摘要
目的探讨过氧化物酶体增殖物激活受体（PPARγ）配体是否通过阻断肝星状细胞的TGF β1型受体（TGF βR1）信号途径，阻止Smad3磷酸化而抑制纤溶酶原激活物抑制剂1（PAI-1）、胶原蛋白1αI的表达，发挥其抗纤维化的作用。方法用促进脂肪细胞分化的培养液预处理人肝星状细胞LX-2，使其呈现脂肪细胞的某些表型，从活化状态转为静止型，分别加用TGF β1或TGF β1加TGF βR1激酶抑制剂SB431542或TGF β1加PPAR γ配体Ciglitazone处理LX-2细胞。分别用荧光实时定量PCR、Western blot、荧光素酶分析的方法检测PPAR γ配体对LX-2细胞用TGF β1诱导前后Smad3磷酸化水平、PAI-1的mRNA与蛋白表达水平和PAI-1启动子活性、胶原蛋白1αI mRNA表达水平的影响。结果诱导脂肪细胞分化的培养液可使LX-2细胞出现脂质沉积与静止型肝星状细胞的标志物之一PPAR γ表达的增加。TGFD1以剂量和时间依赖的方式增加细胞外基质蛋白的表达，胶原蛋白1aI和PAI-1 mRNA的表达在3h内增加3倍，PAI-1蛋白的表达在6h内增加8倍。TGF β1诱导的Smad3磷酸化导致胶原蛋白1aI和PAI-1表达增加。PPARγ配体Ciglitazone与TGF βR1激酶抑制剂SB431542，均以剂量依赖的方式阻断TGF β1的上述作用，10μmol／L SB431542、10μmol／L的Ciglitazone可以阻断TGF β1诱导的Smad3磷酸化、胶原蛋白1αI mRNA、PAI-1 mRNA和蛋白的表达。结论PPAR γ配体Ciglitazone抗纤维化作用可能与其对TGF β1-TGF βR1信号传导、Smad3磷酸化的阻断作用有关，进而抑制胶原蛋白1αI和PAI-1的表达。
Objective We tested a hypothesis that PPAR γ inhibits TGFβl-activation of TGF β receptor （TGF βR）-1 signaling in quiescent stellate cells, thereby abrogating Smad3 phosphorylation and inhibiting PAI-1 and collagen expressions. Methods Human stellate cells （HSC） were cultured in a medium containing isobutylmethylxanthine, dexamethasone and insulin （MDI） to induce a quiescent adipocytic phenotype one, and then they were treated with TGF β1 with or without SB431542, a TGF βR1 kinase inhibitor, or the PPAR γ agonist ciglitazone. Effects on Smad 3 phosphorylation, TGF β-responsive transcriptional activity, and expressions of collagen and PAI-1 were assessed. Results Culturing HSC in MDI induced an adipocytic phenotype characterized by lipid accumulation and increased PPAR γ expression and transcriptional activity. TGF β1 treatment caused dose- and time-dependent increases in ECM gene expression, in-creasing collagen and PAI-1 mRNAs by 3 fold within 3 h and increasing PAI-1 protein levels by 8 fold within 6 h. Treatment with the TGF βR1 kinase inhibitor, SB431542, inhibited all of these responses. The PPAR γ agonist ciglitazone also caused a dose-dependent inhibition of TGF β1＇s fibrogenic actions. 1 mmol/L ciglitazone blocked TGF β1-transcriptional activity and abolished TGF β-mediated induction of collagen and PAI-1 expressions. Conclusions The anti-fibrotic ability of PPAR γ agonist ciglitazone may be related to its ability to inhibit TGF β1-TGF βR1 signaling and blocking pSmad3-dependent induction of PAI-1 and collagen expression.
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2007年第11期840-844,共5页
Chinese Journal of Hepatology
