肾络通调控NR3C2/SGK-1/Smad通路抑制肾间质纤维化的机制研究

Mechanism of Shenluotong regulating NR3C2/SGK-1/Smad pathway in inhibiting renal interstitial fibrosis

目的探讨中药复方肾络通调控NR3C2/SGK-1/Smad通路抑制肾间质纤维化的作用机制。方法 Wistar大鼠随机分为假手术组、模型组、依普利酮组、肾络通组,每组各12只。模型组、依普利酮组和肾络通组均采用单侧输尿管结扎的方法建立肾间质纤维化大鼠模型。术后即开始给药,依普利酮组给予依普利酮混匀于饲料中按100 mg/(kg·d)剂量食入;肾络通组给予肾络通煎剂按照26 g/(kg·d)剂量入水瓶饮用,模型组和假手术组给予等比例生理盐水,每天1次,连续干预10 d。采用激光共聚焦显微镜检测盐皮质激素受体NR3C2表达,采用免疫组化、Westernblotting、q RT-PCR等方法检测大鼠肾组织SGK-1、TGF-β1、Smad4、Smad7表达情况。结果假手术组NR3C2表达于细胞胞质,核内未见表达;模型组小管表达明显增强,核内可见阳性表达;依普利酮组和肾络通组细胞核内可以见到NR3C2表达,但较模型组显著减少。与假手术组比较,其他各组SGK-1、TGF-β1、Smad4表达显著上调,而Smad7表达则显著降低(P<0.05、0.01)。与模型组比较,依普利酮组及肾络通组SGK-1、TGF-β1、Smad4表达范围及强度均显著减弱(P<0.05、0.01),Smad7表达范围及强度均显著增强(P<0.01)。结论肾络通可通过抑制盐皮质激素受体活化,下调SGK-1表达,并通过调控Smads信号蛋白水平,抑制了TGF-β1促纤维化作用,从而阻止了肾间质纤维化的发生发展。

Objective To investigate the mechanism of Shenluotong inhibiting renal interstitial fibrosis by regulating NR3C2/SGK-1/Smad pathway.Methods A total of 48Wistar rats were randomly divided into sham operation group,model group, Eplerenone group,and Shenluotong group (n =12).Model group,Eplerenone group,and Shenluotong group used unilateral ureteral obstruction (UUO)method to establish rat renal interstitial fibrosis model.After the operation,the rats in the eplerenone group were treated with eplerenone at adose of 100mg/(kg·d).Rats in the Shenluotong group were oral given Shenluotong decoction at a dose of 26g/(kg·d)and Sham operation group and model group were administrated equal volume of saline once daily for continuous 10d. Laser confocal microscopy was used to detect mineralocorticoid receptor NR3C2expression.The expressions of SGK-1,TGF-β1, Smad4,and Smad7in renal tissues were detected by immunohistochemistry,Western Blot and Real time-PCR.Results In the sham operation group,NR3C2was expressed in the cytoplasm of renal tubular epithelial cells and was not expressed in the nucleus.The expression of NR3C2in the UUO rat was significantly up-regulated in cytoplasm and positive expression was observed in the nucleus.The expression of NR3C2in the nucleus of cells in the Eplerenone group and Shenluotong group was significantly decreased when compared with the model group.Compared with the sham-operated group,the expression of SGK-1,TGF-β1,and Smad4was significantly up-regulated and the expression of Smad7was significantly decreased (P <0.05,0.01)in the other goups.Compared with model group,the expression range and intensity of TGF-β1and Smad4were significantly decreased in Eplerenone group and Shenluotong group (P <0.05,0.01),and the expression range and intensity of Smad7were significantly increased (P <0.01). Conclusion Shenluotong can inhibit renal interstitial fibrosis through blocking the activation ofmineralocorticoid receptor,reducing the level of SGK-1,and regulating the Smads signal pathway to inhibit the overexpression of TGF-β1.