钩藤碱对自发性高血压大鼠肾脏纤维化的影响

发布时间：2018-03-23 01:34

  本文选题：钩藤碱　切入点：自发性高血压大鼠　出处：《皖南医学院》2016年硕士论文　论文类型：学位论文

【摘要】：目的:观察钩藤碱对自发性高血压大鼠肾脏纤维化的影响,并探讨其机制。方法:取雄性自发性高血压大鼠32只,随机分为钩藤碱高剂量组、钩藤碱低剂量组、卡托普利阳性对照组、模型组,每组8只;另取WKY大鼠8只作为正常对照组。适应性的喂养后,各治疗组分别灌胃给予钩藤碱(10、2.5mg·kg-1·d-1)、卡托普利(17.5 mg·kg-1·d-1),正常对照组和模型组给予蒸馏水(5 ml·kg-1·d-1),共10周。采用尾袖法每2周测定大鼠血压,末次给药后,称量大鼠体重,处死大鼠,钝性分离肾脏,测量左右肾湿重,计算肾脏重量系数;采用肌氨酸氧化酶法测定血肌酐(Scr),脲酶法测定尿素氮含量(BUN);HE染色观察肾脏病理组织学变化,MASSON染色观察肾脏胶原纤维变化;碱水解法测定肾组织羟脯氨酸(HYP)含量;经典的Griess Reagent法测定肾组织一氧化氮(NO)含量;WST-8法测定肾组织超氧化物歧化酶(SOD)含量;比色法测定肾组织丙二醛(MDA)含量;ELISA法检测血清肿瘤坏死因子α(TNF-α)含量;免疫组化法检测肾组织转化生长因子-β_1阳性细胞表达;免疫印迹法检测肾组织转化生长因子-β_1、Smad3、Smad7、MMP-9、TIMP-1、iNOS蛋白表达水平。结果:(1)与WKY组相比,SHR模型组大鼠SBP明显升高(P0.01);给药后,钩藤碱各剂量组的SBP较模型组均有不同程度的降低(P0.01)。(2)与WKY组相比,SHR模型组大鼠肾脏重量指数明显升高(P0.01);钩藤碱高、低剂量组肾脏重量指数与模型组无明显差异(P0.05)。(3)与WKY组相比,SHR组大鼠血清Scr、BUN含量明显升高(P0.01),钩藤碱高、低剂量组较模型组的血清Scr、BUN含量显著降低(P0.05或P0.01)。(4)HE染色和Masson染色显示,SHR组大鼠肾小球基底膜增厚、肾小管萎缩、肾间质纤维化伴炎性细胞浸润,胶原沉积严重增多,而钩藤碱各剂量组对上述病理变化均有不同程度的改善。(5)与WKY组相比,SHR组大鼠肾组织HYP、MDA、NO和血清中TNF-α含量显著升高(P0.01),肾组织SOD含量显著降低(P0.01);给药10周后,钩藤碱各剂量组大鼠肾组织HYP、MDA、NO和血清中TNF-α含量有所降低,肾组织SOD含量显著升高(P0.05或P0.01);(6)与WKY组相比,SHR组大鼠肾组织TGF-β_1、Smad3、iNOS、MMP-9、TIMP-1蛋白表达显著增高,Smad7蛋白表达降低(P0.01);给药10周后,钩藤碱各剂量组能不同程度的降低TGF-β_1、Smad3、iNOS、MMP-9、TIMP-1蛋白表达,升高Smad7蛋白表达(P0.05或P0.01)。结论:钩藤碱能够降低自发性高血压大鼠血压、改善肾脏纤维化,其机制可能与调控TGF-β_1及其下游信号通路、恢复MMP-9/TIMP-1的平衡、抑制氧化应激、降低炎性因子TNF-α含量有关。
[Abstract]:Objective: to observe the effect of leptinine on renal fibrosis in spontaneously hypertensive rats and to explore its mechanism. Methods: Thirty-two male spontaneously hypertensive rats were randomly divided into high dose group and low dose group. Captopril positive control group, model group, 8 rats in each group, and 8 WKY rats as normal control group. Each treatment group was given Leptacine 102.5 mg kg-1 d -1, captopril 17.5 mg kg-1 d -1, and normal control group and model group were given 5 ml kg-1 d -1 of distilled water for 10 weeks. Blood pressure was measured by tail sleeve method every 2 weeks. After the last administration, the rats were weighed and killed. The kidney was separated obtuse, the wet weight of left and right kidneys was measured, the coefficient of kidney weight was calculated, the serum creatinine was measured by creatine oxidase method, the content of urea nitrogen was determined by urease method, the pathological changes of kidney were observed by HE staining and the changes of collagen fibers in kidney were observed by MASson staining. The content of hydroxyproline (Hyp) in renal tissue was determined by alkaline hydrolysis, the content of nitric oxide (no) in renal tissue by classical Griess Reagent method and the content of superoxide dismutase (SOD) in renal tissue by WST-8 method. The content of malondialdehyde (MDA) in renal tissue was determined by colorimetric method, serum tumor necrosis factor 伪 (TNF- 伪) was detected by Elisa, the expression of transforming growth factor 尾 1 (TGF 尾 1) positive cells in renal tissue was detected by immunohistochemical method. The expression of TGF- 尾 1, Smad3, MMP-9, TIMP-1and iNOS in renal tissue was detected by Western blotting. Results compared with WKY group, the expression of SBP in SHR model group was significantly higher than that in SHR model group (P 0.01). Compared with the WKY group, the renal weight index of the rats in the WKY group was significantly higher than that in the WKY group, and the level of leptinine was higher than that of the model group, and the level of leptinine was higher than that in the WKY group, and the renal weight index of the rats in the WKY group was significantly higher than that in the WKY group. There was no significant difference in renal weight index between the low dose group and the model group (P 0.05. 0. 3) compared with the WKY group, the serum Scrn bun content in the SHR group was significantly higher than that in the WKY group, and the level of leptinine was higher in the low dose group than that in the model group. Compared with the model group, the serum levels of Scrnbun in the low dose group were significantly lower than those in the model group. The results of P0.01).(4)HE staining and Masson staining showed that glomerular basement membrane thickened, tubule atrophy, renal interstitial fibrosis accompanied by inflammatory cell infiltration and collagen deposition were significantly increased in SHR group. Compared with the WKY group, the contents of no and TNF- 伪 in the renal tissue and serum TNF- 伪 in the WKY group were significantly higher than those in the control group, and the content of SOD in the renal tissue was significantly decreased after 10 weeks of administration, while the content of TNF- 伪 in the serum of the rats in the WKY group was significantly higher than that in the control group, while the content of SOD in the renal tissue decreased significantly after 10 weeks of administration. Compared with WKY group, the expression of TGF- 尾 1 Smad3NOSMMP-9 TIMP-1 protein decreased significantly in renal tissue of rats with different doses of leptinine, and the content of TNF- 伪 in renal tissue increased significantly (P 0.05 or P0.01) compared with WKY group, the expression of Smad7 protein decreased 10 weeks after administration of Leptacine, the expression of TGF- 尾 1, Smad3iNOSMMP-9, TIMP-1 protein in renal tissue of rats was significantly decreased, and the expression of TGF- 尾 1, Smad3iNOSMMP-9, TIMP-1 protein decreased significantly in renal tissue compared with WKY group. The expression of TGF- 尾 1 Smad3iNOSU MMP-9 and TIMP-1 was decreased in different dosage groups, and the expression of Smad7 protein was increased (P0.05 or P0.01). Conclusion: Leptosine can reduce blood pressure and improve renal fibrosis in spontaneously hypertensive rats. The mechanism may be related to regulating TGF- 尾-1 and its downstream signaling pathway, restoring the balance of MMP-9/TIMP-1, inhibiting oxidative stress and decreasing the content of inflammatory factor TNF- 伪.
【学位授予单位】：皖南医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R259

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