發(fā)布時間：2018-01-04 18:14

  本文關鍵詞：激活FXR下調(diào)visfatin對糖尿病腎病的作用及機制研究　出處：《第三軍醫(yī)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 法尼酯X受體 visfatin 糖尿病腎病 系膜細胞

【摘要】：背景和目的:糖尿病腎病(Diabetic Nephropathy,DN)是糖尿病的重要并發(fā)癥,也是最常見的導致終末期腎臟疾病(End-stage renal disease,ESRD)的病因之一。其主要病理特征為腎臟結(jié)構(gòu)改變包括腎小球肥大、系膜基質(zhì)增厚、腎小球足細胞損傷,導致腎小球硬化和腎小管間質(zhì)纖維化。這些變化共同導致了進行性蛋白尿增多和腎小球濾過率減少,最后發(fā)展為終末期腎病。糖尿病腎病的發(fā)病機制目前并不完全清楚,研究表明高血壓、高血糖、晚期糖基化終末產(chǎn)物、脂質(zhì)代謝異常、脂肪堆積、促纖維化生長因子、炎癥因子、氧化應激等在糖尿病腎病進展中發(fā)揮一定的作用。法尼酯X受體(Farnesoid X receptor,FXR)屬于核受體超家族成員,是一種細胞內(nèi)信號蛋白,作為多功能轉(zhuǎn)錄調(diào)節(jié)因子激活或抑制靶基因表達。FXR在肝臟、腸、腎上腺和腎臟等組織都有高表達�，F(xiàn)有研究表明FXR在糖尿病腎病發(fā)生發(fā)展中發(fā)揮重要作用,可能與FXR參與調(diào)控糖脂代謝基因表達有關,但具體調(diào)控機制仍不清楚。visfatin是新近分離并鑒定的一種分泌型蛋白,亦稱前B細胞克隆增強因子(pre-B cell colony ehancing factor,PBEF),在脂肪組織、肝臟、心臟、骨骼肌、腦、腎臟等組織中均有表達。研究發(fā)現(xiàn)visfatin在糖脂代謝、胰島素抵抗、細胞增殖、分化、凋亡和炎癥反應等過程中發(fā)揮重要作用。研究發(fā)現(xiàn)visfatin在糖尿病患者血清濃度明顯升高,參與了調(diào)控炎癥因子表達、內(nèi)皮功能損傷、胰島素抵抗及動脈粥樣硬化等。體外研究亦發(fā)現(xiàn)visfatin在高糖誘導腎小球系膜細胞及腎小管上皮細胞表達顯著升高,外源性visfatin可促進腎小球系膜細胞葡萄糖轉(zhuǎn)運蛋白GLUT-1的表達上調(diào),促進葡萄糖吸收,并刺激促纖維化基因表達。我們前期研究發(fā)現(xiàn)血清visfatin水平在糖尿病腎病患者明顯升高,且與炎癥因子呈正相關,與腎功能水平呈顯著負相關,提示visfatin很可能與糖尿病腎病的進程及炎癥狀態(tài)有密切相關。本研究擬通過體內(nèi)體外實驗闡明FXR調(diào)控visfatin表達的分子機制及其在糖尿病腎病進展過程中的作用,為深入認識糖尿病腎病發(fā)病機制提供新的思路。方法:1.臨床研究部分:收集2014年6月-2015年12月我科收治經(jīng)腎活檢確診的2型糖尿病腎病患者的腎活檢標本及臨床資料,并根據(jù)病理分期分為早期DN組13例、中期DN組16例、晚期DN組18例;收集我院泌尿外科同期收治的10例腎腫瘤手術患者遠離腫瘤組織的正常組織標本為對照組;對收集的腎組織標本行PAS及visfatin免疫組化染色;患者臨床資料收集包括年齡、性別、24小時尿白蛋白、血肌酐(Scr)、血尿素氮(BUN),并根據(jù)Cockcroft-Grault公式計算腎小球濾過率(e GFR)。并對分析統(tǒng)計結(jié)果進行如下比較:(1)比較各組24小時尿白蛋白、Scr、BUN、e GFR及visfatin表達半定量結(jié)果;(2)選取各期DN組患者24小時尿蛋白、Scr、BUN、e GFR結(jié)果,與visfatin表達半定量結(jié)果進行相關性分析。2.細胞實驗部分:(1)采用FXR激動劑GW4064、FXR拮抗劑Guggulsterone處理人腎小球系膜細胞HMC,Real-time PCR和Western blot檢測visfatin的m RNA及蛋白表達變化;(2)分別給予高糖(HG)誘導HMC轉(zhuǎn)染si RNA沉默visfatin、GW4064或外源性visfatin處理:(1)Real-time PCR檢測visfatin的m RNA表達,Western blot檢測visfatin、NF-κB、IκBα的蛋白表達水平,ELISA法檢測細胞上清MCP-1濃度;(2)Real-time PCR檢測TGF-β1、α-SMA、Collagen IV及FN的的m RNA表達水平,Western blot檢測TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表達水平;(3)cck-8法檢測系膜細胞增殖,Real-time PCR和Western blot檢測增殖相關基因PCNA的m RNA和蛋白表達水平;(3)根據(jù)生物信息學預測結(jié)果構(gòu)建全長及截短visfatin啟動子雙熒光素酶報告基因載體:(1)將全長熒光素酶載體轉(zhuǎn)染至高糖誘導系膜細胞,給予不同濃度的GW4064(0.5μM,1μM,5μM)或溶劑DMSO處理,24小時后檢測visfatin啟動子活性;(2)將FXR表達質(zhì)粒和截短visfatin啟動子雙熒光素酶報告基因載體共轉(zhuǎn)染至293細胞,給予DMSO或GW4064(5μM)處理,24小時后檢測visfatin啟動子活性。3.動物實驗部分:采用db/db小鼠作為糖尿病腎病動物模型,db/m小鼠作為對照,將db/db小鼠分為db/db(12w)組、db/db(16w)組、db/db(20w)組及db/db(20w)+GW4064治療組:(1)檢測各組小鼠血糖、體重、24小時尿白蛋白、Scr、BUN等生化指標;(2)取各組小鼠腎臟組織行PAS、Masson染色,進行visfatin、TGF-β1、α-SMA及FN免疫組化染色;(3)Western blot檢測各組小鼠腎臟組織visfatin、NF-κB、IκBα、TGF-β1、smad2/3、p-smad2/3、α-SMA、Collagen IV及FN的蛋白表達情況。結(jié)果:1.臨床研究部分:(1)免疫組化染色結(jié)果顯示visfatin在DN患者腎臟組織表達,主要定位于腎小球,腎小管間質(zhì)少量表達,且visfatin表達強度隨著DN的分期增加表達增強(P0.01,與對照組比較)。(2)在早、中期DN組,visfatin表達與患者24小時尿白蛋白、Scr、BUN水平呈正相關(R=0.868,P0.01;R=0.913,P0.01;R=0.938,P0.01),與患者e GFR呈負相關(R=-0.979,P0.01);在晚期DN組,visfatin表達與患者24小時尿蛋白呈負相關(R=-0.499,P0.05),與Scr、BUN及e GFR水平無相關性(R=-0.099,P0.05;R=0.281,P0.05;R=0.026,P0.05)。2.細胞實驗部分:(1)激活FXR通過調(diào)控visfatin表達可以抑制高糖誘導系膜細胞增殖及增殖相關基因、炎癥因子、促纖維化基因的表達:與對照組比,HG組細胞增殖顯著增快(P0.01),GW4064組較HG組增殖速度顯著下降(P0.01),GW4064組PCNA蛋白表達水平較HG組顯著下降(P0.01);與GW4064組比,GW4064+visfatin組細胞增殖、PCNA表達顯著回升(P0.01);(2)與對照組比,HG組p-P65蛋白表達、細胞上清MCP-1水平明顯升高(P0.01),而IκBα蛋白表達顯著下降(P0.01);與HG組比,GW4064組p-P65和MCP-1表達水平顯著下調(diào)(P0.01),IκBα表達明顯回升(P0.01);與GW4064組比,visfatin組p-P65蛋白表達、上清MCP-1水平顯著上調(diào)(P0.01),而IκBα表達明顯下降(P0.01);(3)與對照組比,HG組TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達水平明顯升高(P0.01),GW4064組較HG組的TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達水平顯著下降(P0.01);與GW4064組比,GW4064+visfatin組TGF-β1、p-smad2/3、α-SMA、Collagen IV及FN的m RNA和蛋白表達水平明顯回升(P0.01);(4)雙熒光素酶實驗結(jié)果顯示,與對照比,HG組visfatin啟動子活性顯著增強,給予FXR激動劑GW4064可以抑制visfatin啟動子活性,并呈濃度依賴性,其中GW4064(1μM)、GW4064(5μM)與HG組比具有統(tǒng)計學意義(P0.01);截短實驗結(jié)果顯示visfatin啟動子活性在1607bp-1192bp之間明顯增強(P0.01),提示FXR與visfatin啟動子能的結(jié)合位點為visfatin啟動子1607bp-1192bp區(qū)域。3.動物實驗部分:(1)與db/m組比,db/db組小鼠24小時尿白蛋白、Scr、BUN顯著升高(P0.01),且隨著周齡增加而逐漸升高;GW4064治療組小鼠24小時尿白蛋白、Scr、BUN較db/db組顯著下降(與16w及20w比,P0.01);(2)PAS及Masson染色顯示db/db小鼠腎小球明顯增大,球囊擴展,糖原沉積,系膜及基質(zhì)增生,腎小球細胞外基質(zhì)增多,而GW4064治療組腎小球的上述改變明顯減輕;免疫組化染色顯示,visfatin主要表達于腎小球,腎小管間質(zhì)有少量表達,db/db組小鼠visfatin表達隨著小鼠周齡增加逐漸增強,GW4064治療組表達明顯下降,TGF-β1、α-SMA和FN的免疫組化染色結(jié)果顯示其趨勢同visfatin免疫組化染色;(3)Western blot結(jié)果顯示,與db/m組比,db/db組visfatin蛋白表達顯著升高,且隨著周齡增加而逐漸升高(P0.01),p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN表達趨勢同visfatin表達(P0.01,與db/m組比);GW4064組visfatin蛋白表達較db/db組顯著下降(與16w及20w比,P0.01);p-P65、TGF-β1、p-Smad2/3、α-SMA、Collagen IV和FN的表達也明顯下調(diào)(與16w及20w比,P0.01),而IκBα表達趨勢與visfatin表達相反(與16w及20w比,P0.01)。結(jié)論:激活FXR通過調(diào)控visfatin基因啟動子轉(zhuǎn)錄活性下調(diào)其表達,從而抑制系膜細胞增殖、炎癥因子及促纖維化因子的表達,延緩糖尿病腎病的進展;其可能的結(jié)合位點位于visfatin啟動子-1607bp至-1192bp區(qū)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) 緇勫皬榧犺偩鑴忕粍緇囪�孭AS,Masson鏌撹壊,榪涜�寁isfatin,TGF-尾1,偽-SMA鍙奆N鍏嶇柅緇勫寲鏌撹壊;(3)Western blot媯，

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