过氧化物酶体增殖物激活受体γ激动剂对大鼠肾成纤维细胞转化生长因子β1/Smad信号途径的作用研究

Effects of peroxisome proliferators-activated receptor γ agonists on transforming growth factor-β1 and Smads signal pathway: experiment with rat renal fibroblasts

目的研究过氧化物酶体增殖物激活受体γ（PPARγ）激动剂阻断转化生长因子（TGF）β1致肾间质纤维化作用的机制，探讨其抗肾间质纤维化的潜在作用。方法体外培养大鼠肾成纤维细胞株（NRK／49F），观察PPARγ配体15d-PGJ2及其激动剂曲格列酮和齐格列酮对TGFβ1诱导的纤维连接蛋白（FN）mRNA表达的影响。利用Western印迹技术观察PPARγ激动剂对TGFβ1诱导的FN和Smad蛋白表达的影响。结果（1）与1ng／ml TGFβ1组比较，5ng／ml TGFβ1组FN mRNA表达量增加了3．6倍（P〈0．01），5ng／ml TGFβ1刺激24h时较刺激前增加了2．4倍（P〈0．01），TGFβ1诱导FN mRNA表达呈一定范围内的剂量（0—5ne／ml）和时间（0—24h）依赖效应。（2）与5ng／ml TGFβ1组比较，10μmol 15d-PGJ2、曲格列酮和齐格列酮预处理组FN mRNA表达量分别降低37．3％、41．5％和22．7％，FN蛋白表达量分别降低20．6％、38．1％和28．6％。（3）5ng／ml TGFβ1以时间（0．2h）依赖方式诱导p-Smad2／3蛋白表达量增加，作用1h时达到高峰；5ng／ml TGFβ1组p-Smad2／3蛋白表达量较对照组和2ng／ml TGFβ1组分别增加3．42倍和0．97倍。（4）15d-PGJ2、曲格列酮和齐格列酮预处理组p-Smad2／3蛋白表达量与5ng／ml TGFβ1组比较分别降低61．2％、53．0％和59．S％。3种药物干预组之间p-Smad2／3蛋白表达量比较差异无统计学意义，各组Smad2和Smad3蛋白表达量无显著变化。结论PPART激动剂可以抑制TGFβ1诱导的肾间质成纤维细胞细胞外基质合成，其机制可能与阻断TGF-β1／Smad信号途径有关，提示PPARγ激动剂具有抗肾间质纤维化的潜在作用，可能成为延缓终末期肾功能衰竭的治疗新手段之一。

Objective To study the effects of peroxisome proliferators-activated receptor （PPABT） ngonists on transforming growth factor （TGF）-β1-induced fibrotic responses in renal fibroblasts, so as to investigate its effects in prevention of tubulointerstitial fibrosis. Methods Bat renal fibroblasts of the line NRK/49F were cultured and divided into groups. In group 1 TGFβ1 of the concentrations of 0, 1, 2, 5, and 10 ng/ml was added and co-cultured for 24 h. In group 2 TGFβ1 of the concentration of 5 ng/ml was added and co-cultured for0, 6, 12, and 24 h respectively. Groups 3, 4, and 5 were pretreated with 10 μmol/L15d-PGJ2, PPAR-/ligand, 10 μmol/L troglitazone, agonist of, and 10 μmol/L ciglitazone, both PPARγ agonists, respectively for 2 h, then treated with 5 ng/ml TGFβ1. A blank control group was sot up. The cultured cells were collected. RT-PCB was used to detect the mRNA expression of TGF-β1-indued fibronectin （FN）. Western blotting was used to detect the expression of TGF-β1-lnduced FN, Smad, and pbospbory]ated Smad （p-Smad） proteins. Results TGF-β1 enhanced the FN mRNA expression in a doseand time-dependent manner, The FN mRNA expression of the 5 ng/ml TGF-β1 group was 3.7 times that of the control group （P 〈0.05 ）. The FN mRNA expression of the 15d-PGJ2, trnglitazone-, and ciglitasonepretreated groups were lower than that of the 5 ng/nd TGF-β1 group by 37.3%, 41.5%, and 22. 7% respectively （all P 〈 0. 05 ）. The p-Smad2/3 protein expression levels of the TGF-β1 group began to increase 15 minutes after stimulation, increased in a time-dependent manner, peaked 1 hour after, and began to decrease 2 hours later. However, the levels of protein expression of total Smad2 and Smad3 did not change significantly in all groups. Both 2 ng/ml TGFβ1 and 5 ng/mlTGFβ1 significantly induced the increase of protein expression of p-Smad2/3 （ all P 〈 0.05 ）. The levels of protein expression of p-Smad2 and p-Smad3 of the 5 ng/mlTGFβ1 group were 3.42 and 0.97 times those of the 2 ng/mlTGFβ （ beth P 〈 0.05）. The levels of protein expression of p-Smad2/3 of the 15d-PGJ2, troglitazone-, and ciglitazone - pretreated groups were all significantly lower than that of the 5 ng/mlTGFβ group by 61.2%, 53.0%, and 59.5% （all P 〈 0. 05）, However, there was no significant difference among different drug-treated groups （all P 〉 0. 05 ）. Conclusion Possibly through abrogating TGF-β1/Smads signaling pathway, PPARγ agonists inhibit TGF-β1-induced renal fibroblast extracellular matrix synthesis and may play a potential role in preventing tubulointerstitial fibrosis as a novel approach to prevent the progress of end stage renal dysfunction.