糖尿病大鼠肾组织NF-κB、TGF-β的表达及瑞格列奈对其干预作用的研究

发布时间：2018-05-04 18:29

本文选题：糖尿病肾病 + 瑞格列奈　；参考：《河北医科大学》2015年硕士论文

【摘要】：目的:糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病(Diabetes Mellituse,DM)最常见的严重微血管并发症之一。近年来研究发现,氧化应激、炎症反应和纤维化在糖尿病肾病发生发展过程中发挥了重要作用。糖尿病时机体处于氧化应激状态,通过产生过多的活性氧簇(Reactive Oxygen Species,ROS)直接损伤机体蛋白质和核酸等生物大分子,还可通过激活多功能转录调节因子NF-κB(Nuclear Transcription FactorκB),促进多种炎症因子、巨噬细胞及促肾脏纤维化关键因子TGF-β等的过度表达,共同导致肾脏病变。如何阻断或抑制氧化应激及炎症反应,减缓肾脏纤维化成为预防和治疗DN的关键。瑞格列奈(Repaglinide,RE)是一种非磺酰脲类促胰岛素分泌剂,是Ⅱ型糖尿病患者临床一线口服降糖药物,可有效控制餐后血糖。有研究报道,RE还可减缓DN进展,但确切机制尚不明了。本课题拟通过建立链脲佐菌素(Streptozotocin,STZ)诱导的DM大鼠模型,测定大鼠肾脏组织中氧化应激指标丙二醛(MDA)含量及总抗氧化能力(T-AOC)活性,应用免疫组织化学、实时定量荧光PCR(real-time FQ-PCR)等方法检测肾组织中NF-κB、TGF-β表达情况,并予瑞格列奈(Repaglinide,RE)2mg/Kg进行干预,观察DM大鼠肾脏组织内氧化应激、炎症反应及纤维化状况,并探讨瑞格列奈对其存在的干预作用及机制,为人类糖DN的防治提供理论依据。方法:选择健康雄性清洁级SD大鼠36只,体重约200±20g,(购自河北医科大学动物中心),随机分为正常对照(NC组,n=12)、糖尿病模型组(DM组,n=12)、糖尿病瑞格列奈干预组(RT组,n=12)。适应性喂养1周后,空腹12小时,糖尿病模型组大鼠一次性腹腔注射STZ(65mg/kg),72h后尾静脉取血检测血糖≥16.7mmol/L,尿糖+++~++++,视为造模成功。NC组一次性腹腔注射等剂量柠檬酸缓冲液。试验期间所有大鼠均自由进食水,动物房温度(20℃~26℃)、湿度(40%~70%)相对恒定。RT组大鼠给予瑞格列奈(RE)2mg/kg,1/日,植物油溶剂灌胃,NC组、DM组每天予等量植物油灌胃。分别于造模成功后8周、12周末,每组任取6只大鼠,测定24h尿蛋白(24 h UP)、血糖、尿素氮、血肌酐。并收集大鼠肾组织标本,制备组织切片,行苏木素-伊红染色(HE染色)及糖原染色(PAS),光学显微镜下观察大鼠肾脏病理变化;检测肾组织MDA含量、T-AOC活性,免疫组织化学及real-time PCR方法检测TGF-β、NF-κB的表达。实验所得数据用均数±标准差(x±S)表示,应用SPSS 16.0统计分析软件对数据进行单因素方差分析,不满足正态性和方差齐性的数据资料采用非参数检验,P0.05有统计学意义。结果:1一般情况DM组、RT组大鼠腹腔注射STZ后逐渐出现多饮、多食、多尿,反应迟缓,毛发失去光泽,体重逐渐下降,其中DM组症状较RT组明显加重,而NC组大鼠饮食正常,反应灵敏,生长发育良好,体重明显增长。2生化指标2.124小时尿蛋白定量与同期NC组相比,8周末、12周末,DM组、RT组大鼠24小时尿蛋白水平均升高(P0.05);与同期DM组相比,RT组大鼠24小时尿蛋白水平降低(P0.05)。2.2血糖与同期NC组相比,8周末、12周末,DM组、RT组大鼠血糖水平均明显升高(P0.05);与同期DM组相比,RT组血糖变化不明显(P0.05)。2.3血肌酐、尿素氮与同期NC组相比,8周末、12周末,DM组、RT组大鼠血肌酐、尿素水平升高(P0.01);与同期DM组相比,RT组血肌酐、尿素氮水平下降(P0.05)。3氧化应激指标3.1MDA测定与同期NC组相比,8周末、12周末,DM组、RT组大鼠肾组织MDA水平显著升高(P0.05);与同期DM组相比,RT组MDA水平均有不同程度的下降(P0.05)。3.2 T-AOC测定与同期NC组相比,8周末、12周末,DM组、RT组大鼠肾组织内的T-AOC活性显著降低(P0.05);与同期DM组相比,RT组T-AOC活性升高(P0.05)。4肾脏组织变化光镜显示,NC组大鼠肾脏组织结构清晰,肾小管上皮细胞排列整齐,基底膜染色均匀一致,无系膜细胞及系膜基质增多等异常改变。与NC组相比,DM组肾小球体积增大,肾小球系膜基质弥漫增多,系膜细胞增多,基底膜增厚,12周末时病理改变更明显。RT组肾脏组织结构较DM组得到改善。5免疫组织化学检测与同期NC组相比,8周末、12周末,DM组、RT组NF-κB、TGF-β表达均增多(P0.05);与同期DM组相比,RT组NF-κB、TGF-β表达水平不同程度下降(P0.05)。6 Real-time FQ-PCR检测与同期NC组相比,8周末、12周末时,DM组、RT组NF-κB m RNA、TGF-βm RNA表达均增多(P0.05);与同期DM组相比,RT组NF-κB m RNA、TGF-βm RNA表达水平不同程度下降(P0.05)。结论:1 DM大鼠肾脏组织MDA表达增高,T-AOC活性降低,提示处于氧化应激状态,同时NF-κB、TGF-β表达升高,表明有炎症反应及纤维化机制参与。2瑞格列奈可减少DM大鼠蛋白尿的产生、改善肾脏功能;可降低肾脏组织MDA含量,提高T-AOC活性并减少NF-κB、TGF-β表达,从而减轻氧化应激、炎症反应,延缓肾脏纤维化,起到肾脏保护作用。
[Abstract]:Objective: Diabetic Nephropathy (DN) is one of the most common severe microvascular complications of Diabetes Mellituse (DM). In recent years, it is found that oxidative stress, inflammatory reaction and fibrosis play a important role in the development of diabetic nephropathy. Too much Reactive Oxygen Species (ROS) directly damages biological macromolecules, such as protein and nucleic acid, and also activates the multifunction transcription regulator NF- kappa B (Nuclear Transcription Factor kappa B) to promote a variety of inflammatory factors, macrophages and the overexpression of the key factor of renal fibrosis, TGF- beta, etc. Renal lesions. How to block or inhibit oxidative stress and inflammatory response and reduce renal fibrosis is the key to the prevention and treatment of DN. Repaglinide (RE) is a non sulfonylurea secretory agent, an oral hypoglycemic agent in patients with type 2 diabetes, which can effectively control postprandial blood glucose. Research reports, RE also It can slow down the progress of DN, but the exact mechanism is still unknown. By establishing the DM rat model induced by Streptozotocin (STZ), this subject is to determine the content of malondialdehyde (MDA) and the total antioxidant capacity (T-AOC) activity of oxidative stress in kidney tissue of rats, and the application of immunohistochemistry and real-time quantitative fluorescent PCR (real-time FQ-PCR). The expression of NF- kappa B, TGF- beta in renal tissue and the intervention of Repaglinide (RE) 2mg/Kg were used to observe the oxidative stress, inflammatory reaction and fibrosis in the renal tissue of DM rats, and to explore the intervention and mechanism of reaglinide on its existence, and to provide a theoretical basis for the prevention and control of human sugar DN. 36 SD rats, weight about 200 + 20g, were randomly divided into normal control group (group NC, n=12), diabetes model group (DM group, n=12), diabetic regerel intervention group (RT group, n=12). 1 weeks after adaptive feeding, 12 hours after adaptive feeding, rats in diabetic model group were intraperitoneally injected STZ (65mg/kg), 72h rear tail. Blood blood blood glucose more than 16.7mmol/L, urine sugar + + + + + +, considered as a model successful.NC group of one-time intraperitoneal injection of citric acid buffer. All rats during the test were free to eat water, animal room temperature (20 C ~26 C), humidity (40%~70%) relative constant.RT group rats to Yo Ray Glenn Nai (RE) 2mg/kg, 1/ day, vegetable oil solvent gavage, NC group, DM The group was given the same amount of vegetable oil every day. After 8 weeks and 12 weeks after the success of the model, 6 rats in each group were taken to determine 24h urine protein (24 h UP), blood sugar, urea nitrogen and blood creatinine. The specimens of rat kidney tissue were collected and the tissue sections were collected. The pathological changes of kidney pathology were observed under optical microscope. Changes; detection of renal tissue MDA content, T-AOC activity, immunohistochemistry and real-time PCR method to detect the expression of TGF- beta, NF- kappa B. The experimental data were expressed with mean standard deviation (x + S). The data were analyzed by the statistical analysis software of SPSS 16, and the data which did not satisfy the normality and homogeneity of variance were non parameters. Test, P0.05 has statistical significance. Results: 1 general situation DM group, group RT rats after intraperitoneal injection of STZ gradually polydrink, polydipsia, polyuria, slow reaction, hair loss luster, weight gradually decreased, and the DM group symptoms are significantly worse than the RT group, while the NC group rats have a normal diet, the reaction is sensitive, the growth and development are good, the weight of the body weight obviously increases.2 biochemical index. 2.124 hours urine protein quantitative compared with the same period NC group, 8 weekend, 12 weekend, DM group, group RT rats' 24 hours urine protein level increased (P0.05). Compared with the DM group, the 24 hour urine protein level in the RT group decreased (P0.05).2.2 blood sugar compared with the same period NC group, the 8 weekend, the 12 weekend, DM group, RT group rats blood sugar water (P0.05); with the same period Compared with group M, blood glucose in RT group was not significant (P0.05).2.3 blood creatinine, urea nitrogen was compared with NC group, 8 weekend, 12 weekend, DM group, serum creatinine and urea level in RT group increased (P0.01). Compared with group DM, the level of serum creatinine and urea nitrogen in RT group decreased (P0.05) and.3 oxidative stress index was compared with that of the same period, 8 weekend, 12 weekend The MDA level of renal tissue in T group was significantly increased (P0.05). Compared with the DM group, the MDA level in RT group decreased (P0.05).3.2 T-AOC compared with the NC group in the same period. The activity of renal tissue in the renal tissue of the RT group was significantly lower than that in the 8 weekend, the 12 weekend, the DM group and the kidney tissue of the RT group. The change light microscopy showed that the renal tissue structure of the NC group was clear, the epithelial cells of the renal tubules were arranged neatly, the basal membrane staining was uniform, and the number of mesangial cells and mesangial matrix increased. Compared with the NC group, the glomerular volume increased, the glomerular mesangial matrix increased, the mesangial cells increased, the basement membrane thickened, and the pathology at the 12 weekend was pathological. The renal tissue structure of the.RT group was better than that of the DM group, and the.5 immuno histochemical test was improved on the 8 weekend, the 12 weekend, the DM group, the RT group, NF- kappa B, and the TGF- beta expression increased (P0.05). At the 12 weekend, group DM, group RT, NF- kappa B m RNA, TGF- beta m RNA expression increased (P0.05). .2 reagnonide can reduce the production of proteinuria in DM rats and improve renal function, reduce MDA content in kidney tissue, improve T-AOC activity and reduce NF- kappa B, TGF- beta expression, thus alleviating oxidative stress, inflammatory reaction, postponing renal fibrosis and playing the role of renal protection.

【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R587.2

【参考文献】