Research On The Role And Mechanism Of MSC-sEVs In Regulating Mitochondrial Fission In The Treatment Of Diabetic Cardiomyopathy

Objective: Diabetic cardiomyopathy(DCM)is one of the common end-stage complications of diabetes with a high mortality rate,which seriously affects the quality of life of patient Small extracellular vesicles(sEVs),double-layer membrane structures secreted by cells,are small vesicles with a variety of biologically active contents.sEVs derived from mesenchymal stem cells(MSC-sEVs)have been shown to play an important role in the repair process of various tissue injuries.They are novel materials for emerging “cell-free” therapy.The purpose of this study is to explore the protective effect and potential mechanism of umbilical cord MSC-sEVs on DCM.Method: Umbilical cord MSC was isolated by tissue adherent method and culture amplification was carried out.Flow cytometry was used to detect MSC surface marker protein,and osteogenesis and adipogenesis induced culture was used to detect MSC multi-directional differentiation potential.MSC culture supernatants were collected,sEVs was extracted by ultracentrifugation.Transmission electron microscopy,nano-particle tracking analyzer and western blot were used to identify and characterize MSC-sEVs.In vivo experiments,db/db mice and STZ-induced SD rats were used as in vivo model of DCM,and the umbilical cord MSC-sEVs was injected intravenously.Echocardiography detected heart function,biochemical analysis detected serum c Tn I levels,HE staining detected the degree of myocardial tissue damage,Masson staining and Sirius red staining detected the level of myocardial fibrosis,and TUNEL staining detected apoptosis.In vitro experiments,33 m M glucose medium with 300μM palmitic acid(HG/HF)was used to stimulate H9c2 cardiomyocytes to construct a cell model,and then cells were treated with MSC-sEVs and PBS.CCK8,western blot,flow cytometry and DCFH-DA probes were used to detect cell proliferation,apoptosis and ROS levels.High-resolution electron microscopy detected the mitochondrial morphology of cardiomyocytes in db/db mouse,Mito Tracker staining detected the mitochondrial morphology of H9c2 cardiomyocytes,and JC-1 staining detected the mitochondrial membrane potential.Western blot detected the expression levels of key proteins that mediate mitochondrial fission and fusion,and verified them at the cell and tissue levels through immunofluorescence.si RNA was used to knockdown Drp1 and Mdivi-1 was used to inhibit the phosphorylation level of Drp1 at the S616 site,and then apoptosis,ROS,mitochondrial membrane potential and mitochondrial morphology were detected.The expression level of the ROCK1 at the cell and tissue levels was detected,and the Co-IP experiment proved the interaction between ROCK1 and pDrp1(S616).miRNA sequencing detected the enriched miRNA in the umbilical cord MSC-sEVs,the RNAhybrid database predicted the target miRNA that may bind to ROCK1,and the luciferase reporter gene verifies the regulatory relationship between miRNA and ROCK1.7.HG/HF stimulated H9c2 cells transfected with miR-8747 mimics or H9c2 cells transfected with miR-8747 inhibitor and treated with MSCsEVs.The expression levels of ROCK1 and p-Drp1(S616)were detected,and apoptosis,ROS levels,mitochondrial morphology and membrane potential were evaluated.Result: Umbilical cord MSC was successfully isolated and cultured,and it expressed stem cell-specific markers and could induce the osteogenesis and adipogenesis.Transmission electron microscopy observed that MSC-sEVs showed a typical disc shape and was relatively uniform in size.The nano-particle analyzer detected that the median particle size of MSC-sEVs was 145 nm,and western blot showed that MSC-sEVs expressed specific marker proteins.In vivo,umbilical cord MSC-sEVs were able to restore heart function in db/db mice,increase left ventricular ejection fraction and fraction shortening,and reduce serum c Tn I expression levels.Histopathological tests showed that umbilical cord MSC-sEVs intervention restored the morphology of myocardial cells and muscle fibers,reduced inflammation,reduced the level of myocardial fibrosis,reduced the expression of myocardial 8-OHd G,and reduced apoptosis.In the STZ-induced rat DCM model,umbilical cord MSC-sEVs showed similar myocardial protection.In vitro,umbilical cord MSC-sEVs could promote H9c2 cell proliferation,effectively reduce HG/HF-induced apoptosis,reduce cell ROS production,and promote proliferation-related protein expression and reduce apoptosis-related protein expression.Umbilical cord MSC-sEVs inhibited excessive mitochondrial fission and restored mitochondrial morphology in db/db mouse myocardium and H9c2 cells induced by HG/HF,MSC-sEVs could alleviate the decline of mitochondrial membrane potential in H9c2 cells.MSC-sEVs could effectively reduce the expression levels of Drp1 and p-Drp1(S616)in db/db mouse myocardial tissue and HG/HF stimulated H9c2 cells,and inhibit the translocation of Drp1 to mitochondria.Knocking down Drp1 or Mdivi-1 treatment alleviated HG/HF-induced apoptosis,ROS and mitochondrial membrane potential decline,and promoted mitochondrial morphological recovery.ROCK1 highly expressed in db/db mouse myocardial tissue and HG/HF-stimulated H9c2 cells,and was consistent with the expression of p-Drp1(S616).MSC-sEVs could inhibit the expression of both ROCK1 and p-Drp1(S616).Co-IP assay showed that ROCK1 could bind to p-Drp1(S616).According to the expression profile of miRNA in the umbilical cord MSC-sEVs obtained by miRNA sequencing,the binding site to ROCK1 was predicted,and the luciferase reporter gene shows that miR-8747 had a targeted binding to ROCK1.PCR showed that MSC-sEVs treatment significantly increased the expression of miR-8747 in the cell.miR-8747 mimics transfection significantly inhibited the expression of ROCK1 and downstream p-Drp1(S616)in H9c2 cells induced by HG/HF,alleviated apoptosis,ROS and mitochondrial membrane potential decline,and promoted mitochondrial morphology recovery.miR-8747 inhibitor transfection of HG/HF-stimulated H9c2 cells could promote the expression of ROCK1 and downstream p-Drp1(S616)in H9c2 cells,and promote apoptosis,ROS production and mitochondrial membrane potential decline,umbilical cord MSC-sEVs treatment alleviated the above changes.Conclusion: Umbilical cord MSC-sEVs can relieve DCM myocardial injury and improve heart function through targeting and inhibiting ROCK1/Drp-1axis-mediated mitochondrial fission by delivering miR-8747.Our research provides a new experimental basis for the development of cell-free therapies based on sEVs in the treatment of diabetes and its complications.