Effect Of Menstrual Blood-Derived Endometrial Stem Cells On Non-Alcoholic Fatty Liver Disease By Regulating Glucose And Lipid Metabolism

BackgroundNon-alcoholic fatty liver disease（NAFLD）is characterized by fatty storage and degeneration of liver parenchymal cells,and its excessive development leads to liver fibrosis and cirrhosis,causing serious burden to patients and the society^[1-3].The complexity of its pathogenesis and the uncertainty of disease assessment have hindered drug development for NAFLD^[4].Mesenchymal stem cells have been shown to play a role as cell therapy in a variety of diseases.As mesenchymal stem cells,menstrual blood-derived endometrial stem cells（Men SCs）have the advantages of wide sources,short acquisition cycle and strong repeatability^[5-7].However,little is known about the ameliorative effect of Men SCs on NAFLD and related mechanisms.In this study,Men SCs were transplanted and co-cultured to clarify the improvement effect and potential mechanism of Men SCs on NAFLD,and to provide theoretical support for Men SCs treatment of NAFLD and other metabolic diseases.Objectives1.NAFLD and ob/ob mouse models and FFA（oleic acid and palmitic acid mixture）stimulated lipid storage model were modeled by high-fat diet to explore the effect of Men SCs transplantation on NAFLD;2.To clarify that Men SCs transplantation can improve NAFLD by regulating the disorder of glucose and lipid metabolism in NAFLD mouse model.Methods1.Men SCs were isolated,extracted and cultured from female menstrual blood,and cultured to P3 generation for identification of surface markers for subsequent animal and cell experiments;2.NAFLD mouse model was established by high-fat diet（HFD）and lipid storage cell model was established by FFA stimulation;3.The serological biochemical and pathological indexes of the mouse model were detected.Serological indexes include blood glucose,glutamic-propyl/glutamic-oxalacetic transaminase,serum triglyceride,etc.Liver pathological tissues included H＆E staining,Oil Red O staining and PAS staining to verify the success of mouse model construction and the therapeutic effect of Men SCs on NAFLD;4.The mice were divided into four groups,NCD,HFD,NCD/Men SCs and HFD/Men SCs.RNA was extracted from the liver tissues of the mice,and high-throughput transcriptome sequencing was performed to analyze the related signaling pathways and differentially expressed genes;5.Immunohistochemical staining,protein western blot,real-time quantitative PCR and other experimental techniques confirmed the expression of genes related to glucose and lipid metabolism;6.Men SCs were co-cultured with L02 and AML12 cells to demonstrate the effect of Men SCs on glucose absorption and lipid synthesis.Results1.Men SCs were isolated,extracted and verified,and a batch of high-quality and sufficient Men SCs were obtained for subsequent animal and cell experiments;2.The NAFLD mouse model was successfully constructed at the animal level.The lipid storage model of hepatocytes was successfully established at the cellular level;3.Men SCs transplantation could significantly improve serum biochemical indices and liver function of NAFLD mouse model and ob/ob mouse model.The results of pathological staining also showed that it had obvious therapeutic effect;4.High-throughput transcriptome sequencing results showed that inflammatory signaling pathways and glucolipid metabolism signaling pathways were changed before and after treatment;5.Liver proteins were extracted from mouse model to verify that glucose metabolism related genes p-Akt（Ser473）/Akt and p-Gsk3β/Gsk3βwere changed before and after treatment,and lipid synthesis genes Fas,Acc-1 and Srebp1 were also significantly changed before and after Men SCs transplantation.The expression of gluconeose-related genes was verified by real-time quantitative PCR and staining to further confirm the results of western blot;6.After co-culture of Men SCs with hepatocyte lipid model,it was confirmed that Men SCs could significantly improve glucose absorption and lipid synthesis.Conclusions1.Men SCs transplantation can improve the biochemical and pathological characteristics of NAFLD mice;2.Men SCs improved NAFLD by regulating the disorder of glucose and lipid metabolism in NAFLD mouse models.