基于肠道菌群分析探讨葛根减轻T2DM db/db小鼠胰岛素抵抗的作用及机制

Effect and mechanism of Puerariae Lobatae Radix in alleviating insulin resistance in T2DM db/db mice based on intestinal flora

基于肠道菌群分析探讨葛根对2型糖尿病(type 2 diabetes,T2DM)db/db小鼠胰岛素抵抗的作用及可能机制。将50只db/db小鼠随机分为模型组、二甲双胍组以及葛根高、中、低剂量组,另取10只db/m小鼠为正常组。持续给药8周,测量小鼠体质量和血糖;酶联免疫吸附测定法(enzyme linked immunosorbent assay,ELISA)检测糖化血清蛋白(glycosylated serum protein,GSP)、血清空腹胰岛素(fasting serum lisulin,FINS),计算胰岛素抵抗指数(HOMA-IR);HE染色观察胰腺组织病理学变化;免疫组化检测胰腺肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)的表达;16S rRNA技术检测正常组、模型组和葛根中剂量组小鼠粪便肠道菌群结构变化;蛋白免疫印记法检测回肠组织中法尼醇X受体(farnesoid X receptor,FXR)、G蛋白胆汁酸偶联受体5(takeda G protein-coupled receptor 5,TGR5),肝组织中胆固醇7α-羟化酶(cholesterol 7α-hydroxylase,CYP7A1)、甾醇27α-羟化酶(sterol 27α-hydroxylase,CYP27A1)和结肠组织中G蛋白偶联受体41(G protein-coupled receptor 41,GPR41)、G蛋白偶联受体43(G protein-coupled receptor 43,GPR43)的表达。与正常组比较,模型组小鼠体质量、血糖、血清GSP、空腹血糖(fasting blood glucose,FBG)、FINS显著增加,HOMA-IR指数显著上升;胰岛细胞炎性浸润,大量腺泡细胞坏死变性,胰岛细胞和腺泡细胞边界不清晰;肠道菌群发生紊乱;组织中FXR、TGR5、CYP7A1、CYP27A1、GPR41和GPR43蛋白表达显著降低。与模型组比较,二甲双胍组、葛根组小鼠体质量、血糖、血清GSP、FBG、FINS显著降低;胰岛细胞形态完整呈团状边界清晰,少量腺泡细胞坏死,可见较多胰岛细胞;葛根中剂量组肠道菌群从门到属水平均发生了变化,影响肠道菌群代谢物的菌群相对丰度增加;组织中FXR、TGR5、CYP7A1、CYP27A1、GPR41和GPR43蛋白表达显著升高。综合实验结果发现,葛根可改善T2DM db/db小鼠胰岛细胞的炎症损伤,减轻胰岛素抵抗,其作用机制可能与增加肠道中放线菌门、双歧杆菌、拟杆菌属丰度和肠道菌群代谢物相关蛋白表达有关。

This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM)based on the analysis of intestinal flora.Fifty db/db mice were randomly divided into a model group(M group),a metformin group(YX group),a high-dose Puerariae Lobatae Radix group(YGG group),a medium-dose Puerariae Lobatae Radix group(YGZ group),and a low-dose Puerariae Lobatae Radix group(YGD group).Another 10 db/m mice were assigned to the normal group(K group).After continuous administration for eight weeks,body weight and blood sugar of mice were measured.Enzyme linked immunosorbent assay(ELISA)was used to detect glycosylated serum protein(GSP)and fasting serum insulin(FINS),and insulin resistance index(HOMA-IR)was calculated.The histopathological changes in the pancreas were observed by HE staining.Tumor necrosis factor(TNF)-αexpression in the pancreas was detected using immunohistochemistry.The structural changes in fecal intestinal flora in the K,M,and YGZ groups were detected by 16S rRNA.Western blot was used to detect the expression of farnesoid X receptor(FXR)and takeda G protein-coupled receptor 5(TGR5)in the ileum,cholesterol 7α-hydroxylase(CYP7A1)and sterol 27α-hydroxylase(CYP27A1)in the liver,and G protein-coupled receptors 41(GPR41)and 43(GPR43)in the colon.Compared with the K group,the M group showed increased body weight,blood sugar,serum GSP,fasting blood glucose(FBG),and FINS,increased HOMA-IR,inflammatory infiltration of islet cells,necrosis and degeneration of massive acinar cells,unclear boundary between islet cells and acinar cells,disturbed intestinal flora,and down-regulated FXR,TGR5,CYP7A1,CYP27A1,GPR41,and GPR43.Compared with the M group,the YX,YGG,YGZ,and YGD groups showed decreased body weight,blood sugar,serum GSP,FBG,and FINS,islet cells with intact and clumpy morphology and clear boundary,necrosis of a few acinar cells,and more visible islet cells.The intestinal flora in the YGZ group changed from phylum to genus levels,and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased.The protein expression of FXR,TGR5,CYP7A1,CYP27A1,GPR41,and GPR43 increased.The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM.The mechanism of action may be related to the increase in the abundance of Actinobacteria,Bifidobacterium,and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.