PPARγ激动剂对肥胖小鼠肾组织AMPK的影响及其抗氧化应激作用

发布时间：2018-02-25 20:25

  本文关键词： 肥胖相关性肾病 过氧化物酶体增殖物激活受体-γ 脂联素 氧化应激 AMPK NOX-4 小鼠　出处：《天津医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的:肥胖越来越被认为是肾脏疾病的主要危险因素。我们之前的研究表明PPARγ激动剂吡格列酮可通过升高单纯肥胖小鼠血清脂联素水平而起到肾脏保护作用,而有研究表明脂联素有抗氧化应激作用,但PPARγ激动剂的肾脏保护作用是否与抗氧化应激有关尚不清楚。本研究探讨PPARγ激动剂对单纯肥胖小鼠肾组织AMPK的影响及其抗氧化应激作用。方法:6周龄雄性ob/ob小鼠24只和其同系C57BL/6J小鼠8只,适应性饲养2周,测量体重并留尿测量24h尿微量白蛋白,确定ob/ob小鼠体重及24h尿微量白蛋白与C57BL/6J小鼠有显著差异。按照体质量分层抽样,将ob/ob小鼠随机分为3组:模型组(M组,n=8)、小剂量治疗组(T1组,n=8)、大剂量治疗组(T2组,n=8);C57BL/6J小鼠作为对照组(C组,n-8)。治疗组分别给予吡格列酮灌胃,其中T1组为吡格列酮2.25mg·kg-1·d-1,T2组为吡格列酮6.75mg·kg-1·d-1,C组和M组小鼠给予等体积蒸馏水灌胃。每周称量小鼠体重一次,根据体重调整给药剂量,连续12周。灌胃前后分别采集血液标本以测定小鼠血糖,血清ADP,并留尿液测定24小时尿m ALB。12周后处死小鼠,摘取肾脏,称量右肾湿重。光镜下观察小鼠肾脏病理改变,并测量肾小球直径;免疫组化方法检测肾组织内AMPK及NOX-4的表达。比较四组小鼠各个指标间的差异,并将血清ADP、24h尿微量白蛋白、肾组织AMPK及NOX4表达水平分别与其他指标做相关性分析。采用SPSS 17.0统计软件进行数据处理和统计学分析,P0.05为差异具有统计学意义。结果:1小鼠的一般情况1.1实验前两种小鼠体重及尿蛋白的比较:6周龄时ob/ob小鼠平均体质量已明显高于C57BL/6J小鼠(P0.01),适应性饲养2周后,ob/ob小鼠体质量不仅明显高于C57BL/6J小鼠(P0.01),且平均体重已经超过后者的50%(20%),即达到小鼠肥胖标准,ob/ob小鼠尿微量白蛋白明显高于C57BL/6J小鼠(P0.01),虽然两种小鼠血糖存在差异(P0.05),但都尚未达到糖尿病标准,可排除因糖尿病导致的蛋白尿,确定ob/ob小鼠蛋白尿是由肥胖造成。1.2各组小鼠实验前后体重:分组后M组和T1、T2组小鼠体质量均超过C组小鼠平均体质量的20%,即达到小鼠肥胖标准(P0.01),M组、T1组、T2组小鼠体质量无显著差异(P0.05)。实验结束时M组和T1、T2组小鼠体质量超过C组小鼠更为明显(P0.01),T2组较T1组小鼠体质量高(P0.01)。各组小鼠实验后体质量均高于实验前(P0.01)2血清脂联素浓度:2.1各组前后比较:实验后M组和T1、T2组小鼠血清脂联素水平均较实验前降低(P0.01),C组前后无明显变化(P0.05)。2.2各组之间比较:实验前M组和T1、T2组小鼠血清ADP较C组降低(P0.01),实验后T1、T2组小鼠血清ADP高于M组(P0.01),且T2组高于T1组(P0.01)。3 24h尿微量白蛋白:3.1各组前后比较:实验后M组和T1、T2组小鼠24h尿微量白蛋白均较实验前增加(P0.01),C组前后无明显变化(P0.05)。3.2各组之间比较:实验前后M组和T1、T2组小鼠24h尿微量白蛋白均明显高于C组(P0.01);实验后T1、T2组小鼠24h尿微量白蛋白较M组明显降低(P0.01),且T2组明显低于T1组(P0.01)。4肾脏病理改变:4.1肾脏湿重(右肾):实验后M组和T1、T2组小鼠肾脏湿重明显高于C组(P0.05),T1组及T2组右肾湿重明显小于M组(P0.01),T2组右肾湿重明显小于T1组(P0.01)。4.2肾组织病理改变:M组小鼠肾小球鲍曼氏囊增宽,基底膜增厚,系膜细胞和系膜基质增生,肾小球体积增大,部分伴有局灶节段性肾小球硬化,轻度肾小管萎缩和间质纤维化。T1、T2组病变程度较M组减轻。4.3肾小球直径:实验后M组和T1、T2组小鼠肾小球直径明显大于C组(P0.05),T1组及T2组肾小球直径明显小于M组(P0.01),T2组肾小球直径明显小于T1组(P0.01)。5肾组织AMPK表达:可见AMPK在肾小球、肾小管及肾间质均有表达;M组和T1、T2组小鼠肾组织AMPK表达较C组明显减少(P0.01),T1、T2组小鼠肾组织AMPK表达明显高于M组(P0.01),T2组明显高于T1组(P0.05)。6肾组织NOX-4表达:可见NOX-4在肾小球、肾小管及肾间质均有表达;M组和T1、T2组小鼠肾组织NOX-4表达明显高于C组(P0.01),T1、T2组小鼠肾组织NOX-4表达明显低于M组(P0.01),T2组明显低于T1组(P0.05)。7直线相关分析:7.1尿微量白蛋白与体质量、肾小球直径、右肾湿重及NOX-4表达水平呈正相关(r1=0.741;r2=0.936;r3=0.877;r4=0.878,P0.001),与血清ADP及AMPK表达水平呈负相关(r5=-0.915;r6=-0.759,P0.001)。7.2小鼠血清脂联素与体质量、肾小球直径、右肾湿重、尿微量白蛋白、NOX-4呈负相关(r7=-0.772;r8=-0.821;r5=-0.915;r9=-0.958,P0.001),与AMPK表达水平呈正相关(r10=0.878,P0.001)。7.3肾组织AMPK表达水平与体质量、肾小球直径、右肾湿重、尿微量白蛋白及肾组织NOX4表达水平呈负相关(r11=-0.627;r12=-0.711;r13=-0.743;r6=-0.759;r14=-0.884,P0.001),与血清脂联素浓度呈正相关(r10=0.878,P0.001)。7.4肾组织NOX-4表达水平与体质量、肾小球直径、右肾湿重、尿微量白蛋白呈正相关(r15=0.671;r16=0.843;r17=0.794;r4=0.878,P0.001),与血清脂联素浓度及肾组织AMPK表达水平呈负相关(r9=-0.958;r14=-0.884,P0.001)。结论:1.肥胖小鼠血清ADP水平下降,肾组织AMPK表达降低,NOX-4表达增加,氧化应激增强,尿m ALB增加。2.PPARγ激动剂吡格列酮可升高肥胖小鼠血清ADP水平及肾组织AMPK表达,减少NOX-4表达,改善肥胖小鼠的氧化应激状态,从而减少尿蛋白,起到肾脏保护作用。3.相对于小剂量PPARγ激动剂,大剂量PPARγ激动剂更能在肥胖时对肾脏起到保护作用。
[Abstract]:Objective: obesity is increasingly considered as major risk factors for kidney disease. Our previous study showed that PPAR gamma agonist pioglitazone by elevated serum adiponectin levels in simple obese mice and play a role in renal protection, and studies have shown that adiponectin has anti oxidative stress, but the PPAR gamma agonist renal protective effect and oxidative stress is unclear. This study investigated the PPAR gamma agonist on the influence of obesity mice kidney AMPK and oxidative stress. Methods: 6 Zhou Lingxiong 24 ob/ob mice and its homologous C57BL/6J 8 rats, 2 week adaptive feeding, measuring the weight and keep the microalbuminuria measurement of urinary 24h, determine the weight of ob/ob mice 24h and microalbuminuria and C57BL/6J mice have significant differences. According to the body weight of stratified sampling, the ob/ob mice were randomly divided into 3 groups: model group (group M, n=8), low dose treatment Group (group T1, n=8), high dose treatment group (group T2, n=8); C57BL/6J mice as control group (group C, n-8). The treatment group were given pioglitazone orally, group T1, 2.25mg - kg-1 - D-1 pioglitazone group, T2 6.75mg kg-1 D-1 pioglitazone group, C and M mice were given the same volume of distilled water. The weight of mice weighed every week once, according to the weight to adjust the dosage for 12 weeks. Blood samples were collected before and after intragastric administration with serum glucose, serum ADP, m were determined in 24 hour urine urine ALB.12 weeks after the mice were sacrificed, the removal of the kidney, called the right amount the wet weight of kidney. Renal pathological changes were observed under light microscope, and measured the expression of glomerular diameter; immunohistochemical method of AMPK and NOX-4 in renal tissue. The difference of each index in the detection of mice between four groups, and the serum ADP, 24h urinary albumin, renal tissue AMPK and NOX4 levels and other indicators respectively. Do. Correlation analysis. Data processing and statistical analysis were conducted using SPSS 17 statistical software for P0.05, the difference was statistically significant. Results: comparison of body weight and urine protein in mice 1 mice in general 1.1 before the experiment two: 6 week old ob/ob mice have significantly higher than the average body weight of C57BL/6J mice (P0.01) after 2 weeks. Adaptive feeding, the body weight of ob/ob mice not only significantly higher than that of C57BL/6J mice (P0.01), and the average weight of more than 50% of the latter (20%), which reached the mouse obese standard, ob/ob mice urinary albumin was significantly higher than that of C57BL/6J mice (P0.01), while the two differences of blood glucose in mice (P0.05), but have not yet reached the standard of diabetes that can be excluded because of diabetes leads to proteinuria, proteinuria is determined ob/ob mice.1.2 mice before and after the experiment the weight caused by obesity: M group and T1 group, the body mass of mice in T2 group were higher than the average in C group 浣撹川閲忕殑20%,鍗宠揪鍒板皬榧犺偉鑳栨爣鍑，

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