五苓散通过TGF-β1/Smad通路抗大鼠肾间质纤维化的机制研究

Mechanism Study of Wuling Powder on Preventing Renal Interstitial Fibrosis Through TGF-β1/Smad Pathway in Rats

目的探究五苓散(Wuling Power,WLP)防治单侧输尿管梗阻(unilateral ureteral obstruction,UUO)大鼠肾间质纤维化(RIF)的机制。方法将36只大鼠随机分为空白组、模型组、五苓散组,采用单侧输尿管结扎术的方法制备单侧输尿管梗阻的大鼠模型,灌胃给药,连续21 d。用脲酶法测定血清中尿素氮(BUN)含量,用微板法测定血清肌酐(SCr)含量;用HE和Masson染色观察肾脏组织病理学变化;采用免疫组化法检测肾脏组织中α-平滑肌肌动蛋白(α-SMA)、Ⅰ型胶原蛋白(collagenⅠ)和纤连蛋白(Fibronectin)的表达;采用Western Blot法检测肾脏组织中α-SMA、collagenⅠ、Fibronectin、E-钙黏蛋白(E-cadherin)、转化生长因子β1(TGF-β1)、p-Smad2、p-Smad3、Smad4、Smad7的蛋白表达水平。结果与空白组比较,模型组大鼠的血清肌酐、尿素氮含量明显升高(P<0.01),而五苓散组干预后的血清肌酐、尿素氮明显下降(P<0.05)。模型组大鼠梗阻侧肾脏组织部分肾小管上皮细胞空泡变性,部分管腔扩张或萎缩坏死,可见炎性细胞浸润、成纤维细胞增生和肾间质纤维化;五苓散干预治疗后,上皮细胞变形萎缩、炎性细胞浸润及间质纤维化程度均明显改善。免疫组化结果显示,与模型组比较,五苓散组的Fibronectin、α-SMA及collagenⅠ阳性表达水平明显降低(P<0.01)。Western Blot结果显示,与模型组比较,五苓散可以抑制Fibronectin、α-SMA、collagenⅠ、TGF-β1、p-Smad2、p-Smad3和Smad4蛋白的表达(P<0.05,P<0.01),同时可以促进E-cadherin和Smad7蛋白的表达(P<0.05)。结论五苓散具有抗肾间质纤维化的作用,其机制可能与缓解或逆转肾脏组织EMT的发生和ECM的沉积,抑制肾脏组织中TGF-β1/Smad通路蛋白的表达有关。

Objective To explore the mechanism of Wuling Power(WLP)in preventing and treating renal interstitial fibrosis(RIF)of unilateral ureteral obstruction(UUO)rats.Methods Thirty-six rats were randomly divided into blank group,model group and WLP group.UUO model was prepared by unilateral ureteral obstruction and gavaged intragastrically with drug for 21 days.The content of urea nitrogen(BUN)in serum and serum creatinine(SCr)were determined by urease method and microplate method,respectively.Histopathological changes of kidney were observed by HE staining and Masson staining.The expressions ofα-SMA,collagen I and Fibronectin in kidney tissue were detected by immunohistochemistry.Western Blot was used to detect the expression levels ofα-SMA,collagen I,Fibronectin,E-cadherin,TGF-β1,P-Smad2,P-Smad3,Smad4 and Smad7 in kidney tissues.Results Compared with the blank group,the levels of Scr and BUN in model group were significantly increased(P<0.01),and the contents of Scr and BUN in WLP group were significantly decreased(P<0.05).The vacuolar degeneration of renal tubular epithelial cells in the renal tissue of the obstructed side of the model group was observed.Some of the lumens were expanded or atrophied and necrotic,and inflammatory cell infiltration,fibroblast proliferation,and RIF were also observed.After treatment with WLP,the degree of epithelial cell degeneration,inflammatory cell infiltration and interstitial fibrosis were improved obviously.Immunohistochemical showed that compared with the model group,the positive expression levels of Fibronectin、α-SMA and collagenⅠin WLP group were significantly lower(P<0.01).The results of Western Blot showed that compared with rats in the model group,WLP could inhibit the expression of Fibronectin、α-SMA、collagenⅠ、TGF-β1、p-Smad2、p-Smad3 and Smad4 protein(P<0.05,P<0.01),and promote the expression of E-cadherin and Smad7 protein(P<0.05).Conclusion WLP has an anti-fibrotic effect on renal interstitial,which may be related to alleviating or reversing the occurrence of EMT and ECM deposition and inhibiting the expression of TGF-β1/Smad pathway protein in renal tissue.