大黄酸对肾小球硬化肾皮质凋亡蛋白酶-3活性及细胞凋亡的影响

Effects of rhein on activity of caspase-3 in kidney and cell apoptosis on the progression of renal injury in glomerulosclerosis

目的观察大黄酸对肾小球硬化及细胞凋亡的影响,以探讨大黄酸对肾小球硬化的保护作用。方法一侧肾切除加尾静脉注射阿霉素制作肾小球硬化大鼠模型,随机分为对照组、肾病组、大黄酸治疗组和苯那普利治疗组,分别在第6、8、10、12周处死每组6只大鼠,免疫组织化学染色测定肾皮质凋亡蛋白酶3(caspase3),比色法测定caspase3活性,凝胶电泳迁移率转换实验(EMSA)检测核因子κB(NFκB)活性,原位末端转移酶标记法(TUNEL)检测肾组织细胞凋亡,观察caspase3、NFκB的表达与肾组织细胞凋亡情况。结果肾病组出现明显蛋白尿,血白蛋白下降及胆固醇上升,肾小球硬化指数、细胞凋亡指数、NFκB活性及caspase3表达随时间延长均明显高于对照组(P<0.05或P<0.01)。各时点比较,第10周肾病组见大量TUNEL阳性细胞,并略高于12周(9.3±2.3比8.4±1.2,P>0.05),caspase3表达也最为显著,明显高于12周(11.4±2.5比8.2±1.7,P<0.05),主要分布在肾皮质毛细血管周围,与凋亡细胞表达趋于一致。大黄酸或苯那普利治疗后,8周起TUNEL阳性细胞明显减少,并维持在一定水平,NFκB活性和caspase3表达均降低(P<0.05),肾脏病理变化及生化改变明显改善,肾皮质caspase3表达与细胞凋亡指数呈正相关(r=0.836,P<0.01)。结论大黄酸对肾小球硬化有明显防治作用,可能通过影响NFκB、caspase3活性在肾小球硬化早期调控病理改变,caspase3表达下降可能是大黄酸减轻肾小球硬化细胞凋亡的分子机制之一。

Objective To investigate the effects of rhein on the progression of renal injury and cell apoptosis in glomerulosclerosis, and further explore the protective mechanism of rhein on glomerulosclerosis.Methods Glomerulosclerosis models were made for SD rats by unilateral nephrectomy and being injected with Adriamycin into caudal vein, and randomly divided into control group, renal disease group, Rhein treatment group and Benazepril treatment group, and 6 rats in each group were killed at the 6th, 8th, 10th, 12th week respectively. The apoptosis protease-3 (caspase-3) in renal cortex was determined by immunohistochemistry stain method, and the activity of caspase-3 was measured by colorimetry, and the activity of nuclear factor-kappa B(NF-κB) was analyzed by gel electrophoretic mobility shift assay (EMSA), and renal tissue cell apoptosis was tested by terminal -deoxynucleotidyl transferase mediated nick end labeling(TUNEL) in order to observe expressions of caspase-3 and NF-κB and cell apoptosis of renal tissue.Results Renal disease group presented with distinct proteinuria, decreasing of blood albumin content and increasing of cholesterol concentration. Glomerulosclerosis index, apoptosis index, activity of NF-κB and expression of caspase-3 in renal disease group were more significantly higher than those in control group(P<0.05 or P<0.01) as time passed. Compared with the other time points in renal disease group, there were a great number of TUNEL-positive cells observed at the 10th week, slightly higher than that at the 12th week(9.3±2.3 vs 8.4±1.2,P>0.05), the expression of Caspase-3 was also most obvious at the 10th week, significantly higher than that at the 12th week (11.4±2.5 vs 8.2±1.7, P<0.05), which mainly located around capillary vessel in renal cortex, tending to be consistent with apoptosis cells expression. After the 8 weeks treatment of rhein or Benazepril, The number of TUNEL-positive cells significantly decreased and maintained at a certain level, and the activity of NF-κB and expression of caspase-3 decreased(P<0.05), and renal pathological changes and biochemical changes improved magnificently, moreover, the expression of caspase-3 showed positive correlation with apoptosis index(r=0.836,P<0.01). Conclusion Rhein could have significant protective effects on the progression of renal injury, and might regulate pathological changes by influencing the activities of NF-κB and caspase-3 in the early phase of glomerulosclerosis. Therefore, down-regulating caspase-3 expression in kidney might be one of the molecular mechanisms in the way that rhein could alleviate renal tissue cell apoptosis in glomerulosclerosis.