激活FXR下调visfatin对糖尿病肾病的作用及机制研究

发布时间：2018-01-04 18:14

  本文关键词：激活FXR下调visfatin对糖尿病肾病的作用及机制研究　出处：《第三军医大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 法尼酯X受体 visfatin 糖尿病肾病 系膜细胞

【摘要】：背景和目的:糖尿病肾病(Diabetic Nephropathy,DN)是糖尿病的重要并发症,也是最常见的导致终末期肾脏疾病(End-stage renal disease,ESRD)的病因之一。其主要病理特征为肾脏结构改变包括肾小球肥大、系膜基质增厚、肾小球足细胞损伤,导致肾小球硬化和肾小管间质纤维化。这些变化共同导致了进行性蛋白尿增多和肾小球滤过率减少,最后发展为终末期肾病。糖尿病肾病的发病机制目前并不完全清楚,研究表明高血压、高血糖、晚期糖基化终末产物、脂质代谢异常、脂肪堆积、促纤维化生长因子、炎症因子、氧化应激等在糖尿病肾病进展中发挥一定的作用。法尼酯X受体(Farnesoid X receptor,FXR)属于核受体超家族成员,是一种细胞内信号蛋白,作为多功能转录调节因子激活或抑制靶基因表达。FXR在肝脏、肠、肾上腺和肾脏等组织都有高表达。现有研究表明FXR在糖尿病肾病发生发展中发挥重要作用,可能与FXR参与调控糖脂代谢基因表达有关,但具体调控机制仍不清楚。visfatin是新近分离并鉴定的一种分泌型蛋白,亦称前B细胞克隆增强因子(pre-B cell colony ehancing factor,PBEF),在脂肪组织、肝脏、心脏、骨骼肌、脑、肾脏等组织中均有表达。研究发现visfatin在糖脂代谢、胰岛素抵抗、细胞增殖、分化、凋亡和炎症反应等过程中发挥重要作用。研究发现visfatin在糖尿病患者血清浓度明显升高,参与了调控炎症因子表达、内皮功能损伤、胰岛素抵抗及动脉粥样硬化等。体外研究亦发现visfatin在高糖诱导肾小球系膜细胞及肾小管上皮细胞表达显著升高,外源性visfatin可促进肾小球系膜细胞葡萄糖转运蛋白GLUT-1的表达上调,促进葡萄糖吸收,并刺激促纤维化基因表达。我们前期研究发现血清visfatin水平在糖尿病肾病患者明显升高,且与炎症因子呈正相关,与肾功能水平呈显著负相关,提示visfatin很可能与糖尿病肾病的进程及炎症状态有密切相关。本研究拟通过体内体外实验阐明FXR调控visfatin表达的分子机制及其在糖尿病肾病进展过程中的作用,为深入认识糖尿病肾病发病机制提供新的思路。方法:1.临床研究部分:收集2014年6月-2015年12月我科收治经肾活检确诊的2型糖尿病肾病患者的肾活检标本及临床资料,并根据病理分期分为早期DN组13例、中期DN组16例、晚期DN组18例;收集我院泌尿外科同期收治的10例肾肿瘤手术患者远离肿瘤组织的正常组织标本为对照组;对收集的肾组织标本行PAS及visfatin免疫组化染色;患者临床资料收集包括年龄、性别、24小时尿白蛋白、血肌酐(Scr)、血尿素氮(BUN),并根据Cockcroft-Grault公式计算肾小球滤过率(e GFR)。并对分析统计结果进行如下比较:(1)比较各组24小时尿白蛋白、Scr、BUN、e GFR及visfatin表达半定量结果;(2)选取各期DN组患者24小时尿蛋白、Scr、BUN、e GFR结果,与visfatin表达半定量结果进行相关性分析。2.细胞实验部分:(1)采用FXR激动剂GW4064、FXR拮抗剂Guggulsterone处理人肾小球系膜细胞HMC,Real-time PCR和Western blot检测visfatin的m RNA及蛋白表达变化;(2)分别给予高糖(HG)诱导HMC转染si RNA沉默visfatin、GW4064或外源性visfatin处理:(1)Real-time PCR检测visfatin的m RNA表达,Western blot检测visfatin、NF-κB、IκBα的蛋白表达水平,ELISA法检测细胞上清MCP-1浓度;(2)Real-time PCR检测TGF-β1、α-SMA、Collagen IV及FN的的m RNA表达水平,Western blot检测TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表达水平;(3)cck-8法检测系膜细胞增殖,Real-time PCR和Western blot检测增殖相关基因PCNA的m RNA和蛋白表达水平;(3)根据生物信息学预测结果构建全长及截短visfatin启动子双荧光素酶报告基因载体:(1)将全长荧光素酶载体转染至高糖诱导系膜细胞,给予不同浓度的GW4064(0.5μM,1μM,5μM)或溶剂DMSO处理,24小时后检测visfatin启动子活性;(2)将FXR表达质粒和截短visfatin启动子双荧光素酶报告基因载体共转染至293细胞,给予DMSO或GW4064(5μM)处理,24小时后检测visfatin启动子活性。3.动物实验部分:采用db/db小鼠作为糖尿病肾病动物模型,db/m小鼠作为对照,将db/db小鼠分为db/db(12w)组、db/db(16w)组、db/db(20w)组及db/db(20w)+GW4064治疗组:(1)检测各组小鼠血糖、体重、24小时尿白蛋白、Scr、BUN等生化指标;(2)取各组小鼠肾脏组织行PAS、Masson染色,进行visfatin、TGF-β1、α-SMA及FN免疫组化染色;(3)Western blot检测各组小鼠肾脏组织visfatin、NF-κB、IκBα、TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表达情况。结果:1.临床研究部分:(1)免疫组化染色结果显示visfatin在DN患者肾脏组织表达,主要定位于肾小球,肾小管间质少量表达,且visfatin表达强度随着DN的分期增加表达增强(P0.01,与对照组比较)。(2)在早、中期DN组,visfatin表达与患者24小时尿白蛋白、Scr、BUN水平呈正相关(R=0.868,P0.01;R=0.913,P0.01;R=0.938,P0.01),与患者e GFR呈负相关(R=-0.979,P0.01);在晚期DN组,visfatin表达与患者24小时尿蛋白呈负相关(R=-0.499,P0.05),与Scr、BUN及e GFR水平无相关性(R=-0.099,P0.05;R=0.281,P0.05;R=0.026,P0.05)。2.细胞实验部分:(1)激活FXR通过调控visfatin表达可以抑制高糖诱导系膜细胞增殖及增殖相关基因、炎症因子、促纤维化基因的表达:与对照组比,HG组细胞增殖显著增快(P0.01),GW4064组较HG组增殖速度显著下降(P0.01),GW4064组PCNA蛋白表达水平较HG组显著下降(P0.01);与GW4064组比,GW4064+visfatin组细胞增殖、PCNA表达显著回升(P0.01);(2)与对照组比,HG组p-P65蛋白表达、细胞上清MCP-1水平明显升高(P0.01),而IκBα蛋白表达显著下降(P0.01);与HG组比,GW4064组p-P65和MCP-1表达水平显著下调(P0.01),IκBα表达明显回升(P0.01);与GW4064组比,visfatin组p-P65蛋白表达、上清MCP-1水平显著上调(P0.01),而IκBα表达明显下降(P0.01);(3)与对照组比,HG组TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表达水平明显升高(P0.01),GW4064组较HG组的TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表达水平显著下降(P0.01);与GW4064组比,GW4064+visfatin组TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表达水平明显回升(P0.01);(4)双荧光素酶实验结果显示,与对照比,HG组visfatin启动子活性显著增强,给予FXR激动剂GW4064可以抑制visfatin启动子活性,并呈浓度依赖性,其中GW4064(1μM)、GW4064(5μM)与HG组比具有统计学意义(P0.01);截短实验结果显示visfatin启动子活性在1607bp-1192bp之间明显增强(P0.01),提示FXR与visfatin启动子能的结合位点为visfatin启动子1607bp-1192bp区域。3.动物实验部分:(1)与db/m组比,db/db组小鼠24小时尿白蛋白、Scr、BUN显著升高(P0.01),且随着周龄增加而逐渐升高;GW4064治疗组小鼠24小时尿白蛋白、Scr、BUN较db/db组显著下降(与16w及20w比,P0.01);(2)PAS及Masson染色显示db/db小鼠肾小球明显增大,球囊扩展,糖原沉积,系膜及基质增生,肾小球细胞外基质增多,而GW4064治疗组肾小球的上述改变明显减轻;免疫组化染色显示,visfatin主要表达于肾小球,肾小管间质有少量表达,db/db组小鼠visfatin表达随着小鼠周龄增加逐渐增强,GW4064治疗组表达明显下降,TGF-β1、α-SMA和FN的免疫组化染色结果显示其趋势同visfatin免疫组化染色;(3)Western blot结果显示,与db/m组比,db/db组visfatin蛋白表达显著升高,且随着周龄增加而逐渐升高(P0.01),p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN表达趋势同visfatin表达(P0.01,与db/m组比);GW4064组visfatin蛋白表达较db/db组显著下降(与16w及20w比,P0.01);p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN的表达也明显下调(与16w及20w比,P0.01),而IκBα表达趋势与visfatin表达相反(与16w及20w比,P0.01)。结论:激活FXR通过调控visfatin基因启动子转录活性下调其表达,从而抑制系膜细胞增殖、炎症因子及促纤维化因子的表达,延缓糖尿病肾病的进展;其可能的结合位点位于visfatin启动子-1607bp至-1192bp区域。
[Abstract]:Background and objective: diabetic nephropathy (Diabetic Nephropathy DN) is an important complication of diabetes mellitus, which is the most common cause of end-stage renal disease (End-stage renal, disease, ESRD) one of the causes. The main pathological features of renal structural changes including glomerular hypertrophy, thickening of glomerular mesangial matrix, podocyte injury, leading to glomerular sclerosis and tubulointerstitial fibrosis. These changes lead to the increase of proteinuria and glomerular filtration rate decreased, and finally develop into end-stage renal disease. The pathogenesis of diabetic nephropathy is not completely clear, studies show that high blood pressure, high blood glucose, advanced glycation end products, lipid metabolism, fat accumulation, promoting growth factors, inflammatory fibrosis, oxidative stress plays a role in the progression of diabetic nephropathy. The farnesoid X receptor (Farnesoid X, receptor, FXR) belongs to the nuclear receptor Body superfamily, is an intracellular signaling protein, as a multifunctional transcription factor activation or inhibition of target gene expression of.FXR in liver, intestine, kidney and adrenal tissues had high expression. The current study shows that FXR in diabetic nephropathy plays an important role in the development, may be related to FXR gene involved in the regulation of lipid metabolism the expression, but the specific regulatory mechanism is still not clear that.Visfatin is a secreted protein recently isolated and identified the factor B cell clone (pre-B cell colony also called enhanced ehancing, factor, PBEF) in adipose tissue, liver, heart, skeletal muscle, brain, kidney and other tissues. The expression of visfatin in lipid research found metabolism, insulin resistance, cell proliferation, differentiation, apoptosis and inflammation play an important role in the process. The study found that visfatin increased significantly in serum concentration in patients with diabetes mellitus, participate in the regulation of inflammation The expression of inflammatory factor in endothelial dysfunction, insulin resistance, and atherosclerosis. In vitro studies have found that Visfatin in high glucose induced glomerular mesangial cells and renal tubular epithelial cells was significantly increased, the exogenous visfatin can promote the increase of mesangial cell glucose transporter GLUT-1 expression, promote the absorption of glucose, and stimulated profibrotic gene expression. Our previous study found that serum visfatin levels were significantly increased in patients with diabetic nephropathy, and a positive correlation with inflammatory factors, was negatively correlated with the level of renal function, suggesting that visfatin may be closely related to the process and the inflammatory state and diabetic nephropathy. In this study the in vitro and in vivo experiments to elucidate the molecular mechanism of FXR regulate the expression of visfatin and the progression of diabetic nephropathy in the process, to provide a new way of understanding the pathogenesis of diabetic nephropathy. Methods: 1. clinical research: from June 2014 -2015 year in December in our hospital and the clinical data of renal biopsy specimens of patients with type 2 diabetic nephropathy renal biopsy, and the pathological stages were divided into DN group of 13 cases of early stage, 16 cases in DN group, DN group of 18 cases collected late; the Department of Urology in our hospital during the period 10 cases of patients with renal tumors from tumor normal tissues as the control group; the collection of renal tissue specimens of PAS and visfatin by immunohistochemical staining; the clinical data were collected including age, gender, 24 hours urinary albumin, serum creatinine (Scr), blood urea nitrogen (BUN), and glomerular filtration rate calculation according to the Cockcroft-Grault formula (E GFR). And the statistical analysis of results are as follows: (1) comparison comparison of 24 hours urinary albumin, Scr, BUN, e, GFR and visfatin expression of semi quantitative results; (2) the DN group of patients 24 hours urine protein, Scr, BUN, e, G The results of FR, the expression of visfatin and semi quantitative analyzed the correlation between.2. cell experiment: (1) the FXR agonist GW4064, FXR antagonist Guggulsterone HMC in human mesangial cells, the expression changes of M protein and RNA Real-time PCR and Western blot detection of visfatin; (2) were treated with high glucose (HG) induced by HMC transfection of Si RNA silencing visfatin, GW4064 or exogenous visfatin treatment: (1) the expression of M RNA Real-time PCR visfatin Western blot detection, visfatin detection, NF- kappa B, the expression level of I kappa B alpha protein, detection of supernatant MCP-1 concentration by ELISA; (2) the detection of TGF- Real-time PCR 1 beta, alpha -SMA. Collagen IV and FN m RNA expression, Western blot detection of TGF- beta 1, smad2/3, p-smad2/3, alpha -SMA, the expression level of Collagen IV and FN protein; (3) detection of mesangial cell proliferation by CCK-8 m RNA, Real-time PCR and Western blot to detect the proliferation related gene PCNA And the protein expression level; (3) according to the bioinformatics prediction results of full-length and truncated visfatin promoter luciferase reporter gene vector: (1) the full-length luciferase vector was transfected into glucose induced mesangial cells treated with different concentrations of GW4064 (0.5 M, 1 M, 5 M) or solvent DMSO visfatin, detection of promoter activity after 24 hours; (2) the FXR expression plasmid and truncated visfatin promoter luciferase reporter vector was transfected into 293 cells treated with DMSO or GW4064 (5 M) treatment, detection of visfatin promoter activity in.3. animal experiment after 24 hours: using db/db mice as diabetic nephropathy the animal model of db/m mice as control group, db/db mice were divided into db/db group (12W), db/db (16W) group, db/db (20W) group and db/db (20W) +GW4064 treatment group: (1) the blood glucose, body weight of mice were detected, 24 hours urinary albumin, Scr, BUN and other biochemical indicators; (2 take each) 缁勫皬榧犺偩鑴忕粍缁囪�孭AS,Masson鏌撹壊,杩涜�寁isfatin,TGF-尾1,伪-SMA鍙奆N鍏嶇柅缁勫寲鏌撹壊;(3)Western blot妫，

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