【作者】 隆献；

【导师】 何泽云；

【作者基本信息】 湖南中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的：1.研究5/6肾切除大鼠术后HGF/c-met信号通路的激活和六味地黄汤的干预机制；2.研究六味地黄汤不同组方对5/6肾切除大鼠肾功能的保护作用。方法：实验1：60只SD健康雄性大鼠随机分为空白组、假手术组、模型组、六味地黄汤组。模型组和六味地黄汤组均实施5/6肾切除术。术后稳定1周,六味地黄汤组采用六味地黄汤灌胃干预,其他组均以等体积蒸馏水灌胃。灌胃8周后处死动物。分别检测大鼠24小时尿蛋白定量、血清BUN和CR水平、残肾重量、残肾病理变化,和残肾组织HGF、c-met和CoL-Ⅳ在蛋白和mRNA水平的表达差异。实验2：60只SD健康雄性大鼠随机分为六味地黄汤组、三补组、三泻组。造模方法同实验1。术后稳定1周,六味地黄汤组采用六味地黄汤灌胃干预,三补组用六味地黄汤三补方(熟地、山莱萸、山药)灌胃,三泻组用六味地黄汤三泻方(泽泻、茯苓、牡丹皮)灌胃。灌胃8周后处死动物。检测方法同实验1。结果：1.5/6肾切除术后,大鼠残肾出现肾小球硬化、肾小管损伤和间质纤维化等病理改变,血清尿素氮、肌酐和24小时尿蛋白定量均升高(P<0.01)。六味地黄汤干预后,残存肾病理变化改善,24小时尿蛋白定量、血清肌酐和尿素氮水平明显降低(P<0.01)2.5/6肾切除术后,大鼠残肾组织HGF蛋白、c-met蛋白和Col-Ⅳ蛋白表达均升高(P<0.01)。六味地黄汤干预后,HGF蛋白和c-met蛋白表达进一步升高(P<0.05)、Col-Ⅳ蛋白表达减少(P<0.05)。5/6肾切除术后,残肾组织HGFmRNA表达升高(P<0.05),c-metmRNA和Col-ⅣmRNA表达也升高(P<0.01)。六味地黄汤干预后HGFmRNA表达进一步升高(P<0.01),c-metmRNA表达也升高(P<0.05),而Col-ⅣmRNA表达减少(P<0.05)。3.六味地黄汤三补方和六味地黄汤三泻方干预5/6肾切除大鼠后,残肾病理改变较重,24小时尿蛋白、血清肌酐和尿素氮水平均高于六味地黄汤组,HGF和c-met在蛋白和mRNA水平上表达均低于六味地黄汤组,而Col-Ⅳ在蛋白和mRNA水平上均高于六味地黄汤组。结论：1.5/6肾切除术后,大鼠残存肾出现肾小球硬化、肾小管损伤和间质纤维化等病理改变,血清尿素氮、肌酐含量水平和24小时尿蛋白定量升高,提示5/6肾切除大鼠模型复制成功。2.六味地黄汤干预可以减轻5/6肾切除大鼠病理改变,改善肾功能。3.5/6肾切除术后大鼠残肾存在HGF/c-met信号通路激活。4.六味地黄汤可以减少5/6肾切除大鼠残肾组织中Ⅳ型胶原的表达,减轻肾纤维化程度,这可能与六味地黄汤提高5/6肾切除大鼠残肾组织中HGF/c-met的表达有关。5.六味地黄汤原方改善5/6肾切除大鼠病理改变,保护肾功能的作用优于六味地黄汤三补方和六味地黄汤三泻方更多还原

【Abstract】 Objective:1. To study the activation of HGF/c-met pathway in5/6neph-rectomy rats and the treatment mechanism of Liuwei Dihuang Decoction.2.To study the protectionof different combinations of Liuwei Dihuang Decoction on kidney function in5/6Nephrectomy rats.Methods:Experiment1:The60healthy male Sprapue-Demey rats were randomly divided into normal group, sham operation group, model group and Liuwei Dihuang decoction group. Model group and Liuwei Dihuang decoction group both adopted5/6nephrectomy. A week after operation. Liuwei Dihuang decoction group were administrated with Liuwei Dihua-ng decoction, other groups all received equivoluminal distilled water. All animals were killed after8weeks. The24hour urinary protein, serum creatinine and blood ureanitrogen in rats were tested, the remnant kidney were weighted, the pathological changes were observed, the expression level of protein and mRNA of HGF、c-met and Col-Ⅳ in reremnant kidney were measured.Experiment2:The60healthy male Sprapue-Demey rats were randomly divided into Liuwei Dihuang decoction group, three tonifying ingredients group and three reducing ingredients group.Used the same way to set up rat model. A week after operation,Liuwei Dihuang decocti-on group were administrated with Liuwei Dihuang decoction, three tonifying ingredients group were administrated with three tonifying ingredients of Liuwei Dihuang decoction(Ehmannia,Fructus corni, Chine-se yam),and three reducing ingredients were administrated with three reducing ingredients group of Liuwei Dihuang decoction(Rhizoma alismatis, Poria cocos, Cortex Moutan). All animals were killed after8weeks. The observation methods are the same as experiment1.Results:l.After5/6neSphrectomy, the remnant kidney tissues of rats appeared pathological changes,such as glomerular sclerosis, renal tubular lesions and interstitial fibrosis, the levels of serum creatinine, blood urea-nitrogen and24h urinary protein all raised(P<0.01).After the intervene-tion of Liuwei Dihuang decoction, the pathological changes of remnant renal tissues improved, the levels of serum creatinine, blood ureanitrogen and24h urinary protein all obviously reduced (P<0.01)2. After5/6nephrectomy, the expression level of protein of HGF、 c-met and Col-Ⅳall increased in the remnant renal tissues of rats (P<0.01). After the intervention of Liuwei Dihuang decoction,the expression level of HGF protein and c-met protein levels further increased(P<0.05), while the expression level of Col-Ⅳprotein decreased (P<0.05). After5/6nephrectomy, the expression level of of HGFmRNA increased in the remnant renal tissues of rats (P<0.05), the expression level of c-met mRNA and Col-Ⅳ mRNA also further increased (P<0.01). After the intervention of Liuwei Dihuang decoction, the expression level of HGFmRNA further increased (P<0.01), the expression level of c-metmRNA further increased too (P<0.05), however,the expression level of Col-IV mRNA decreased (P<0.05)3. After the intervention of three tonifying ingredients of Liuwei Dihuang decoction,and three reducing ingredients of Liuwei Dihuang dec-oction, the pathological changes were more serious in the remnant renal tissues of rats, the levels of serum creatinine,blood urea nitrogen and24h urinary protein are higher than Liuwei Dihuang decoction group, the expression level of HGF protein, HGF mRNA, c-met protein and c-met mRNA are all less than Liuwei Dihuang decoction group,however, the expression level of Col-Ⅳ protein and Col-Ⅳ mRNA are higher than Liuwei Dihuang decoction group.Conclusion:1. After5/6nephrectomy, the remnant renal tissues of rat appeared pathological changes,such as glomerular sclerosis, renal tubular lesions and interstitial fibrosis, the levels of serum creatinine、 blood urea nitrogen and24h urinary protein all raised. Suggested success-fully copied5/6nephrectomy rat model.2. Liuwei Dihuang decoction can reduce pathological changes and improve renal function in5/6nephrectomy rats. 3. After5/6nephrectomy, HGF/c-met pathway was activated in the remnant renal tissues of rats.4. Liuwei Dihuang decoction can reduce the expression level of Col-Ⅳ, alleviate renal fibrosis. Which might be relative to the function that Liuwei Dihuang decoction can improve the expression level of HGF/c-met in the remnant renal tissues of rats.5. Liuwei Dihuang decoction can reduce pathological changes and protect renal function in5/6nephrectomy rats. Its roles were better than th-Ree the tonifying ingredients of Liuwei Dihuang decoction and the three reducing ingredients of Liuwei Dihuang decoction.更多还原