红景天甙对HSC-T6细胞株Rho／ROCK信号通路影响的研究

Effects of salidroside on signal transduction of Rho/ROCK in the hepatic stellate cell

目的:观察红景天甙对大鼠肝星状细胞株HSC-T6细胞增殖活化、胶原合成、Rho相关卷曲螺旋形成蛋白激酶-Ⅰ(Rhoassociated coiled coil forming protein kinase-Ⅰ,ROCK-Ⅰ)及基质金属蛋白酶抑制剂-1(Tissue inhibitor of metalloproteinases-1,TIMP-1)表达的影响并以ROCK特异性抑制剂Y-27632作为阳性对照,探讨红景天甙抗肝纤维化作用的分子机制。方法:MTT法检测HSC-T6细胞生长增殖;酶联免疫吸附(ELISA)法检测HSC-T6细胞上清Ⅰ型胶原含量;实时荧光定量PCR(Re-al-time PCR)检测细胞α-平滑肌肌动蛋白(Alpha-smooth muscle actin,α-SMA)、ROCK-Ⅰ、TIMP-1mRNA的表达;Western blot检测细胞α-SMA、ROCK-Ⅰ、TIMP-1蛋白表达.结果:MTT检测显示红景天甙和Y-27632对HSC-T6细胞的生长增殖具有抑制作用,并呈剂量依赖性;ELISA结果提示红景天甙和Y-27632可抑制HSC-T6细胞Ⅰ型胶原的分泌;Real-time PCR和West-ern blot结果表明红景天甙和Y-27632均能下调HSC-T6细胞ROCK-Ⅰ、TIMP-1和α-SMA的表达。结论:红景天甙可抑制HSC-T6细胞活化增殖,减少其Ⅰ型胶原分泌,其机制可能与抑制HSC-T6细胞的Rho/ROCK信号通路有关。

Objective： To investigate the effects of salidroside on the proliferation, activation, Collagen Ⅰ secretion as well as expression of ROCK- Ⅰ and TIMP-1 in HSC-T6 cell, and to elucidate the molecular mechanism of salidroside against hepatic fibrosis with Y-27632 as the positive control. Methods： HSC-T5 cell line was employed as the model for following study. MTT colorimetric assay was used to detect the proliferation of HSC-T6 cell. ELISA was employed to value the content of collagen I in supernatant of HSC-T6 cells treated with salidroside and Y-27632. The effects of salidroside and Y-27632 on expressions of smooth muscle actin （α-SMA） ,ROCK-Ⅰ and TIMP-1 expression were analyzed by Real-time PCR and Western blot. Results：（ 1 ） Both Salidroside（3,6,12 mmol/L）and Y-27632 （5, 10,20 μmool/L） had dose-dependent inhibitory effects on α -SMA expression which represents the activated state of HSC-T6 （P〈0.05） （2）The expression of ROCK-Ⅰ could be downregulated by the 6 mmol/L salidroside and 10 μmol/L Y-27632 in HSC-T6 （P〈0.05）. （3） The 6mmol/L salidroside and 10 μmol/L Y-27632 both had inhibitory effects on TIMP-1 expression and collagen Ⅰ output in HSC-T6 cell （P〈0.05）. Conclusion：Salidroside can inhibit activation, TIMP-1 expression and collagen Ⅰ secretion of HSC-T6 cell, the mechanisms of which may be involved with its inhibitory effect on Rho/ROCK signal transduction pathway.