姜黄素衍生物对原代大鼠肝星状细胞作用的体外研究

目的：观察姜黄素衍生物（Curc-OEG）抑制原代肝星状细胞（HSC）增殖、活化以及诱导其凋亡的作用,探讨其可能的作用机制。方法：采用IV型胶原酶、DNA酶消化SD雄性大鼠肝脏,percoll梯度离心得到纯化的肝星状细胞。细胞分离后1d分别加入0、6.25、12.5μg/mL的姜黄素衍生物,作用7d后用RT-PCR及Western blot检测细胞α-SMA、TGF-β1、Smad2的mRNA水平和蛋白表达量。活化的肝星状细胞在第14天分别加入0、6.25、12.5、25、50、75μg/mL的姜黄素衍生物,作用24h后RT-PCR检测凋亡基因Bax、Bcl-2的mRNA水平和纤维化相关基因TGF-β1、collagen I、NF-κB及TIMP-1的mRNA水平。结果：药物作用7d后,6.25μg/mL和12.5μg/mL浓度药物处理组与对照组相比,细胞数目分别减少了56%和86%。在12.5μg/mL浓度药物作用下,原代肝星状细胞α-SMA、TGF-β1及Smad2的mRNA表达水平分别下调83%、85%及75%,蛋白表达水平分别下调94%、92%及73%（P〈0.05）。25μg/mL浓度药物作用24h后,细胞凋亡明显。在50μg/mL浓度药物作用下,活化的肝星状细胞Bax的mRNA表达水平上调约2.3倍,Bcl-2的mRNA表达水平下调约5.6倍;TGF-β1、collagen I、NF-κB及TIMP-1的mRNA表达水平分别下调90%、83%、74%、65%（P〈0.05）。结论：姜黄素衍生物可以明显抑制原代肝星状细胞的增殖和活化,促进活化的肝星状细胞凋亡及减少细胞外基质的沉积。

Effects of Curc-OEG on primary rat hepatic stellate cells in vitro

AIM：To investigate the effects and mechanisms of Curc-OEG on the proliferation,activation and apoptosis of hepatic stellate cells（HSCs）. METHODS：The HSCs were isolated from male SD rats by collagenase IV and DNase digestion of liver,and further purified by percoll gradient centrifugation.Freshly isolated cells at day 2 were treated with Curc-OEG at concentrations of 0,6.25 and 12.5μg/mL for 7days.The expression ofα-SMA,TGF-β1 and Smad2 was confirmed by RT-PCR and Western blot.Activated HSCs at day 14 were treated with Curc-OEG at concentrations of 0,6.25,12.5,25,50 and 75μg/mL for 24 h.The expression of Bax and Bcl-2 and other genes associated with fibrogenesis including TGF-β1,collagen I,NF-κB and TIMP-1 was observed with RT-PCR.RESULTS：After 7days cultivation,Curc-OEG caused a significant concentration-dependent reduction in cell numbers of 56% and 86%a t 6.25 and 12.5μg/mL respectively compared with the control.At 12.5μg/mL,CurcOEG reducedα-SMA,TGF-β1 and Smad2 mRNA levels by 83%,85% and 75%,and protein levels by 94%,92% and 73%,respectively（P〈0.05）.Cell apoptosis was significantly increased at a concentration of 25μg/mL CurcOEG.At 50 μg/mL,Curc-OEG significantly upregulated the mRNA levels of the proapoptotic Bax by 2.3-fold,downregulated the antiapoptotic Bcl-2,TGF-β1,collagen I,NF-κB and TIMP-1 by 5.6-fold,90%,83%,74% and 65%,respectively（P〈0.05）.CONCLUSION：Curc-OEG not only significantly inhibited the proliferation and activation of primary HSCs,but also induced apoptosis of activated HSCs and reduced ECM accumulation.