The Effect Of Mesenchymal Stem Cell Conditioned Medium On The Inflammation And Insulin Resistance In Islet ? Cells And Its Mechanism Study

BackgroundT2DM is a chronic and low grade inflammation disease,characterized by dysfunction of pancreatic ? cells and insulin resistance.However,pancreatic a cells play a critical role in glucose homeostasis owing to their function in releasing glucagon and stimulating glucose production from the liver.Dysregulation of a cells gives rise to fasting and postprandial hyperglycemia in T2DM,accompanied by higher glucagon-to-insulin ratio and augment of pancreatic alpha-to beta-cell area ratio.In vivo,a cells is not only regulated by its own factors,but also by environmental factors,such as insulin and somatostatin secreted by adjacent islet ?cells and ? cells,inflammatory factors in circulation,glucose,lipids,amino acids and so on.Inhibition of glucagon secretion by insulin is an important condition for maintaining blood glucose homeostasis.This inhibition could decline or even disappear once the insulin signal in a cells weakened,which leads to hypersecretion of glucagon and producing "insulin resistance".A high-fat diet plays an important role in the production of insulin resistance.High-fat-fed obese rats have elevated blood levels of insulin and glucagon,but elevated insulin doesn't inhibit glucagon secretion,suggesting that a cells are not sensitive to insulin inhibition.High FFA levels can also activate NF-?B inflammatory pathways by inducing oxidative stress in vivo,leading to the development of peripheral insulin resistance.In vitro,insulin receptor phosphorylation and intracellular insulin signal in a cells was greatly reduced by PA.Upregulation of PTEN activity and transcription by PA mediated its inhibitory effects on insulin signal.PTEN deletion in pancreatic a-cells could protect against high-fat diet-induced hyperglucagonemia and insulin resistance in T2DM rats.Recently,several clinical trials supposed that mesenchymal stromal cell(MSC)-based therapies are effective in decreasing blood glucose and alleviating some complications.MSCs or their conditioned medium not only ameliorate pancreatic ?cell injury and islet endothelium apoptosis and functional impairment,but also enhance insulin sensitivity in insulin target tissues.Additionally,in a variety of cells such as cardiomyocytes and nerve cells,MSCs also reduce ROS production and maintain their function.Increasing studies indicate that the rationale for MSC therapy is based on its paracrine action rather than differentiation mechanism.Studies have shown that miR-181 was highly expressed in MSC exosome.The absence of miR-181 leads to increased PTEN activity,inhibiting PI3K pathway activity and therefore AKT activation.The miR-181 family has been a metabolic rheostat in vivo through the nonredundant regulation of PTEN.Overall,MSCs can regulate functions of different kinds of cells,but there are few researches showing the direct effect of MSCs on islet a cell dysfunction.Therefore,exploring the mechanism involved in it is expected to provide new ideas and targets for regenerative therapy.PurposeThis study was to investigate the effect of MSC-CM on inflammation and insulin resistance of islet a cells in vitro,and the mechanism involved in it.Methods1.Isolation and identification of BM-MSCs:BM-MSCs were obtained by isolating the femurs of rats and flushing the marrow.Flow cytometry analysis was performed to identify MSCs according to characteristics that they are positive for CD90 and CD44 and negative for CD34 and CD45.The BM-MSCs were analyzed for their ability to differentiate into adipocytes and osteoblasts by Oilred O stain for the lipid droplets and Alizarin Red S stain for the calcium nodes.2.PA induced inflammation and insulin resistance in a cells:InR1-G9 cells were stimulated by 0.4mmol/l PA for 48h.Glucagon level in supernatants was detected by ELISA.Western blot was performed to investigate the expression of inflammatory factors(TNF-a and IL-1?),PTEN and insulin signal(IR/IRS1/AKT).Intracellular ROS production was evaluated by DCFH-DA fluorescent probe and flow cytometry.3.Effects of MSC-CM on PA-induced inflammation and insulin resistance in a cells:InR1-G9 cells were simultaneously stimulated by PA and MSC-CM.Glucagon level,expression of inflammatory factors,PTEN and insulin signal,and intracellular ROS were respectively detected by ELISA,Western blot and DCFH-DA fluorescent probe.4.Investigation on the mechanism of MSCs action:(1)Comparison of miR-181a expression in MSCs and a cells:RNA was extracted from BM-MSCs?InR1-G9 cells and InR1-G9 cells pretreated by MSC-CM.RT-qPCR was performed to detected expression of miR-181a.(2)Verification of miR-181a mimics and inhibitor transfection efficiency:Lipo2000 was used to transfect miR-181a mimics and inhibitor and their NC to InR1-G9 cells.Expression of miR-181a was detected by RT-qPCR.(3)Effects of miR-181a overexpression on a cells:InR1-G9 cells were transfected by miR-181a mimics followed by PA stimulation.Western blot was performed to investigate the expression of PTEN and insulin signal.Glucagon level in supernatants was detected by ELISA.(4)Effects of miR-181a inhibiton on a cells:InR1-G9 cells were transfected by miR-181a mimics followed by MSC-CM treatment.Western blot and ELISA were performed to investigate the expression of PTEN and insulin signal and glucagon level in supernatants respectively.Results1.BM-MSCs were fusiform,swirling arrangement.They were positive for stem cell markers CD90 and CD44 and negative for hematopoietic markers CD34 and CD45.Cells could differentiate toward adipocytes and bone cells.2.PA promoted production of intracellular ROS and upregulated expression of TNF-??IL-1? and PTEN,while transduction of insulin signal was obstructed in InR1-G9 cells.In addition,PA increased secretion of glucagon.3.MSC-CM reduced ROS production and down-regulated expression of TNF-??IL-1? and PTEN,whereas rescued transduction of insulin signal.Moreover,MSC-CM inhibited glucagon secretion.4.miR-181a expression in MSCs was much higher than that in InR1-G9 cells,and MSC-CM increased its expression in InRl-G9 cells.miR-181a mimics transfection lead to overexpression of miR-181a,which reduced PTEN expression,rescued transduction of insulin signal and subsequently decreased secretion of glucagon.By contrast,miR-181a inhibitor reduced expression of miR-181a,which upregulated level of PTEN,obstructed transduction of insulin signal and increased glucagon secretion.ConclusionMSC-CM could mitigate PA-induced inflammation and insulin resistance of InRl-G9 cells.miR-181a secreted by MSCs could participate in this process by regulating PTEN and insulin signal.