【作者】 吴汉利；

【导师】 孙伟；

【作者基本信息】 南京中医药大学 ， 中西医结合临床， 2010， 博士

【摘要】 目的：本实验研究观察雷至胶囊治疗肾病综合征的疗效及减毒增效作用；并探讨雷至胶囊对蛋白尿、肾小球系膜、肾小管的影响及其细胞分子机制。方法：将健康雌性SD大鼠60只,随机分为正常组(10只)、阿霉素肾病模型组(10只)、雷公藤多甙组(10只)、雷至胶囊低剂量组(10只)、雷至胶囊中剂量组(10只)、雷至胶囊高剂量组(10只),尾静脉注射法(第一天4mg/kg体重,第七天3mg/kg体重)制作肾病综合征模型。用药剂量及方法：正常组和模型组：生理盐水1ml.100g-1.d-1；雷公藤多甙组：2mg.100g-1.d-1体重；雷至胶囊低剂量组：0.4g.100g-1.d-1；雷至胶囊中剂量组：0.8g.100g-1.d-1;雷至胶囊高剂量组：1.2g／100g-1.d-1,上述用药均为第一次造模2周后开始,每日一次,疗程3周。①观察一般情况及体重,造模前、第一次造模后第14天、给药第7、14、21天,测24HUPQ、尿NAG酶②末次治疗给药后24h后,眼眶取血检测Scr、Bun、ALB、TG和CHO、ALT和AST。③末次给药24h后,West-Blot法检测大鼠肾组织nephrin、podocin、podocalyxin、TGF-β、MMP-9的表达；RT-PCR法检测大鼠肾组织nephrin. podocin、podocalyxin;实时定量PCR法检测大鼠肾组织Dicer酶的表达；免疫荧光法检测大鼠肾组织podocalyxin的表达。④末次给药24h后,将摘除右肾组织,在Jem-1011透射电镜下观察；其余部分肾组织及部分肝脏组织经PAS染色,光镜下观察。(注：分子生物学指标及病理仅检测了正常组、模型组、雷公藤多甙组及雷至胶囊中剂量组)。结果：①雷至胶囊对阿霉素肾病大鼠血液生化学及尿蛋白的影响：给药后第7天、给药后第14天、给药后第21天,各治疗组24HUPQ均显著低于模型组(P＜0.01)；给药后第14天、给药后第21天,雷至胶囊低、中、高剂量组明显低于雷公藤多甙组(P均＜0.01)。给药三周后各治疗组血清白蛋白均显著高于模型组(P＜0.01)；雷至胶囊低、中、高剂量组血清白蛋白明显高于雷公藤多甙组(P＜0.01)。血脂结果显示：各治疗组血清TG, CHO含量显著低于模型组(P＜0.01)；雷至胶囊低、中、高剂量组血清TG、CHO显著低于雷公藤多甙组(P＜0.01)；雷至胶囊低、中、高剂量组间无统计意义。肝功结果：雷公藤多甙组血清ALT、AST显著高于其它各组(P＜0.01)。其它各组间BUN、Scr水平比较无显著性差异(P＞0.05)。②雷至胶囊对阿霉素肾病大鼠病理学的影响：光镜下观察,模型组大鼠可见肾组织：弥漫性肾小球系膜区增宽,系膜细胞、系膜基质增生,肝组织：雷公藤多甙组肝细胞排列紊乱,基质明显增多,其他各组组肝脏结构无明显变化。透射电镜检测结果,模型组可见肾小球足细胞足突的广泛融合,系膜基质增多,肾小球基底膜增厚等；各治疗组病变程度明显较模型组轻,仅见足突部分融合。③雷至胶囊对阿霉素肾病大鼠肾小球系膜基质的影响：TGF-β在治疗组显著低于模型组(P＜0.01),雷至胶囊组与雷公藤多甙组间无显著性差异；MMP-9在治疗组显著高于模型组(P＜0.01),在雷至胶囊组也显著高于雷公藤多甙组(P<0.01)。另外相关性研究显不,TGF-β、24HUPQ、TG、CHO与系膜基质指数呈正相关(P<0.01), MMP-9与系膜基质指数呈负相关(P＜0.01)。④雷至胶囊对阿霉素肾病大鼠肾小管的影响：给药后第7天、给药后第14天、给药后第21天,治疗组NAG酶量均显著低于模型组(P均＜0.01),雷至胶囊组NAG酶量也明显低于雷公藤多甙组(P＜0.05)。另外相关性研究发现,TGF-β、24HUPQ与NAG呈正相关(P<0.01), MMP-9与NAG呈负相关(P＜0.01)。⑤雷至胶囊对阿霉素肾病大鼠肾脏组织SD分子nephrin、podocin的影响：模型组nephrin、podocin蛋白表达降低,而基因表达nephrin下降、podocin上升,与正常组相比均有明显差异(P均＜0.01)；与模型组相比,各治疗组明显抑制nephrin、podocin模型组改变(P均＜0.01)；相关性分析显示,nephrin蛋白和基因、podocin蛋白与24HUPQ呈负相关(P均＜0.01),podocin基因与24HUPQ呈正相关(P＜0.01)。⑥雷至胶囊对阿霉素肾病大鼠肾脏组织足细胞分子podocalyxin的影响：模型组podocalyxin蛋白表达明显降低,基因表达明显上升,与正常组相比均有明显差异(P均<0.01)；与模型组相比,各治疗组明显抑制podocalyxin蛋白和基因模型组改变(P均＜0.05)。相关性分析显示,Podocalyxin蛋白与24HUPQ呈负相关,而基因与24HUPQ呈正相关。⑦雷至胶囊对早期阿霉素肾病大鼠肾脏组织足细胞Dicer酶的影响：模型组Dicer酶mRNA表达显著低于正常组(P＜0.01)；与模型组相比,雷至胶囊组Dicer酶mRNA表达显著高于模型组(P＜0.05)；雷至胶囊组与雷公藤多甙组间比较无统计学意义。相关性分析显示Dicer酶mRNA与pococin mRNA、podocalyxin mRNA、24HUPQ呈负相关,Dicer酶mRNA与nephrin mRNA呈正相关。结论：1本研究证明雷至胶囊能有效降低阿霉素肾病大鼠蛋白尿,且在改善阿霉素肾病大鼠营养状况、调节蛋白质代谢及脂质代谢方面的良好作用,与雷公藤多甙相比,功效明确,无明显肝脏等毒副作用,体现了减毒增效的效果。2雷至胶囊治疗后大鼠病理改变明显减轻,为雷至胶囊治疗阿霉素肾病大鼠、减轻蛋白尿提供了组织病理学上的依据。3雷至胶囊可通过①降低尿蛋白②降低高脂血症③抑制TGF-β和提高MMP-9表达④保护足细胞作用,抑制肾小球系膜基质增生。4雷至胶囊可通过降低TGF-β蛋白表达、24HUPQ,提高MMP-9蛋白表达,保护阿霉素肾病大鼠肾小管免受损伤。5雷至胶囊可通过调控炎症因子(TGF-β、MMP-9)、Dicer酶、足细胞分子nephrin、podocin、podocalyxin等多靶点治疗阿霉素肾病大鼠蛋白尿。更多还原

【Abstract】 Objective:Based on the theories of Traditional Chinese Medicine and modern medicine. This experimental research is designed to study the impact of Leizhi capsule on Adrianmycin-inducded nephritic syndrome in rats and part of the mechanism of action from the perspectives of Hemo-Biochemistry,Histopathology and Molecular Biology.Methods:60 female SD rats were divided at random into the following groups:the normal group(n=10), the model group(n=10), the high-dosed Leizhi capsule group(n=10), the middle-dosed Leizhi capsule group(n=10), the low-dosed Leizhi capsule group(n=10), the tripterygium glycosides group (n=10). The nephritic syndrome mode induced by etail intravenous injection of adriamycin in day 1 and day 7. The administrations of medicin were as follows:the normal and model groups, saline lml.100g-1.d-1; the tripterygium glycosides 2mg.100g-1.d-1; the high-dosed Leizhi capsule group 1.2mg.100g-1.d-1; the middle-dosed Leizhi capsule group 0.8g.100g-1.d-1; the low-dosed Leizhi capsule group 0.4g.100g-1.d-1. All the administrations metioned above began two week after the models had been made, each medicine was administered once a day. The period of treatment was three weeks.①Observe the general conditions and weight of body;check 24 hour urea protein quantity(24HUPQ), before and 14 days after the model had been made first, and on the 7th,14th,21th day after theadministation of related medieine.②24 hours after the last administration of medieine, check Scr, Bun, ALB, TG,CHO.③24 hours after the last administration of medicine, the expression of nephrin,podocin, podocalyxin mRNA and protein of the kidney tissues of rats were examined through RT-PCR and West-Blot method. The expression of TGF-βand MMP-9 protein of the kidney tissues of rats were examined through West-Blot method.The expression of Dicer mRNA of the kidney tissues of rats were examined through real-time PCR method. The expression of podocalyxin of the kidney tissues of rats were examined through indirect immunofluorescence stainin method.④24 hours after the last administration of medicine, the removed kidney tissues were observed under Jem-1011 penetrating electric microscope; The part of kidney tissues and hepatic tissues, which were dyed with PAS after being dealt with, were observed under light microscope. (Note:The indicators of molecular biology and pathology detected only in the middle-dosed Leizhi capsule group).Results:①The research of the impact of the high-dosed Leizhi capsule group on the Hemo-biochemistry and urea protein of the Adrianmycin-induced rats with kidney disease. On the 7 th, 14th.,21th day after the administration of medicine,24HUPQ of the treatment groups was significantly lower than that of the model group(P<0.01),24HUPQ of the high-dosed,the middle-dosed, the low-dosed group was significantly lower than that of the the tripterygium glycosides group(P<0.01)。3 weeks after the administration of medicine, the serum ALB of the treatment groups were examined to be significantly higher than that of model group(P<0.01);the serum level of ALB the high-dosed,the middle-dosed, the low-dosed groups was examined to be higher than that of the tripterygium glycosides group(P<0.01). The content of TG and CHO in the treatment groups was significantly lower than that in the model group(P<0.01); The level of TG and CHO in the high-dosed,the middle-dosed,the low-dosed groups was significantly lower than that in the tripterygium glycosides group(P<0.01); There was no significant difference between the high-dosed,the middle-dosed,the low-dosed groups (p>0.05). There was no significant difference when the levels of Bun and Scr in different groups were compared with each other(p>0.05).②The pathological research of the effect of Leizhi capsule on the Adrianmycin-induced rats with kidney disease.Under the light microscope, the diffuse hyperplasia of mesangium was seen in the model group. It could be seen under penetrating electric microscope that the main pathological changes of Adrianmycin induced rats with kidney disease were the widespread fusion of foot protrusion in the glomerulus of kidney and hyperplasia of mesanguim matrix, thickening of glomerular basement membrane;In the treated groups the degree of pathological changes in rats was obviously slighter than that in the model group, and only part of the fusion of foot protrusion could be seen.③The research of the impact of Leizhi capsule on the glomerular mesangial matrix of Adrianmycin-induced rats with kidney disease.The TGF-βin the treated groups were significantly lower than that in the model group(P<0.01).There was no significant difference when the levels of TGF-βin different groups were compared with each other(p>0.05). The MMP-9 in the treated groups were significantly higher than that in the model group(P<0.01);The MMP-9 in the Leizhi capsule groups were significantly higher than that in the the tripterygium glycosides group(P<0.01). Correlation studies have shown that TGF-β,24HUPQ, TG, CHO and mesangial matrix index were positively correlated (P<0.01), MMP-9 and mesangial matrix index was negatively correlated (P<0.01.④The research of the impact of Leizhi capsule on the tubular cells of Adrianmycin-induced rats with kidney disease. On the 7 th,14th.,21th day after the administration of medicine, the NAG in the treated groups were significantly lower than that in the model group(P<0.01), the NAG in the Leizhi capsule groups were significantly lower than that in the tripterygium glycosides group(P<0.05). Correlation studies have shown that TGF-β,24HUPQ and NAG were positively correlated (P<0.01), MMP-9 and NAG was negatively correlated (P <0 .01).④The research of the impact of Leizhi capsule on nephrin,podocin of Adrianmycin-induced rats with kidney disease. The protein expression of nephrin, podocin was reduced, while gene expression of nephrin was decreased, gene expression of podocin was increased in the model group. There was significant difference when the levels of nephrin, podocin in model and nomal glycosides groups were compared with each other(p<0.01). Leizhi capsule and tripterygium was significantly inhibited changes of nephrin, podocin in the model group(p<0.01). Correlation studies have shown that protein and mRNA of nephrin,protein of podocin and 24HUPQ were negatively correlated (P<0.01), mRNA of podocin and 24HUPQ was positively correlated (P <0 .01).⑥The research of the impact of Leizhi capsule on podocalyxin of Adrianmycin-induced rats with kidney disease. The protein expression of podocalyxin was reduced, while gene expression of podocalyxin was increased in the model group. There was significant difference when the levels of podocalyxin in model and nomal glycosides groups were compared with each other(p<0.01). Leizhi capsule and tripterygium was significantly inhibited changes of podocalyxin in the model group(p<0.01). Correlation studies have shown that protein of podocalyxin,protein of podocin and 24HUPQ were negatively correlated (P<0.01), mRNA of podocalyxin and 24HUPQ was positively correlated (P<0.01).⑦The research of the impact of Leizhi capsule on Dicer of Adrianmycin-induced rats with kidney disease. The mRNA of Dicer in the model group were significantly higher than that in the nomal group(P<0.01); The mRNA of Dicer in the Leizhi capsule groups were significantly higher than that in the model group(P<0.01); There was no significant difference in dicer expression between Leizhi capsule group tripterygium glycosides group. Correlation studies have shown that mRNA of dicer and pococin mRNA、podocalyxin mRNA,24HUPQ were negatively correlated (P<0.01), mRNA of dicer and nephrin mRNA was positively correlated (P <0 .01).Conclusion:1.This research proved that Leizhi capsule could effeetively bring down the protein uria of the Adrianmycin-induced rats with kidney disease, and play a wholesome role in improving the nutrient conditions of Adrianmycin-induced rats and regulating the metabolism of protein and lipid. Compared with tripterygium glycosides, Leizhi capsule has the obviouse anti-proteinuria effectiveness and no obvious side effects such as the liver which reflects the attenuated, synergistic effect.2.After the treatment with Leizhi capsule, the pathological change were significantly relieved, this provided a histo-pathological evidence for the suggestion that Leizhi capsule could be used to treat adrianmycin-induced rats with kidney disease and reduce the proteinuria.3.Leizhi capsule inhibit proliferation of glomerular mesangial matrix probably through①reducing urinary protein②reducing hyperlipidemia③inhibitting TGF-βand enhance MMP-9 expression④protecting podocyte.4.Leizhi capsule protect renal tubular cells probably through reducing urinary protein,inhibitting TGF-βand enhancing MMP-9 expression.5. Leizhi capsule could bring down the proteinuria of the Adrianmycin-induced rats through the multi-target regulation of inflammatory factors (TGF-P, MMP-9), Dicer enzyme, nephrin, podocin, podocalyxin etc.更多还原