| Objective The purpose of this study was to observe the protective effects of three kinds of total glycosides of Yiqi Huoxue prescription on OGD/R-induced reperfusion injury in H9c2 cardiomyocytes and their effects on mitochondrial autophagy.At the same time,to explore whether the mechanism of paeoniflorin,the main component of the three total glycosides,on cardiomyocyte reperfusion injury is related to the regulation of PINK1/parkin-mediated mitochondrial autophagy pathway by SIRT1.Methods 1.The crude glycosides of three kinds of Yiqi Huoxue prescriptions were extracted by water extraction and alcohol precipitation and extraction.The main effective components of the three crude glycosides were detected.H9c2 was randomly divided into normal control group,myocardial ischemia-reperfusion group(OGD/R)and total glycosides pre-administration group.The model of cardiomyocyte reperfusion injury in vitro was established by hypoxia reoxygenation in anoxic chamber.The safe dose range of Yiqi Huoxue prescription to H9c2 cells and the effective drug concentration stimulated by OGD/R were screened and determined by MTT experiment.The expression levels of mitochondrial autophagy related signals in SIRT1,Beclin1,LC3-Ⅱ/Ⅰ,p62,PINK1 and parkin were detected by Westernblotting and Q-PCR assay.The effect of total glycosides of Yiqi Huoxue prescription on mitochondrial membrane potential of H9c2 cells injured by OGD/R was observed by fluorescence microscope.The effects of total glycosides of Yiqi Huoxue prescription on MDC autophagy fluorescence and ROS oxidative damage of H9c2 cells were detected by flow cytometry.The levels of MDA and SOD were also detected by kit.2.The effect of paeoniflorin on the proliferation rate of H9c2 cells was detected by MTT method,with or without paeoniflorin treatment before modeling.The expression levels of ROS,MDC autophagy fluorescence,mitochondrial membrane potential and PINK1/parkin pathway related signals were detected to observe whether the protective effect of paeoniflorin on myocardium was related to mitochondrial autophagy.In order to further clarify whether paeoniflorin-induced SIRT1 activation is necessary for autophagy and its potential mechanism,we detected the level of autophagy in H9c2 cells and the expression of signals related to mitochondrial autophagy pathway with or without SIRT1 inhibitor(EX527).Results 1.Among the three glycosides of Yiqi Huoxue prescription,the content of astragaloside was the most,paeoniflorin was the second,and amygdalin was the least.It accords with the composition characteristics of Astragalus membranaceus as monarch medicine in all three kinds of compound prescription.Among them,the total glycosides of astragalus in Naoxintong total glycosides is the most among the three compound prescriptions,followed by Yangyin Tongnao glycosides,and finally Buyang Huanwu decoction total glycosides.Among the three kinds of compound prescription,the content of paeoniflorin in total glycosides of Buyang Huanwu decoction is the most.Among the three compound glycosides,only the total glycosides of Buyang Huanwu decoction detected amygdalin.2.The results of cell experiment showed that all the three compound glycosides could promote autophagy of cardiomyocytes and improve mitochondrial dysfunction.We observed that three kinds of compound glycosides had obvious regulatory effects on the changes of related indexes of oxidative stress injury such as ROS,MDA and SOD after myocardial reperfusion.In addition,the total glycosides of Buyang Huanwu decoction and Yangyin Tongnao could significantly inhibit the important signals SIRT1 and Beclinl in autophagy and promote the classical mitochondrial autophagy pathway mediated by PINK 1/parkin,but the regulatory effect of Naoxintong glycosides on autophagy and related pathways was not obvious.3.paeoniflorin preconditioning can significantly improve the viability of cardiomyocytes after reperfusion injury and inhibit the excessive expression of ROS induced by OGD/R.The increase of MDC autophagy fluorescence and mitochondrial membrane potential suggest that the myocardial protective effect of paeoniflorin may also be related to mitochondrial autophagy.Then the related signals in autophagy and PINK1/parkin classical mitochondrial autophagy pathway were detected by Q-PCR and Western blot.The results showed that paeoniflorin pretreatment could promote the levels of SIRT1,Beclin1,PINK1,parkin and LC3Ⅱ/Ⅰ,inhibit the level of P62 and increase autophagy flow.In order to further clarify whether the activation of SIRT1 induced by paeoniflorin is directly related to mitochondrial autophagy,we detected the autophagy level of H9c2 cells with or without SIRT1 inhibitor.The results showed that the protective effect of paeoniflorin on oxidative damage and autophagy pathway of cardiomyocytes was significantly decreased after EX527 pretreatment,and the mechanism was related to SIRT1-PINK 1/parkin mitochondrial autophagy pathway.Conclusion 1.The analysis of the three main active components in the total glycosides of the three prescriptions for replenishing qi and activating blood circulation basically accords with the composition characteristics of the three prescriptions.2.Buyang Huanwu decoction and Yangyin Tongnao glycosides can improve the oxidative damage of cardiomyocytes after reperfusion injury.at the same time,they can also protect myocardium by promoting mitochondrial autophagy after reperfusion injury.The underlying mechanism is closely related to the regulation of the classical mitochondrial autophagy pathway by PINK1/parkin.Naoxintong glycosides also have obvious inhibitory effect on myocardial oxidative injury after reperfusion,but the effect on PINK1/pakin-mediated mitochondrial autophagy is not obvious,and its specific mechanism of anti-myocardial reperfusion injury needs to be further studied.3.Paeoniflorin pretreatment can prevent H9c2 cardiomyocytes from OGD/R-induced injury,and its mechanism may be through activating the upstream signal SIRT1 of autophagy,thus promoting the PINK1/parkin pathway.To provide further experimental basis for paeoniflorin in the treatment of myocardial reperfusion injury. |
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