| BackgroundCardiovascular disease is known to have high morbidity and mortality worldwide.Its treatment and prevention have always been an important topic for researchers.Ischemic injury is one of the leading causes of cardiovascular disease.When the coronary arteries are blocked,heart tissue will be exposed to ischemic and hypoxic,which may cause irreversible damage.Timely recovery of blood flow can remove hydrogen ions that accumulate outside the cell during ischemia,and provide the oxygen and substrate needed for aerobic respiration ATP production.However,many studies have shown that timely reperfusion only save hypoxic tissue to a certain extent.More severe damage response will occur in the damaged tissue after overdyed reperfusion,which is called ischemia/reperfusion injury(I/R injury).Ischemia-reperfusion injury will cause a series of damaging cellular changes such as intracellular calcium overload,reactive oxygen and nitrogen production,endoplasmic reticulum stress and mitochondrial damage,leading to cell apoptosis and necrosis.Interestingly,studies have shown that transiently harmless I/R can improve the tolerance of heart tissue to subsequent exposure to long-term ischemic damage,resulting in a significant reduction in post-ischemic injury,demonstrating that I/R could act as a beneficial pretreatment for potential injury.It was found that autophagy,apoptosis,and necrosis all participate in the process of I/R injury.Autophagy is a biochemical process that encapsulates damaged organelles and erroneous proteins by autophagosome formation and transports them to lysosomal degradation.Therefore,autophagy performs housekeeping functions in normal cells,mainly to remove damaged and aging macromolecular proteins and organelles.On the one hand,it provides amino acids,nucleotides and other substances for energy recycling,and promotes cell hunger survival.On the other hand,it may also protect damaged cells as a cellular defense mechanism.During the I/R process,autophagy pathways are activated due to insufficient energy supply,oxidative stress,and endoplasmic reticulum stress,and are promoted by producing amino acids and fatty acids to maintain cellular function,or by removing damaged organelles.Furthermore,since ischemia causes cellular starvation at the ischemic site,decomposition of autophagy-regulated cellular components can promote cell survival by providing a substrate that maintains cellular energy levels.Paradoxically,although moderate autophagy plays an important role in maintaining cell homeostasis,excessive autophagy can cause dysfunction of the corresponding tissue organs and progress to various clinical diseases.Therefore,autophagy has always been the focus of cytology research.MicroRNAs(miRNAs)are a series of single-stranded,non-coding RNAs consisting of 20-24 nucleotides with highly conserved sequences.Among the many miRNAs,microRNA-210(miR-210)is a well-known hypoxia miRNA and produces very stable biological effects in a variety of cell lines.MiR-210 regulates many genes,but only a few of them have been shown to function in cardiovascular-related models.These genes regulate the protective response of the cardiovascular system to hypoxia from the aspects of apoptosis,autophagy,proliferation,cell migration,energy metabolism,angiogenesis,and improve the adaption of cells exposed to hypoxia.What's more,miR-210 is known as a biomarker,detecting significant changes in the plasma of patients with various cardiovascular diseases,such as atherosclerosis,acute coronary syndrome,heart failure,valvular heart disease,and diabetic cardiomyopathy.Therefore,miR-210 has a wide application prospect in the cardiovascular system.Among these genes regulated by miR-210,Bcl-2/adenovirus E1 B interacting protein 3(BNIP3)is a target gene that participates in various of biochemical processes.BNIP3 is a member of the BH3-only family of proteins as a typical hypoxia-regulated protein.Because of its structural and functional specificity of other proteins in the BH3-only family,its functions in the process of apoptosis are more diverse.Its BH3-only region can specifically bind to anti-apoptotic protein Bcl-2 for its pro-apoptotic function.In addition,its C-terminal transmembrane domain can also regulate mitochondrial membrane potential and other related functions,leading to the destruction of mitochondria,thereby further exacerbating cell damage.There are also some researches proved that BNIP3 regulates autophagy in different cell lines.Studies have shown that BNIP3 can be up-regulated in cardiovascular disease,and inhibition of BNIP3 can improve myocardial damage.Therefore,the diagnosis and therapeutic significance of BNIP3 in cardiovascular diseases needs further study.ObjectsIn this study,we used a hypoxic culture of rat cardiomyocytes H9c2 to construct a myocardial ischemia model.In the model,we identified the activation of apoptosis and autophagy signaling pathways by detecting proteins related to apoptosis and autophagy.The effect of miR-210 expression on BNIP3 and autophagy,the role of miR-210 in myocardial ischemia and its regulation of autophagy and BNIP3 were clarified by detecting the expression changes of miR-210 and BNIP3 and other related protein in this process.In addition,we also used H9c2 cells to construct an ischemia-reperfusion model to detect changes in apoptosis and autophagy-related pathways during reperfusion injury and prove the function of autophagy in this process.These results will provide new theoretical support for the protective effects of miR-210 on the cardiovascular system.Methods1.Hypoxia culture of H9c2 cells were performed to construct a myocardial ischemia model.Cell viability was measured by CCK-8 activity detection kit,LDH release amount was detected by lactate dehydrogenase detection kit,and ROS content in cells was detected by DCFH-DA probe.Western blot was used to detect the expression of apoptosis-related proteins Caspase3,Bak,Bax and anti-apoptotic protein Bcl-2,and to assess the damage of cells in ischemia model.The expressions of autophagy-related proteins LC3 B and p62 in ischemia model were also detected by Western blot.The aggregation of LC3 B was observed by immunofluorescence staining to support the result of western blot.2.Real-time quantitative PCR was used to detect the expression of miR-210 in the ischemia model.Lentiviral transfection was used to construct a high expression and low expression model of miR-210.After transfection,the expression of miR-210 was detect by real-time quantitative PCR again.The cell viability of H9c2 cells,and the expression of apoptosis-related protein Caspase3,Bak,Bax and anti-apoptotic protein Bcl-2 were detected again,demonstrating miR-210 function in hypoxia.The aggregation of LC3 B was observed by immunofluorescence staining,which confirmed the effect of miR-210 to autophagy.Real-time quantitative PCR and Western blot were used to detect the expression of BNIP3 mRNA and protein in X ischemia cells and the regulation of miR-210 on BNIP3 in miR-210 high expression and low expression model.The siRNA was used to construct the BNIP3 knockout model.The cell viability of H9c2 cells and apoptosis-related protein Caspase3 were detected in the ischemia cells for apoptosis,and Western blot was used to observe the changes of LC3 B,which could illuminate the role of BNIP3 in the ischemia model and its influence to autophagy.3.The ischemia-reperfusion model was built by H9c2 cells by reoxygenate culture after hypoxia.The expressions of autophagy-related proteins LC3 B,p62 and Beclin1 in ischemia model were also detected by Western blot.The aggregation of LC3 B was observed by immunofluorescence staining to observe the formation of autophagosomes.To evaluate the role of autophagy during I/R,3-MA was used to inhibit autophagosome formation.CCK were used to evaluate the damage to cells during this process.The expression of apoptosis-related protein Caspase3 was detected by Western blot to prove the function of autophagy.Results1.During hypoxia,the cell viability of H9c2 cells decreased,the release of LDH increased,and the content of ROS increased,suggesting that oxidative damage occurred in H9c2 cells under hypoxia.By detecting apoptosis-related proteins,it was found that the expression of pro-apoptosis protein Bak and Bax increased,the expression of pro-apoptosis protein Bcl-2 decreased,and increased Caspase3 cleavage fragment indicated that the apoptotic pathway was activated.The expression of LC3 B,p62 and Beclin1 in H9c2 cells increased during hypoxia,indicating that autophagy was activated.2.During hypoxia of H9c2 cells,miRNA-210 was significantly up-regulated.High expression of miR-210 can increase the activity of H9c2 cells under hypoxic conditions,decrease the release of LDH,inhibit the expression of apoptosis-related proteins Caspase3,Bax and Bak,and up-regulate the expression of anti-apoptotic protein Bcl-2,thereby playing a certain protective effect on hypoxic cells.Low expression of miR-210 reduces the activity of H9c2 cells under hypoxia,promotes the release of LDH,and exacerbates cell damage.BNIP3 is a gene directly regulated by miR-210.BNIP3 expression increased in the H9c2 cell hypoxia-reoxygenation model and played a pro-apoptotic role.Inhibition of BNIP3 expression can inhibit apoptosis in H9c2 cell hypoxia model and partially offset the increase in apoptosis caused by low expression of miR-210.In addition,inhibition of BNIP3 can reduce the occurrence of autophagy during hypoxia.3.The process of reoxygenation further promoted the expression of apoptosis-related proteins and autophagy-related proteins.3-MA inhibited t autophagosome formation in our model.The inhibition of autophagy during hypoxia increased the cell viability in H9c2 cells.The results indicate that autophagy has an adverse effect on H9c2 cells in the absence of oxygen.Inhibition of autophagy during reoxygenation further aggravated apoptosis,and may prove that autophagy played a positive role in the survival of H9C2 cells.ConclusionIn ischemia and ischemia-reperfusion injury model of H9c2 cell,the signaling pathways of apoptosis and autophagy are activated.In the process of cell hypoxia injury,miR-210 exerts cytoprotective effects by inhibiting the target gene BNIP3 in turn inhibiting the apoptotic and autophagy signaling pathway.In the process of ischemia-reperfusion,the activated autophagy plays a cytoprotective role by inhibiting the apoptotic signaling pathway,and the elevated miR-210 also exerts a certain cytoprotective effect by inhibiting apoptosis.Innovation and significanceThis study demonstrated for the first time to the different effects of autophagy on cell viability and apoptotic pathway during cardiomyocyte ischemia and reperfusion,and also the regulation of miR-210 on apoptosis and autophagy in this process.We further demonstrated that miR-210 exert cytoprotective effects by inhibiting BNIP3,a typical pro-apoptosis and anti-autophagy protein.This study provides new theoretical support for the mechanism of cardiovascular ischemia-reperfusion injury and the role of autophagy in it,providing a new theoretical basis for the protective role of miR-210 during ischemia-reperfusion injury.We hope these can prove new ideas for clinical treatment and prevention of cardiovascular ischemia-reperfusion injury. |
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