基于“脾瘅”理论探讨葛根芩连汤通过CAP/Cb1信号通路对2型糖尿病大鼠骨骼肌的作用机制

Exploration on the mechanism of Gegen Qinlian Decoction on skeletal muscle of type 2 diabetic rats through CAP/Cb1 signaling pathway based on‘Pi-dan'theory

目的:探讨葛根芩连汤(GQD)治疗2型糖尿病的可能作用机制。方法:36只SD大鼠随机分为空白组,模型组,二甲双胍组,GQD高、中、低剂量组,每组6只。除空白组外,其余各组大鼠给予高脂饲料联合链尿佐菌素(STZ)诱导建立2型糖尿病模型。药物灌胃干预后,血糖仪检测空腹血糖(FBG);ELISA法检测血清空腹胰岛素(FINS)、游离脂肪酸(FFA)、全血糖化血红蛋白(HbA1c);HE染色观察骨骼肌病理形态;免疫荧光法检测骨骼肌组织Cb1相关蛋白(CAP)、磷酸化大麻素受体1(p-Cb1)、小GTP结合蛋白(TC10)蛋白表达定位;免疫组化法检测骨骼肌组织中葡萄糖转运蛋白4(GLUT4)、小结合蛋白(CrkⅡ)、鸟苷酸交换因子(C3G)蛋白定位表达;Western Blot法检测骨骼肌组织CrkⅡ、C3G蛋白表达水平;qRT-PCR法检测骨骼肌组织CrkⅡ、C3G mRNA表达。结果:与模型组比较,二甲双胍组和GQD高剂量组FBG、FINS、FFA、HbA1c含量显著下降(P<0.01),GQD中剂量组FBG、FINS含量显著下降(P<0.01,P<0.05),GQD低剂量组FBG含量显著下降(P<0.01);二甲双胍组及GQD高、中剂量组CAP、p-Cb1、TC10、GLUT4蛋白平均光密度值显著升高(P<0.01,P<0.05),CrkⅡ、C3G蛋白平均光密度值显著降低(P<0.01,P<0.05);GQD低剂量组GLUT4蛋白平均光密度值显著升高(P<0.01);各给药组CrkⅡ、C3G蛋白及mRNA表达显著降低(P<0.01)。结论:GQD可以调节糖脂代谢,改善胰岛素抵抗,其作用机制可能与激活骨骼肌CAP/Cb1信号通路有关。

Objective:To explore the possible mechanism of Gegen Qinlian Decoction(GQD)in the treatment of type 2 diabetes mellitus(T2DM).Methods:A total of 36 SD rats were randomly divided into blank group,model group,metformin group and GQD high,medium and low doses groups,with an average of 6 animals in each group.Except of the blank group,the rats were given a high-fat diet combined with streptozotocin(STZ)to induce the model of T2DM,after drug intragastric intervention.Fasting blood glucole(FBG)was detected by blood glucose meter.Serum fasting insulin(FINS),free fatty acid(FFA)and whole blood glycated hemoglobin(HbAlc)were determined by ELISA.HE staining was used to observe the pathological morphology of skeletal muscle.The localized expression of Cbl-associated protein(CAP),phosphorylated cannabinoid receptor 1(p-Cbl),small GTP binding proteins(TC10)in skeletal muscle was detected by immunofluorescence method,the localized expression of glucose transporter 4(GLUT4),small binding protein(Crk Ⅱ),guanylate exchange factor(C3G)in skeletal muscle was detected by immunohistochemistry method.The protein expressions of CrkIl and C3G in skeletal muscle tissue were detected by Western Blot.The mRNA expression of Crk Ⅱ and C3G in skeletal muscle tissue was detected by qRT-PCR.Results:Compared with the model group,the levels of FBG,FINS,FFA and HbAlc in metformin group and GQD high dose group were significantly decreased(P<0.01),the levels of FBG and FINS in GQD medium dose group were significantly decreased(P<0.01,P<0.05),the level of FBG in GQD low dose group was significantly decreased(P<0.01).The average optical density of CAP,p-Cb1,TC10 and GLUT4 protein in metformin group and GQD high,medium dose group were significantly increased(P<0.01,P<0.05),the average optical density of Crk Ⅱ and C3G protein decreased significantly(P<0.01,P<0.05);the average optical density of GLUT4 protein in GQD low dose group increased significantly(P<0.01).The expression of Crk Ⅱ,C3G protein and mRNA in metformin group and GQD high,medium and low dose groups were significantly decreased(P<0.01).Conclusion:GQD can regulate glucose and lipid metabolism and improve insulin resistance,and its mechanism may be related to the activation of skeletal muscle CAP/Cbl signaling pathway.