| The state of autoimmune plays a very important role in the pathogenesis of type 1diabetes(T1DM).Mesenchymal stem cells(MSCs)have been shown to be effective in ameliorating autoimmune conditions in previous studies,providing new insights into the treatment of T1 DM.Objective: By using combination therapy of umbilical cord mesenchymal stem cells(h UCMSCs)and resveratrol(Res)in NOD mice with T1 DM,we observed the effect of combined use of h UCMSCs and resveratrol on the autoimmune conditions and improving the islet function of T1 DM mice.Methods:After diagnosing T1 DM,the experimental mice were randomly divided into the following groups,and given the corresponding intervention measures.(1)Group Diabetes Control.There are not any interventions after the onset of the disease in this group.(2)Group h UCMSCs.Mice in this group were treated with an intravenous infusion of h UCMSCs through vena caudalis at the 3rd day after diagosis,the cell number was 1×106/mouse and cells were suspended in 0.3 ml PBS.(3)Group Res.Mice in this group were treated with resveratrol 200mg/kg·d by oral from the 3rd day after diagosis.(4)Group Combined Therapy.Mice in this group were treated with an intravenous infusion of h UCMSCs through vena caudalis at the 3rd day after diagosis,the cell number was 1×106/mouse and cells were suspended in 0.3 ml PBS.Meanwhile,mice in this group were treated with resveratrol200mg/kg·d by oral.(5)Group Insulin.Mice in this group were treated with Glargine,according to the situation of blood glucose to adjust the amount.(6)Group Normal Control.NOD mice without diagosis were selected as normal control group after 9 weeks of detection.The day of onset as day 0,and the body weight was weighed and the blood glucose was measured at the 7th,14 th,21st,28 th,35th,42 th,49th and 56 th day.Collect the blood at day 0 and day 56.Fasting serum C peptide were detected by ELISA.The serum cytokines Th1/Th2 were detected by Flow cytometry.At the 56 th day of the study,all mice were sacrificed and their spleen were collected for Flow cytometry,detectingproportion of CD4+CD5+Foxp3+T cells(Treg cells)and CD4+T cells,CD8+Tcells.The pancreases were dyed by HE to observe the islet structure and inflammatory cell infiltration condition.Result:1.General observation After diagnosis of diabetes,each group of mice were polydipsia and polyuria.Messy fur,lags in response were observed as expected among mice in diabetic groups.2.Weight The weight of diabetes control group appears more substantial,rapid decline.In the combined treatment group and the resveratrol group,the body weight did not appear to fluctuate after the onset of the disease,and remained stable in the relatively stable state.In h UCMSCs group,the body weight decreased significantly at the beginning of the disease(<35 days),reached the lowest level at 35 days,and then the body weight recovered.At 49 days,the body weight was restored at the time of onset and was equal to that of the normal group.3.Glucose Glucose in hUCMSCs group,resveratrol group and combination therapy group began to decrease gradually after 7 days of onset,and there was significant difference compared with untreated diabetic group(P<0.05).HUCMSCs group and combination therapy group had obvious effect on blood glucose control and gradually close to normal group,which had been similar to normal group after 14 days(P>0.05).The therapeutic effect lasted until 56 days.The resveratrol group had a tendency to control blood glucose,but the effect was not as good as the above two groups,and the maintenance time was only about 28 days.After 28 days,the blood glucose began to increase gradually in the resveratrol group,and there was no significant difference with the diabetic control group at56 days.4.Fasting serum C peptide There was no significant difference in fasting serum C-peptide between the five groups at the onset of diabetes(P>0.05).After the corresponding treatment,the levels of fasting C-peptide in serum were significantly decreased(P<0.05).The fasting serum C peptide in the combination therapy group was higher than that before treatment,and the difference was significant(P<0.05).5.The serum cytokines Th1/Th2 Compared with the diabetic control group,the levels of IL-5 and IL-6 in the serum of the combined treatment group and h UCMSCs group were significantly increased(P<0.05),while IL-2,TNF-α,IFN-γ and IL-17 were significantly decreased(P<0.05).IL-2,TNF-α,IFN-γ and IL-17 were significantly decreased in resveratrol group(P<0.05),while we cannot observe a significantly change in IL-5 and IL-10(P>0.05).6.Proportion of Treg cells and CD4+T cells,CD8+Tcells.The levels of Treg in the h UCMSCs group,resveratrol group and combination therapy group were higher than those in the dibetes control group,and compared with the other groups,there was significant difference in the level of Treg in the combined treatment group.CD4+T cells and CD8+T cells in the diabetic control group mice were significantly higher than those in the normal group at 56 th day(P<0.05),while other group show significantly decrease trend.There were no significant difference in the level of CD4+T cells and CD8+T cells in h UCMSCs group,resveratrol group and combination therapy group(P>0.05).7.Islet structive and inflammatory cell infiltration In the normal control group,the islets were structurally well formed and had no inflammatory cell infiltration.Insulin treatment group,a large number of islet structure damage,inflammatory cell infiltration was obvious.HUCMSCs treatment group is relatively complete structure of the islets,with well shape and fewer inflammatory cells.Resveratrol group is a certain damage to the structure of the islets,cells arranged slightly disorder,surrounded by inflammatory cell infiltration.The formation of islets in the combination therapy group was relatively complete and the infiltration of inflammatory cells was mild.We can not find complete pancreatic islet structure under the mirror in diabetes control group and a large number of inflammatory cell infiltration can be observed.Conclusion: Application of hUCMSCs combined with resveratrol treatment can significantly improve the reduction of weight of the state,ameliorate hyperglycemia,improve the level of fasting serum C-peptide.We believe that resveratrol can serve as an adjunct to increase the therapeutic effect of h UCMSCs,reduce β cell damage,maintain residual β cell function. |
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