| ObjectivesAcute myocardial infarction(AMI)is a critical cardiovascular disease,with a high mortality rate.Effectively and timely recover blood supply to the ischemic myocardium is a key step to save the heart.It can effectively reduce the area of myocardial infarction,improve long-term heart function and reduce mortality.However,the process of recovering blood supply to ischemic myocardium is accompanied by myocardial ischemia-reperfusion injury(MIRI),which will lead to further increase in the area of myocardial infarction and poor clinical prognosis.Therefore,finding an endogenous method to combat MIRI may become a new idea for the prevention and treatment of ischemic cardiomyopathy.Hypoxia-inducible factors(HIFs),as one of the nuclear transcription complexes that mediate the hypoxia response in mammalian cells,are the most important regulators of the cell to coordinate hypoxia changes at the gene transcription level.The hypoxia-inducible factor 2-alpha(HIF2?)subunit is a functional subunit that regulates HIF-2 activity.Previous reports have reported that HIF2? plays a protective role in the process of MIRI.HIF2a also have been reported to dampens MIRI by inducing AREG expression in cardiac myocytes.The myocardial necrosis area of HIF2a knockout mice increased significantly.However,the mechanism between HIF2a and MIRI has not yet been fully elucidated.Its downstream regulation mechanism needs to be further explored.Interleukin 6(IL-6),as a pleiotropic cytokine,plays a core regulatory role in inflammatory response,regulating immune response,acute response,inflammation and hematopoiesis.Moreover,it plays an important role in the body's immune response against infection.It is worth mentioning that the final effect of IL-6 signaling pathway is closely related to the duration of the signal and the downstream cascade activation signal.IL-6 often exerts an anti-inflammatory effect in the acute phase of inflammation.However,with the progress of the inflammatory response,this anti-inflammatory effect is gradually converted into a pro-inflammatory effect,which has an adverse effect on the body.In the acute phase,IL6 preserves cardiac tissue by inducing an anti-apoptotic program in the myocyte and triggers the preconditioning response.When IL-6 signaling continues chronically,these protective responses become pathogenic and induce depressed myocyte function.There is decreased contractility and left ventricle enlargement occurs.Clinical studies showed a correlation between the area of myocardial necrosis and plasma IL-6 levels in patients with acute myocardial infarction.Elevated IL-6 serum levels in patients with coronary heart disease especially acute myocardial infarction may be predictive of poor outcomes.In vitro studies have suggested that IL-6 is involved in MIRI.Futhermore,myocardial secretion of IL-6 also plays an important role in cardiac dysfunction due to ischemia-reperfusion injury.However,the upstream regulatory mechanism of IL-6 involved in MIRI has not been elucidated.The relationship between HIF2? and IL-6 has been reported in the fields of tumor and osteoarthritis.Studies have shown that Il-6 is the direct target gene of HIF2a in mouse articular chondrocytes.HIF2a can induce the upregulation of IL-6 specific receptors in chondrocytes.However,there is no related research on ischemia-reperfusion injury in acute myocardial infarction.The purpose of this study is to explain the following questions:(1)Clarify the protective effect of HIF2a in myocardial ischemia-reperfusion and its regulation of IL-6.(2)Determine that IL-6 is involved in the protective role of HIF2a in myocardial ischemia-reperfusion.MethodsAccording to the content and purpose of this study,corresponding research was carried out through an in vitro myocardial cell hypoxia/reoxygenation(H/R)model and a mouse in vivo ischemia/reperfusion(I/R)model.H/R model was established using H9c2 rat myocardial cell line and AC 16 human myocardial cell line.Hypoxia for 6 hours and Reoxygenation for 6 hours was used as a H/R model to detect the expression of Hif2a,IL-6(protein and mRNA),myocardial cell necrosis and apoptosis.Construction of lentivirus expressing shRNA for rat HIF2a.After knocking down endogenous HIF2?,the myocardial cell line was subjected to H/R treatment,and AnnexinV/PI dual staining was used to detect early cell apoptosis.CCK8 was used to detect cell survival,while western blot and QPCR were used to detect changes in IL-6 protein and mRNA levels.Chromatin Immunoprecipitation(ChIP)verified that the transcription level of HIF2a directly regulated the expression level of IL-6.We constructed a type 9 adeno-associated virus vector(AAV9-shHIF2a)that specifically down-regulated the HIF2a gene.For animals,male C57BL/6 mice was used.Surgical ligation of left coronary artery(LAD)was performed to establish an I/R model(ischemia 2 hours,reperfusion for 24 hours)4 weeks later.Western blot was used to detect changes in the expression of IL-6 protein in the two groups.TTC(triphenyltetrazolium chloride)and Evans blue was used to assess the area of myocardial infarction in mice.Besides,the release of serum lactate dehydrogena(LDH)and troponin I(TnI)were detected To clarify the protective effect of HIF2a on myocardial ischemia-reperfusion and its regulation on IL-6.To explore that IL-6 participates in the protective effect of HIF2a on myocardial ischemia-reperfusion,a type 9 adeno-associated virus vector that specifically down-regulated the IL-6 gene(AAV9-shIL-6)was constructed to knocking down IL-6 in vivo.After establishing an I/R model 4 weeks later,the area of myocardial infarction was evaluated,and the serum LDH and TnI levels were measured.After mice were infected with AAV9-shIL-6 and AAV9-shHIF2a respectively,exogenous IL-6 was supplemented to detect wheteher it could reverse MIRI in mice,and to detect the phosphorylation level of the protein-serine-threonine kinase(AKT)signaling pathway.ResultsIn vitro experiments showed that for the cardiomyocytes knocking down endogenous HIF2a,the proportion of apoptosis after H/R were increased,while the expression of IL-6 protein and mRNA decreased.By sequence analysis,two original HIF transcription factor response elements were found in the IL-6 gene promoter regions.The results from chromatin immunoprecipitation experiments show that the DNA in the promoter region of the IL-6 gene recruited by the HIF2a antibody was richer under hypoxic conditions than under normoxic conditions.However,we found no significant differences in terms of the DNA in the promoter region pulled by IgG antibody.Taken together,Chip and promoter activity experiments confirmed that HIF2? has a direct regulatory effect on IL-6 transcription levels.After I/R treatment in mice with down-regulated HIF2a genome,TTC and Evans blue staining results showed that compared with the control group,the risk areas of the two groups were basically the same,but the area of myocardial infarction of HIF2? knock-down mice was significantly increased.The expression of LDH and TnI increased significantly,while the expression of IL-6 decreased significantly.After the I/R treatment,the area of myocardial necrosis of the endogenous IL-6 knock-down mice increased,the release of LDH and Tnl increased.The area and extent of myocardial infarction decreased after the administration of exogenous IL-6,while p-AKT expression level increased,and myocardial AKT activity was significantly increased.Similar results was seen in endogenous HIF2a knock-down mice with exogenous IL-6.Western blot results also showed that p-AKT expression level increased,and myocardial AKT activity increased significantly.ConclusionThe above results indicate that HIF2a plays a protective role in the process of myocardial ischemia-reperfusion and has a transcriptional regulation effect on IL-6.IL-6 participates in the protective effect of HIF2a on myocardial ischemia and reperfusion.During this process,the PI3K/AKT signaling pathway is activated.It is suggested that HIF2a transcription regulates the expression of IL-6 in cardiomyocytes and plays a role in preventing MIRI. |
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