SIRT1在糖尿病肾病足细胞凋亡中的作用及机制研究

发布时间：2018-03-11 12:41

本文选题：糖尿病肾病　切入点：血管紧张素Ⅱ　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及目的糖尿病肾病是导致终末期肾病的最主要病因,超40%的肾脏替代治疗患者是由糖尿病引起。临床主要表现为大量蛋白尿。研究认为蛋白尿的产生与肾小球足细胞损伤密切相关。在糖尿病肾病中,有多种因素如高血糖、血管紧张素Ⅱ(Ang-Ⅱ)等导致足细胞凋亡。严格控制血糖及应用肾素-血管紧张素系统(RAS)阻断剂能够明显减少尿蛋白的产生。SIRT1是一种沉默信息调节因子,具有NAD+依赖的去乙酰化酶活性,参与了多种疾病的发病过程,其中也包括糖尿病肾病。在糖尿病肾病中,SIRT1参与了炎症、细胞凋亡、纤维化等多个病理过程。在细胞凋亡方面,既往研究发现,高糖和Ang-Ⅱ可以通过不同的信号通路影响SIRT1的表达及活性,Ang-Ⅱ诱导内皮细胞凋亡并激活了P38/SIRT1通路。高糖培养可以激活足细胞中的AMPK/SIRT1通路介导细胞凋亡。在临床应用中,奥美沙坦能够减少糖尿病肾病患者尿蛋白,但是在血糖控制不佳的患者中,奥美沙坦降低尿蛋白的作用并不明显。说明高糖状态下奥美沙坦的治疗会受到影响。也提示高糖和Ang-Ⅱ可能通过不同通路诱导了足细胞凋亡。因此我们设想,在糖尿病肾病中,Ang-Ⅱ和高糖可能通过不同的信号通路(P38/SIRT1通路与AMPK/SIRT1通路)影响SIRT1的表达,从而导致了足细胞的凋亡,而奥美沙坦只能对P38/SIRT1通路起作用。奥美沙坦是临床常用的血管紧张素Ⅱ抑制剂,能够抑制Ang-Ⅱ与血管紧张素Ⅱ受体的结合。既往认为Ang-Ⅱ主要通过丝裂原活化蛋白激酶(MAPK),尤其是ERK1/2信号通路诱导足细胞凋亡。而Ang-Ⅱ是否也能通过P38/SIRT1通路介导足细胞凋亡目前缺乏相关报道。研究方法1.动物实验:糖尿病肾病模型db/db小鼠为糖尿病组,并分成两组:安慰剂组(db/db+CMC)和奥美沙坦组(db/db+OM),C57BL小鼠作用为非糖尿病组(WT),每组10只。在小鼠10-12周龄时,开始药物灌胃处理,安慰剂为0.5%羧甲基纤维素钠(CMC),奥美沙坦剂量为每天20mg/kg。在处理的第0、2、4、6、8、10、12周测尾静脉血糖并收集24小时尿液测尿蛋白。第12周处死动物,留取肾组织做病理切片及提取组织蛋白,通过western blot分析蛋白通路变化。2.细胞实验:在33℃条件下,条件永生化小鼠足细胞可以进行传代培养,并且需要在培养基(RPMI1640)中添加10%牛血清和1×104U/L的γ干扰素;在37℃条件下细胞开始分化,分化完成需要14天,分化时培养基中不加γ干扰素。分化完成后给予葡萄糖、Ang-Ⅱ、奥美沙坦等刺激处理,收集细胞用流式细胞仪检测凋亡或提取蛋白用WesternBlot方法分析通路变化。3.统计学处理:所有数据结果用均数±标准差(±s)表示,利用Graphpad Prism5.0软件进行数据统计分析和制图。组间的差异分析方法采用one wayANOVA,定义P值0.05为差异有统计学意义。研究结果1.奥美沙坦治疗12周后可以减少db/db小鼠的尿蛋白,而不影响小鼠的体重和血糖。在db/db小鼠肾脏组织中,奥美沙坦明显抑制p38/SIRT1通路,减少了足细胞的凋亡,但对AMPK没有影响。2.奥美沙坦能部分抑制高糖激活的AMPK/SIRT1通路,减少足细胞凋亡。3.Ang-Ⅱ激活p38/SIRT1通路诱导足细胞凋亡,而奥美沙坦明显的抑制这条通路,保护足细胞;P38抑制剂s8307具有类似作用。结论本课题验证了SIRT1在糖尿病肾病足细胞中具有重要的保护作用。高糖通过AMPK途径抑制SIRT1活性诱导足细胞凋亡;血管紧张素II通过p38/SIRT1通路触发了足细胞的凋亡。奥美沙坦能够抑制Ang-Ⅱ/p38/SIRT1通路,减少足细胞凋亡,降低糖尿病肾病小鼠尿蛋白;P38抑制剂s8307具有类似作用。
[Abstract]:Background and objective: diabetic nephropathy is the main cause leading to end stage renal disease, over 40% of the renal replacement therapy patients is caused by diabetes. The main clinical manifestations of proteinuria. Studies suggest that podocyte injury and proteinuria are closely related. In diabetic nephropathy, there are many factors such as high blood glucose, blood pressure angiotensin II (Ang- II) as a result of podocyte apoptosis. Strict blood glucose control and application of the renin-angiotensin system (RAS) blocking agent can significantly reduce the production of.SIRT1 urine protein is a kind of silent information regulator, has NAD+ dependent deacetylase activity is involved in the pathogenesis of many diseases, including including diabetic nephropathy. In diabetic nephropathy, SIRT1 is involved in inflammation, apoptosis, fibrosis and other multiple pathological processes. In apoptosis, previous studies have found that high glucose and Ang- II The expression and activity of different signaling pathways affect SIRT1, Ang- II induced endothelial cell apoptosis and activation of the P38/SIRT1 pathway. High glucose can activate AMPK/SIRT1 pathway in podocytes apoptosis. In clinical application, olmesartan can reduce urinary protein in diabetic nephropathy patients, but in patients with poor glycemic control. Olmesartan reduces urinary protein, the effect is not obvious. The treatment under high glucose condition olmesartan will be affected. Also suggested that high glucose and Ang- could be induced by different pathways of podocyte apoptosis. So we suppose, in diabetic nephropathy, Ang- II and glucose may through different signaling pathways (P38/SIRT1 pathway with the AMPK/SIRT1 pathway) affect the expression of SIRT1, thus leading to podocyte apoptosis, and olmesartan can only work on P38/SIRT1 pathway. Olmesartan is commonly used in clinical blood Angiotensin II inhibitor, can inhibit Ang- II and angiotensin II receptor binding. Previous studies showed that Ang- II mainly through mitogen activated protein kinase (MAPK), especially induced podocyte apoptosis and ERK1/2 signaling pathway. Ang- II can the current lack of relevant reports of apoptosis mediated by P38/SIRT1 signaling in podocytes. Research methods 1. animal experiment: db/db mice model of diabetic nephropathy in diabetic group, and divided into two groups: placebo group (db/db+CMC) and olmesartan group (db/db+OM), C57BL mice as non diabetes group (WT), 10 rats in each group. The mice at the age of 10-12 weeks, starting drug treatment by gavage, placebo was 0.5% cm sodium carboxymethyl cellulose (CMC), olmesartan dose was 20mg/kg. per day in the treatment of tail vein blood glucose measurement in 0,2,4,6,8,10,12 weeks and collected 24 hours urine test urine protein. Twelfth weeks animal, nephridial tissue pathological slices and Extraction of tissue protein, through the western analysis of blot protein signaling pathway in.2. cells: experiment under the condition of 33 DEG C, conditionally immortalized mouse podocytes can be passaged, and in the culture medium (RPMI1640) supplemented with 10% bovine serum and 1 x 104U/L interferon gamma; under the condition of 37 DEG C cells differentiation, differentiation 14 days, the differentiation culture medium without interferon gamma. After differentiation to glucose, Ang- II, olmesartan stimulation treatment, cells were collected through the analysis of path changes statistically.3. WesternBlot method with flow cytometry was used to detect the apoptosis or protein extraction: all the data with the standard deviation (SD s) said, statistical data analysis and mapping using Graphpad Prism5.0 software. Differences between groups were analyzed by one wayANOVA method, the definition of the P value is 0.05. There was a significant difference on the results of the 1. olmesartan 12 After a week can reduce urinary protein in db/db mice, and the mice did not affect body weight and blood glucose. In the kidneys of db/db mice, olmesartan inhibited p38/SIRT1 pathway, reduce podocyte apoptosis, but did not affect.2. AMPK/SIRT1 pathway of olmesartan could partially inhibit high glucose on activation of AMPK, reduce the apoptosis of podocyte.3.Ang- II induced podocyte apoptosis and activation of the p38/SIRT1 pathway, and olmesartan significantly inhibit this pathway, protect the podocyte; P38 inhibitor s8307 has similar effect. Conclusion the study proved that SIRT1 has an important protective role in diabetic nephropathy in podocytes. High glucose induced podocyte apoptosis by AMPK pathway inhibits SIRT1 activity; angiotensin II through the p38/SIRT1 pathway triggered podocyte apoptosis. Olmesartan can inhibit Ang- II /p38/SIRT1 pathway, reduce podocyte apoptosis, reduce diabetic nephropathy rat urine and small eggs White; P38 inhibitor s8307 has a similar effect.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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