枇杷叶熊果酸对大鼠肝星状细胞增殖抑制作用及对PPAR-γ、TGF-β1表达的影响

Effects of ursolic acid from loquat leaves on proliferation inhibition and expression of PPAR-γ,TGF-β1 in rat hepatic stellate cells

目的研究枇杷叶熊果酸对大鼠肝星状细胞增殖的抑制作用及对PPAR-γ、TGF-β1表达的影响,探讨枇杷叶熊果酸抗肝纤维化的可能作用机制。方法体外培养HSC-T6细胞,随机分为空白组,罗格列酮对照组,枇杷叶熊果酸低、中、高浓度组,分别干预24、48、72 h后,运用CCK-8法检测各组细胞的增殖情况;ELISA法检测各组细胞培养上清液中的Ⅰ型胶原蛋白含量;Real-time PCR法检测各组PPAR-γ、TGF-β1 mRNA的表达;免疫细胞化学法检测各组PPAR-γ、TGF-β1蛋白的表达。结果 CCK-8结果显示,随着熊果酸作用时间的延长,药物对细胞的增殖的抑制率增高(P<0.01);ELISA结果显示,随着熊果酸药物浓度的增加,上清中的Ⅰ型胶原蛋白含量下降(P<0.01);Real-time PCR结果显示,随着熊果酸药物浓度增加,PPAR-γmRNA的表达量增加,TGF-β1 mRNA的表达量下降(P<0.01);免疫细胞化学结果显示,随着熊果酸药物浓度增加,PPAR-γ蛋白的表达增强,TGF-β1蛋白的表达减弱(P<0.01)。以上作用具有剂量-效应关系,高浓度组作用较对照组强。结论枇杷叶熊果酸能够抑制HSC-T6细胞增殖,降低细胞外基质分泌,该机制可能与上调PPAR-γ表达,下调TGF-β1表达有关。

Aim To investigate the effects of ursolic acid from loquat leaves on proliferation inhibition and expression of PPAR-γ 、TGF-β1 in rat hepatic stellate cells,so as to explore the mechanism of anti-hepatic fibrosis of UA.Methods HSC-T6 cells were randomly divided into blank group,rosiglitazone control group,the low,medium and high concentration of UA group to detect the cell proliferation inhibition by CCK-8 after24,48,72 h.The content of type collagen Ⅰ in cell culture supernatant of each group was examined by ELISA.The expression of PPAR-γ mRNA,TGF-β1mRNA in HSC-T6 cells exposured were examined by real-time quantitative PCR.Effects on HSC-T6 PPAR-γ and TGF-β1 protein from each group were detected by immunocytochemical method.Results CCK-8 results showed that the inhibitory rate of UA on cell proliferation increased with the prolongation of drug action time（P 〈 0.01）.ELISA results showed that with the increase of the concentration of UA,the content of typeⅠ collagen content decreased（P 〈 0.01）.Real-time PCR results showed that with the increase of the concentration of UA,and the expression of PPAR-γ mRNA increased,the expression of TGF-β1 mRNA decreased（P 〈 0.01）.The results of immunocytochemistry showed that the expression of PPAR-γ protein was increased,and the expression of TGF-β1 protein was decreased with the increase of the concentration of UA（P 〈 0.01）.All effects mentioned above were dose-dependent.Moreover,the effects in the high concentration groups were stronger than those in control group.Conclusion UA can inhibit the proliferation of HSCT6 cells,Which may be associated with the up-regulation of PPAR-γ expression and the down-regulation of TGF-β1 expression.