鳖甲煎改良方对肝星状细胞生长及TGFβ1、Smad3与Smad7表达的影响

The effect of modified turtle shell decoction on the expression of TGFβ1, Smad3 and Smad7 in rat hepatic stellate cell

目的探讨鳖甲煎改良方(modified turtle shell decoction,MTSD)对肝星状细胞T6(hepatic stellate cell T6,HSC-T6)TGFβ1、Smad3及Smad7表达的影响。方法用含生药量为0.71 g/ml的MTSD处理HSC-T6作为实验组,与MTSD剂量相同的鳖甲煎丸(turtle shell pills,TSP)处理的HSC-T6作为阳性对照组,常规培养的HSC-T6作为正常对照组,倒置显微镜下观察比较各组HSC-T6的形态变化。CCK-8检测各组HSC-T6细胞的生长情况,并绘制生长曲线;采用Real time-PCR、免疫荧光细胞化学及Western blotting技术分别检测各组HSC-T6的TGFβ1、Smad3及Smad7基因及其编码蛋白的表达变化。结果倒置显微镜下观察显示,与阳性对照细胞比较,MTSD处理48 h的HSC-T6,胞体变小,部分细胞死亡碎裂。CCK-8检测证实,MTSD对HSC-T6生长具有抑制作用,与2个对照组比较,MTSD处理的HSC-T6的生长减慢,从48 h开始差异具有统计意义(P<0.01)。Real time-PCR、免疫荧光细胞化学及Western blotting检测显示,MTSD能在m RNA及蛋白水平明显下调HSC-T6的TGFβ1、Smad3的表达,上调其Smad7的表达,与2个对照组相比,差异均具统计学意义(P<0.01)。结论 MTSD能有效抑制HSC-T6的生长,下调其TGFβ1、Smad3的表达,同时使Smad7的表达上调。MTSD抑制HSC-T6的生长可能与干扰TGFβ1/Smad3信号通路密切有关。

Hepatic fibrosis（HF） will develop into cirrhosis when it continues for long periods of time.Activated hepatic stellate cells（HSC） are involved in the formation of HF and structural remodeling of the liver through the proliferation and secretion of extracellular matrix. Modified turtle shell decoction（MTSD） has a certaininhibitory effect on liver fibrosis in rats, but its mechanism is not clear. Then we plan to investigate the effect of MTSD on the expression of TGFβ1, Smad3 and Smad7 in hepatic stellate cell line T6（HSC-T6） of rat. In this study,HSC-T6 was treated with MTSD containing crude drug 0.71 g/ml（experimental group）, treated with turtle shell pills（TSP） at the same dose（positive control）, or cultured routinely（normal control）. The morphological changes of HSC-T6 in different group were observed and compared under the inverted microscope; the growth of HSC-T6 cells in each group was tested by cell count kit-8（CCK-8）, and the growth curve was draw. Real time-PCR,immunofluorescence and Western blot were employed to detect the expression changes of TGFβ1, Smad3 and Smad7 genes and their coding proteins in each group of HSC-T6, respectively. Inverted microscope observation showed that HSC-T6 treated with MTSD for 48 h became small, and showed partial cell death and fragmentation, ascompared with the control cells; CCK-8 assay identified that MTSD could inhibit HSC-T6 cellsgrowth, and the growth of the HSC-T6 cell line treated with MTSD were lower than that of controls and the differences were statistically significant after the cells were treated for 48 h（P〈0.01）. Real time-PCR, immunofluorescence and Western blotting revealed that MTSDcould significantly down-regulate the expression of TGFβ1 and Smad3 in mRNA and protein levels, butup-regulated the expression of Smad7 in HSC-T6（P〈0.01）. In conclusion, MTSD can significantly inhibit thegrowth of HSC-T6, down-regulate the expression of TGF beta 1 and Smad3, and up-regulated the expression ofSmad7 at the same time, and the inhibition effects of MTSD on the growth of HSC-T6 may be closely related tointerference of the TGF beta 1/Smad3 signaling pathway.