(6)Compared with Control,MMP in Model was significantly lower(P<0.01).Compared with Model,MMP in G-L,G-M,G-H and TMZ significantly increased(P<0.01).(7)Compared with Control,the ratio of NAD+/NADH and ATP expression in Model significantly decreased(P<0.01).Compared with Model,the ratio of NAD+/NADH in G-L,G-M,G-H and TMZ increased in different levels(P<0.05,P<0.05,P>0.05,P>0.05),the expression of ATP in G-L,G-M,G-H and TMZ increased distinctly(P>0.05,P<0.05,P>0.05,P<0.05).(8)Compared with Control,the average fluorescence intensity of Mito TrackerTM Red in Model significantly decreased(P<0.01).Compared with Model,that in G-L,G-M,GH and TMZ increased in different contents(P<0.01,P<0.01,P<0.05,P<0.01).(9)Compared with Control,the ratio of Bax/Bcl2 in Model was significantly higher(P<0.01).Compared with Model,the ratio in G-L,G-M,G-H and TMZ decreased by different degrees(P<0.01,P<0.01,P<0.05,P<0.01).(10)Compared with Control,the SIRT3 deacetylase activity and protein expression in Model were significantly lower(P<0.01).Compared with Model,SIRT3 enzyme activity and protein expression level of mitochondria of cardiomyocytes in G-L,G-M,G-H and TMZ increased differently(P>0.05,P<0.01,P<0.05,P<0.05).(11)Compared with Control,the degree of FoxO1 acetylation significantly increased in Model(P<0.01).Compared with Model,the degree of FoxO1 acetylation in G-L,G-M,G-H and TMZ significantly decreased(P<0.05).Compared with Control,Fisl protein expression in Model significantly increased(P<0.01).Compared with Model,the expression of Fis1 protein in G-L,GM,G-H and TMZ significantly decreased(P<0.05).Compared with Control,the relative expression level of SOD2 protein in Model significantly decreased(P<0.01).Compared with Model,the protein expression level in G-L,G-M,G-H and TMZ increased to varying degrees(P<0.05,P<0.01,P<0.01,P<0.05).Compared with Control,the expression of Mfn1 and Mfn2 in Model were significantly lower(P<0.01).Compared with Model,the expression level of Mfnl protein in G-L,G-M,G-H and TMZ increased to varying degrees(P<0.05,P<0.05,P>0.05,P>0.05),and the expression level of Mfn2 protein also increased to various degrees(P<0.05,P<0.01,P<0.05,P<0.05).Compared with Control,the expression of PGC-1α protein in Model significantly decreased(P<0.01).Compared with Model,G-L,G-M,G-H and TMZ increased the relative expression of this protein to varying degrees(P<0.05,P<0.05,P<0.05,P>0.05).Conclusion:Ginsenoside Re can improve mitochondrial productivity,enhance mitochondrial function,stabilize MMP,reduce oxidative stress injury and inhibit the apoptosis of H9c2 cardiomyocytes by maintaining mitochondrial homeostasis.The mechanism may be related to the regulation of FoxO1 deacetylation activity by SIRT3 to optimize MQC system.