糖肾方调控FXR/vimentin/α-SMA通路减轻糖尿病肾脏疾病肾脏纤维化的研究

Tangshen Formula ameliorates renal fibrosis of diabetic kidney disease through regulating the FXR/vimentin/α-SMA pathway

目的通过体内及体外实验,观察益气养阴、活血通络中药复方糖肾方对法尼醇X受体(FXR)/波形蛋白(vimentin)/α-平滑肌肌动蛋白(α-SMA)通路介导的糖尿病肾脏疾病肾脏纤维化的作用。方法(1)体内实验。8周龄雄性db/db小鼠分为模型组和糖肾方组,同遗传背景db/m小鼠为对照组。糖肾方组灌胃糖肾方配方颗粒(3.6 g/kg),1次/d,连续给药12周。观察小鼠的一般状态和体质量,监测血糖,检测血清肌酐(Scr)、尿素氮(BUN),计算尿白蛋白肌酐比(UACR)。过碘酸希夫染色、Masson染色观察肾脏形态结构变化,使用蛋白质印迹法及激光共聚焦显微镜检测肾脏FXR、vimentin及α-SMA蛋白表达水平。(2)体外实验:构建棕榈酸钠诱导的肾小管上皮细胞(mTECs)损伤模型,给予糖肾方(250或500 mg/L)干预后,使用蛋白质印迹法及激光共聚焦显微镜检测mTECs中FXR、vimentin、α-SMA蛋白表达水平,并用FXR干扰小RNA(FXR-siRNA)验证糖肾方激活FXR来抑制mTECs纤维化的分子机制。结果(1)糖肾方可降低db/db小鼠血清Scr水平;改善肾脏病理损伤,上调肾脏FXR蛋白表达,同时抑制肾脏vimentin及α-SMA蛋白表达。(2)糖肾方可上调棕榈酸钠诱导的mTECs中FXR蛋白表达,同时抑制vimentin及α-SMA蛋白表达;FXR-siRNA干预后减弱了糖肾方上调FXR蛋白表达、抑制vimentin及α-SMA表达的作用。结论糖肾方可减轻糖尿病肾脏疾病小鼠肾脏纤维化,其作用机制与调控FXR/vimentin/α-SMA通路有关。

Objective We aimed to analyze the effects of the traditional Chinese medicine(TCM)Tangshen Formula,according to TCM method of benifiting qi and nourishing yin,promoting blood circulation for removing obstruction in collaterals,on renal fibrosis of diabetic kidney disease through farnesoid X receptor(FXR)/vimentin/α-smooth muscle actin(α-SMA)pathway in vivo and in vitro.Methods(ⅰ)Eight-week-old male db/db mice were randomly divided into the model group and the Tangshen Formula group(3.6 g/kg),once a day for 12 weeks,and db/m mice with the same genetic background were used as the control group.The general state and body weight of the mice were observed,blood glucose was monitored,and serum creatinine(Scr),blood urea nitrogen(BUN),and urine albumin-to-creatinine ratio were calculated.PAS staining and Masson staining were used to observe the changes in kidney morphology and structure.Western blotting and laser confocal microscopy were used to detect the expression levels of FXR,vimentin,andα-SMA in kidneys.(ⅱ)Sodium palmitate(PA)-stimulated mouse tubular epithelial cell(mTEC)injury model was constructed in vitro,and after intervention with Tangshen Formula(250 or 500 mg/L),the expression levels of FXR,vimentin,andα-SMA in mTECs were detected by Western blotting and laser confocal microscopy.Moreover,FXR was silenced to explore the molecular mechanism by which Tangshen Formula inhibits cellular fibrosis in mTECs.Results(ⅰ)Tangshen Formula significantly reduced Scr levels and renal pathology in db/db mice.Western blotting analysis and laser confocal microscopy showed that Tangshen Formula upregulated FXR and downregulated vimentin andα-SMA in the kidneys of db/db mice.(ⅱ)Western blotting analysis and laser confocal microscopy also showed that Tangshen Formula significantly upregulated FXR and downregulated vimentin andα-SMA in PA-induced mTECs.FXR silencing significantly suppressed the effects of Tangshen Formula on cellular fibrosis and the expression of vimentin andα-SMA.Conclusion Tangshen Formula can reduce renal fibrosis in diabetic nephropathy,and its mechanism is related to the regulation of the FXR/vimentin/α-SMA pathway.