Integrin-Linked Kinase-Modified Bone Marrow Mesenchymal Stem Cells Improve Cardiac Function After Infarction In Rats Via Exosomal Pathway

Objective:Based on the previous experiments,this study observed the effect of Integrin-linked kinase（ILK）-modified Bone marrow mesenchymal stem cells（BMSC）and their secreted exosomes on improving cardiac function in a rat Myocardial infarction（MI）model,and screened the differentially expressed Micro RNAs（mi RNAs）in the exosomes by gene microarray,in order to explore the efficacy-related molecular mechanisms of exosomes and provide a provide a more effective method and means to improve the efficacy of stem cell transplantation to improve cardiac function after infarction.Methods:1.Animal experiment:Preparation of a model of rat myocardial infarction by left anterior descending ligation of the coronary artery,and BMSC transplantation as well as exosome injection were performed.Rats were divided into sham group（sham）,infarct model group（MI）,empty carrier BMSC group（MI+Null-BMSC）,empty carrier BMSC-secreted exosome group（MI+Null-Exo）,wild-type ILK-modified BMSC-secreted exosome group（MI+WT-ILK-Exo）,high-kinase activity ILK-modified BMSC-secreted exosome group（MI+SD-ILK-Exo）.At 4w postoperatively,left ventricular（LV）function was examined by echocardiography;HE and Masson staining examined myocardial tissue morphology and myocardial fibrosis,and ELISA was used to determine serum c Tn I and CK-MB,and Western blotting was used to determine Caspases3 in myocardial tissue.2.Cellular experiments:The exosome inhibitor GW4869 was applied to inhibit BMSC exosome secretion,and the effects of BMSC medium on cell viability and apoptosis of cardiomyocytes（H9c2）were observed by CCK-8and DAPI assays.Wild-type ILK-modified BMSC exosomes（WT-ILK-Exo）and high-kinase activity ILK-modified BMSC exosomes（SD-ILK-Exo）were extracted for high-throughput sequencing of mi RNAs microarrays to screen MI-related differentially expressed mi RNAs.Results:1.Rat models were successfully established;2.Animal experiments:echocardiographic results showed that the application of BMSC or exosome treatment after infarction significantly improved left ventricular cardiac function in rats.There are the best treatment outcomes in the MI+SD-ILK-Exo group,whose left ventricular end-systolic diameter（LVESD）and left ventricular ejection fraction（LVEF）were（88.00±1.94）%and（1.12±0.02）times（P<0.05）times（P<0.05）of the MI+WT-ILK-Exo group,respectively;HE and Masson staining yielded similar results,with more regular myofascicular arrangement,fewer myocardial fiber breaks,and a smaller proportion of collagen in the MI+SD-ILK-Exo group than in the MI+WT-ILK-Exo group（P<0.05）,indicating that exosomes secreted by wild-type and high-kinase activity ILK-modified BMSC both improved cardiac function in post-infarction rats,and the latter had the most pronounced effect.The serum c Tn I and CK-MB measurements and WB results were consistent with the above trend;3.Cellular experiments:Both CCK-8 and DAPI results showed that BMSC medium could reverse the decrease in H9c2cell viability and apoptosis caused by H₂O₂,and SD-ILK modification was better than WT-ILK,and cell viability decreased after the application of exosome inhibitor GW4869,indicating that the above regulatory effects were related to exosomes;4.High-throughput sequencing and mi RNAs screening:mi RNAs sequencing screened 47 differentially expressed mi RNAs in SD-ILK-Exo group and WT-ILK-Exo group.combined with OMIM、DRUGBANK and other databases screened the infarct-related target genes Tnf,Postn,Vegfa,Igf1 and Olr1,and then predicted the related 4 mi RNAs（mi R-181d-5p,mi R-203a-3p,mi R-466b-2-3p,and mi R-466b-3p）.Conclusions:1.ILK especially high kinase activity ILK-modified BMSC-secreted exosomes improve post-infarction cardiac function and myocardial fibrosis in rats.2.ILK-modified BMSC medium ameliorates H₂O₂-induced decrease in H9c2 cell viability and apoptosis through the exosome pathway,and which is associated with kinase activity at the ILK343 locus.3.High kinase activity ILK-modified BMSC secreted exosomes may improve post-infarction cardiac function in rats via mi R-181d-5p,mi R-203a-3p,mi R-466b-2-3p and mi R-466b-3p.