【作者】 姚琼；

【导师】 邱幸凡； 张六通；

【作者基本信息】 湖北中医药大学 ， 中医基础理论， 2010， 博士

【摘要】 目的：肾病综合征(Nephrotic syndrome, NS)是临床上常见多发的疾病,西医治疗难度大,而中医治疗取得了显著成果,故从新的理论总结开始,开展了本课题研究。本课题分为理论探讨和实验研究两大部分,以邱幸凡教授提出的“脏虚络痹”病机理论为基础,拟定了温阳利湿通络方,运用现代医学研究方法,深入研究了该方对阿霉素诱导的肾病综合征大鼠模型的治疗作用及其部分机制,目的在于为临床NS的治疗提供新的理论和实验依据。理论探讨：1、基于络脉络病理论及“脏虚络痹”的病机理论,提出“肾脾阳虚,湿瘀阻络”是NS的基本病机：(1)肾脾阳虚是致病之本：肾脾阳虚是NS的基本病机；(2)湿瘀阻络为致病之标：湿瘀阻络是NS发生的病理关键；(3)脏虚络痹相互影响,互为因果。2、根据病机,提出“温肾补脾,利湿泄浊,化瘀通络”是NS的基本治疗法则：(1)补脏以治其本：以补脾肾之阳为基本；(2)通络以治其标：“利湿泄浊,化瘀通络”是治疗NS的关键；3、提出治疗NS的方药为“温阳利湿通络方”。该方由熟附片10g、黄芪30g、党参10g、白术10g、泽泻10g、车前子15g、水蛭6g和蝉蜕10g组成。方法：本研究采用尾静脉一次性注射阿霉素6mg/kg的方法建立阿霉素诱导的肾病综合征大鼠模型,即按Bertani法造模,进行了温阳利湿通络方干预该模型的实验研究。将40只Wistar雄性大鼠,随机分为正常组(n=10)、模型组(n=10)、中药组(n=10)、西药组(n=10)。造模成功后,正常组和模型组灌生理盐水(1ml/100g体重)；中药组灌温阳利湿通络方(1ml/100g体重)；西药组灌盐酸贝那普利溶液(0.3mg/100g体重),均每日一次,连续灌胃四周。给药期间每天观察食量、体毛、大便、精神状态、体重及水肿情况。尿标本检测24小时尿蛋白定量(24HUPQ)、β2微球蛋白(β2-MG)、尿白蛋白(ALB),采用干化学法检测；血标本检测血肌酐(Scr)、尿素氮(Bun)、总蛋白(TP)、白蛋白(ALB)、甘油三酯(TG)和胆固醇(CHOL),采用全自动生化分析仪测定。ET-1、VEGF及TGF-β1的变化采用免疫组化法研究。TGF-β1mRNA的表达采用RT-PCR方法检测。结果：1、对大鼠一般情况的观察：正常组大鼠活动灵活,反应较快,皮毛致密、有光泽,没有出现水肿；模型组大鼠造模后3天出现活动减低,精神差、体毛翻转凌乱、大便稀,食量减少,3周后出现水肿并逐渐加重,尤以腹水明显；中药组与西药组大鼠经治疗后,精神较模型组好,食量好,大便呈颗粒状,活动情况好转,且水肿较轻。模型组、中药组、西药组在造模成功时,体重较正常组显著下降(P<0.01)；实验结束时,中药组和西药组大鼠体重显著高于模型组(P<0.05),而中药组与西药组之间无显著性差异；2、对大鼠蛋白尿的影响：模型组、中药组、西药组于造模后第7天开始,24HUPQ均明显高于正常组(P<0.01),说明造模成功。治疗后第14、21、28天,中药组的24HUPQ明显低于模型组(P<0.01),中药组与西药组比较无显著性差异。实验结束时,中药组的尿β2-MG明显低于模型组(P<0.01),尿ALB低于模型组(P<0.05)。中药组与西药组比较无显著性差异；3、对大鼠血脂的影响：实验结束时,模型组大鼠TG、CHOL结果明显高于正常组(P<0.01),中药组和西药组的TG结果低于模型组(P<0.01),中药组与西药组比较无显著性差异；中药组和西药组的CHOL结果低于模型组(P<0.05)。中药组与西药组比较无显著差异；4、对大鼠肾功能的影响：实验结束时,模型组、中药组、西药组与正常组比较,血Sci、BUN的结果均无显著性差异；5、对大鼠血清蛋白的影响：实验结束时,模型组、中药组、西药组大鼠的TP、ALB结果均明显低于正常组(P<0.01),中药组和西药组大鼠的TP、ALB结果高于模型组(P<0.05),而中药组与西药组之间无显著性差异;6、对大鼠ET-1、VEGF、TGF-β1及TGF-β1mRNA水平的影响:模型组与中药组的ET-1、VEGF、TGF-β1及TGF-β1mRNA水平均明显升高(与正常组相比,P<0.01),但中药组的ET-1、VEGF、TGF-β1及TGF-β1mRNA水平较模型组显著降低(P<0.05),中药组与西药组无显著性差异；7、病理学观察：在光镜下,模型组大鼠HE及PAS染色病理切片有轻度系膜增生,其它各组肾小球及肾小管均未见明显病理变化。在电镜下,正常组大鼠肾小球基底膜未见增厚,系膜未见增生,足突清晰完整,未见融合。模型组大鼠肾小球基底膜未见增厚,局部系膜细胞、基质增生,足突间隙消失,足突广泛融合,尚可见部分内皮细胞异染色质染色加深、成块、趋边,核孔增宽,出现细胞凋亡；模型组大鼠可见部分肾小管细胞周边水肿,溶酶体增多。中药组和西药组,肾小球病变程度相似,基底膜平滑,未见增厚,系膜未见增生,足突部分融合,肾小管细胞结构正常,与模型；组比较,病变程度明显较轻。结论：本研究结果提示：温阳利湿通络方能显著改善阿霉素诱导的肾病综合征大鼠的一般营养状况；提高阿霉素诱导的肾病综合征大鼠血清白蛋白、总蛋白水平,降低大鼠蛋白尿、甘油三酯、胆固醇水平。该方对阿霉素诱导的肾病综合征大鼠肾组织的超微结构有改善作用。其治疗机制可能与下调ET-1、VEGF、TGF-β1及-TGF-β1mRNA水平有关,在降低ET-1、VEGF、TGF-β1及TGF-β1mRNA水平的过程中,减少了中性粒细胞和单核巨噬细胞浸润,抑制炎症细胞的聚集活化和炎症介质的释放,从而减轻足突细胞损伤,减少足突融合,达到保护肾小球滤过膜的结构和功能的作用,,最终降低尿蛋白,并且,因其减轻了肾脏免疫损伤,从而能调节蛋白和脂质代谢,抑制脂质过氧化物的形成,达到升高血清白蛋白,降低血脂的作用,因此,对ET-1、VEGF、TGF-β1及TGF-β1mRNA的研究,从细胞免疫及分子角度更深入阐明了温阳利湿通络方对NS治疗作用的分子机制。提示：温阳利湿通络法及其方是中医治疗肾病综合征的一种优势方法。更多还原

【Abstract】 Objective:Nephrotic syndrome (Nephrotic syndrome, NS) was a common disease, treatment with Western medicine was difficult, and traditional Chinese medical treatment had achieved significant results, so to sum up the new theory, carried out research of this topic. This topic was divided into two parts:theoretical studies and experimental studies. According to Professor Xingfan Qiu proposed "ZangXuLuoBi" pathogenesis theory, developed OnyangLishiTongluoFang.In this study, using modern detection methods, to study the OnyangLishiTongluoFang for Adriamycin-induced nephrotic syndrome in rats of the therapeutic effect and mechanism. The purpose of this study was to provide for the treatment of nephrotic syndrome in new theoretical and experimental basis.Theory probing:1.Based on a Basic Theory of TCM and the "ZangXuLuoBi" sick machine theory, "ShiYuZuLuo" was the basic pathogenesis of NS.①"ShenPiYangXu" was the root cause of the disease. "ShenPiYangXu" was the basic pathogenesis of NS.②"ShiYuZuLuo" as the external factors caused disease. "ShiYuZuLuo" was an important factor in NS occurs.③"ZangXu" and "LuoBi" mutual influenced reciprocal causation.2. According to the patient machine, that "WenshenBupi, LishiXiezhuo, HuayuTongluo" was the basic treatment of NS.①"BuZang" to treat its fundamental:"BuZang" referred to compensation "yin and yang, qi and blood." Compensation "Yang of Shen and Pi" was the basic.②"TongLuo" to treat the symptoms: "TongLuo" was an important part of treatment of NS; "LiShiHuaYu" was the key to treatment of NS.Methods:In this study, single tail vein injection of Adriamycin 6mg/kg method to establish model of adriamycin-induced nephropathy, according to Bertani’s modeling method, conducted the OnyangLishiTongLuoFang to intervene in the model experiments.40 Wistar male rats were randomly divided into normal group (n=10), model group (n=10), TCM group (traditional Chinese medicine group, n=10), western medicine group (n=10). Modeling was successful, normal group and model group were gavaged with normal saline (1ml/100g body weight); TCM group was gavaged with OnyangLishiTongluoFang (1ml/100g body weight); Western medicine group was gavaged Benazepril hydrochloride (0.3mg/100g body weight), all of the rats were gavaged once a day, administered orally 4 weeks. During treatment, observed their daily food intake, body hair, stool, mental state, body weight and edema. Urine specimens:a 24-hour urinary protein (24HUPQ),β2 micro globulin (β2-MG), urinary albumin (ALB), dry chemical method detection; blood samples:serum creatinine (Scr), blood urea nitrogen (Bun), total protein (TP), albumin (ALB), triglyceride (TG) and cholesterol (CHOL), using automatic biochemical analyzer. ET-1, VEGF, and TGF-β1 changes studied by immunohistochemistiy. The expression of TGF-β1mRNA was observed using RT-PCR.Results:1. On the general observation in rats:Normal group rats of the activities were flexible; they responded freely, their dense fur shined without edema. Model group rats, after modeling in the first 3 days appeared to reduce activity, mental difference, turning messy hair, loosed stool, food intake, after 3 weeks arid gradually increased edema, ascites was particularly significant; After treatment, TCM group and western medicine group rats, compared with model group, the spirit was better, ate better, stool granular, activities were better, lighter, and edema. Model group, TCM group and western medicine treatment group after the success inmodeling, body weight decreased significantly compared with the normal group (P＜0.01); At the end of the experiment, TCM group and western medicine group body weight were significantly higher than model group;(P＜0.05), while the TCM group and western medicine group there iwas no significant difference;2.The impact of proteinuria in rats:model group, TCM group; western medicine group at 7 days after modeling,24HUPQ were significantly higher than the normal group (P＜0.01), shows the model was successful.14,21,28 days after treatment, TCM group 24HUPQ significantly lower than model group (P＜0.01),TCM group and western medicine group showed no significant difference.At the end of the experiment, the Medicines for urinaryβ2-MG was significantly lower than model group (P＜0.01), urinary ALB compared with model group (P＜0.05). TCM group and western medicine group showed no significant difference;3. On serum lipids in rats:At the end of the experiment, the model group TG, CHOL results were significantly higher than the normal group (P＜0.01), Western medicine group and TCM group were significantly lower than the model group TG (P＜0.01),TCM group and western medicine group showed no significant difference; Western medicine group and TCM group the results of CHOL were significantly lower than model group (P＜0.05). TCM group and western medicine group showed no significant difference;4.On renal function:At the end of the experiment, the model group,TCM group, Western group compared the results with the normal group,,blood Scr, BUN results were not significantly different;5.On serum protein: At the end of the experiment,the model group, TCM group, Western group of rats TP, ALB results were significantly lower than normal group (P＜0.01), TCM group and western medicine group rats TP, ALB results than model group (P<0.05), TCM group and western medicine group showed no significant difference;6.Rat ET-1,VEGF,TGF-β1 and TGF-β1mRNA levels:Model group and TCM group of ET-1, VEGF, TGF-β1 and TGF-β1mRNA levels were significantly increased (compared with the normal group,P＜0:01); but the TCM group ET-1, VEGF,TGF-β1 and TGF-β1mRNA levels were significantly higher than the model group lower (P＜0.05), TCM group and western medicine group there was no significant difference;7. In the pathological study, in the light microscope could be observed:Model group rats of HE and PAS staining biopsy had mild mesangial proliferation, glomerular and renal tubular other groups were no significant pathological changes. In the electron microscope could be observed:the normal group was not thickened glomerular basement membrane, mesangial proliferation was not enough to suddenly clear and complete, not integration. Model group rat glomerular basement membrane was not thickened, partial mesangial cells, stromal hyperplasia, foot processes of the gap disappeared, extensive foot process fusion, was still visible part of endothelial cells in heterochromatin stain deeper into the block, trend edge, nuclear pore widened, there apoptosis; Model group visible part of the renal tubular cells in rat peripheral edema, increased lysosome. TCM group, western medicine group had a similar level of glomerular lesions, basement membrane smooth, no thickening, mesangial no hyperplasia, some foot process fusion, renal tubular cell structure of normal, compared with model group, lesions significantly lesser degree.Conclusion:The results suggested that OnyangLishiTongluoFang could be significantly improved in rats with adriamycin nephropathy general nutritional status; could enhance adriamycin-induced nephropathy rat serum albumin, total protein levels, reduced proteinuria in rats, triglyceride cholesterol level; their treatment might be associated with reduced ET-1, VEGF, TGF-β1 and TGF-β1mRNA related to the level; the party treated adriamycin nephrotic rats were morphological evidence, there might be a network mechanism for immune regulation. Prompt "OnyangLishiTongluo" treatment was an effective traditional Chinese medicine treatment of nephrotic syndrome.更多还原