| Objective:Umbilical Cord Mesenchymal Stem Cells derived Exosomes (UMSCS-EXO)can promote myocardial repair after myocardial infarction,but the specific mechanism needs further investigation.This study aims at investigating the role of macrophage in UMSCsExo mediated cardiac repair after myocardial infarction,and further exploring the possible molecular mechanisms.Methods:(1) UMSCs were isolated by enzyme digestion.Flow cytometry analysis was used to detect cell surface markers of UMSCs.To evaluate multilineage differentiation potency,UMSCs were induced to differentiate into osteocytes,adipocytes and chondrocytes.And the UMSC culture supernatant was collected to extract exosomes by ultrahigh-speed centrifuge.The identification of exosomes was carried out as follows:The morphology of exosomes was observed by Transmission electron microscopy (TEM);The size distribution was measured by Nanoparticle tracking analysis (NTA);And exosomal protein markers(CD63 and TSG101) were detected by western blot.(2) UMSCs-Exo were transplanted into myocardium of the infarcted border zone,and the influence of UMSCs-Exo on inflammatory response,myocardial fibrosis,angiogenesis and cardiac function was detected by western blot,tissue section and echocardiography after MI.(3) To evaluate the effect of the macrophage in UMSCs-Exo mediated cardiac repair,macrophages were cleared with intraperitoneally injected clodronate liposomes (Cl2MDP).Then MI models were established and transplanted UMSCs-Exo,the heart function was detected by echocardiography.(4) Murine macrophages were co-cultured with UMSCs-Exo and stimulated by lipopolysaccharide (LPS).The M2 polarization of macrophages was detected by flow cytometry,real-time fluorescence quantitative PCR (q-PCR) and Western blot.At the same time,the hearts of mice were extracted on the third day after MI,and M2 polarization was detected by flow cytometry,western blot and immunofluorescent staining.(5) The nucleic acid and protein components of UMSCs-Exo were extracted,and co-cultured with macrophages respectively,so as to explore the main functional components in exosomes that regulate the polarization of macrophages.With combination of macrophage mRNA sequencing and UMSCs-Exo miRNA sequencing results,further bioinformatics analysis was carried out to analyze the key pathway in regulating macrophage polarization by UMSCs-Exo.The accuracy of the predicted target gene was detected by dual luciferase assay and western blot.Flow cytometry and western blot were used to verify the possible mechanism of regulating macrophage M2 polarization by UMSCs-Exo.Results:(1) UMSCs were successfully isolated and cultured;The UMSCs-Exo extracted by ultra-high speed centrifugation method were consistent with the characteristics of exosomes as previous reported.(2) UMSCs-Exo could significantly improve the cardiac function of MI mice by reducing inflammatory response,inhibiting myocardial fibrosis and promoting angiogenesis.(3) After depleting the macrophages of MI mice with Cl2MDP,the therapeutic effects of UMSCs-Exo were disrupted.(4) Experiments both in vitro and in vivo have confirmed that UMSCs-Exo could up-regulate the expression of M2-type related genes and inhibit the expression of M1-type related genes of macrophages in inflammatory microenvironment,which promoting M2 polarization of mouse macrophages.(5) We confirmed that the nucleic acid of UMSCs-Exo plays a major role in promoting M2 polarization of macrophages.Bioinformatic analysis suggested that UMSCs-Exo regulate macrophage M2 polarization through miR-24-3p targeting Plcb3. Subsequent experiments further found that UMSCs derived exosomal miR-24-3p inhibits the expression of Plcb3 in macrophages and NF-κB signal pathway,thus promoting macrophages M2 polarization.Conclusion:This study confirmed that UMSCs-Exo could reduce the inflammatory response,inhibit myocardial fibrosis,promote angiogenesis,and improve cardiac function after MI.Macrophages play vital roles in UMSCs-Exo mediated cardiac repair after MI.Further,in inflammatory microenvironment,UMSCs-Exo promote macrophage M2 polarization by inhibiting Plcb3/NF-κB signaling activation through miR-24-3p. |
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