Hypoxia-induced Exosomes Protect H9c2 Cells Against Oxidative Stress Injury Through MicroRNA-21/ATG12

BackgroundCardiovascular diseases such as ischemic heart disease(IHD)are the leading cause of death worldwide.Oxidative stress can promote the development of IHD by causing irreversible damage and death in cardiomyocytes.Oxidative stress activates cardiomyocyte autophagy via various pathways.While moderate autophagy can maintain cardiomyocyte function,excessive autophagy can induce cardiomyocyte death.The ATG12 protein is an important part of the ubiquitin-like coupling system in autophagy and is directly involved with autophagosome membrane formation.Therefore,in-depth studies of the role of autophagy in oxidative stress and regulatory molecules based on ATG12 could help to prevent oxidative stress injury in cardiomyocytes.Exosomes are important mediators of information exchange between cells that can carry a variety of signaling molecules to regulate the physiological function or pathological state of cardiomyocytes.Hypoxia can induce exosome secretion and enhance their targeting ability while altering the expression of their internal signaling molecules.Micro RNAs(mi RNAs)are abundant in exosomes and can target and regulate gene expression.Micro RNA-21(mi R-21)is a hypoxia-responsive mi RNA that can affect cell survival through multiple pathways and is significantly enriched in hypoxia-induced exosomes.Therefore,it is important to study its role in oxidative stress injury and clarify its regulatory mechanism in autophagy to improve the potential clinical applications of exosomes.Objectives1.To explore the effect of hydrogen peroxide(H₂O₂)-simulated oxidative stress on myocardial cell injury and clarify the effect of hypoxia-induced exosomes on myocardial cell oxidative stress injury.2.To explore the effect of oxidative stress on autophagy and clarify the role of autophagy in oxidative stress injury,as well as the effect of hypoxia-induced exosomes on oxidative stress-mediated autophagy.3.To clarify whether hypoxia-induced exosomes affect oxidative stress injury through mi R-21/ATG12.Methods1.A myocardial cell model of oxidative stress injury was constructed using H₂O₂.Cell viability was detected using a cell counting kit-8(CCK-8)and cell proliferation was detected using an Ed U cell proliferation kit.Cell injury was detected using an lactate dehydrogenase(LDH)cytotoxicity assay kit and the levels of cellular reactive oxygen species(ROS)were detected using a ROS kit to evaluate the effect of oxidative stress on cardiomyocytes.2.A cellular model of hypoxia was constructed using an anaerobic gas production bag and hypoxia-induced exosomes were extracted by ultracentrifugation.Exosome structure was observed by transmission electron microscopy and particle diameter was analyzed by dynamic light scattering.The levels of exosome marker proteins,such as CD9,TSG101,and HSP70,and the negative control protein calnexin,were detected by western blotting.HIF-1?,RAB27a,and SYTL4 levels were measured to clarify the effect of hypoxia on exosome secretion by cardiomyocytes.Exosomes were labeled with PKH67 and co-cultured with cardiomyocytes.Exosome uptake was observed by confocal microscopy and changes in cell viability,proliferation,and LDH were detected to explore the effect of hypoxia-induced exosomes on myocardial cell oxidative stress injury.3.H9c2 cells were stimulated with H₂O₂and changes in intracellular autophagosomes were observed using transmission electron microscopy.Changes in autophagic flux were detected after transfection with m Cherry-GFP-LC3 recombinant adenovirus and western blotting was used to detect the levels of autophagy-related proteins(Beclin-1,LC3,P62,ATG12,and ATG5).The autophagy inhibitor,3-MA,was added to detect changes in cell activity,proliferation,and LDH levels.Punctate LC3 aggregation was observed by immunofluorescence staining and Beclin-1,LC3,and P62 levels were detected using western blotting.The same assays were repeated after the addition of hypoxia-induced exosomes to detect ATG12 levels and explore their effect on oxidative stress-mediated autophagy.4.ATG12 and mi R-21 binding sites were predicted using bioinformatics and the targeting relationship between the two was verified using dual-luciferase reporter gene experiments.Cells were transfected with mi R-21 mimics and inhibitors to construct high-and low-mi R-21 expression models,and cell viability,LDH,LC3,P62,and ATG12 levels were detected.A cell model with high ATG12 expression was also constructed and changes in cell activity and LC3 and P62 levels were detected.Real-time quantitative PCR was used to detect mi R-21 levels in cardiomyocytes after H₂O₂stimulation and in cardiomyocytes and exosomes under normoxic and hypoxic culture conditions,and after co-culture with hypoxia-induced exosomes.Hypoxia-induced exosomes were extracted from cardiomyocytes with low mi R-21 expression,and mi R-21 expression was detected using real-time quantitative PCR.Changes in cell activity and LDH,LC3,and P62 levels were detected after exosomal mi R-21 inhibition.Results1.H₂O₂stimulation decreased the activity and proliferation of H9c2 cells in a time-and concentration-dependent manner and increased LDH activity and ROS levels in a time-dependent manner.2.Hypoxic conditions activated the HIF-1?/RAB27a/SYTL4 pathway and significantly increased the secretion of exosomes by H9c2 cells.Following the uptake of hypoxia-induced exosomes,the activity and proliferation of H9c2 cells increased significantly and LDH activity decreased significantly.3.H₂O₂stimulation upregulated autophagosomes and autophagic flux in H9c2cells while significantly increasing the levels of Beclin-1 and LC3-II/LC3-I and decreasing P62 levels.3-MA treatment significantly increased cell activity and proliferation,P62 levels,and the expression of ATG12 and ATG5,but significantly decreased LDH activity,punctate LC3 aggregation,and the levels of Beclin-1 and LC3-II/LC3-I.In addition,co-incubation with hypoxia-induced exosomes downregulated autophagosomes and autophagic flux,significantly decreased the levels of LC3-II/LC3-I and ATG12,and significantly increased P62 levels.4.The mi R-21 sequence can target ATG12.We found that mi R-21 expression decreased in H9c2 cells stimulated with H₂O₂.High mi R-21 expression increased cell viability after H₂O₂stimulation while decreasing LDH and LC3-II/LC3-I levels,increasing P62 levels,and inhibiting autophagy and ATG12 expression,whereas the opposite effects were observed in H₂O₂-stimulated cells with low mi R-21 expression.In addition,high ATG12 expression antagonized the regulatory effects of high mi R-21expression on autophagy.Under hypoxic conditions,mi R-21 expression was significantly increased in H9c2 cells and exosomes,and exosomal mi R-21 was taken up by H9c2 cells.When the expression of hypoxia-induced exosomal mi R-21 was inhibited,exosomes decreased cell activity and P62 levels while increasing the levels of LDH and LC3-II/LC3-I after H₂O₂stimulation.ConclusionsH₂O₂can induce oxidative stress injury and activate autophagy in H9c2 cells.Hypoxia induces cardiomyocytes to secrete exosomes that can inhibit autophagy through mi R-21/ATG12 and alleviate oxidative stress-mediated cell damage.Innovation and significanceThis study addressed the role of autophagy in oxidative stress injury and examined the intermediary role of ATG12 in oxidative stress-mediated autophagy.We found that hypoxia-induced cardiomyocyte exosomes can reduce cell damage caused by oxidative stress and could therefore provide a new avenue for treating oxidative stress damage.In addition,we found that exosomal mi R-21 can act as an upstream regulator of ATG12to inhibit its expression and thereby inhibit autophagy.Together these findings provide a theoretical basis for the clinical application of exosomes and new therapeutic targets.