Panax Notoginseng Saponins Regulate Mitophagy Against Myocardial Ischemia-Reperfusion Injury Via PINK1/Parkin Pathway

Objective To observe whether Panax notoginseng saponins（PNS）can regulate mitophagy through PINK1/Parkin pathway and affect mitochondrial dynamics to maintain mitochondrial homeostasis synergistically and reduce myocardialischemia-reperfusion injury on rat’s myocardial ischemia-reperfusion（I/R）and cardiomyocyte hypoxia/reoxygenation（H/R）models.Methods 40 SD rats were randomly divided into 3 groups:sham operation group（Sham,n=11）,myocardial ischemia-reperfusion group（I/R,n=17）,myocardialischemia-reperfusion+PNS group（I/R+PNS,n=12）.The myocardial I/R model was established by ligation of the left anterior descending coronary artery for 40 min and reperfusion for 120 min.The hemodynamic parameters were measured at the end of reperfusion.The myocardial pathological changes were observed under lightmicroscope and electron microscope.The myocardial infarct size was detected byTTC staining.Serum LDH and CK levels were detected,and the protein expression of mitochondrial PINK1,Parkin and LC3 in myocardial tissue were detected by Western Blot.H9c2 cells were pretreated with different concentrations of PNS,and the H/model was established by hypoxia for 6 h and reoxygenation for 2 h.CCK8 was used to show cell viability and LDH was detect to indicate cell damage.After determining the optimal dose of PNS as 100μg/ml,the experiment was carried out in thefollowing groups:normal control group（Control）,hypoxia/reoxygenation group（H/R）,hypoxia/reoxygenation+PNS group（H/R+PNS）,hypoxia/reoxygenation+PNS+carbonyl cyanide-3-chlorophenylhydrazine group（H/R+PNS+CCCP）,hypoxia/reoxygenation+Mito-TEMPO group（H/R+Mito-TEMPO）,hypoxia/reoxygenation+Mito-TEMPO+PNS group（H/R+Mito-TEMPO+PNS）.Mitochondrial ROS and mitophagy were observed by laser microscopy.Western Blot was used to detect the proteins levels of mitochondrial PINK1,Parkin,LC3,Mfn1,Mfn2,Opa1,Drp1 and Fis1.Results1.PNS increased the levels of m LVSP,DP,+dp/dt_max（p<0.05～0.01）,decreased m LVDP、-dp/dt_max levels（p<0.05）,improving cardiac function after I/R.2.PNS alleviated the pathological changes and mitochondrial morphological structure in MIRI.3.PNS reduced MIRI myocardial infarction area（p<0.001）,as well as LDH and CK levels in rat’s serum（p<0.01）.4.PNS decreased the expression of mitochondrial PINK1,Parkin and LC3 in reperfused myocardium（p<0.05～0.001）.5.PNS increased H9c2 cell activity after H/R（p<0.001）and reduced LDH leakage（p<0.001）.6.PNS could inhibit H9c2 cell mitophagy and the production of autophagy lysosome,as well as down-regulating the expression of mitochondrial PINK1,Parkin and LC3in H/R cardiomyocytes（p<0.05～0.001）,similar to Mito-TEMPO.CCCP couldreverse the role of PNS in inhibiting mitophagy by PINK1/Parkin pathway（p<0.05～0.01）.7.PNS decreased mitochondrial ROS levels in H/R cardiomyocytes.8.PNS increased the protein expression of CII and CIII in the mitochondrial respiratory chain and reduced ROS generation（p<0.05）,which was similar to Mito-TEMPO and could be reversed by CCCP（p<0.05～0.01）.9.PNS up-regulated the expression of Mfn1,Mfn2 and Opa1 in H/R cardiomyocytes（p<0.01～0.001）,reduced the expression of Drp1 and Fis1（p<0.05～0.001）,whichcould be reversed by CCCP（p<0.01～0.001）.Conclusion PNS can inhibit myocardial mitophagy through PINK1/parkin pathway during ischemia-reperfusion,and affect mitochondrial dynamics（division/fusion）,maintain mitochondrial homeostasis,and finally play a cardioprotective role in MIRI.