消渴方基于CN/NFAT信号通路对2型糖尿病大鼠的干预效果研究

Study on the intervention effect of Xiaoke Decoction on type 2 diabetic rats based on CN/NFAT signaling pathway

目的探讨消渴方对2型糖尿病模型大鼠钙调磷酸酶(CN)/活化T细胞因子(NFAT)信号通路的影响。方法选取45只清洁级Wistar大鼠,随机分为正常组、模型组、消渴方组各15只,模型组、消渴方组建立2型糖尿病模型。建模成功后,消渴方组大鼠给予消渴方灌胃,正常组和模型组大鼠给予等体积生理盐水灌胃,均连续15 d。灌胃结束后,取血检测血清糖脂代谢指标[空腹血糖(FPG)、总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)]、胰岛功能指标[空腹胰岛素(FINS)、胰岛β细胞功能指数(HBCI)、胰岛β细胞分泌功能指数(FBCI)];取胰腺组织,采用HE染色法进行病理组织观察,采用免疫组化SP法进行胰岛β细胞胰岛素分泌情况观察,采用酶联免疫法检测胰腺组织中氧化应激损伤指标[丙二醛(MDA)、总超氧化物歧化酶(T-SOD)、肿瘤坏死因子-α(TNF-α)],体外检测胰岛β细胞生长活力、存活率、凋亡率,采用Western blot法检测胰腺组织中CN/NFAT信号通路蛋白表达量。结果与正常组比较,模型组大鼠血清FPG、TC、TG、LDL-C水平及胰腺组织中MDA、TNF-α均明显升高(P均<0.05),血清HDL-C、FINS、HBCI、FBCI及胰腺组织中T-SOD均明显降低(P均<0.05);与模型组比较,消渴方组大鼠血清FPG、TC、TG、LDL-C水平及胰腺组织中MDA、TNF-α均明显降低(P均<0.05),血清HDL-C、FINS、HBCI、FBCI及胰腺组织中T-SOD均明显升高(P均<0.05)。模型组大鼠胰岛组织出现萎缩,胰岛素分泌量显著减少;消渴方组大鼠胰岛组织形态与正常组相似,胰岛素分泌量增加。与正常组比较,模型组大鼠胰岛β细胞生长活力、存活率及胰腺组织中PDX-1、Bcl-2蛋白表达量均明显降低(P均<0.05),胰岛β细胞凋亡率及胰腺组织中CN、NFATc1、Bax蛋白表达量均明显升高(P均<0.05);与模型组比较,消渴方组大鼠胰岛β细胞生长活力、存活率及胰腺组织中PDX-1、Bcl-2蛋白表达量均明显升高(P均<0.05),胰岛β细胞凋亡率及胰腺组织中CN、NFATc1、Bax蛋白表达量均明显降低(P均<0.05)。结论消渴方可改善2型糖尿病大鼠胰岛功能,减轻胰腺氧化应激反应,促进胰岛β细胞再生分化成熟,抑制胰岛β细胞凋亡,其机制可能与阻断CN/NFAT信号通路相关。

Objective It is to investigate the effect of Xiaoke Decoction on calcineurin(CN)/nuclear factor of activated T-cells(NFAT)signaling pathway in type 2 diabetic rats.Methods Forty-five clean Wistar rats were selected and randomly divided into normal group,model group,and Xiaoke Decoction group with 15 rats in each group.The type 2 diabetes models were established in the model group and Xiaoke Decoction group.After successful modeling,the rats in the Xiaoke Decoction group were given Xiaoke Decoction by gavage,and the rats in the normal group and model group were given an equal volume of normal saline by gavage,all treated for 15 days.After the gavage,the blood was taken to detect serum glucose and lipid metabolism indicators[fasting blood glucose(FPG),total cholesterol(TC),triacylglycerol(TG),high-density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C)],islet function indexes[fasting insulin(FINS),HOMA beta cell function index(HBCI),fasting beta-cell function index(FBCI)];the pancreatic tissue was taken for pathological tissue observation by HE staining method,the insulin secretion of pancreaticβ-cells was observed by immunohistochemical SP method,the oxidative stress damage indicators in pancreatic tissue[malondialdehyde(MDA),total superoxide dismutase(T-SOD),tumor necrosis factor-α(TNF-α)]were detected by enzyme-linked immunoassay,the growth viability,survival rate,and apoptosis rate of isletβcells were detected in vitro,and the protein expression of CN/NFAT signaling pathway in pancreatic tissue was detected by Western blot.Results Compared with the normal group,the levels of FPG,TC,TG,LDL-C in serum and MDA and TNF-αin pancreatic tissue in the model group were significantly increased(P<0.05),and the levels of HDL-C,FINS,HBCI,FBCI in serum and T-SOD in pancreatic tissue were significantly reduced(all P<0.05);compared with the model group,the levels of FPG,TC,TG,LDL-C in serum and MDA and TNF-αin pancreatic tissue in the Xiaoke Decoction group were significantly decreased(P<0.05),and the levels of HDL-C,FINS,HBCI,FBCI in serum and T-SOD in pancreatic tissue were significantly increased(all P<0.05).The pancreatic islet tissue of rats in the model group was atrophic,and the amount of insulin secretion was significantly decreased;the morphology of the pancreatic islets of the rats in the Xiaoke Decoction group was similar to that of the normal group,and the amount of insulin secretion was increased.Compared with the normal group,the growth viability and survival rate of pancreatic isletβ-cells and the expression of PDX-1 and Bcl-2 protein in pancreatic tissue in the model group were significantly reduced(all P<0.05),and the apoptosis rate of pancreatic isletβ-cells and the expression levels of CN,NFATc1 and Bax protein in pancreatic tissue were significantly increased(all P<0.05);compared with the model group,the growth viability and survival rate of rat pancreatic isletβcells and the protein expression levels of PDX-1 and Bcl-2 in pancreatic tissues in the Xiaoke Decoction group were significantly increased(all P<0.05),the apoptotic rate of pancreaticβ-cells and the expression levels of CN,NFATc1,and Bax proteins in pancreatic tissue were significantly reduced(all P<0.05).Conclusion Xiaoke Decoction can improve islet function,reduce pancreatic oxidative stress,promote the regeneration,differentiation and maturation of isletβ-cells,and inhibit isletβ-cell apoptosis in type 2 diabetic rats.The mechanism may be related to blocking CN/NFAT signaling pathway.