转化生长因子β细胞内信号转导负调控蛋白Smad6,7在肾间质纤维化大鼠肾脏表达与益气活血法的影响

Effect of qi supplement and blood activation therapy on the expressions of Smad6 and Smad7 in rats with renal interstitial fibrosis

目的:观察肾间质纤维化大鼠肾组织中转化生长因子β细胞内信号转导负调控蛋白Smad6,7表达变化与益气活血法中药制剂的影响。方法:实验于2006-12/2007-06在首都医科大学解剖与组织胚胎学系实验室完成。①实验动物:Wistar雄性大鼠30只,采用随机数字法随机分为模型组、中药大剂量组、中药中剂量组、中药小剂量组、西药蒙诺组、假手术组,每组5只。益气活血法中药提取液(生黄芪15g,当归10g,赤白芍10g,丹参30g,黄芩10g,车前草15g,牛膝15g等)经水提醇沉得4.4kg/L生药提取液。实验过程中对动物处置符合动物伦理学要求。②实验方法:其中前5组行单侧输尿管梗阻致肾小管间质纤维化模型手术。假手术组打开大鼠腹腔后分离左侧输尿管,并不结扎即关闭腹腔。中药大剂量组、中药中剂量组、中药小剂量组、西药蒙诺组,于手术前2d给予灌胃给药0.072mL/(kg·d),0.036mL/(kg·d),0.018mL/(kg·d),10mg/(kg·d),1次/d,连续2周。模型组及假手术组用相同体积的生理盐水灌胃。③实验评估:6组大鼠于术后14d麻醉后处死,观察梗阻肾组织病理改变,用免疫组化方法检测肾组织Smad6,7蛋白表达,通过医学病理图像分析系统对Smad6,7蛋白的积分光密度进行统计学分析。结果:30只大鼠全部进入结果分析。苏木精-伊红染色显示模型组肾小管上皮细胞萎缩,大部分肾小管管腔扩张,间质纤维组织增生,大量炎性细胞浸润,肾小球数目明显减少。中药大剂量组、中剂量组、西药蒙诺组较模型组肾间质炎性细胞浸润、纤维组织增生、肾小管扩张情况明显减轻。Smad6,7蛋白主要在肾皮质肾小管上皮细胞内表达,在模型组它们的表达较假手术组明显减弱且分布面积减少,两组间积分光密度差异具有统计学意义(P<0.05);中药中剂量组、大剂量组、西药蒙诺组Smad6,7蛋白的表达较模型组明显增强、面积增加,积分光密度差异具有统计学意义(P<0.05)。结论:益气活血法可以通过诱导肾组织Smad6,7蛋白表达而抑制肾间质纤维化。

AIM： To investigate the effect of qi supplement and blood activation therapy with traditional Chinese medicine on the expression of Smad6 and Smad7, the signal transduction negative modulin of transforming growth factor 13 cell, in rats with renal interstitial fibrosis. METHODS： The experiment was performed in the Department of Anatomy and Histology, Capital Medical University from December 2006 to June 2007.①Thirty male Wistar rats were selected and randomly divided into model group, traditional Chinese medicine high, middle and low dose groups, western medicine （Monopril） group and sham operation group with 5 rats in each group. Qi supplement and blood activation therapy with traditional Chinese medicine was consisted of 15 g milkvetch root, 10 g angelica root, 10 g paeoniae radix, 30 g salvia miltiorrhiza, 10 g baical skullcap root, 15 g plantain and 15 g achyranthis. 4.4 kg/L crude drug extract was obtained by water extraction and alcohol precipitation. ②Rat model of renal interstitial fibrosis was established in the former 5 groups by the unilateral ureteral obstruction. The left ureter in rats of the sham operation group was separated after abdomen opening without ligating the ureter. At two days before operation, rats in the high, middle and low dose groups and western medicine group were administrated with corresponding medicine at the dose of 0,072, 0.036, 0.018 mL/kg·d, and 10 mg/kg.d, once daily for 2 weeks. While rats in model group and sham operation group were infused with matching normal saline, ③Rats were executed under anesthesia at 14 days after operation, and the histopathological changes of nephridial tissues were evaluated by HE staining, The expression of Smad6 and Smad7 in renal tissue was examined by immunohistochemistry after 14 days, Medical pathology image analysis was performed to detect the integral photodensity for Smad-6 and Smad-7. All treatments for animal were accorded with the animal ethical requirements. RESULTS： All 30 rats were involved in the result analysis. HE staining studies showed that the renal tubular epithelial cells in model group got atrophy; many lumens of the renal tubule expanded; there were lots of fibroplasias and inflammatory cell infiltrate in renal interstitium, and the number of glomerulus lessened. In the traditional Chinese medicine high and middle dose groups and western medicine group, the histopathological changes in inflammatory cell infiltrate, fibroplasia and tubular ectasia of renal interstitium were obviously better than the model group, Smad6 and Smad7 were mainly expressed in the renal tubular cells of renal cortex, They were weakly expressed in model group compared to the sham operation group, and significant differences were found in the integral photodensity between them （P 〈 0.05）. Smad6 and Smad7 were strongly expressed in the traditional Chinese medicine middle and high dose groups and western medicine group compared to the model group, and the differences in integral photodensity were statistically significant （P 〈 0.05）, CONCLUSION： Qi supplement and blood activation therapy can suppress the development of renal interstitial fibrosis via inducing the expression of Smad6 and Smad7.