鳖甲水煎液药物血清对肝星状细胞的作用

Turtle Carapace Decoction and Drug Containing Serum on Hepatic Stellate Cells

目的:实验研究鳖甲水煎液药物血清对肝星状细胞的影响和作用机制。方法:双抗夹心ELISA法检测鳖甲水煎液药物血清对TGF-β1刺激的LX-1细胞胶原及调节因子表达的影响,MTS法检测鳖甲水煎液药物血清对TGF-β1刺激的LX-1细胞增殖的影响,Western blot检测鳖甲水煎液药物血清对TGF-β1刺激的LX-1细胞活化、胶原合成及细胞外基质等表达的影响。结果:鳖甲水煎液药物血清能明显抑制TGF-β1刺激的LX-1细胞的增殖;显著下调TGF-β1刺激的LX-1细胞Ⅰ、Ⅲ、Ⅳ型胶原、HA与α-SMA蛋白表达;并作用于TGF-β1下游信号通道蛋白,抑制Smad 3及P-smad3等因子表达,从而阻断TGF-β1等因子的生物学效应;能显著上调MMP 1、MMP 3蛋白表达,同时通过抑制TIMP-1蛋白表达、更有利于改善胶原酶活性,从而促进ECM的降解。在实验浓度范围内,鳖甲水煎液药物血清对肝星状细胞的作用呈现量效关系,作用显著优于目前有效的抗肝纤维化药物或细胞因子IFN-γ,与扶正化瘀胶囊作用相当。结论:鳖甲水煎液药物血清能抑制肝星状细胞活化增殖及细胞外基质合成分泌、促进细胞外基质降解吸收,调控细胞因子水平及信号传导通路,从而发挥抗肝纤维化的作用。本实验进一步验证了鳖甲抗肝纤维化药效学的有效性,为鳖甲抗肝纤维化的药效物质基础研究及其活性组分抗纤维化新药研发、保健品开发和临床医疗提供科学依据和研究思路。

Objective ：To evaluate the Turtle Carapace Decoction and drug containing serum on hepatic stellate cells （HSC）. Methods ： Double - antibody sandwich ELISA assay was used to detect the effect of Turtle Carapace Decoction and drug containing serum on TGF- β1 -induced collagen and regulatory factors expression in LX -1 cells; MTS assay was used to detect the effect of Turtle Carapace Decoction and drug containing serum on TGF - β1 - induced LX - 1 cell pro- liferation ; Western blotting analysis was used to detect the effect of Turtle Carapace Decoction and drug containing serum on TGF -β1 - induced LX - 1 cell activation, collagen synthesis and extracellular matrix expression. Results：Turtle Cara- pace Decoction and drug containing serum can significantly inhibit HSC proliferation and down - regulate TGF - β1 - in- duced type Ⅰ, type Ⅲ and type Ⅳ collagen, α - SMA and HA expressions in LX - 1 cells, and for the TGF -β1 down- stream signal pathway proteins, it can inhibit the expressions of Smad 3, P - smad3 and other cytokines, which act on the TGF - β1 feedback loop in all stages of blocking TGF - β1. Moreover, Turtle Carapace Decoction and drug containing serum can significantly strengthen the expressions Of MMP - 1 and MMP - 3 proteins. By inhibiting TIMP - 1 protein ex- pression, it was conducive to improve collagen enzyme activity, and to promote ECM degradation. Within the experimen-tal concentrations, the effect of Turtle Carapace Decoction and drug containing serum on hepatic stellate cells was in a dose- dependent manner, and this effect was significantly better than those of γ - interferon, and was comparable to those of Fuzheng Huayu capsule, and generally accepted effective anti - liver fibrosis drugs or cytokine. Conclusion ： Turtle Carapace Decoction and drug containing serum can inhibit hepatic stellate cells activated multiplication and extra cellular matrix （ECM） synthesis or secretion, promote ECM degradation, regulate cell factor level and regulate signaling path- way, and significantly block or treat liver fibrosis. This experimental result is verifying the validity of turtle carapace for anti - liver fibrosis, providing scientific evidences and research ideas for basic investigation on material basis for its anti - liver fibrosis effect as well as development and clinical application of its active components.