| Objective: To study the intervention effects of Gualou Xiebai Banxia decoction(GXBD)on the PINK1/Parkin signaling pathway during myocardial ischemia-reperfusion injury,and to identify the therapeutic target and mechanism of this decoction on mitochondrial autophagy in myocardial ischemia-reperfusion injury(MI/RI).Methods: 1.Fifty male SD rats were randomly divided into sham operation group,MI/RI model group,mdivi-1 inhibitor group,high-dose and low-dose groups of GXBD,each group 10 only.Each group was fed with drugs for three weeks in advance,and the MI/RI model was established by reversible ligation of the left anterior descending coronary artery for 30 min ischemia and reperfusion for 2 h.The content of serum cTnI was detected by enzyme-linked immunosorbent assay(ELISA),the content of serum LDH was detected by lactic acid substrate method,the serum CK-MB was determined by immunosuppression.The content of myocardial ATP was determined by bioluminescence.Fluorescence probe was used to detect the changes of myocardial mitochondrial membrane potential.The protein expressions of Beclin-1,p62,Drp1,PINK1 and Parkin in cardiac mitochondria were determined by Western Blot.2.Sixty male SD rats were randomly divided into sham operation group,MI/RI model group,3-MA inhibitor group,high-dose and low-dose groups of GXBD,each group 12 only.Each group was fed with drugs for three weeks in advance,and the MI/RI model was established by reversible ligation of the left anterior descending coronary artery for 30 minischemia and reperfusion for 2 h.The protein expressions of Beclin-1,p62,Bax,Bcl-2,PINK1 and Parkin in myocardial tissue were determined by Western Blot.Results:(1)Under the transmission electron microscope,the morphology of myocardial myofilaments in the sham operation group were complete and closely arranged,and the structure of mitochondrial membrane and ridge were basically normal,there was no obvious swelling in the whole,and the arrangement was good.In the MI/RI model group,the myocardial filaments were obviously broken or loose,the mitochondria were swelled or vacuolated,the internal structure was loose,and autophagosomes were formed in the myocardial tissues and mitochondria.Although the myocardial myofilament was fractured or loose in the 3-MA inhibitor group,high-dose and low-dose groups of GXBD,the degree of myocardial myofilament was significantly less than that of the MI/RI model group.The mitochondria were also swollen,but the morphology was relatively intact.The degree of ridge process loosening was lighter than that of the model group,there was no significant vacuolization,and there were a few autophagosomes in myocardial tissue and mitochondria.(2)Compared with the sham operation group,the contents of serum CK-MB,LDH and cTnI in MI/RI model group were significantly increased(P<0.05),while the content of myocardial ATP and mitochondrial membrane potential were significantly decreased(P<0.01 or P<0.05).Compared with the MI/RI model group,the contents of the LDH and CK-MB in the mdivi-1 inhibitor group,high-dose and low-dose groups of GXBD were significantly decreased,while the ATP contents and membrane potential were significantly increased(P<0.01 or P<0.05),and content of the cTnI in the mdivi-1 inhibitor group and high-dose group of GXBD was significantly decreased(P<0.01).(3)Compared with the sham operation group,the expression of mitochondrial Drp1 protein in the MI/RI model group was significantly increased(P<0.05),the expression of Beclin-1 and p62 proteins in the myocardial tissue and mitochondria were significantly increased(P<0.01 or P<0.05),the expression of Bax protein in the myocardial tissue was significantly increased and the expression of Bcl-2 protein was significantly decreased(P<0.01 or P<0.05).Compared with the MI/RI model group,the expressions of Beclin-1,p62 and Drp1 proteins in the mitochondria were significantly decreased in the mdivi-1inhibitor group,high-dose and low-dose groups of GXBD(P<0.05).The expressions of Beclin-1,p62 and Bax proteins in the myocardium were significantly decreased in 3-MA inhibitor group,high-dose and low-dose groups of GXBD(P<0.01 or P<0.05)while the expression of Bcl-2 protein was significantly increased(P<0.05).(4)Compared with the sham operation group,the expressions of PINK1 and Parkin proteins in myocardial tissue and mitochondria were significantly increased in the MI/RI model group(P<0.05).Compared with the MI/RI model group,the expressions of mitochondrial PINK1 and Parkin proteins in the mdivi-1 inhibitor group,high-dose and low-dose groups of GXBD were all significantly reduced.The expressions of myocardial tissue PINK1 protein were significantly decreased in the 3-MA inhibitor group,high-dose and low-dose groups of GXBD were all significantly reduced(P<0.01 or P<0.05).The expression of cardiac Parkin protein was significantly decreased in the 3-MA inhibitor group and in the high-dose groups of GXBD(P<0.01 or P<0.05).Conclusion: MI/RI can lead to the destruction of myocardial mitochondrial structure,the significant decrease of mitochondrial membrane potential and ATP content,the increase of serum myocardial enzyme,and the activation of mitochondrial autophagy mediated by the PINK1/Parkin pathway,and the aggravation of myocardial cell injury.GXBD can inhibit the release of serum CK-MB,LDH and cTnI,Partial recovery of myocardial ATP content and mitochondrial membrane potential level,reduce the damage of myocardial tissue structure and maintain the relative integrity of mitochondrial function,Inhibit the over-activation of mitochondrial autophagy mediated by the PINK1/Parkin pathway,and thus have a better cardiac protection effect on MI/RI rats. |
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