温阳活血利水法对足细胞骨架蛋白信号通路影响的研究

发布时间：2018-11-21 15:42

【摘要】：目的：观察温阳活血利水法对阿霉素肾病大鼠肾小球足细胞的骨架蛋白CD2AP、 a-actinin-4、 synaptopodin、Arp3及cortactin表达的影响。探讨温阳活血利水方如何通过Nephrin到足突骨架蛋白及接头蛋白,以及信号转导途径治疗儿童肾病综合征的作用机制。方法：SPF级SD雄性大鼠42只,体重115±15克,将其适应性喂养十天,使其体重增至200±30克,按随机分层分组的原则将其分为4组,其中中药组12只、正常组6只、强的松组12只、模型组12只,除正常组外,其余各组均采用阿霉素(ADR)5.5mg/kg一次性尾静脉注射,诱导一种类似人类微小病变型肾病综合征的大鼠肾病模型,正常组尾静脉一次性注射生理盐水1毫升。注射ADR一周后开始治疗,中药组每日一次经胃灌服温阳活血利水方颗粒剂混悬液3毫升,强的松组按12mg/kg·d,经胃灌服强的松悬液3毫升(强的松剂量同前),正常组和模型组每日灌服3毫升生理盐水,均持续4周。观察实验大鼠的体重、二便、水肿、舌质等一般情况；造模前、造模后第1周末及第5周末分别测大鼠24小时尿蛋白定量；实验第6周末处死大鼠取材。(1)麻醉后心脏取4m1血,分离血清,检测总蛋白(TP)、总胆固醇(CHOL)、甘油三酯(TG)的水平。(2)采用免疫荧光染色测定各组大鼠肾小球足细胞中骨架蛋白CD2AP、 a-actinin-4、 synaptopodin、 Arp3及cortactin的表达水平。(3)取肾组织光镜检测组织形态学变化；电镜下检测超微结构变化。结果：(1)一般情况：正常组大鼠毛发润泽,活动自如,精神状态较好,食欲佳。其余各组大鼠注射阿霉素10～14天后大鼠出现体重不增,反应迟钝,活动明显减少,弓背蜷缩,聚堆,体毛稀疏,甚至片状脱落,进食量及饮水量明显减少,时有便溏。注射阿霉素15天后大鼠开始出现四足水肿,足、尾红紫,舌质紫暗。治疗期间,各治疗组分别用中药、强的松治疗2周后大鼠反应逐渐好转,进食及饮水量较前增加,活动增加；其中中药组尿量明显增多,体重缓慢回升。强的松组大鼠表现为躁动不安,易激惹,攻击性强。 (2)尿液及血液检测结果：造模后1周,各造模组大鼠24小时尿蛋白定量与注射前比较有所升高,两周后增加明显。温阳活血利水方和强的松在治疗4周后能显著降低阿霉素肾病大鼠的尿蛋白水平,而中药组和强的松组之间无明显差异。阿霉素肾病大鼠存在明显的低蛋白血症和高脂血症,温阳活血利水方和强的松都能在治疗5周后升高阿霉素肾病大鼠的血清蛋白水平,但仅温阳活血利水方能降低其血清脂质水平。 (3)免疫荧光染色：阿霉素肾病大鼠肾小球足细胞蛋白CD2AP、 synaptopodin、 Arp3及cortactin表达降低,而温阳活血利水方和强的松能在一定程度上抑制四种骨架蛋白表达的下降,而模型组及治疗组a-actinin-4的表达均无改变,但其分布存在差异。 (4)病理：温阳活血利水方可以有效改善阿霉素肾病大鼠肾组织足细胞的足突融合。结论：温阳活血利水方可以降低阿霉素肾病大鼠的蛋白尿；控制胆固醇、血脂；增加血清总蛋白；减轻其肾组织足细胞的足突融合；抑制阿霉素肾病大鼠肾组织足细胞蛋白CD2AP、synaptopodin、 Arp3及cortactin表达的下降；
[Abstract]:Objective: To observe the effect of Wenyang Huoxue water method on the expression of bone frame protein CD2AP, a-actin-4, synaphodin, Arp3 and cortin in adriamycin-induced nephropathy rats. Objective To study the mechanism of the effect of Wenyang Huoxue Lishui Recipe on the treatment of children's nephrotic syndrome through the treatment of the protein and its protein, and the signal transduction pathway in the treatment of children's nephrotic syndrome. Methods: 42 SD male rats, weighing 115 to 15 g, were fed for 10 days, the weight was increased to 200 to 30 g, and the body weight was divided into 4 groups according to the principle of random stratified grouping, 12 of the traditional Chinese medicine group, 6 in the normal group and 12 in the strong group, and 1 in the model group. 2. In addition to the normal group, the other groups were treated with adriamycin (ADR) of 5. 5 mg/ kg for one-time tail vein to induce a rat kidney disease model similar to the human micro-pathological type nephrotic syndrome, and the normal group tail vein was injected with normal saline at one time. One week after the injection of ADR, the treatment was started, and the traditional Chinese medicine group was given 3 ml of suspension liquid of Wenyang Huoxue Huoxue prescription granule once daily through the stomach, and the prednisone group was administered with 3 ml of strong suspension liquid (the same dose as before), the normal group and the model group with 3 ml of physiological saline in the normal group and the model group. Brine, continuous 4 weeks. The body weight, edema, tongue and other general conditions of the experimental rats were observed. The 24-hour urine protein was measured on the first weekend and the fifth weekend before the model, and the rats were sacrificed at the end of the first week. Materials are obtained. (1) After anesthesia, the heart takes 4m1 blood, separates serum, and detects total protein (TP), total cholesterol (CHOL), and triglyceride (TG). level. (2) The expression of bone frame protein CD2AP, a-actin-4, synaptopdin, Arp3 and cortin in the rat's glomerular foot cells was determined by immunofluorescence staining. level. (3) the morphological changes of the tissue were detected by the light microscope of the renal tissue; the ultrastructures were examined under the electron microscope. Change. Result: (1) General situation: the normal group of rats' hair is moist, the activity is free, the mental state is higher, Good appetite. The rest of the rats were injected with adriamycin for 10 to 14 days. The rats showed no increase in body weight, slow response, and reduced activity. After 15 days of injection of adriamycin, the rats began to have four-foot edema, foot, and red violet. and the urine volume of the traditional Chinese medicine group is obviously increased, and the volume of the traditional Chinese medicine group is obviously increased, The heavy and slow recovery of the rats. The rats in the high-pressure group showed restlessness and irritability. (2) The results of urine and blood test: 1 week after the model, the 24-hour urine protein of each model group was higher than that of the pre-injection. and the level of the urinary protein in the adriamycin-induced nephropathy rats can be significantly reduced after 4 weeks of treatment by the Wenyang Huoxue Lishui prescription and the prednisone, and the traditional Chinese medicine group and the prednisone group There was no significant difference between the groups. There were obvious hypoproteinemia and hyperlipoidemia in the rats with adriamycin nephrosis. The serum protein level of adriamycin-induced nephropathy rats could be increased after 5 weeks of treatment. (3) Immunofluorescence staining: the expression of CD2AP, synaptopdin, Arp3 and cortin in the glomerular podocyte protein of the adriamycin-induced nephropathy rats decreased, while the Wenyang Huoxue and the prednisone could inhibit the decrease of the expression of the four kinds of skeletal proteins to a certain extent, while the model group and the treatment group a-actin None of the in-4 expression Change, but there is a difference in its distribution. (4) Pathology: Wenyang Huoxue Lishui Prescription can effectively improve the adriamycin kidney Conclusion: Wenyang Huoxue Lishui prescription can reduce the albuminuria in the adriamycin-induced nephropathy rats, control the cholesterol and blood lipid, increase the serum total protein, reduce the apophysis fusion of the renal tissue foot cells, and inhibit the renal tissue foot cell protein CD2AP, synaptopdin, Ar in the adriamycin-induced nephropathy rats.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R272

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