Effect Of GPR41 On Type 1 Diabetes Mellitus And Its Mechanism

In this study,GPR41 knockout mice were used to construct Streptozotocin(STZ)-induced type 1 diabetes mellitus(T1D)model and the spontaneous T1D of NOD mice.The role of T1D in pathogenesis and its underlying mechanism provide new therapeutic targets for T1D patients and theoretical basis forintestinal flora metabolites in regulating the development of T1D.The pancreatic and colonic tissues of NOD mice at different ages were extracted,and the expression of GPR41 was observed at both the protein level and mRNA level by Western blot and qPCR,respectively.Then,mice in the model group and the GPR41 knockout group were intraperitoneally injected with STZ.The fasting blood glucose level,body weight,and drinking water of mice in each group were observed.The expression of cell surface markers(CD11c,CD80,CD86,CD40),and cells secret cytokines(IL-12,IL-4,IL-10,TGF-?)in pancreas tissues were observed using qPCR at the mRNA level,analysis of short-chain fatty acids in colon contents was measured by using high-performance gas chromatography-mass spectrometry(GC-MS),pathological observation and scoring of pancreas and colon were detected by hematoxylin-eosin staining,meanwhile,total macrophages,M1 macrophages,M2macrophages,dendritic cells,CD4~+T cells,CD8~+T cells,and regulatory T cells in the pancreas of each group were analyzed by flow cytometry.CD80 and CD86 mRNAs were measured by qPCR.The levels of IL-12,IL-23,IL-10 and TGF-?were determined at the gene level by qPCR,Western blot was used to determine the phosphorylation levels of the critical signaling pathway proteins STAT3 and NF-?B that affect the secretion of inflammatory cytokines in dendritic cells and the expression of the cytokine signal inhibitors SOCS1 and SOCS3.Then the molecular mechanism of adoptive transfer experiments was carried out to investigate the maturation status of GPR41-deficient dendritic cells in the T1D immune environment and the effect on T cell activation.Results of the experiment have shown that GPR41 expression in pancreas and colon tissues of NOD mice decreased significantly.We observed that the absence of GPR41 significantly promoted the development of T1D from the STZ model.The specific manifestations were as follows:fasting blood glucose in the GPR41 knockout group increased and weight decreased significantly.The expression of immune cell surface markers at the gene level in pancreas shows that the absence of GPR41 promotes the increase of the proinflammatory cytokine IL-12,and the expression of cytokines(IL-10,IL-4,TGF-?)were significantly reduced,the total content of short-chain fatty acids in the intestine indicated that the loss of GPR41 affects the abundance of intestinal probiotics,and the content of short-chain fatty acids was reduced.The pathological evaluation of colon tissues and pancreas can be disordered:The absence of GPR41reduced the number of?cells,the intestinal inflammation and infiltration,and the barrier destruction.Analysis of immune cells in the pancreas shows that the absence of GPR41promoted the maturation of dendritic cells.Not only that,the deletion in GPR41 also promoted the activation of CD4~+T cells and CD8~+T cells and inhibited the regulatory T cells in the pancreas,In vitro studies have shown that the loss of GPR41 resulted in inhibiting SOCS3expression,which promotes the increase in phosphorylation of STAT3 leads to increased secretion of IL-12,and promoted the maturation of dendritic cells in vitro.Adoptive transfer experiments showed that the inflammatory microenvironment of T1D can easier to be accelerated by the maturation of GPR41-deficient dendritic cells,and promoted the differentiation of na?ve T cells into CD4~+T cells and CD8~+T cells,and increase the secretion of IFN-?,accelerated infiltration of islets and destruction in?cells,thereby promoting the pathogenesis of T1D.This study confirms,for the first time,that GPR41 has reduced during the development of T1D,and the absence of GPR41 promotes the onset of T1D,destroyed the pancreatic immune microenvironment,and leaded to the maturation of dendritic cells and the activation of effector T cells.And the maturation of dendritic cells mediated by the SOCS3/STAT3 signaling pathway,thereby promotes the activation of T cells,leading to an imbalance of pancreatic immune cells and accelerating the death of?cells.Therefore,GPR41 may be a potential target for effective prevention and treatment of T1D,and an essential medium for studying the relationship between intestinal and T1D,providing a new direction for understanding the intestinal flora in regulating the onset of T1D.