Protective Effect And Mechanism Of Vitexin On Isolated Hearts Induced By Ischemia-reperfusion Injury Via Regulating Autophagy And Autophagic Flow In Rats

ObjectiveTo study the protective effect and mechanism of vitexin on isolated hearts induced by ischemia-reperfusion injury via regulating autophagy and autophagic flow in rats.Methods1. Isolated heart perfusion was used to simulate ischemia reperfusion in vivo.The experiment was falled into control group,I/R（ischemia/reperfusion）group,I/R+Vitexin low dose（I/R+VT-1μM）group,I/R+Vitexin medium dose（I/R+VT-3μM）group,VT+Vitexin high dose（I/R+VT-10μM）group five groups in total.Biological function system detec hemodynamic index.LDH detect damage of isolated cardiac myocytes.TTC staining detec the infarct size of each group.Hematoxylin and eosin（H&E）staining detec the Pathology change of heart tissues.The number of cardiomyocyte apoptosis was detected by TUNEL staining.Western Blot detec observe LC3、P62、Beclin 1、Cleaved caspase-3（CC-3）and Cleaved caspase-9（CC-9）expression.2.Hypoxia/reoxygenntion（H/R）was used to simulate ischemia reperfusion.The experiment was falled into control group,H/R（hypoxia/reoxygenation）group,H/R+Vitexin low dose（H/R+VT-1μM）group,H/R+Vitexin medium dose（H/R+VT-3μM）group,H/R+Vitexin high dose（H/R+VT-10μM）group five groups in total.LDH detec damage of primary rat cardiomyocyte.ROS kit for detecting reactive oxyfen species.Autophagy double-labeled adenovirus（m RFP-GFP-LC3）detect of autophagy in hypoxic reoxygenation primary cardiomyocytes.Western Blot detec observe LC3、P62、Beclin 1、Cleaved caspase-3（CC-3）and Cleaved caspase-9（CC-9）expression.Result1.Compared with the control group,the HR,CF,LVSP,+d P/dt_max,-d P/dt_min,and Cardiac contraction tension were significantly reduced,the LVEDP was markedly increased;the HR,CF,LVSP,+dp/dt_max,-dp/dt_min and systolic tension of the vitexin group were significantly increased,and LVEDP was significantly decreased.The LDH test results showed that the LDH value of the perfused rat heart in the model（I/R）group was significantly increased,while the LDH value of the vitexin group was significantly decreased.The TTC results showed that the myocardial infarct size of the isolated heart of the model group（I/R）group was significantly increased,while the myocardial infarct size of the isolated heart of the vitexin group was significantly decreased.The results of HE showed that the myocardial tissue of the control group was uniformly stained,and the normal myocardial fibers and cell structure were not destroyed,and the arrangement was neat and orderly.In the model（I/R）group,the myocardial tissue was uniformly stained,the normal myocardial fiber and cell structure were obviously destroyed,the arrangement was disordered,and a large number of inflammatory cells could be seen.The vitexin group can significantly alleviate the rats.Pathological damage of myocardial tissue improves myocardial cell morphology.The results of TUNEL staining showed that the number of positive cells in the isolated heart tissue of the model（I/R）group was significantly increased,while the number of positive cells in the isolated myocardial tissue of the vitexin group was decreased to some extent.Western Blot results showed that compared with the normal control group,the expression of autophagy protein Beclin 1,LC3Ⅱprotein increased,the ratio of LC3Ⅱ/Ⅰincreased,and the expression of P62 protein decreased in the model group（P<0.05 or P<0.01）,Suggesting that ischemic myocardial autophagosomes accumulate and ischemic myocardial cells have increased autophagy;at the same time,the expression of Cleaved caspase-3 and Cleaved caspase-9 proteins in ischemic myocardial tissues increases,and cardiomyocyte apoptosis increases.Compared with the model group,the doses of vitexin reduced the expressions of Beclin 1,and LC3Ⅱto varying degrees,the ratio of LC3Ⅱ/Ⅰdecreased,and the expression of P62 was increased,this reduces the accumulation of ischemic myocardial autophagosomes,inhibits ischemic myocardial autophagy enhancement,and at the same time inhibits the expression of Cleaved caspase-3 and Cleaved caspase-9 proteins,reducing myocardial cell apoptosis.2. After hypoxia and reoxygenation（H/R）of primary cardiomyocytes,the LDH value of primary cardiomyocytes in the model（H/R）group was significantly higher than that in the control group,and the ROS level was significantly increased,the doses of vitexin could significantly inhibit the release of LDH from H/R cardiomyocytes and reduce the ROS level in cardiomyocytes.The primary cardiomyocytes were transfected with m RFP-GFP-LC3 virus,and the results showed that compared with the control group,the cardiomyocytes in the H/R group showed significant autophagy and autophagy flow was enhanced,It is suggested that autophagosomes accumulate in myocardial cells after H/R,and autophagolysosomes increase accordingly;The vitexin dose groups can inhibit autophagy and increase autophagy flow in cardiomyocytes after H/R,and reduce the accumulation of autophagosomes in cardiomyocytes.In addition,Western Blot results showed that compared with the normal control group,the expression of autophagy proteins Beclin 1,and LC3Ⅱin primary myocardial cells of the H/R group increased,and the ratio of LC3Ⅱ/Ⅰincreased,The down-regulation of P62 protein（P<0.05 or P<0.01）,it also accelerated the expression of Cleaved caspase-3 and Cleaved caspase-9 in cardiomyocytes;Compared with the H/R group,vitexin at each dose group inhibited Beclin 1 up-regulation and decreased the expression of LC3Ⅱprotein,the ratio of LC3Ⅱ/Ⅰwas reduced,and the expression of P62 protein was also increased,it also inhibits the expression of Cleaved caspase-3and Cleaved caspase-9 proteins in cardiomyocytes.ConclusionVitexin has obvious protective effect on isolated cardiac I/R and H/R cardiomyocyte injury in rats,the mechanism may be related to its inhibition of autophagy and autophagy flow enhanced after cardiomyocyte I/R,reducing the accumulation of cardiomyocyte autophagosomes and inhibiting cardiomyocyte apoptosis,and reducing the accumulation of cardiomyocyte autophagosomes and inhibiting cardiomyocyte apoptosis.