The Function And Mechanism Of Mesenchymal Stem Cell-derived Exosome In The Therapeutic Effects Of ? Cell Function In Type 2 Diabetes

BackgroudDiabetes is a chronic metabolic disorder with a complex etiology.It currently affects approximately 463 million patients worldwide(https://www.idf.org/).As the population ages and lifestyle changes,the number of people affected by the disease will increase in the coming decades,and is expected to exceed 600 million by 2040.Given its high incidence and repeated complications,diabetes treatment has become a major public health challenge.Liver is the main organ that regulates blood glucose concentration.The abnormal output and storage of liver grapes are the main causes of hyperglycemia in type 2 diabetes(T2DM)patients.The main manifestations are reduced glycolysis,decreased glycogen synthesis capacity,and increased gluconeogenesis.The main causes of T2DM are pancreatic ?-cell hypofunction and insulin resistance.The failure of ? cell function is mainly due to the decrease in the number and quality of ? cells.The traditional view is that the decrease in the number of ? cells is the result of premature programmed cell death,and early research efforts have focused on increasing ? cell survival.However,several recent studies have challenged the ?-cell apoptosis hypothesis.These data suggest that ?-cell dedifferentiation is a new mechanism to explain the loss of functional ?-cell.In the process of dedifferentiation,? cells do not undergo apoptosis,but with key ? cell markers(such as pancreatic-duodenal homeobox-1[PDX1],forkhead box-containing protein O1[Foxo1],myofascial fibrosarcoma oncogene A[MAFA])reduces expression and lose the identity of the original mature beta cells,then transforms into expression of transcription factors such as nerve element 3(Ngn3)and octamer binding protein-4(Oct4)of endocrine precursor cells,resulting in decreased ? cells and decreased insulin secretion.Therefore,improving hepatic glucose metabolism,preventing or reversing the progressive decline in islet ? cell population and function is an important strategy for the treatment of T2DM.Stem cells are a type of cells that can self-renew and differentiate into many types,mainly divided into three types,including embryonic stem cells(ESCs),adult stem cells(ASCs),and induced pluripotent stem cells(iPSCs).Mesenchymal stem cells(MSCs)belong to ASCs with multipotential differentiation potential.MSCs can be derived from almost all types of tissues,including bone marrow,umbilical cord,adipose tissue,skeletal muscle tissue,liver tissue and blood.In recent years,the efficacy and safety of MSCs in cell-based therapies have drawn great attention.It has avoided the ethical and carcinogenic risks of ESCs and iPSCs.It is currently the most studied cell in T2DM cell therapy.Bone marrow mesenchymal stem cells(bmMSCs)are the first MSCs to be discovered and thoroughly studied.The first clinical study of retrospective T2DM was the combination of intrapancreatic bmMSCs infusion with hyperbaric oxygen therapy(HOT).The trial found that during the 1-year follow-up,metabolic parameters of T2DM patients gradually improved,and insulin demand was significantly reduced.Subsequent clinical trials and basic studies have also confirmed that MSCs play a therapeutic effect in lowering glucose,improving islet function and hepatic glucose and lipid metabolism in T2DM.However,the research on how MSCs play a therapeutic role is relatively insufficient.Exosome is about 100 nm in diameter and is enveloped by lipid bilayers.It comes from a wide range of sources in the body(mainly from body fluids such as plasma/serum,urine,cerebrospinal fluid,saliva,etc.)Isolation and extraction in vitro(from cell conditioned medium).Exosome communicates between cells(mRNA,miRNA,and other RNAs),proteins,and lipids contained in vesicles for intercellular communication,affecting a variety of life activities including cytokine production,cell proliferation,apoptosis and metabolism.Previous studies have suggested that MSCs can reach damaged pancreatic tissues after infusion,perform in situ repair of islet ? cells and promote regeneration.More and more evidence shows that the effect of MSCs on T2DM treatment is not due to direct differentiation into functional ? cells,but is mediated by paracrine,especially exosome.The RNA carried by Exosome is mainly miRNA,and it has been confirmed that miRNA plays a vital role in regulating the function of islet ? cells.Based on the above research results,we make the following hypotheses:1.Exosome derived from MSCs can play a role in the treatment of T2DM by improving hepatic glucose metabolism and pancreatic islet function;2.Exosome mainly affects the downstream differentiation of pancreatic islet cells through miRNA carried on downstream signaling pathways.Part I Effects of mesenchymal stem cell-derived exosome on hepatic glucose metabolism and islet function in type 2 diabetesObjectiveTo investigate the effects of bone marrow mesenchymal stem cells(bmMSCs)and its derived exosomes(bmMDEs)on T2DM islet function and hepatic glucose metabolism.Methods1.After extraction and identification of bmMSCs and bmMDEs,Cy7 and PKH67 labeled bmMDEs were used to verify the exosome uptake in vitro and in vivo.2.A T2DM rat model was established by high-fat diet+low-dose STZ induction.T2DM group rats were randomly divided into 3 groups.The tail vein was infused with PBS,bmMSCs and bmMDEs,respectively.The body weight and blood glucose of rats were measured weekly.3.After bmMSCs and bmMDEs intervention,abdominal glucose tolerance test(IPGTT)and intraperitoneal insulin tolerance test(IPITT)were performed,then serum insulin levels and ?/? cell were measured.4.Western blotting was used to detect glycolysis-related enzymes(GCK,PFK and PK),glycogen synthesis-related enzymes(p-GSK3?/GSK3?)and hepatic gluconeogenesis-related enzymes(PEPCK and G-6-P)in SD rats before and after intervention with bmMDEs.expression.PAS staining detects the accumulation of glycogen.Results1,The cultured bmMSCs were long-fusiform and vortex when viewed under a light microscope.Directed differentiation showed that bmMSCs could differentiate into adipocytes and osteoblasts.Flow cytometry was used to detect bmMSCs with high expression of stem cell markers CD90 and low expression of hematopoietic markers CD45 and CD34.The isolated bmMDEs were about 100 nm in diameter and were "cup-shaped" or "disc-shaped".Western blot results showed that exosomal markers CD9 and TSG101 were highly expressed,and cell markers Calnexin were low-expressed.Cy7-labeled bmMDEs in vivo showed strong fluorescence signals in all organs(heart,lung,liver,pancreas,spleen,kidney,and small intestine),suggesting that bmMDEs in the tail vein could successfully reach damaged pancreatic tissues.In vitro tracer results of PKH67-labeled bmMDEs proved that after co-culturing bmMDEs with INS-1 cells for 24 hours,a large amount of green dot-like fluorescence was detected in the cells.2.After the high-fat diet and STZ injection modeling,the fasting blood glucose of SD rats in the model group was all>16.7 mmol/L.With the intervention of bmMSCs and bmMDEs,symptoms such as polydipsia and polyuria and thirst were significantly improved in T2DM rats.Compared with the T2DM group,fasting blood glucose of rats was significantly reduced and the weight was relatively increased after bmMSCs intervention.bmMDEs also showed similar therapeutic effects.3.IPGTT and IPITT experiments suggest that bmMSCs and bmMDEs treated T2DM rats have significantly improved impaired glucose tolerance and decreased insulin sensitivity.Compared with the T2DM group,serum insulin levels increased significantly after treatment with bmMDEs,the islet volume of rats increased,the proportion of Insulin-positive cells increased,and the proportion of Glucagon-positive cells decreased.4.BmMDEs-treated T2DM rats had higher expression levels of glycolytic enzymes and p-GSK3?/GSK3?,whereas hepatic gluconeogenic enzymes were decreased compared with those in T2DM group.BmMDEs also increased PAS-positive staining in T2DM group.Conclusions1.bmMSCs can significantly reduce blood glucose and improve insulin resistance in T2DM rats.2.In addition to lowering blood glucose and improving insulin resistance in T2DM rats,bmMDEs can also increase serum insulin levels and reverse islet ?/? cell ratio.3.bmMDEs can improve hepatic glucose metabolism,mainly to promote glycolysis and glycogen synthesis,while inhibiting gluconeogenesis.Part ? Effects and mechanism of mesenchymal stem cell-derived exosome on diabetic P cell dedifferentiationObjectiveTo investigate the effects and mechanism of bmMDEs on T2DM islet P cell dedifferentiation.Methods1.Immunofluorescence staining was used to detect the dedifferentiation of islet ? cells in each group,and qRT-PCR was used to detect dedifferentiation-related indicators in primary islets isolated from each group.2.Isolated primary islets and INS-1 cells were stimulated with 33.3 mmol/L glucose for 72 hours.The GSIS test was used to detect insulin secretion levels after glucose loading.Immunofluorescence and qRT-PCR were used to detect ? cell dedifferentiation.3.bmMDEs and exosome controls(INS-1 cell-derived exosomes,IDEs)were used to interfere with high glucose-induced primary islets and INS-1 cells,respectively,and the changes in insulin secretion levels and ?-cell dedifferentiation before and after the intervention were measured.4.INS-1 cell-derived exosomes(IDEs),HG-treated INS-1 cell-derived exosomes(HG-treated INS-1 cell-derived exosomes,HIDEs),and bmMDEs were subjected to high-throughput miRNA sequencing.GSIS functional experiments were used to screen the target miRNAs.5.Combining miRNA mimics and inhibitors,the effects of the target miRNA on high glucose-induced ?-cell dedifferentiation were measured by GSIS experiments and qRT-PCR,respectively.6.Target miRNA downstream target gene prediction and functional verification.7.Lentiviral transfection was used to establish stable MSCs with the target miRNA knockdowm.T2DM rats were intravenously infused with bmMDEsmiRNA KD,and the therapeutic effect of bmMDEs after inhibiting the target miRNA was observed.Results1.Immunofluorescence staining showed that compared with normal values,the expression of islet ?-cell markers(PDX1,FOXO1)decreased,and the expression of islet progenitor cell markers(NGN3,OCT4)increased significantly;after the intervention of bmMDEs,the expression of PDX1 and Foxol was up-regulated NGN3,OCT4 expression is down-regulated.PCR detection of primary islet dedifferentiation from each group showed similar results of immunofluorescence.2.72 hours after high glucose induced INS-1 cells and primary islets,the GSIS experiment showed that the insulin secretion capacity of the induced group decreased after high glucose load.Immunofluorescence and PCR results showed that high glucose induced islets dedifferentiated,and bmMDEs intervened in INS-1 The expression of Pdxl and Foxol decreased after intervention of high glucose,and the expression of bmMDEs was up-regulated after intervention.The expression of Ngn3 and Oct4 was not significantly different from the normal increase.3.According to the result map generated by high-throughput miRNA,qRT-PCR was performed to verify the top 10 miRNAs.GSIS experiments show that the top 10 mi miRNAs mimic INS-1,and then add high glucose medium to continue culturing for 72 hours.miR-146a-5p(R-146a)mimics other miRNAs and significantly increases the replication after glucose load.This is further proven in the island.So we are initially committed to miR-146a for further research.4.The qRT-PCR results showed that in the primary cultured islets,the miR-146a simulated intervention increased the expression levels of Pdxl and Foxol mRNA,and decreased the levels of Ngn3 mRNA.The miR-146a inhibitor attenuated the effect of bmMDEs on inhibiting ?-cell dedifferentiation.5.Through TargetScan target gene prediction database and luciferase reporter gene detection,we found that Numb is a direct downstream target gene of miR-146a,and the expression of increased in vitro plasmids in T2DM rats and high glucose-induced primary islets and INS-1 cells After overexpression of Numb transfection,the expression levels of Pdxl and Foxol were significantly reduced,and the expression levels of Ngn3 and Oct4 were significantly increased.After siRNA transfection inhibited Numb,the expression of ? cell marker gene increased,and the expression of progenitor cell marker gene decreased..Then we further proved that ?-catenin,as a downstream signaling molecule of NUMB,also participated in the transformation of dedifferentiation of ? cells.6.Lentiviral transfection knocked down the expression of miR-146a in bmMSCs,extracted exosomes(bmMDEs miR-146a KD)and then infused T2DM rats.Guide island function and verify that the role of guide island ? cell dedifferentiation is significantly reduced.Conclusions1.bmMDEs can inhibit diabetic islet ? cell dedifferentiation.2.bmMDEs can reduce blood glucose,improve pancreatic islet function and inhibit ?-cell dedifferentiation through miR-146a.3.The miR-146a carried by bmMDEs activates the NUMB/?-catenin signaling pathway,thereby improving the insulin secretion ability after glucose load and inhibiting the dedifferentiation of ? cells.