基于Notch1-RBP-Jk/Msx2信号通路探讨大黄酸对糖尿病肾病主动脉钙化的作用及机制研究

Effectiveness and its mechanism of chrysophanic acid for aortic calcification with diabetic nephropathy based on the Notch1-RBP-Jk/Msx2 signaling pathway

目的基于人跨膜受体蛋白Notch-1通路(Notch1-recombining binding protein suppressor of hairless,Notch1-RBP-Jk)/相互作用蛋白Msx2(Msx2-interacting nuclear target protein,Msx2)信号通路,探讨大黄酸对糖尿病肾病主动脉钙化的控制作用。方法取32只大鼠建立糖尿病肾病主动脉钙化模型并随机分为4组(模型对照组、低剂量组、中剂量组、高剂量组),另取8只正常大鼠记为正常对照组,其中低剂量组、中剂量组和高剂量组分别给予50 mg/kg、100 mg/kg、150 mg/kg剂量的大黄酸灌胃,模型对照组和正常对照组给予等量生理盐水灌胃,每天1次,持续4周。对比干预前后肾功能变化;干预后将各组大鼠处死,取腹主动脉观察其钙化情况;取腹主动脉组织检测钙含量;采用实时聚合酶链反应检测各组腹主动脉组织Notch1、RBP-JK、Msx2、α-肌动蛋白(α-smooth muscle aorta,α-SMA)、Runt相关转录因子2(Runt-related transcription factor 2,Runx2)的信使核糖核酸(mRNA)表达;采用蛋白质免疫印迹法检测腹主动脉组织Notch1、RBP-JK、Msx2、α-SMA、Runx2蛋白表达。结果与模型对照组比较,3剂量组干预后肾功能指标均改善,腹主动脉钙化情况均减轻,腹主动脉组织钙含量减少,且3剂量组Notch1、RBP-JK、Msx2、Runx2 mRNA和蛋白表达均下降,α-SMA mRNA和蛋白表达升高。在3个剂量组中,高剂量组各项指标改善情况均为最佳。结论大黄酸能够保护糖尿病主动脉钙化大鼠的肾功能,减轻钙化,推测其作用机制与下调Notch1、RBP-JK、Msx2、Runx2 mRNA和蛋白表达,上调α-SMA mRNA和蛋白表达有关。

Objective To explore the effectiveness of chrysophanic acid for aortic calcification in diabetic nephropathy based on the Notch1-recombining binding protein suppressor of hairless(Notch1-RBP-Jk)/Msx2 signaling pathway.Methods Forty adult SD rats were selected and 32 were used to establish the model of aortic calcification with diabetic nephropathy and randomly divided into 4 groups(model control group,low-dose group,middle-dose group and high-dose group).The remaining 8 rats were recorded as normal control group.The rats in the low,medium and high dose groups were administered gastrically with 50 mg/kg,100 mg/kg and 150 mg/kg chrysophanic acid respectively;and the rats in the model control and normal control groups were given the same amount of saline,once a day for 4 weeks.Renal function changes before and after intervention were compared.After intervention,the rats in each group were sacrificed,and the abdominal aorta was taken to observe the calcification.The calcium contents in abdominal aorta tissues were detected.RT-PCR was used to detect the expressions of Notch1,RBP-JK,Msx2,α-SMA(α-smooth muscle actin)and Runt-related transcription factor 2(Runx2)mRNA in the abdominal aorta of each group.The related expressions of Notch1,RBP-JK,Msx2,α-SMA and Runx2 proteins in abdominal aorta tissues were detected by Western blotting.Results Compared to the model control group,in the three dose groups,after intervention the renal function indexes were improved,abdominal aortic calcification was alleviated and calcium content in abdominal aortic tissues was reduced.The expressions of Notch1,RBP-JK,Msx2,Runx2 and protein in the three dose groups were all decreased,while the expression ofα-SMA and protein were increased.Among the three dose groups,in the high dose group various indices were improved best.Conclusions Chrysophanic acid can protect renal function and reduce calcification in diabetic aortic calcification rats,and it is expected that its mechanism may relate to down-regulation of Notch1,RBP-JK,Msx2,Runx2 mRNA and protein expressions,and up-regulation ofα-SMA mRNA and protein expressions.