单侧输尿管梗阻大鼠肾脏组织基质金属蛋白酶3、基质金属蛋白酶2和金属蛋白酶组织抑制因子2的表达及益气活血法的影响

Effect of qi supplement and blood activation therapy on the expression of matrix metalloproteinase-3, 2 and tissue inhibitor of metalloproteinases-2 in rats with renal interstitial fibrosis induced by unilateral ureteral obstruction

目的:肾间质纤维化是肾脏疾病进展到肾功能衰竭的共同通路。通过观察益气活血法中药制剂对肾间质纤维化大鼠肾组织基质金属蛋白酶3,基质金属蛋白酶2和金属蛋白酶组织抑制因子2表达的影响,探讨其抗肾间质纤维化的作用机制。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,采用随机数字法分为模型组、西药蒙诺组、中药小剂量组、中药中剂量组、中药大剂量组和假手术组。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,分离其左侧输尿管,不结扎即关闭腹腔。益气活血法中药制剂主要由生黄芪,当归,赤白芍,丹参,黄芩,车前草,牛膝等组成。西药组、中药小剂量组、中药中剂量组和中药大剂量组于手术前2d开始灌胃给药,1次/d,其药量分别为:西药组蒙诺10mg/(kg·d)、小剂量组0.018mL/(kg·d)、中剂量组0.036mL/(kg·d)、大剂量组0.072mL/(kg·d),连续给予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,观察梗阻肾脏病理改变,并用免疫组织化学方法检测肾组织对基质金属蛋白酶2、基质金属蛋白酶3和金属蛋白酶组织抑制因子2的表达,通过医学病理图像分析系统对基质金属蛋白酶2、基质金属蛋白酶3和金属蛋白酶组织抑制因子2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。肾组织切片免疫组织化学方法结果显示:基质金属蛋白酶2、基质金属蛋白酶3和金属蛋白酶组织抑制因子2主要表达部位在肾小管上皮,少量表达在肾间质和肾小球。基质金属蛋白酶2和基质金属蛋白酶3在模型组的表达较假手术组明显减弱,两组间积分光密度比较差异有显著性意义(P<0.05);中药大、中剂量组及西药蒙诺组的表达较模型组显著增强,积分光密度差异有显著性意义(P<0.05)。金属蛋白酶组织抑制因子2在模型组的表达较假手术组显著增强,两组间积分光密度比较差异有显著性意义(P<0.05);中药大、中剂量组及西药蒙诺组的表达较模型组有所减弱,两组间积分光密度比较差异有显著性意义(P<0.05);中药小剂量组无论基质金属蛋白酶2和基质金属蛋白酶3还是金属蛋白酶组织抑制因子2的表达与模型组相比均较为相似,两组间积分光密度比较差异无显著性意义(P>0.05)。结论:大鼠输尿管梗阻后呈现出的病理损害可能和肾组织基质金属蛋白酶3,基质金属蛋白酶2与金属蛋白酶组织抑制因子2的表达失衡有关,益气活血法可以上调基质金属蛋白酶2,基质金属蛋白酶3的表达,下调金属蛋白酶组织抑制因子2的表达,显示在抑制或延缓肾间质纤维化的进程中具有一定的作用。

AIM: Renal interstitial fibrosis is the common development process of renal diseases to renal failure. In this study, the effect of qi supplement and blood activation therapy with traditional Chinese medicine on the expression of matrix metalloproteinase-3 (MMP-3), metalloproteinases-2 (TIMP-2) and tissue inhibitor of TIMP-2 in rats with renal interstitial fibrosis was investigated, so as to explore the mechanisms of anti-renal interstitial fibrosis. METHODS: The experiment was performed in the Department of Anatomy and Histology in Capital Medical University from December 2006 to June 2007. ①Thirty male Wistar rats were selected and randomly divided into model group, western medicine group (Monopril), traditional Chinese medicine high, middle and low dose groups, and sham operation group with 5 rats in each group. All treatment during the experiment was accorded with the animal ethical standards. ②Rat models of renal interstitial fibrosis were established in the former 5 groups by unilateral ureteral obstruction, while in the sham operation group, the left ureter of the rats were separated after abdomen-opening without deligating the ureter. Qi supplement and blood activation therapy with traditional Chinese medicine was consisted of milkvetch root, angelica root, paeoniae radix, salvia miltiorrhiza, baical skullcap root, plantain, achyranthis radix. Rats in the high, middle and low dose groups were administrated with the traditional Chinese medicine at 0.072, 0.036, 0.018 mL/kg·d, and in western medicine group with 10 mg/kg·d Monopril for 2 weeks. Rats in the 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. The expressions of MMP-3, MMP-2 and TIMP-2 in renal tissue were examined by immunohistochemistry. Pathological image analysis was performed to detect the integral photodensity for MMP-3, MMP-2 and TIMP-2. RESULTS: All 30 rats were involved in the result analysis. Immunohistochemistry staining studies indicated MMP-3, MMP-2 and TIMP-2 were mainly expressed in renal tubular cells of renal cortex, and rarely in renal interstitium and renal glomerulus. MMP-3 and MMP-2 were weakly expressed in model group compared to the sham operation group, and the differences in the integral photodensity between them were statistically significant (P < 0.05). MMP-3 and MMP-2 were strongly expressed in the traditional Chinese medicine middle and high dose groups and western medicine group compared to the model group. The differences in the integral photodensity among them were statistically significant (P < 0.05). TIMP-2 was strongly expressed in model group compared to the sham operation group, and the differences in the integral photodensity between them were statistically significant (P < 0.05). TIMP-2 was weakly expressed in the traditional Chinese medicine middle and high dose group and western medicine group compared with the model group. The differences in the integral photodensity among them were statistically significant (P < 0.05). There was no statistical difference in the integral photodensity about MMP-3, MMP-2 and TIMP-2 between low dose group and model group (P > 0.05). CONCLUSION: The pathological lesion after unilateral ureteral obstruction is concerned with the expression misbalance of MMP-3, MMP-2 and TIMP-2 in kidney tissue. Qi supplement and blood activation therapy can up-regulate the expression of MMP-3 and MMP-2, and down-regulate the expression of TIMP-2, indicating it plays a role in suppressing or delaying the development of renal interstitial fibrosis.