当归补血汤对2型糖尿病大鼠IRS-1/PI3K/Akt2信号通路的影响研究

Effect of Danggui Buxue Decoction on the expression of IRS-1,PI3K,Akt2 proteins and their substrates in the rats with type 2 diabetes

目的探讨当归补血汤对2型糖尿病大鼠胰岛素受体底物1(IRS-1)、磷脂酰肌醇3激酶p85亚基(PI3Kp85)、蛋白激酶B(Akt2)表达的影响。方法取8只雄性ZDF(fa/+)大鼠作为对照组;另取24只雄性ZDF(fa/fa)大鼠给予3周高脂饲料建立2型糖尿病模型,然后将造模成功大鼠随机分为模型组、盐酸二甲双胍组、当归补血汤组,每组8只。盐酸二甲双胍组给予盐酸二甲双胍肠溶片300 mg/kg灌胃,当归补血汤组给予当归补血汤(配方颗粒加蒸馏水配制)12.8 g/kg灌胃,对照组和模型组给予等量生理盐水灌胃,均1次/d,连续8周。末次灌胃后,禁食14 h进行口服葡萄糖耐量试验,计算胰岛素生成指数、胰岛素抵抗指数(HOMA-IR)和胰岛β细胞功能指数(HOMA-β);糖耐量试验结束后隔天禁食12 h抽取腹主动脉血,检测丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、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)水平;取各组大鼠肝脏组织,采用RT-qPCR法检测IRS-1、PI3Kp85和Akt2 mRNA表达情况,采用Western blot法检测IRS-1、p-IRS1、PI3Kp85、p-PI3Kp85、Akt2、p-Akt2蛋白表达情况。结果模型组大鼠空腹血糖水平、空腹胰岛素水平、血糖曲线下面积、HOMA-IR均明显高于对照组(P均<0.05),胰岛素生成指数、HOMA-β均明显低于对照组(P均<0.05);盐酸二甲双胍组和当归补血汤组空腹血糖水平、空腹胰岛素水平、血糖曲线下面积、HOMA-IR均明显低于模型组(P均<0.05),胰岛素生成指数、HOMA-β均明显高于模型组(P均<0.05),当归补血汤组各指标改善情况均明显优于二甲双胍组(P均<0.05)。与对照组比较,模型组大鼠血清ALT、AST、TC、TG、LDL-C水平均明显升高(P均<0.05),HDL-C水平明显降低(P<0.05);与模型组比较,盐酸二甲双胍组和当归补血汤组大鼠血清ALT、AST、TC、TG、LDL-C水平均明显降低(P均<0.05),HDL-C水平均明显降低(P均<0.05);除ALT、TC外,其余指标当归补血汤组改善情况均明显优于盐酸二甲双胍组(P均<0.05)。模型组大鼠肝脏组织中IRS-1、PI3Kp85、Akt2 mRNA表达量和IRS-1、p-IRS1、PI3Kp85、p-PI3Kp85、Akt2、p-Akt2蛋白表达量均明显低于对照组(P均<0.05),盐酸二甲双胍组和当归补血汤组上述各指标表达量均明显高于模型组(P均<0.05);除Akt2 mRNA、p-Akt2外,其余指标当归补血汤组均明显高于盐酸二甲双胍组(P均<0.05)。结论当归补血汤可能通过调节IRS-1/PI3K/Akt2信号通路发挥治疗2型糖尿病的作用。

Objective It is to explore the effects of Danggui Buxue Decoction on the expression of insulin receptor substrate 1(IRS-1),phosphatidylinositol 3-kinase p85 subunit(PI3Kp85),protein kinase B(Akt2)proteins and their substrates in type 2 diabetic rats.Methods Thirty-two male rats were divided into 4 groups.8 male ZDF(fa/+)rats were set as the control group,and 24 male ZDF(fa/fa)rats were fed with high-fat diet for 3 weeks to establish type 2 diabetic models,and were randomly divided into the model group,metformin hydrochloride group and Danggui Buxue Decoction group,8 rats in each group.The metformin hydrochloride group was given metformin hydrochloride enteric-coated tablets 300 mg/kg by gavage,the Danggui Buxue Decoction group was given Danggui Buxue Decoction(prepared with granules and distilled water)12.8 g/kg by gavage,and the control group and model group were given the same amount of normal saline by gavage,all once per day,continuously treated for 8 weeks.After the last gavage,fasting for 14 hours,oral glucose tolerance test was performed to calculate insulin production index,insulin resistance index(HOMA-IR)and isletβcell function index(HOMA-β);fasting for 12 hours on the next day after the glucose tolerance test,abdominal aortic blood was drawn to detect the levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triacylglycerol(TG),high density lipoprotein cholesterol(HDL-C)and low density lipoprotein cholesterol(LDL-C).The liver tissue of the rats in each group was taken to detect the mRNA expression of IRS-1,PI3Kp85 and Akt2 by RT-qPCR method;the pancreas tissue of the rats in each group was collected to detect the expression of IRS-1,p-IRS1,PI3Kp85 and p-PI3Kp85,Akt2,p-Akt2 protein by Western blot method.Results The levels of fasting blood glucose and fasting insulin,area under curve of blood glucose and HOMA-IR of the rats in the model group were significantly higher than those in the control group(all P<0.05),while the insulin production index and HOMA-βwere significantly lower than those in the control group(all P<0.05);the levels of fasting blood glucose and fasting insulin,area under curve of blood glucose and HOMA-IR of the rats in the metformin hydrochloride group and Danggui Buxue Decoction group were significantly lower than those in the model group(all P<0.05),and the insulin production index and HOMA-βwere significantly higher than those in the model group(all P<0.05),the improvement of each index in the Danggui Buxue decoction group was significantly better than that in the metformin group(all P<0.05).Compared with the control group,the serum levels of ALT,AST,TC,TG and LDL-C in the model group were significantly increased(P<0.05),and the HDL-C level was significantly decreased(P<0.05).The levels of serum ALT,AST,TC,TG and LDL-C in the metformin hydrochloride group and Danggui Buxue Decoction group were significantly decreased(all P<0.05),and the levels of HDL-C were significantly decreased(all P<0.05),the improvement of all indexes except for ALT and TC,in Danggui Buxue Decoction group was significantly better than that in metformin hydrochloride group(all P<0.05).The mRNA expressions of IRS-1,PI3Kp85 and Akt2 in the liver tissue and the protein expressions of IRS-1,p-IRS1,PI3Kp85,p-PI3Kp85,Akt2 and p-Akt2 in the pancreatic tissue of the rats in the model group were significantly lower than those in the control group(all P<0.05),the expression levels of the above indexes in the metformin hydrochloride group and Danggui Buxue Decoction group were significantly higher than those in the model group(all P<0.05),all the indexes except for Akt2 mRNA and p-Akt2 in Danggui Buxue decoction group were significantly higher than those in metformin hydrochloride group(all P<0.05).Conclusion Danggui Buxue Decoction may play a role in the treatment of type 2 diabetes by regulating the IRS-1/PI3K/Akt2 signaling pathway.