Protective Effect And Mechanism Of Resveratrol On Hypoxic Cardiomyocytes At High Altitude

Objective:To reveal the protective effect and mechanism of resveratrol on hypoxic cardiomyocytes at high altitude,and to provide theoretical basis for seeking key targets and effective drugs for the treatment of high altitude myocardial injury.Methods:1.In vivo experiment:45 healthy male Wistar rats aged 8 weeks were randomly divided into 3 groups:normal oxygen control group,hypoxic exposure group,and resveratrol intervention group.Animals in each group were given gavage for 15 days in advance according to the experimental arrangement(resveratrol intervention group was given gavage of resveratrol at a concentration of 400 mg/kg;the normal oxygen control group and the hypoxic exposure group were given gavage with 5%sodium carboxymethyl cellulose solution of the same volume).Subsequently,rats in the hypoxic exposure group and the resveratrol intervention group were transferred to low-pressure oxygen chamber,and the height was set at 6000 m.The patient was boarded for 20 hours a day and chronically hypoxic intervention lasted for 30 days.The control group was fed normally under normal oxygen.Gavage,feeding and water change were performed daily at non-hypoxic intervention times.After the last gavage and hypoxia intervention,the rats were weighed and killed immediately.Abdominal aorta blood was collected from the collection vessel,serum was separated and frozen for subsequent index determination.The hearts of rats were taken,washed with normal saline,dried and weighed,and then fixed in tissue fixating solution or frozen at low temperature and liquid nitrogen for subsequent index determination.Index detection:(1)Echocardiography:The echocardiography was measured by the color Doppler ultrasound imaging system of small animals,and the image results were processed and analyzed on the instrument.The experiment was measured once at the beginning and then at 15-day intervals.(2)Heart index:The heart of the rat was separated,cleaned with normal saline,dried with absorbent paper,and the heart weight was measured.The ratio of heart weight to the body weight of the rat was calculated as heart index.(3)Serum biochemical indexes:Superoxide Dismutase(SOD),Glutathione peroxidase(GSH-Px),Tumor necrotic factor-α(TNF α),Interleukin 6(IL-6).(4)Histopathological detection:myocardial cross-section area was detected by Wheat Germ Agglutinin(WGA)staining,myocardial fibrosis degree was detected by Masson staining,and reactive oxygen species(ROS)level was detected by DHE(Dihydroethidium)staining.(5)Mitochondrial structure observation:Transmission electron microscopy was used to observe the structure of cells,mitochondria,and the formation of autophagosomes and autophagolysosomes.(6)Protein expression level:autophagy and mitophagy marker proteins were detected by immunofluorescence.2.In vitro experiment:(1)Rat H9c2 cardiomyocytes were placed in a hypoxic workstation at 37℃,0.5%O2,and the intervention time was set to 0,12,24,36,48 h to establish an in vitro hypoxic injury model of cardiomyocytes.(2)Cells were pretreated with different concentrations of resveratrol(0,10,25,50,100 μM)for 24 h to determine cell viability and screen out the optimal concentration of resveratrol.This concentration of resveratrol was used for follow-up intervention to observe the protective effect of resveratrol on hypoxic cardiomyocytes.(3)H9c2 cells were pretreated with 10 mM autophagy inhibitor(3-methyladenine,3MA)for 12 h and treated with hypoxia for 48 h;H9c2 cells were pretreated with 5 μM Mdivi-1(Mdivi1)mitophagy inhibitor for 6 h and treated with hypoxia for 48 h.To discuss the role of autophagy and mitophagy in myocardial hypoxia injury.(4)Mitophagy receptor protein BNIP3L has been knocked down with Small interfering RNA(siRNA)for 48 h to investigate the effect of BNIP3L on myocardial hypoxic injury.Subsequently,BNIP3L was overexpressed by overexpression plasmid in resveratrol pretreated cells,and the effect of BNIP3L on myocardial injury induced by resveratrol was explored after 48 h hypoxia intervention.Index detection:(1)Cell damage detection:Cell viability was detected by cell Counting Kit-8(CCK-8).DCFH-DA probe(2’,7’Dichlorodihydrofluorescein diacetate,DCFH-DA)to detect cellular ROS levels;Cell apoptosis was detected by flow cytometry.(2)Mitochondrial function detection:JC-1 fluorescent probe was used to detect mitochondrial membrane potential;ATP content was detected by luciferin-luciferase assay.MitoSOX probe detected the level of Mitochondrial reactive oxygen species(mROS).(3)Autophagy associated proteins(LC3,P62)and mitophagy associated proteins(BNIP3L,BNIP3,NLRX1,TOMM20)were detected by Western Blot(WB).(4)Autophagy was detected by MDC staining.Results:1.In vivo experiment:(1)Heart index and echocardiogram results showed that resveratrol significantly alleviated ventricular wall thickening and ventricular volume reduction caused by hypoxia exposure in rats structurally;In terms of function,it significantly improved myocardial contractility,ejection fraction and cardiac output(P<0.05).(2)Serum biochemical results showed that,compared with hypoxia exposure group,resveratrol significantly increased the levels of serum antioxidant enzymes SOD and GSH-Px in hypoxia exposed rats(P<0.05);The levels of TNF-α and IL-6 were significantly decreased(P<0.05).(3)WGA staining showed that resveratrol significantly improved cardiomyocyte hypertrophy induced by hypoxic exposure(P<0.05).Masson staining showed that resveratrol significantly reduced the fibrosis level of hypoxic exposed rats(P<0.05).(4)DHE staining showed that resveratrol could significantly reduce ROS levels in myocardial tissue of hypoxic exposed rats(P<0.05).(5)Transmission electron microscopy showed that the mitochondrial structure of the heart was seriously damaged after hypoxia exposure,and the number of autophagosomes and autophagolysosomes increased.In resveratrol intervention group,mitochondrial damage was alleviated,and autophagosomes and autophagolysosomes were rare.(6)Immunofluorescence results showed that resveratrol reversed the expression levels of LC3,TOMM20 and other proteins caused by hypoxic exposure(P<0.05),and significantly reduced the levels of hypoxia induced autophagy and mitophagy.2.In vitro experiment:(1)With the extension of hypoxic exposure time,H9c2 cell vitality decreased significantly,ROS content and apoptosis rate increased significantly,and cell damage was serious after hypoxia(P<0.05).In addition,with the extension of hypoxia time,the intracellular ATP content decreased significantly(P<0.05),and hypoxia damaged the mitochondrial function of myocardial cells.(2)Compared with the hypoxia group,the H9c2 cell activity in the resveratrol intervention group was significantly increased,and the ROS level was significantly decreased(P<0.05);ATP content and mitochondrial membrane potential levels were significantly increased(P<0.05),while mROS levels were significantly decreased(P<0.05).Resveratrol could significantly relieve the damage of cell and mitochondrial function caused by hypoxia.(3)WB results showed that autophagy and mitophagy were enhanced during hypoxia exposure(P<0.05),and resveratrol significantly inhibited autophagy and mitophagy of hypoxia cardiomyocytes.(4)Compared with hypoxia group,myocardial cell activity was significantly increased after 3MA and Mdivi-1 administration,and ROS level was significantly decreased(P<0.05);ATP content and mitochondrial membrane potential levels were significantly increased(P<0.05),while mROS levels were significantly decreased(P<0.05),indicating that hypoxia induced autophagy and mitophagy caused myocardial injury.Resveratrol can protect hypoxic myocardium by inhibiting autophagy and mitophagy.(5)After knockout of target gene BNIP3L,the myocardial cell activity was significantly increased,and the ROS level was significantly decreased(P<0.05);ATP level and mitochondrial membrane potential were significantly increased(P<0.05),while mROS level was significantly decreased(P<0.05).After overexpression of BNIP3L,compared with resveratrol intervention group,myocardial cell viability decreased significantly,and ROS level increased(P<0.05).ATP level and mitochondrial membrane potential were significantly decreased(P<0.05).Overexpression of BNIP3L reversed the protective effect of resveratrol on hypoxic cardiomyocytes.Resveratrol alleviates myocardial injury caused by hypoxia exposure by regulating mitophagy mediated by BNIP3L.Conclusion:These results suggest that autophagy and mitophagy may play a major role in seriously damaging the cardiac function of rats after long-term exposure to high altitude hypoxia.Resveratrol can protect hypoxic cardiomyocytes by inhibiting autophagy and mitophagy.Mitophagy receptor BNIP3L plays a key role in myocardial injury caused by hypoxia exposure.Resveratrol alleviates myocardial injury caused by hypoxia by inhibiting BNIP3L-mediated mitophagy.