肾间质纤维化大鼠肾脏细胞外基质成分变化与益气活血治法的影响

Effect of qi supplement and blood activation therapy on extracellular matrix in rats with renal interstitial fibrosis

目的:观察益气活血法中药制剂对肾间质纤维化大鼠肾组织细胞外基质成分纤维连接蛋白、层粘连蛋白和胶原蛋白Ⅳ表达的影响,分析其抗肾间质纤维化的机制。方法:实验于2007-07/10在首都医科大学解剖与组织胚胎学系实验室完成。①材料:Wister雄性大鼠30只随机分为模型组,中药大、中、小剂量组,西药组和假手术组6组,每组5只。益气活血法中药生黄芪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。②方法:假手术组手术但不结扎输尿管,其他5组制备单侧输尿管梗阻致肾小管间质纤维化模型。手术前2d开始灌胃给药:中药大、中、小剂量组分别给予益气活血中药提取液0.072,0.036,0.018mL/(kg·d),给予福辛普利钠10mg/(kg·d),模型组及假手术组给予相同体积的生理盐水,1次/d,连续2周。③评估:术后14d处死大鼠,观察梗阻肾组织病理改变,免疫组化法检测肾组织纤维连接蛋白、层粘连蛋白和胶原蛋白Ⅳ的表达,通过医学病理图像分析系统对其积分吸光度进行统计学分析。结果:30只大鼠全部进入结果分析。①模型组层粘连蛋白、纤维连接蛋白和胶原蛋白Ⅳ的积分吸光度高于假手术组(P<0.05)。②中药小剂量组胶原蛋白Ⅳ的积分吸光度低于模型组(P<0.05),层粘连蛋白和纤维连接蛋白积分吸光度与模型组比较差异无显著性意义(P>0.05)。③中药大、中剂量组和西药组层粘连蛋白、纤维连接蛋白和胶原蛋白Ⅳ的积分吸光度均低于模型组(P<0.05),中药大、中剂量组胶原蛋白Ⅳ的积分吸光度低于西药组(P<0.05)。结论:益气活血法可以通过下调层粘连蛋白,纤维连接蛋白和胶原蛋白Ⅳ在肾组织的表达,从而减少细胞外基质的过度聚集,抑制或延缓肾间质纤维化。

AIM： To investigate the effect of qi supplement and blood activation therapy with traditional Chinese medicine （TCM） on expression of the extracellular matrix （ECM）, including fibronectin （FN）, laminin （LN） and collagen Ⅳ （Col-Ⅳ） in rats with renal interstitial fibrosis, and analyze the mechanisms of anti-renal interstitial fibrosis. METHODS： The experiment was carried out the Department of Anatomy, Histology and Embryology in Capital University of Medical Sciences from July to October in 2007.① Thirty male Wister rats were selected and randomly divided into the model group, the high, middle and low dose TCM groups, western medicine group and sham operation group with 5 rats in each group. Qi supplement and blood activation therapy was consisted of 15 g Shenghuangqi, 10 g Danggui, 10 g Chibaishao, 30 g Danshen, 10 g Huangqin, 15 g Cheqiancao and 15 g Niuxi. 4.4 kg/L crude drug extract was obtained by water extraction and alcohol precipitation.② Rat model of renal interstitial fibrosis was established in the 5 groups except sham group by the unilateral ureteral obstruction. The left ureter in rats of sham operation group was separated after abdomen opening without deligating the ureter. At two days before operation, rats in the high, middle and low dose TCM groups, and western medical group were administrated with corresponding medicine at the dose of 0.072, 0.036, 0.018 mL/kg.d and 10 mg/kg.d Monopril, 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 nephridial tissues of operating side were detected for pathology. The expressions of FN, LN and Col-Ⅳ in renal tissue were examined by immunohistochemistry. Medical pathology image analysis was performed to detect the integral photodensity for FN, LN and Col-Ⅳ. RESULTS： All 30 rats were involved in the results analysis.①Significant differences were found in the integral photodensity for FN, LN and Col-Ⅳ in model group compared to the sham operation group （P 〈 0.05）.②The integral photodensity of Col-Ⅳ in the low dose TCM group was lower than that in model group （P 〈 0.05）, while no significant difference was detected regarding the integral photodensity of FN and LN （P 〉 0.05）.③The difference in the integral photodensity for FN, LN and Col-Ⅳ were statistically significant in the high and middle TCM groups and western medicine group compared to the model group （P 〈 0. 05）. Moreover the integral photodensity in the higher and middle TCM groups was lower than that in western medicine group （P 〈 0.05）. CONCLUSION： Qi supplement and blood activation therapy can suppress or delay the development of renal interstitial fibrosis, because it can decrease the excessive aggregation of ECM via lessening expressions of FN, LN and Col-Ⅳ.