Comparison Of The Therapeutic Effects On Type 2 Diabetic Rats Through Intravenous Infusion Of Different Types Of Stem Cells And Its Mechanism Study

BackgroundType 2 diabetes mellitus(T2DM)is a chronic inflammatory disease characterized by insulin resistance and progressive functional recession of islet ? cells,which is also involved immune imbalance and hyperfunction of a cells.Stem cells are pluripotent cells which can be able to secret all sorts of growth factors and regulatory factors and differentiate into other cell types.Because of this,more and more clinical and basic studies focus on treatment of diabetes by infusion of stem cells.However,how to select proper stem cells to achieve the best therapeutic efficacy,which is the most concerned issue by researchers.Bone marrow hematopoietic stem cell or bone marrow mononuclear cell-based therapies have been proven to be effective in various clinical trials for both T1DM and T2DM,which is shown to alleviate hyperglycemia and improve islet function.But meanwhile,it has inevitable deficiency.Bone marrow stem cells(BMSCs)are acquired by invasive surgery,and proliferation and secretion of autologous bone marrow hematopoietic stem cells in patients may be impaired by prolonged exposure to high glucose.In addition,if using allogeneic BMSCs,we' d be faced with the use of immunosuppressive agents and drug side effects and so on.So more and more studies focus on other types of stem cells in addition to BMSCs,such as bone marrow mesenchymal stem cells(bm-MSCs)with lower immunogenicity,a wider range of adipose mesenchymal stem cells and noninvasive access to umbilical cord mesenchymal stem cells and umbilical cord blood mononuclear cells.The existing clinical studies have shown that the above several stem cells can play a certain role in the balance of blood glucose,but there is a certain degree of inconsistency between different studies,especially in improving islet function.So far there is no study focusing on the comparison among the effects of different stem cells on blood glucose control and islet function under the same test criteria.Considering inconsistency among the previous clinical findings of single stem cell infusion and in order to develop the precise treatment for different patients and screen the best stem cell type,we compared the effectiveness of different stem cell infusions and their respective emphasized efficacy under the same study criteria,which has a great clinical significance.The islets are highly vascularized miniorgans,with their combined 1-2%of the pancreatic volume receiving 10-20%of the total pancreatic blood flow.Because of this unique structure,the islets are able to respond rapidly to glucose and hormone fluctuations but are also vulnerable to unfavorable stimuli such as oxidative stress.Once the microvascular integrity of the islets are impaired,together with the disturbance of the NO-mediated endothelium vasodilation and the up-regulation of adhesion molecules,it becomes easier for the inflammatory cells to adhere and migrate into the islet and cause further islet destruction.Considering the importance of islet microvascular endothelium,preserving its integrity and proper function might be a novel target in islet protection.Mesenchymal stem cell(MSC)-based therapies have been proven to be effective for improvement of glucose intolerance and islet function in various clinical trials as well as in diet-induced or genetically modified experimental diabetic animal models.But the specific factors and pathways achieving the above effect are little reported.Wnt is an important factor associated with growth and development,cell proliferation and migration and stemness maintenance and differentiation regulation of stem cells,which is secreted by stem cells and tumor cells with low differentiated degree.Mature cells under normal condition only secreted little Wnt,which can be up-regulated in the progression of tissue repair.In different cell types and different concentrations,Wnt can activate different downstream pathways,including the ?-catenin-dependent classical Wnt pathway,and ?-catenin dependent nonclassical Wnt pathway.Previous studies have confirmed that activation of the Wnt pathway promotes pathologic or physiological vascularization,and knockout of ?-catenin or its downstream TCF7L2 in pancreatic ?cells can lead to islet function and morphological differences and often lead to impaired glucose tolerance.Wnt3a and Wnt4 can promote ? cell proliferation by activating the classical ?-catenin-dependent Wnt pathway,while Wnt4 has no significant effect on ? cell secretion.Based on the above findings,we make reasonable assumptions that MSC can secrete Wnt protein,activate islet ? cells and ?-catenin pathway in endothelial cells of islet microcirculation,and then improve islet function and microcirculated endothelial function.Objective:The aim of this study was to assess the efficacy of single or double infusion of bmMSC,hucMSC and CBSC and compare the effects of each treatment regimen on glucose status,islet function,insulin resistance and chronic inflammatory response in diabetic rats,and then investigate treatment focus of all kinds of stem cells,which would provide the best method for the clinical application of stem cells and offer evidence for the preparation of individual stem cell treatment program.Additionally,we co-cultured bmMSC with islet microcirculation endothelial cells MS-1 and isolated islets in vitro,and investigate the effects of Wnt protein secreted by MSC and the ?-catenin-dependent Wnt pathway on the function of MS-1 and islet under oxidative stress injury.Methods:Part1 Comparison of the therapeutic effects on type 2 diabetic rats through intravenous infusion of different types of stem cells1.We used a high-fat,high-sugar diet and low-dose STZ to induce type 2 diabetes mellitus SD model and monitored blood glucose weekly to determine the success of the model.After 7days from modeling success,the T2DM rats were randomly divided into four groups:T2DM,T2DM + bmMSC,T2DM + hucMSC,and T2DM + CBSC.For T2DM + hucMSC rats,the second stem cells infusion were performed 2 weeks after the first treatment,with infusion of HucMSC or CBSC.After 2w,4w,8w since the first stem cell injection,IPGTT were performed and tail tip blood glucose,insulin,glucagon were detected.After three days,we put rats to death and took fasting blood and various organs.2.Comparison of blood glucose control in different treatment groups:weekly test tail blood glucose.IPGTT was performed by administering glucose intraperitoneally(1.5g/kg),anesthetizing rats with isoflurane,and cutting the tail to take blood.3.The islet function and morphological changes of different treatment groups were compared by detecting fasting insulin/glucagon and IPGTT insulin/glucagon curve using radioimmunoassay.Pancreatic paraffin section and HE staining were used to observe the changes of islet morphology and size.Pancreas paraffin free immunohistochemical staining was used to observe the size and proportion changes of insulin positive region.Pancreatic paraffin immunofluorescence staining was used to observe the proportion of insulin(+)/glucagon(+)region and the ratio of PCNA and insulin double positive cells in insulin-positive cells.4.Comparison of changes in insulin resistance status in different treatment groups:HOMA-IR,Insulin Sensitivity Index(Matsuda index)and Insulin resistance index(Belfiore index)were used to assess insulin resistance condition of rats.Meanwhile insulin pathway-related proteins P-AKT/t-AKT and p-Erk/t-Erk in liver were detected by Western blotting.5.Comparison of changes in liver glucose metabolism in different treatment groups:we extracted hepatic protein and detected the level of GSK3,GLUT2,PK,PFK,PDK and G-6-Pase using Western blotting.And we stained the liver tissue paraffin sections with glycogen to observe the changes of glycogen storage.6.Changes in lipid metabolism in different treatment groups were compared:TC,TG,LDL,HDL and FFA were detected by biochemical method.7.Comparison of the differences in serum metabolic index and inflammatory factors among different treatment groups:IL-1b,IL-6,IL-17,IL-22,TNF-a and IFN-g were detected by ELISA.Part2 Studies on the Mechanisms of MSC in Improving the Islet Function and Islet Microcirculation Endothelial Cells1.Oxidative stress-induced rat isolated islets and MS-1 cells injury was established by exogenous administration of H202,and co-cultured them with bmMSC using Transwell system.The activity changes of islet and MS-1,apoptosis,insulin secretion levels,eNOS activity and other functional indicators were detected.2.QPCR was used to compare the expression of Wnt protein in isolated rat pancreatic islets and islet microcirculation endothelial cells MS-1 and bmMSC,and to observe the activation of ?-catenin-dependent Wnt pathway in islets and MS-1 after Transwell culture.3.XAV-939 was used to specifically block ?-catenin in islets or MS-1,and we observed whether the effect of bmMSC co-culture on H202 induced oxidative stress injury was canceled.4.Wnt4 and Wnt5a in bmMSC were knocked out respectively,and the knocked bmMSC was co-cultured with islet and MS-1.And then we observed the effect of Wnt4 and Wnt5a knockout on pancreatic islets and MS-1 activity changes,apoptosis and insulin secretion,eNOS activity and other functional indicators under oxidative stress.5.BmMSCs were co-cultured with islets and MS-1 respectively,after Wnt4 and Wnt5a were knocked out.The activation of the ?-catenin-dependent Wnt pathway in the islets and MS-1 was detected.Results:Part1 Comparison of the therapeutic effects on type 2 diabetic rats through intravenous infusion of different types of stem cells1.Once the T2DM model was set,the blood glucose of SD rats increased significantly.However,after the infusion of stem cells,the fasting blood glucose were lower than before,and the effects can last for 8 weeks since the infusion.There were no significant differences in fasting blood glucose between groups of infusing different stem cells and groups of different infusion times.The results of IPGTT showed no significant improvement in postprandial blood glucose at 2w after stem cell infusion.However,the blood glucose of each group in different time point was significantly lower than that of T2DM at 4w.At 8w after the infusion,although the fasting and half-hour postprandial blood glucose could still maintain good in stem cell intervention groups,the one-to three-hour postprandial blood glucose in each group,except the hucMSC+CBSC double infusion group,had no significant differences compared with the T2DM group.2.Stem cell infusion could significantly inhibit the increased fasting glucagon in T2DM rats,and there was no significant difference between each group.BmMSC or CBSC alone could significantly improve the fasting insulin level in T2DM rats,and this index was not obviously improved in other groups.The results of insulin release test showed that postprandial insulin levels in the bmMSC infusion group were significantly increased and it had no obvious change in the other groups at 2 weeks after transfusion,and the release curves were quite irregular.At 4w,the insulin release curve in each group was regular and basically could be reduced to the baseline level at 3h postprandial.The postprandial insulin levels of bmMSC group or CBSC group were significantly higher than that of T2DM untreated group.At 8w,the release curve of bmMSC group was further close to normal,whereas the hucMSC + CBSC double infusion group showed a significant increase in postprandial insulin release.In addition to bmMSC group,the other groups can significantly inhibit postprandial glucagon secretion.3.At 2w and 4w after stem cell infusion,stem cells could significantly preserve the islet area.And the bmMSC had a more favorable effect on the preservation of ? cell area.There was no significant difference in improving the ratio of ? and ? cells at 2w after stem cell infusion.At 4w,the bmMSC group showed a significant increase in the proportion of ? cells,and the a-cell area showed an obvious decrease in all stem cell infusion groups.And there was no significant difference between each two groups.The lasting decreasement of a-cell area in all stem cell groups were significant at 8w,while the significant increase in ? cell proportion in CBSC alone group,hucMSC + CBSC group and hucMSC double infusion group suggesting that CBSC alone or stem cell injecting twice can maintain the therapeutic effect longer in improving the proportion of ? cell.4.At 2w and 4w after the stem cell infusion,a significant increase of PCNA positive ? cells could be observed in CBSC group,hucMSC + CBSC group and hucMSC double infusion group,indicating a proliferation of ?cells.And at 8w,the proliferation was significantly reduced,and only in hucMSC+CBSC group a significant proliferation could still be observed.5.The study also observed the double-staining of insulin and glucagon in part of the pancreatic islet cells in the CBSC group,while the phenomenon did not appear in other groups,suggesting that CBSC may promote the transformation of ? to ? cells.6.Insulin sensitivity index suggested that hucMSC double infusion could significantly improve insulin resistance in diabetic rats,while a single infusion of mesenchymal cells(bmMSC single infusion group and hucMSC single infusion group)might also be effective.In the liver of overnight fasting diabetic rats,Western blotting indicated that bmMSC single infusion significantly promoted Akt phosphorylation,whereas stem cells double infusion(T2DM + hucMSC&CBSC group and T2DM + hucMSC*2group)not only increased the phosphorylation of Akt,but also obviously upregulated the level of Erk phosphorylation under fasting condition,and then activated Akt and Erk signaling pathways to improve insulin resistance at the cellular level.7.There were reduced glycogen synthesis and glycolysis and increased gluconeogenesis in the liver of fasting T2DM rats.However,stem cells infusion could selectively influenced different key enzymes and eventually played a regulatory role.In terms of improving liver glycogen synthesis and increasing GLUT2 expression,all stem cell infusion regimens referred in this article were all effective.BmMSC promoted glycolysis,whereas in the two groups with multiple times of stem cells infusion,inhibition of gluconeogenesis in the liver was more reflected.8.TC and TG decreased could be observed significantly in CBSC single infusion group,whereas other groups had no significant improvement in blood lipid levels.9.HucMSC+CBSC and hucMSC*2 infusion could significantly improve the immune-related indicators,such as IL-1b,IL-6,IL-17,IL-22 and IFN-g.Part2 Studies on the Mechanisms of MSC in Improving the Islet Function and Islet Microcirculation Endothelial Cells1.Co-culturing bmMSCs and MS-1 cells ameliorated H202-induced apoptosis,the reduction of eNOS phosphorylation and increased expression of adhesion molecule VCAM.Meanwhile it Improved insulin secretion in isolated islets.2.There was a significant increase in the expression of Wnt4 and Wnt5a in the bmMSCs compared to that of the isolated islets and MS-1 cells.The nuclear translocation of ?-catenin in the islets and MS-1 cells was significantly elevated after co-culturing with MSCs,which indicated the activation of classic Wnt pathway.3.After XAV-939 treatment,the above protective roles of bmMSCs in islets and MS-1 cells was significantly weakened,which demonstrated that the beneficial effects of MSCs were partially conducted by the activation of the classic ?-catenin-dependent Wnt signaling pathway.4.Wnt4 and Wnt5a were knocked out in MSC and co-cultured with H202-stimulated MS-1 cells and isolated islets.The results suggested that after knocking down Wnt4 in the MSCs,the ameliorative effects of MSCs were hampered,while Wnt5a knock-down seemed to show the opposite effects:the protective effects were reinforced.Wnt4 knock-down weakened the activation of canonical Wnt signaling pathways,whereas Wnt5a knock-down strengthened them.The above results indicated that MSC could secrete different Wnt proteins,whose roles in the islet were complex,and sometimes may be antagonistic,but finally they had beneficial effects on ameliorating oxidative stress injury.Conclusion:In our study,we first compared therapeutic efficacy of single or double infusion of bmMSC,hucMSC and CBSC in the model of T2DM rats.1.All kinds of stem cells were equivalent in reducing fasting blood glucose,but only hucMSC+ CBSC group could effectively improve postprandial blood glucose until 8 weeks after infusion.2.BmMSC and CBSC group improved fasting insulin significantly,and bmMSC and hucMSC+CBSC double injection group could ameliorate insulin secretion in response to glucose until 8 weeks after infusion.3.CBSC single injection or stem cell double injections could maintain a longer period for improving the proportion of ? cells,which was possibly related to sustaining ? cell proliferation.4.All sorts of stem cell infusion obviously inhibited fasting and postprandial glucagon secretion,and suppressed a cell proliferation.5.BmMSC single infusion and stem cell twice infusion(T2DM + hucMSC&CBSC group and T2DM + hucMSC*2 group)could prominently ameliorate the state of insulin resistance.6.All of the stem cell infusion protocols involved in this study improved hepatic glycogen synthesis and increased the expression of GLUT2 in hepatocytes.BmMSC could promote fasting glycolysis.And infusion of multiple stem cells in two groups depressed fasting gluconeogenesis effectively.7.CBSC single infusion decreased TC and TG obviously.8.Double injection of stem cell was able to ameliorate the body inflammation significantly.9.After stem cells infusion,there were different degrees of classical Wnt pathway activation in endocrine part of pancreas and islet microvascular endothelial cells.10.BmMSC could secrete Wnt4 and subsequently activated classic ?-catenin-dependent Wnt pathway in isolated islets and microvascular endothelial cells,which improved islet function and reduced oxidative stress-induced endothelial cell dysfunction and apoptosis.