基于Nrf2/Keap1信号通路研究黄芩甲苷对糖尿病肾病模型小鼠肾损伤的作用

Effects of Baicalin Ⅳ on Renal Injury in Diabetic Nephropathy Mice Based on Nrf2/Keap1 Signaling Pathway

目的:基于核转录因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)/Kelch样环氧氯丙烷相关蛋白1(Kelch-like ech-associated protein 1,Keap1)信号通路研究黄芩甲苷对糖尿病肾病(diabetic nephropathy,DN)小鼠肾损伤的作用。方法:从60只C57BL/6小鼠中随机选择20只,分为对照组、正常+黄芩甲苷组,每组10只,其余40只小鼠制备DN模型。将建模成功的小鼠随机分为DN组、DN+黄芩甲苷组、DN+LV-Keap1组、DN+LV-control组,每组10只,药物持续干预10周。采用高糖诱导SV40-Mes-13细胞后,进行pcDNA-Keap1、si-Keap1、miR-142 inhibitor、miR-Keap1mimic转染,转染后的细胞分组并进行药物干预。化学比色法检测SV40-Mes-13细胞培养上清液中SOD的活性;RT-qPCR检测肾组织中miR-142 mRNA的表达水平;Western Blot检测Nrf2、Keap1、HO-1蛋白表达水平;流式细胞仪检测SV40-Mes-13细胞凋亡。结果:与对照组比较,DN组miR-142 mRNA表达水平明显升高,Keap1、Nrf2蛋白表达水平明显降低;与DN组比较,DN+黄芩甲苷组miR-142 mRNA表达水平明显降低,Keap1、Nrf2蛋白表达水平明显升高;与DN+LV-control组比较,DN+LV-Keap1组Keap1、Nrf2、HO-1蛋白表达水平明显升高;与葡萄糖+NC组比较,葡萄糖+miR-142 inhibitor组Keap1、Nrf2蛋白表达水平及SOD水平明显升高,细胞凋亡率明显降低;与葡萄糖+miR-142 inhibitor组比较,葡萄糖+miR-142 inhibitor+si-Keap1组Keap1、Nrf2蛋白表达水平及SOD水平明显降低,细胞凋亡率明显升高;与葡萄糖+黄芩甲苷+pre-NC组比较,葡萄糖+黄芩甲苷+miR-142 mimic组Keap1、Nrf2蛋白表达水平及SOD水平明显降低,细胞凋亡率明显升高;与葡萄糖+黄芩甲苷+miR-142 mimic+pcDNA组比较,葡萄糖+黄芩甲苷+miR-142 mimic+pcDNA-Keap1组Keap1、Nrf2蛋白表达水平及SOD水平明显升高,细胞凋亡率明显降低,差异均具有统计学意义(P<0.05)。结论:黄芩甲苷可逆转miR-142对Nrf2/Keap1信号通路的抑制作用进而改善糖尿病肾病肾损伤。

Objective:To study the effect of BaicalinⅣbased on the nuclear factor E2-related factor 2(Nrf2)/Kelch-like ech-associated protein 1(Keap1)signaling pathway on renal injury in diabetic nephropathy mice.Methods:Twenty mice were randomly selected from 60 C57BL/6 mice and divided into control group,normal+baicalinⅣgroup,10 mice in each group,and the remaining 40 mice were used to prepare DN model.The successfully modeled mice were randomly divided into DN group,DN+baicalinⅣgroup,DN+LV-Keap1 group and DN+LV-control group,with 10 mice in each group,and the drug continued to intervene for 10 weeks.After induction of SV40-Mes-13 cells with high glucose,pcDNA-Keap1,si-Keap1,miR-142 inhibitor,and miR-Keap1mimic were transfected,and the transfected cells were grouped and subjected to drug intervention.The activity of SOD in SV40-Mes-13 cell culture supernatant was detected by chemical colorimetry;RT-qPCR was used to detect the expression level of miR-142 mRNA in kidney tissue;Western Blot was used to detect the protein expression levels of Nrf2,Keap1 and HO-1;The apoptosis of SV40-Mes-13 cells was detected by flow cytometry.Results:Compared with the control group,the expression levels of miR-142 mRNA in the DN group were significantly increased,and the protein expression levels of Keap1,Nrf2 were significantly decrdased;Compared with the DN group,the miR-142 mRNA expression level in the DN+baicalinⅣgroup was significantly decreased,and the protein expression levels of Keap1 and Nrf2 were significantly increased;Compared with the DN+LV-control group,the protein expression levels of Keap1,Nrf2 and HO-1 in the DN+LV-Keap1 group were significantly increased;Compared with the glucose+NC group,the protein expression levels of Keap1 and Nrf2 and the activity of SOD in the glucose+miR-142 inhibitor group were significantly increased,and the apoptosis rate was significantly decreased;Compared with the glucose+miR-142 inhibitor group,in the glucose+miR-142 inhibitor+si-Keap1 group;the protein expression levels of Keap1,Nrf2 and the activity of SOD were significantly decreased,and the apoptosis rate was significantly increased;Compared with the glucose+baicalinⅣ+pre-NC group the protein expression levels of Keap1,Nrf2 and the activity of SOD in the glucose+baicalinⅣ+miR-142 mimic group were significantly decreased,and the apoptosis rate was significantly increased;Compared with the glucose+baicalinⅣ+miR-142 mimic+pcDNA group,the expression levels of Keap1 and Nrf2 proteins and the activity of SOD in the glucose+baicalinⅣ+miR-142 mimic+pcDNA-Keap1 group were significantly increased,and the apoptosis rate was significantly decreased,and the differences were statistically significant(P<0.05).Conclusion:Baicalin can reverse the inhibitory effect of miR-142 on Nrf2/Keap1 signaling pathway and improve renal injury in diabetic nephropathy.