Mechanism Of Targeting Cardiomyocyte HMGB1 Signals By Exosomes Derived From Mesenchymal Stem Cells To Protect Ischemic Myocardium

Background and Objective:Background:Acute myocardial infarction(AMI),which is myocardial necrosis caused by acute coronary artery occlusion Clinically,AMI provides the body with a large amount of oxygen and electron acceptors,resulting in the explosive growth of oxygen free radicals in a short time,producing a large number of reactive oxygen species,which attack cell membranes and lead to lipid peroxidation.Causing irreversible Myocardial ischemia reperfusion injury(MI/RI),which is the major barrier to patients benefiting from ischemia.Exosome(Exo)is a kind of vesicle secreted by most cells.It has a phospholipid bilayer structure,with a diameter of 40-150 nm.The membrane carries RNA,protein and other substances,and plays an important role in the transmission of information between cells.Recent studies have shown that Exo may have a certain preventive and protective effect on MI/RI,but the specific mechanism of its effect remains unclear.Purpose:Mesenchymal stem cell derived exosome,mesenchymal stem cell derived exosome,Mesenchymal stem cell derived exosome,hypoxia/reoxygenation(H/R)model was used in this study.The molecular mechanism of MSC-Exo to prevent MI/RI,this study will help clarify the anti-Mi /RI action mechanism of MSC-Exo,discover new intervention targets,and provide experimental support for clinical MI/RI treatment.Methods:(1)Exo were separated from MSC by ultracentrifugation,and verified by Transmission electron microscopy(TEM)at the morphological level.Exo were identified by Western blot(WB)and Nanoparticle tracking analysis(NTA).(2)Exo was labeled with Di I membrane dye and incubated with H9c2 cardiomyocytes for 6h at 37°C.The uptake efficiency of Exo in H9c2 cardiomyocytes were observed with an upright fluorescent microscope.(3)The H/R model of H9c2 cardiomyocytes was constructed by using Anaero Pack hypoxia system.Before hypoxia,H9c2 cardiomyocytes were co-cultured with MSC-Exo.The apoptosis levels of Control group,H/R group,H/R+Exo group were detected by CCK-8,flow cytometry,WB,immunofluorescence,HE staining and other methods to verify whether MSC Exo can reduce the apoptosis rate of MI/RI cardiomyocytes.(4)By ferroptosis specific indicators,sush as DCFH-DA,ferroptosis promoting protein ACSL4 and ferroptosis inhibiting protein GPX4,as well as GSH and MDA representing the level of lipid peroxidation.Through the detection of the above indicators,it was discussed whether the model built by our research group can induce ferroptosis and whether MSC-Exo can reduce the ferroptosis level in MI/RI.(5)Si RNA was used to transfect the silencing signal protein HMGB1,Exo were used to pretreat cardiomyocytes,after H/R,the apoptosis rate of cardiomyocytes in NCsi RNA group,NCsi RNA+Exo group,HMGB1 si RNA group and HMGB1 si RNA+Exo group were detected by flow cytometry with Annexin V-FITC/PI double staining and the expression of anti apoptosis protein Bcl-2.The apoptosis rate of H9c2 cells in the above four groups were detected by AO/EB staining and HE staining,the level of ROS in each group after transfection were detected by DCFH-DA probe according to flow cytometry,the expression of ferroptosis specific protein after transfection were measured by WB,and the content of GSH and MDA in each group were detected by micro method.Results(1)The Exo extracted by ultracentrifugation were 40-150 nm in diameter,and expressed Exo markers TSG101 and CD9,but did not express GM130.(2)MSC-Exo can be taken up by H9c2 myocardial cells.(3)WB detected the expression level of anti apoptosis protein Bcl-2 in normal group,H/R group and Exo group.Compared with normal group,the expression level of Bcl-2 protein in H/R group decreased,after MSC-Exo intervention,the expression of Bcl-2 protein in H9c2 cardiomyocytes increased.Flow cytometry and immunofluorescence and HE staining were used to detect the apoptosis rate of H9c2 cells,which were consistent with WB results.MSC-Exo pretreatment could significantly reduce the apoptosis rate of myocardial cells.(4)Compared with the Control group,the promoting indexes ACSL4,MDA and ROS in the H/R model group were highly expressed,while the inhibiting indexes GPX4 and GSH were lower,indicating that the model constructed in this study induced ferroptosis.(5)Compared with H/R group,ACSL4 protein expression,MDA and ROS levels were decreased in Exo group and Fer-1 group.However,GPX4 protein expression and GSH level were increased in Exo group and Fer-1 group.These results indicated that MSCExo pretreatment could significantly reduce iron death in MI/RI cardiomyocytes.(6)The expression level of anti-apoptotic protein Bcl-2 decreased in the knocked HMGB1 group compared with the non-knocked HMGB1 group.Flow cytometry showed increased apoptosis rate of cardiomyocytes.HE staining showed an increase in apoptotic corpuscles.AO/EB staining showed a decrease in cell number and an increase in red fluorescence.ACSL4 was highly expressed,and GPX4 was low.At the same time,with the decrease of GSH level and the increase of MDA and ROS levels,the knockdown of HMGB1 protein inhibited the protective effect of MSC-Exo.Conclusions:(1)After co-culture with H9c2 cells,MSC-Exo can significantly reduce H/ R-induced apoptosis,improve cell survival rate,improve cell morphology,and protect ischemic myocardium.(2)MSC-Exo targets HMGB1 signal to protect ischemic myocardium.MSC-Exo upregulates the expression of HMGB1 to reduce the apoptosis rate and the degree of ferroptosis.(3)MSC-Exo can enhance the protective effect of MSC-Exo on heart by up-regulating the expression of HMGB1 protein,reducing the level of myocardial apoptosis,resisting ferroptosis,and alleviating the damage of MI/RI.