Mechanism Of Shuangshen Ningxin Capsule Regulating Mitochondrial Autophagy Against Myocardial Ischemia-reperfusion Injury In Miniature Pigs

Ⅰ.PurposeTo study the protective effect of Shuangshen Ningxin Capsule(SSNX)on myocardial ischemia reperfusion injury(MIRI)in miniature pigs,and reveal its mechanism of regulating mitochondrial autophagy in myocardial cells to protect MIRI from PINK 1/Parkin pathway1.Part Ⅰ Protective effect of Shuangshen Ningxin capsule on MIRI in miniature pigs1.1 Animal group and administration Male Bama miniature pigs were randomly divided into sham operation group,model group,positive drug group,SSNX preventive treatment group and SSNX treatment group,with 6 pigs in each group.Except SSNX preventive treatment group(20mg·kg-1),which was given in advance for 7 days,the other 4 groups were given routine feeding for 7 days.Seven days later,except for the sham operation group,the other groups were given interventional balloon blocking the proximal blood flow structure reperfusion model of the left anterior descending branch;The animals in treatment group and preventive treatment group were fed with SSNX(20 mg·kg-1)for 7 days after operation.The positive drug group was fed with nicorandil(1.2 mg·kg-1)for 7 days,while the sham operation and model group were fed with ordinary feed for 7 days.1.2 MIRI model evaluation 15min minutes after operation 12-lead ECG was detected,and the model construction was evaluated by ECG changes.1.3 Analysis of tongue picture and determination of biochemical indexes Blood was collected before and 7 days after operation,and the tongue picture of miniature pigs was detected by macroscopic characterization and photographed.PT image analysis software was used to analyze the r,g and b value indexes of color classification of tongue picture of miniature pigs.Enzyme-linked immunosorbent assay(ELISA)was used to detect serum cardiac troponin(cTnT),IFCC method was used to detect serum lactate dehydrogenase(LDH),colorimetric method was used to determine serum creatine kinase isoenzyme(CK-MB),and immunosuppressant method was used to determine creatine kinase MB isoenzyme.Automatic blood coagulation instrument monitors coagulation indexes and evaluates coagulation function;Nitric oxide(NO)was measured by nitrate reductase method,and endothelin-1(ET-1)was measured by enzyme-linked immunosorbent assay to evaluate the damage of vascular endothelial function.Detection of brain natriuretic peptide(BNP)by enzyme-linked immunosorbent assay was used to diagnose and evaluate the degree of heart failure in miniature pigs.The levels of superoxide dismutase(SOD)and malondialdehyde(MDA)in serum were detected by microanalysis to evaluate the oxidative stress level of myocardium in miniature pigs.1.4 Evaluation of cardiac function by imaging echocardiography was performed at 30min and 7 days after operation.Seven days after operation,myocardial infarction in miniature pigs was observed by cardiac magnetic resonance imaging(MRI).1.5 Myocardial infarction area calculation and pathomorphological observation 7 days after the experiment,all animals were killed and the myocardium in the proximal distribution area of the left anterior descending coronary artery was taken out;Myocardial infarction area was measured by MPIAS-500 multimedia color pathological graphic analysis system software after NB-T staining.Pathological changes of myocardial vessels and tissues were observed by HE staining and Masson staining.2.Results2.1 Analysis of tongue picture The tongue of small pigs in normal group is light red and the coating is thin and white.Compared with the normal group,the tongue quality of the model group was darker,the coating was darker and white,the R value of the tongue image decreased,and the G and B values of the tongue image increased Compared with the model group,SSNX miniature pigs have improved the dark red tongue and dark white fur,increased the r value of the tongue picture,and significantly decreased the g and b values of the tongue picture.2.2 Comparison of biochemical indexes of mini-pigs in each group the serum CK,CK-MB,LDH and cTnT contents in model group were higher than those in sham operation group While the serum CK,CK-MB,LDH and cTnT contents in SSNX group were lower than those in model group.The indexes related to coagulation function showed that PT and APTT time of model group was longer than that of sham operation group,and FIB value was decreased.Compared with the model group,PT and APTT time in SSNX group was shortened,and FIB value was increased.Oxidative stress index showed that SOD activity decreased and MDA value increased in model group compared with sham operation group.Compared with the model group,SOD activity increased and MDA value decreased in each treatment group.Endothelial function index showed that serum ET-1 in sham operation group was higher than that in model group,while NO value was lower.Compared with the model group,serum ET-1 decreased and NO increased in each administration group.The index of heart failure showed that BNP in model group was higher than that in sham operation group,while BNP in SSNX group was lower than that in model group.2.3 Compared with the normal group,the cardiac ultrasound results of the model group showed that many cardiac function indexes of miniature pigs changed Ejection fraction and short axis shortening fraction decreased,and end systole inner diameter,end systole volume,end diastole inner diameter and end diastole volume increased.Compared with the model group,the ejection fraction and short axis shortening fraction in Shuangshen Ningxin group increased,and the end diastolic volume decreased significantly.MRI results showed that the heart structure of miniature pigs in sham operation group was intact,and there was no abnormality in blood flow and opening and closing of each valve.In the model group,a large area of high density shadow appeared in the left ventricle and endocardium of miniature pigs,and even ventricular septal rupture occurred,which indicated that the degree of myocardial ischemia,myocardial necrosis and myocardial injury were serious in the model group miniature pigs.Some high-density shadows can also be seen in SSNX administration group,but the area of high-density shadows is smaller,the heart shape is relatively complete,and the damage degree is lighter than that in model group.2.4 Pathomorphological observation HE and Masson staining showed that myocardial fibers in normal group were arranged neatly,without degeneration,necrosis,swelling and fracture,and without inflammatory cell infiltration.In the model group,large areas of myocardial fibers were necrotic and calcified,myocardial fibers were degenerated and broken,fibrous tissues proliferated,inflammatory cells infiltrated,and necrotic areas were congested and bleeding.In the positive drug group,the muscle fibers were arranged neatly,the morphology of myocardial cells was normal,and the patchy myocardial fibers were necrotic and steatosis,accompanied by some tissue hyperplasia and inflammatory cell infiltration Most of the myocardial fibers in SSNX groups were arranged neatly,and the nucleus of the myocardium was round and located in the center of the cells.Some of the myocardial fibers were patchy and necrotic,accompanied by a small amount of hyperplasia and infiltration of inflammatory cells.2.5 Comparison of myocardial infarction area Compared with the model group The myocardial infarction rate of SSNX preventive treatment group and SSNX treatment group decreased significantly,and the infarction rate of positive drug group also decreased compared with the model group.3.SummaryThe MIRI model of miniature pigs was successfully constructed.SSNX can reduce serum myocardial enzyme level and improve vascular endothelial function and coagulation function in MIRI miniature pigs It can also increase SOD activity and reduce MDA level to resist oxidative stress;It can significantly reduce the myocardial infarction area of MIRI miniature pigs.Imaging and pathomorphology showed that myocardial injury was the most serious in model group,followed by positive drugs,followed by SSNX treatment group and SSNX preventive treatment group.The above results prove that SSNX can improve the heart function of MIRI miniature pigs and has a definite protective effect on MIRI.2.Part Ⅱ2.1 Animal grouping and administration methods are the same as those in the first part.2.2 Detection of ATP and Δψ Evaluation of Autophagy Level The content of adenosine triphosphate(ATP)in ischemic myocardium was detected by phosphomolybdic acid colorimetry.The changes of mitochondrial membrane potential(Δψ m)in myocardial cells were detected by fluorescence probe method.2.3 Ultrastructural observation of myocardium Ultrastructural changes of mitochondria and mitochondrial autophagy were observed under transmission electron microscope.2.4 qRT-PCR was used to detect the expression of related genes,Western Blot was used to detect the expression level of related autophagy proteins Real-time fluorescence quantitative PCR was used to detect the transcription level of autophagy related genes in myocardial tissue.The protein LC3-B,Cleave Caspase-3,PINK1 and Parkin in myocardial mitochondria were determined by Western Blot Express the situation.2.Part Ⅱ Study on autophagy mechanism of Shuangshen Ningxin capsule on MIRI mitochondria in miniature pigs2.1 Comparison of autophagy level in each groupCompared with sham operation group,ATP content and mitochondrial membrane potential in model group decreased significantly;Compared with the model group,myocardial ATP content and mitochondrial membrane potential in SSNX groups were significantly increased.Although the positive drug group had an increasing trend,the increasing value was not as obvious as that in SSNX groups.2.2 Myocardial ultrastructureUnder transmission electron microscope,it can be seen that the myocardial myofilaments in the sham-operated group are complete and closely arranged,and the mitochondrial membrane structure and ridge structure are basically normal,with no obvious swelling on the whole and good arrangement;In the model group,the myocardial myofilaments were obviously broken or loose,the mitochondria were swollen or vacuolated,the internal structure was loose,and autophagy was formed in the myocardial tissue and mitochondria In SSNX treatment group,SSNX preventive treatment group,and positive drug group,although myocardial myofilaments were broken or loose,the degree was significantly reduced compared with that in model group.Mitochondria became swollen but the shape was relatively intact,the degree of cristae looseness decreased,and there was no significant cavitation in the whole.There were a few autophagy structures in myocardial tissue and mitochondria.2.3 Comparison of gene expression levels of related proteinsqRT-PCR results showed that the mRNA gene expression levels of PINK1,Parkin,Cleaved caspase-3 and LC3B were higher than those of the model group.Compared with the model group,the expression level of each protein gene in the administration group showed a significant downward trend,and the decrease value was the most obvious in SSNX preventive treatment group,followed by SSNX treatment group and positive drug group.2.4 Comparison of expression levels of related autophagy pathway proteinsThe results of Western Blot showed that the expression levels of PINK 1/Parkin pathway,Cleaved caspase-3 autophagy pathway and LC3 Ⅱ/LC3 Ⅰ ratio in model group were higher than those in sham operation group.Compared with the model group,the expression level of each protein in the administration group showed a significant downward trend,and the decline value of SSNX preventive treatment group was the most obvious,followed by SSNX treatment group,and the decline value of positive drug group was relatively small,which was consistent with the trend of qRT-PCR.SummarizeMitochondrial autophagy is over-activated in MIRI stage.SSNX can reduce mitochondrial membrane potential and ATP content,and protect myocardium after ischemia-reperfusion injury.Its mechanism may be achieved by inhibiting PINK1/Parkin autophagy pathway,inhibiting the over-activation of autophagy level,and reducing myocardial apoptosis and mitochondrial damage.Ⅳ.ConclusionShuangshen Ningxin Capsule has protective effect on myocardial tissue of mini-pigs after myocardial ischemia-reperfusion injury,which can reduce the damage of myocardial tissue structure and maintain the relative integrity of mitochondrial function.Its mechanism may be through inhibiting the excessive activation of mitochondrial autophagy mediated by PINK)/Parkin pathway,thus protecting the heart function of mini-pigs after myocardial ischemia-reperfusion injury.