过氧化物酶体增殖物活化受体γ减少高糖诱导系膜细胞外基质的积聚及其机制  

Protective effect and mechanisms of overexpressed PPARγ against fibrosis induced by high glucose in cultured mesangial cells

作　　者：孙晶[1] 马骥[1] 顾勇[1] 林善锬[1] 

机构地区：[1]复旦大学附属华山医院肾脏科,上海200040

出　　处：《中华肾脏病杂志》2004年第4期255-259,共5页Chinese Journal of Nephrology

基　　金：国家自然科学基金(30200125);上海市科学委员会基金(03JC14084)

摘　　要：目的探讨过氧化物酶体增殖物活化受体(PPAR)γ对高糖诱导肾小球系膜细胞(GMC)外基质(ECM)积聚的抑制作用及其机制。方法以表达野生型小鼠PPARγ1的质粒pIRES2-EGFP-mPPARγ1/WT转染系膜细胞(WT),然后予30mmol/L的高糖(HG)刺激48h,以ELISA法检测细胞上清液中TGF-β1、纤连蛋白(FN)的浓度。GMC中c-fos、c-jun、葡萄糖转运蛋白1(GLUT-1)mRNA的表达检测采用半定量RT-PCR法,并以Western印迹检测胞浆中核因子抑制物(Ⅰ-κB)、核因子(NF-κB)及胞核中NF-κB、磷酸化细胞外调节激酶(p-ERK)的蛋白表达水平。同时以2μmol/L的PPARγ激活剂吡咯列酮(Pio)、转染表达功能缺陷的突变型(dominantnegative,DN)PPARγ1的质粒pIRES2-EGFP-mPPARγ1/DN(DN)或空白质粒pIRES2-EGFP作为对照。PPARγ的活性以其与PPARγ反应元件(PPRE)的结合能力表示。结果HG培养时系膜细胞的PPARγ活性较正常糖浓度(5mmol/L)培养时明显上升(P<0.01),HG+WT组的PPARγ活性显著高于HG+DN、HG+pIRES2-EGFP和HG+Pio组。与HG+DN、HG+pIRES2-EGFP相比,WT转染所致的PPARγ1高表达能显著抑制高糖诱导GMC的TGFβ-1、FN生成增多,减轻c-fos和c-jun的mRNA表达的上调,改善p-ERK蛋白水平的增加、胞浆Ⅰ-κB表达的降低以及NF-κB由胞浆向胞核转移的增加(P均<0.Objective To study the inhibitory effect and underlying mechanis ms of PPAR?on increased extracellular matrix (ECM) production from cultured mesan gial cells induced by high glucose. Methods Plasmid expressing functional wild-type mouse PPAR?1, pIRES2-EGFP-mPPAR?1/WT (WT) was transfected into rat mesa ngial cells cultured in high glucose (HG) condition, while transfection with pla smid expressing non-functional dominant negative type of mPPAR?1, pIRES2-EGFP-mPPAR?1/DN (DN), and blank plasmid, pIRES2-EGFP (Blank), were used as contro ls. The activity of PPAR?was assessed by its binding capacity to PPRE. Concentr ations of TGF-a1 and fibronectin in culture medium were examined by ELISA. Sem i-quantitative RT-PCR was used to determine mRNA expression of GLUT-1, c-fos and c-jun. Protein levels of p-ERK, Ⅰ-êB and nucleus/cytosol ratio of NF-êB were estimated by Western blot. Effect of 2 ìmol/L PPAR?agonist pioglitazo ne (Pio) was also studied. Results PPAR?activity was significantly higher in ce lls treated with HG.WT transfection showed stronger activity than DN or Blank tr ansfection and Pio treatment(P >0 05).Among them,WT but neither DN, Blank, nor Pio significantly ameliorated the increased concentrations of TGF-a1 and fibro nectin in culture medium induced by HG. Compared with DN and Blank, WT transfect ion significantly attenuated high glucose-caused elevation in c-fos, c-jun and p-ERK expression, reduction in Ⅰ-êB level, and incretio n in nucleus/cytosol ratio of NF-êB, while Pio demonstrated similarly signific ant changes only in p-ERK and NF-êB. No significant difference of GLUT-1 mRN A level was detected between HG groups. Conclusions Overexpressed PPAR?1 can su ppress increased ECM production from glomerular mesangial cells cultured in HG c ondition. Its inhibitory effects on activation of ERK/AP-1 and NF-êB pathways are involved. The further increase of PPAR?activity may have direct anti-fibr otic effect in diabetic kidney.

关 键 词：过氧化物酶体增殖物 活化受体γ 高糖 系膜细胞外基质 积聚作用 激活蛋白 

分 类 号：R692[医药卫生—泌尿科学]