Transfer Of MiR-27b-3p From Hypoxia Induced-Cardiac Microvascular Endothelial Cells-derived Exosomes Attenuates Myocardial I/R Injury

Background:Myocardial Infarction(MI)is an important cause of death.In recent years,the diagnosis and treatment of MI is not satisfactory.Exosomes derived from Cardiac Microvascular Endothelial Cells(CMECs),as a newly discovered cell-cell communication mode,play an important role in the diagnosis,treatment and prognosis of cardiovascular diseases.In addition,the abundance of proteins,m RNAs and miRNAs encapsulated by exosomes is higher under hypoxic conditions.Therefore,in-depth investigation of the role and mechanism of miRNA in the hypoxic exosomes of CMECs in myocardial I/R injury can provide new strategies for the prevention and treatment of cardiovascular diseases.Methods:Firstly,exosomes were extracted from the supernatant of CMECs cultured in hypoxia environment,and identified by transmission electron microscopy and Nano Sight NTA particle size analyzer.The myocardial I/R injury model of rats was established by ligating coronary arteries with wire plugs,and the hypoxic exosomes of CMECs were injected into multiple myocardium to detect the changes of cardiac tissue structure and cardiac function.Including myocardial infarct size,Creatine kinase isozyme(CK-MB)and lactate dehydrogenase(LDH)levels in serum of rats in each group.The changes of related inflammatory factors and myocardial pyroptosis related factors(NLRP3,GSDMD and caspase-1).Secondly,miRNAs in the tissues surrounding myocardial infarction were screened.The expression of miR-27b-3p in CMECs cells was inhibited by mir NA-inhibitor,and the H/ R-induced H9C2 cells were treated with exosomes to observe the fusion of exosomes and H9C2 cells,and the degree of injury of H9C2 cells was detected.The results included the degree of cytoskeleton damage of H9C2,cell viability,changes in the levels of IL-1β and IL-18 in culture supernatant,and changes in the expression levels of NLRP3,GSDMD and caspase-1 in cells.Subsequently,3.In order to explore whether miR-27b-3p in CMECs hypoxic exosomes plays a role by targeting Foxo1 regulation,H9C2 cardiomyocytes were transfected with Foxo1 overexpression plasmid before H/R induction.Then,specific inhibitors were used to inhibit the hypoxic exosomes of CMECs cells after miR-27b-3p.The fusion of exosomes in H9C2 cells and the changes of H9C2 cytoskeleton were observed under Confocal.The viability of H9C2 cells was detected by CCK8.The levels of LDH,IL-1β and IL-18 in the supernatant of H9C2 cells were detected by ELISA.The expression of pyroptosis related proteins was detected by Western blot.H9C2 cells overexpressing Foxo1 and rats overexpressing Foxo1 in myocardium were constructed,and the effects of CMECs derived exosomes with low expression of miR-27b-3p on H/R injury in Foxo1 overexpressing H9C2 cells and I/R injury in myocardium of Foxo1 overexpressing rats were detected.;Cell proliferation was detected by CCK8.The degree of myocardial infarction and tissue injury(including TTC staining and H&E staining)were analyzed by histology.LDH in the supernatant of cell culture medium and serum of rats in each group were detected by enzyme activity kit.The levels of pyroptosis related inflammatory cytokines IL-1β and IL-18 were detected by ELISA.RT-q PCR was used to detect the expression of miR-27b-3p and Foxo1 in H9C2 cells and myocardial tissue of rats in each group.Western blot was used to detect the changes in the expression levels of pyoptosis related proteins(Foxo1,NLRP3,GSDMD,pro-caspase-1 and caspase-1)in each group and rat myocardial tissue.To further verify whether miR-27b-3p in CMECs hypoxic exosomes plays a protective role through Foxo1 in I/ R-induced cardiomyocyte injury.Finally,the binding sites between miR-27b-3p and Foxo1 3’-UTR were predicted by Target Scan and miRanda system,and the binding sites between Foxo1 and GSDMD gene promoter were predicted by JASPAR database query.The 3’-UTR luciferase reporter plasmid of wild-type and mutant Foxo1 was constructed.In addition,the luciferase reporter plasmid was constructed with the wild-type and mutant promoter of GSDMD,and the relationship between miR-27b-3p and Foxo1 3’-UTR and the interaction between Foxo1 and GSDMD promoter were verified by double luciferase reporter assay.Ch IP assay was used to detect the interaction between Foxo1 and GSDMD gene promoter region.Results:TEM and Nano Sight NTA particle size analysis showed that CMECs cells could secrete exosomes with complete structure under hypoxia induction,and highly expressed CD63,CD81 and TSG101 exosome marker proteins.In terms of function,CMEC-derived exosomes can improve H/ R-induced injury of H9c2 cardiomyocytes,reduce the levels of CK-MB,LDH,IL-1β and IL-18 in serum,and demonstrate that CMEC-derived exosomes can regulate the expression levels of GSDMD,NLRP3 and caspase-1 in H9c2 cardiomyocytes.In addition,CMEC-derived exosomes cultured under hypoxic conditions ameliorated I/ R-induced H9c2 cardiomyocyte injury more significantly than those cultured under normal conditions.2.RT-q PCR results showed that the expression level of miR-27b-3p in the tissues surrounding myocardial infarction of rats was significantly decreased.Functionally,miR-27b-3P-inhibitor reduced the ability of CMECs hypoxic-derived exosomes to protect H9C2 cytoskeleton structure,leading to the weakened proliferation ability of H9C2 cells and increased the levels of IL-1β and IL-18.In addition,miR-27b-3P-inhibitor significantly increased the expression levels of GSDMD,NLRP3,pro-caspase-1 and caspase-1 in H9C2 cells.3.Experiments were conducted to further determine whether miR-27b-3p in CMECs hypoxic exosomes inhibited pyroptosis by regulating the expression of Foxo1.It was found that overexpression of Foxo1 decreased the protective ability of CMECs hypoxic exosomes to H9C2 cytoskeleton structure and cell proliferation ability,resulting in increased levels of IL-1β and IL-18,and upregulation of Foxo1,GSDMD,NLRP3,pro-caspase-1 and caspase-1 in H9C2 cells.4.Adenovirus was used to overexpress the protein expression level of Foxo1 in cardiac tissue,and the results showed that adenovirus-transfected Foxo1 overexpression reduced the protective effect of CMECs hypoxic exosomes on rat tissues.Furthermore,the expression of Foxo1,GSDMD,NLRP3,pro-caspase-1 and caspase-1 in myocardial tissue was significantly increased.5.Luciferase reporter assay showed that miR-27b-3p could target Foxo1 degradation.The results also suggested that the GSDMD gene promoter sequence contained a direct binding site to Foxo1.The results of dual luciferase reporter gene and Ch IP assay showed that miR-27b-3p affected the methylation of GSDMD gene promoter region and inhibited the transcription of GSDMD.Conclusion:1.Under hypoxia,CMECs-derived exosomes protect the pyrophosis during the I/R induced myocardial injury in rats.2.Under hypoxic conditions,CMECs-derived exosomes reduce pyrophosis of H/R H9C2 cells by mediating miR-27b-3p.3.In hypoxia condition,CMECs-derived exosomes alleviates pyrophosis during I/R induced myocardial injury through regulating miR-27b-3p /Foxo1/GSDMD axis.