肿瘤坏死因子α对肾小管上皮细胞PPARγ及辅调节因子表达的影响

Regulation of the expression of PPARγ and its coregulators in renal tubular cells by tumor necrosis factor-α

目的：探讨在肿瘤坏死因子α（TNF-α）介导的炎症反应中PPARγ及其辅调节因子,包括辅激活因子SRC/p160家族（steroid receptor coactivators/p16family,包括SRC-1、SRC-2、SRC-3）、PGC-1（PPARγcoactivator-1）以及辅抑制因子NCoR（Nuclear receptor corepressor）和单核细胞趋化因子（monocyte chemotactic protein,MCP-1）的表达水平变化,分析其中相互作用机制。方法：体外培养人肾小管上皮细胞（HK-2）。用TNF-α分别在不同浓度和不同时间点刺激HK-2细胞,收集细胞总mRNA和胞核蛋白。应用Real-time QPCR法和Western蛋白印迹法分别从mRNA水平和蛋白质水平检测PPARγ及其辅调节因子和单核细胞趋化因子（MCP-1）的表达变化。结果：在不同浓度的TNF-α（0~20ng/ml）刺激24h后,PPARγ、SRC-1、SRC-2、SRC-3和PGC-1均呈总体下调趋势（P〈0.05）,同时NCoR和MCP-1呈显著上调（P〈0.05和P〈0.01）。选择10ng/ml TNF-α为最适刺激浓度,作用不同的时间（0~16h）。发现PPARγ、SRC-1和SRC-2的mRNA在刺激2h后出现显著下调（P〈0.05）,分别是37%、35%和41%（P〈0.05）;而SRC-3和PGC-1mRNA水平仅在刺激1h后就出现显著下调（P〈0.05）,分别是53%和46%（P〈0.05）;NCoR则在刺激16h后出现显著上调（约为对照组的2.16倍,P〈0.01）,之后又缓慢下降;MCP-1在刺激0.5h时出现明显上调（为对照组的2.74倍,P〈0.05）,4h时达到高峰（为对照组的11倍,P〈0.01）。West-ern蛋白印迹法观察到PPARγ和SRC-2在10ng/ml TNF-α刺激4h和2h后出现明显下降。结论：TNF-α可不同程度的抑制HK-2细胞PPARγ及几种辅激活因子（SRC-1、SRC-2、SRC-3、PGC-1）的表达,同时上调辅抑制因子（NCoR）和炎症介质MCP-1的表达。提示PPARγ及其辅调节因子积极参与炎症反应,并且可能在炎症过程中对PPARγ的表达发挥共同的调节作用。

ObjeCtive：To investigate the changes of PPARγ expression and its coregulators and monocyte chemotactic factor （MCP-1） treated with tumor necrosis factor （TNF-α）, thus to analysis the mechanism of interaction of these factors, nethodology：Renal tubular ceils （HK-2 ceils） were cultured in vitro. The total cellular RNA was isolated for real-time quantitative polymerase chain reaction （real-time-QPCR） and nuclear extracts were prepared for Western blot analysis after treated with TNF-α in different concentrations and time points. Results ： Under stimulus of different concen- tration of TNF-α （0 -20ng/ml） for 24 hours, all of PPARγ,SRC-1 ,SRC-2,SRC-3 and PGC-1 mRNA expression showed obvious descending trend （ P 〈 0. 05 ）, meanwhile, NCoR presented light increase （ P 〈 0. 05 ） and MCP-1 presented significant up-regulation （P 〈 0. 01 ）. In time-dependent experiment, the levels of PPARγ,SRC-1 and SRC-2 mRNA were decreased 37% ,35% and 41% at 2 hours after treating with 10 ng/ml TNF-α （P 〈 0. 05）. The levels of SRC-3 and PGC-1 mRNA were decreased 53% and 46% only after 1 h （ P 〈 0. 05 ）. NCoR was not found obviously change until stimulated with TNF-α for 16 hours （nearly 2. 16 folds, P 〈0. 01）, then decreased slowly. MCP-1 was increased at 0. 5 h and reach the peak at 4h （ nearly eleven folds, P 〈 0. 01 ）. Western blot analysis showed that the proteins of PPARγ/and SRC-2 were significant decrease at 4h and 2h treated with TNF-α at 10ng/ml （ P 〈 0. 05 ）. Conclusion： These data demonstrate that TNF-α can transparently down-regulate the expression of PPARγ/and PPAR＇s coactivators, meanwhile up-regulate the expression of PPAR＇s corepressor NCoR and MCP-1. It indicates that PPAR＇s coregulators may actively participate the inflammarion in kidney.