地黄饮子通过PI3K/Akt和MAPK信号通路双重调节db/db小鼠视网膜胰岛素抵抗和糖代谢异常的机制研究

Study on Mechanism of Dihuang Yinzi(地黄饮子) Regulating Insulin Resistance And Abnormal Glucose Metabolism in Retina of Db/Db Mice through PI3K/Akt and MAPK Signal Pathway

目的探讨地黄饮子通过磷脂酰肌醇三激酶-蛋白激酶B(phosphoinositide 3-kinase/protein kinase B,PI3K/Akt)和丝裂原激活蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路双重调节db/db小鼠视网膜胰岛素抵抗和糖代谢异常,探究地黄饮子多途径发挥治疗糖尿病视网膜病变(diabetic retinopathy,DR)作用的分子机制。方法10只db/m小鼠作为空白组,30只db/db小鼠随机分为模型组、地黄饮子组(地黄饮子,30.03 g/kg)、阳性对照组(二甲双胍,0.61 g/kg)。药物干预4周,每周检测小鼠体质量、空腹血糖。末次给药后进行口服糖耐量试验(oral glucose tolerance test,OGTT)及胰岛素耐量试验(insulin tolerance test,ITT)并计算曲线下面积(area under the curve,AUC);葡萄糖氧化酶法检测小鼠视网膜葡萄糖含量;酶联免疫吸附法(ELISA)检测小鼠视网膜胰岛素含量;苏木素-伊红(HE)染色观察小鼠视网膜组织病理改变;免疫组化法检测视网膜组织胰岛素受体底物1(insulin receptor substrate 1,IRS1)、葡萄糖转运蛋白4(glucose transporter 4,GLUT4)表达;实时荧光定量聚合酶链式反应法(RT-qPCR)检测视网膜PI3K、Akt、细胞外调节蛋白激酶(extracellular regulated kinase,ERK)、c-Jun氨基末端激酶(c-Jun N-terminal protein kainse,JNK)、丝裂原活化蛋白激酶p38抗体(p38 mitogen-activated protein kinase,p38MAPK)mRNA表达量。结果与空白对照组相比,模型组小鼠空腹血糖显著升高(P<0.01);OGTT、ITT试验各时间点血糖均升高(P<0.05,P<0.01);视网膜组织水肿,新生血管生成;视网膜葡萄糖、胰岛素含量升高(P<0.01);IRS1蛋白表达量显著升高而GLUT4蛋白表达量显著降低(P<0.01);PI3K、Akt mRNA表达量降低(P<0.05,P<0.01)、ERK、JNK、p38MAPK mRNA表达量升高(P<0.05,P<0.01)。地黄饮子和阳性对照药物均可降低小鼠OGTT、ITT试验AUC(P<0.01);改善视网膜水肿,减少新生血管;降低视网膜葡萄糖、胰岛素含量(P<0.05,P<0.05);降低小鼠视网膜IRS1蛋白表达,提高GLUT4蛋白表达(P<0.05,P<0.01)、升高PI3K、Akt mRNA表达量,降低ERK、JNK、p38MAPK mRNA表达量(P<0.05,P<0.01)。结论地黄饮子可通过激活PI3K/Akt信号通路和抑制MAPK信号通路双重调节GLUT4表达从而改善db/db小鼠视网膜胰岛素抵抗和糖代谢异常。

Objective To explore the dual regulation of retinal insulin resistance and glucose metabolism abnormalities in db/db mice through phosphoinositide 3-kinase/protein kinase B(PI3K/Akt) and mitogen-activated protein kinase(MAPK) signaling pathways by Dihuang Yinzi(地黄饮子) and the molecular mechanism of multi-pathway therapeutic effect of Dihuang Yinzi on diabetic retinopathy(DR). Methods Ten db/m mice were used as blank group, 30 db/db mice were randomly divided into model group, Dihuang Yinzi group(30.03 g/kg) and positive control group(metformin, 0.61 g/kg). The drug intervention was performed for 4 weeks, and the body mass and fasting blood glucose of mice were tested weekly. After the last dose, oral glucose tolerance test(OGTT) and insulin tolerance test(ITT) were performed and the area under the curve(AUC) was calculated. The glucose oxidase method was used to detect the glucose content of mouse retina. The enzyme-linked immunosorbent assay(ELISA) was used to detect the insulin content of mouse retina and hematoxylin-eosin(HE) staining was used to observe the histopathological changes of mouse retina. The immunohistochemistry method was used to detect retinal tissue IRS1 and GLUT4 expressions. The real-time fluorescence quantitative polymerase chain reaction(RT-qPCR) was used to detect retinal PI3K,Akt, extracellular regulated protein kinase(ERK),c-Jun amino-terminal kinase c-Jun N terminal protein kainse(JNK),mitogen-activated protein kinase(p38MAPK) mRNA expressions. Results Compared with those of the blank control group, the level of fasting blood glucose was significantly higher in the model group(P<0.01). The levels of blood glucose were higher at all time points of OGTT and ITT tests(P<0.01). There was retinal tissue edema and neovascularization. The levels of retinal glucose and insulin increased(P<0.01). The expression of IRS1 protein significantly increased while the expression of GLUT4 protein significantly decreased(P<0.01). The mRNA expressions of PI3K and Akt decreased(P<0.05,P<0.01),while the mRNA expressions of ERK,JNK and p38MAPK increased(P<0.05,P<0.01). Both Dihuang Yinzi and positive control reduced the AUC values of OGTT and ITT test in mice(P<0.01),improved retinal edema and reduced neovascularization, reduced the levels of retinal glucose and insulin(P<0.05,P<0.05). Both Dihuang Yinzi and positive control drugs reduced retinal IRS1 protein expression and increased GLUT4 protein expression in mice(P<0.05,P<0.01),elevated PI3K and Akt mRNA expressions, and decreased ERK,JNK and p38MAPK mRNA expressions(P<0.05,P<0.01). Conclusion Dihuang Yinzi can improve retinal insulin resistance and glucose metabolism abnormalities in db/db mice by dual regulation of GLUT4 expression through activation of PI3K/Akt signaling pathway and inhibition of MAPK signaling pathway.