翻白草总黄酮调控Glp-1介导的MAPK通路修复胰岛β细胞研究

Effect of Total Flavonoids from Potentilla Discolor on Renovating IsletβCell through Adjusting Glp-1 Mediated MAPK Pathway

目的:研究翻白草总黄酮调控胰高血糖素样肽-1(glucagon-like peptide-1,Glp-1)介导丝裂原活化蛋白激酶(mitogen-activitived protein kinases,MAPK)信号通路修复2型糖尿病(T2MD)大鼠胰岛β细胞的作用机制。方法:复制T2MD大鼠模型后,将造模成功的大鼠随机分为模型对照组、翻白草总黄酮108 mg/kg和216 mg/kg组、二甲双胍20.8 mg/kg组,另设正常对照组。各组分别予等体积相应药物或生理盐水灌胃,连续灌胃8 w,实验前和每周末测定大鼠体重,8 w后处死大鼠。葡萄糖氧化酶法检测血糖、血脂和游离脂肪酸(FFA)含量;HE染色法观察胰岛病理形态的改变;实时荧光定量聚合酶链式反应(Real-time,PCR)检测肌肉组织Glp-1、蛋白激酶B(protein kinase B,Akt)、细胞外信号调节激酶(extracellular signal-regulated kinase,Erk)和半胱氨酸天冬氨酸蛋白酶-9(Caspase-9)mRNA水平的表达;免疫组化法检测胰岛β细胞的Glp-1、Akt、Erk、Caspase-9蛋白表达。结果:与正常对照组比较,模型对照组大鼠造模8 w后体重明显降低,血糖、TC、LDL-C、FFA含量明显升高,HDL明显降低(P<0.05),肌肉组织中Glp-1、Akt mRNA及胰岛中蛋白表达明显下调,Erk、Caspase-9 mRNA及胰岛中蛋白明显上调(P<0.05)。与模型对照组比较,翻白草总黄酮216 mg/kg组和二甲双胍20.8 mg/kg组大鼠的体重上升、血清中血糖、总胆固醇(TC)、FFA、LDL-C含量明显降低,高密度脂蛋白(HDL-C)含量明显上升,胰岛结构较完整(P<0.05);肌肉组织Glp-1、Akt mRNA表达明显上调,Erk、Caspase-9 mRNA表达明显下调;胰岛β细胞Glp-1、Akt蛋白表达上调,胰岛β细胞Erk、Caspase-9蛋白含量表达明显下调(P<0.05)。结论:翻白草总黄酮修复2型糖尿病大鼠胰岛β细胞,其机制可能是通过激活Glp-1介导的MAPK信号通路,上调肌肉组织Glp-1、Akt mRNA表达,下调肌肉组织Erk、Caspase-9 mRNA表达,上调胰岛β细胞Glp-1、Akt蛋白表达,下调胰岛β细胞Erk、Caspase-9蛋白表达,从而修复胰岛细胞,增强胰岛β细胞功能。

Objective:To study the mechanism of total flavonoids from potentilla discolor on renovating isletβcell in type 2 diabetic rats through adjusting glucagon-like peptide-1(Glp-1)to mediate(mitogen-activitived protein kinases)MAPK pathway.Methods:After type 2 diabetic rat model was established,the rats successfully modeled were randomly divided into the model group,108 mg/kg and 216 mg/kg total flavonoids from potentilla discolor groups,20.8 mg/kg metformin group.Healthy rats were set as the control group.Rats in each group were given an equal volume of corresponding drugs or saline by gavage for 8 weeks.The weights of rats were measured before the experiment and every weekend during the experiment,all rats were sacrificed after 8 weeks.Glucose oxidase method was used to detect the blood glucose,blood lipids and free fatty acids,HE staining method was used to observe the pathological changes of islet,Real-time(PCR)method was used to detecte muscle tissue glucagon-like peptide-1(Glp-1),protein kinase B(Akt),extracellular signal-regulated kinase(Erk)and caspase-9(Caspase-9)mRNA expression.The contents of Glp-1,Akt,Erk and Caspase-9 in isletβcell were detected by immunohistochemistry.Results:Compared with the control group,the rat weights were significantly decreased after 8 weeks of administration in the model group,the contents of blood glucose,total cholesterol(TC),LDL and FFA levels were significantly increased,the level of high-density lipoprotein(HDL)was significantly decreased(P<0.05).The expressions of Glp-1 and Akt mRNA in muscle tissues and the protein in isletβcells were significantly down-regulated,and the expression of Erk and Caspase-9 mRNA in the muscle tissue and the protein ofβ-cells were significantly up-regulated(P<0.05).Compared with the model group,the weights of rats in 216 mg/kg potentilla discolor and 20.8 mg/kg metformin group were increased,the levels of blood glucose,TC and FFA in the serum were significantly reduced,and the level of HDL was significantly increased(P<0.05).The structure of pancreatic islets was relatively complete(P<0.05).The expressions of Glp-1 and Akt mRNA in muscle tissues were significantly up-regulated,and the expressions of Erk and Caspase-9 mRNA were significantly down-regulated(P<0.05).The protein expressions of Glp-1 and Akt in isletβcell were significantly up-regulated,while the protein expressions of Erk and Caspase-9 were significantly down-regulated(P<0.05).Conclusion:The total flavonoids from potentilla discolor can renovate the isletβcell of type 2 diabetic rats.The mechanism may be related to the activation of Glp-1 mediated MAPK signaling pathway,up-regulating the levels of Glp-1 and Akt mRNA in muscle tissues,down-regulating the levels of Erk,Caspase-9 mRNA in muscle tissues,up-regulating the protein expression of Glp-1 and Akt in isletβcell,and down-regulating the protein expressions of Erk and Caspase-9 in isletβcell,in order to protect islet cell and enhance the function of isletβcell.