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p3 8抗体(p38 mitogen-activated protein kinase，p38 MAPK)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。
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
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)sig-naling 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 glu-cose 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),mito-gen-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 glu-cose and insulin increased(P＜0.01).The expression of IRS1 protein significantly increased while the expression of GLUT4 pro-tein significantly decreased(P＜0.01).The mRNA expressions of PI3K and Akt decreased(P＜0.05,P＜0.01),while the mR-NA 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 expres-sions,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 ac-tivation of PI3K/Akt signaling pathway and inhibition of MAPK signaling pathway.
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