| Background:Type 2 diabetes mellitus(T2DM)is a complex disease characterized by hyperglycemia caused by insufficient insulin secretion of pancreatic ? cells or insensitivity of target cells to insulin.Long-term hyperglycemia will lead to vascular and nerve damage and a series of inflammatory reactions.Therefore,T2DM is also considered as a chronic low-grade inflammatory condition that is regulated by the immune system.Infiltrating immune cells in the pancreas can interfere with insulin signal transduction in peripheral tissues or induce cell damage by autocrine or paracrine means,but due to the complexity of the pathogenesis of T2DM,the specific role and mechanism of immune cells in T2DM are not completely clear.Objective:In this study,the db/db mouse model was used to explore the regulation effect and internal mechanism among pancreatic myeloid-derived suppressor cells(MDSCs),CD8+T and ? cells in T2DM,and partially revealed the process and mechanism of gastrodine to repair ? cell.Methods:SPF grade male db/db mice and the same type control mice were used as the research objects,and the body weight,fasting blood glucose,glucose tolerance and insulin tolerance of the mice within 4-16 weeks were measured.The expression of inflammatory cytokines in peripheral blood was detected by ELISA.The number and function of ? cells were measured by insulin antibody staining.Flow cytometry was used to analyze the proportion of MDSCs,CD8+T cells and? cells in peripheral blood,liver,spleen and pancreas of mice.Through CD8 T cell proliferation inhibition experiment and cell co-culture experiment in vitro,adoptive transfer experiment in vivo,confirmed the interaction between pancreatic MDSCs,CD8+T and ? cells.In vitro,MTT assay was used to detect the effect of various components in 70%of Spiranthes sinensis on pancreatic MDSCs;cell proliferation assay,Western blot and cell slide assay were used to detect the effect of gastrodin on the proliferation of MDSCs and the changes in the expression of node proteins in the signaling pathway.Results:1.Inflammation and ? cell injury in T2DM:(1)Compared with WT mice,weight and blood glucose of db/db mice were increased;glucose and insulin tolerance were impaired;the proportion of MDSCs in pancreas decreased significantly(P<0.01);the proportion of CD8 T cells increased(P<0.001).(2)At 16 weeks,the levels of inflammatory cytokines IL-6,IL-?,TNF-?and TGF-? in peripheral blood of db/db mice were significantly higher than those in the control group(P<0.05).At the same time,the number of ? cells inand the secretion of insulin decreased.2.Effect of MDSCs on CD8+T cell mediated ? cell injury in the pancreas of T2DM:(1)In the pancreas,the gene expression of MDSCs chemokine CCL2 and the ratio of pancreatic MDSCs in db/db mice were significantly lower than that in WT mice(P<0.001).The pancreas MDSCs are not in contact with ? cells,while some CD8+T cells are infiltrated in the islet,adjacent to ? cells.(2)In vitro experiments showed that pancreatic MDSCs inhibited the proliferation of CD8+T.When co-cultured with CD8+ T cells,the number of ?cells decreased.The survival rate of ? cells increased after the addition of pancreatic MDSCs(P<0.001).3.Gastrodin can significantly enhance the activity of pancreatic MDSCs cells,and the most significant at the concentration of 0.25 ng/ml(P<0.01).Gastrodin has no direct effect on ? cell.4.After the treatment with gastrodin(0.25 ng/ml),the proliferation of pancreatic MDSCs were significantly increased.Western blot results showed that the expressions of p38,P-p38,ERK and p65 proteins in pancreatic MDSCs treated with gastrodin(0.25 ng/ml)were significantly increased(P<0.01).After TNFR2 receptor knockout on the surface of pancreatic MDSCs cells,gastrodin had no effect on the expression of p38 and p65 proteins.Conclusion:1.Pancreatic MDSCs improves CD8+ T-cell-mediated islet cell injury by inhibiting the proliferation of CD8+T cells.2.Gastrodin stimulates the TNFR2/MAPK signaling pathway and promotes the proliferation of MDSCs cells for repairing ? cell. |
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