索拉菲尼抑制肝星状细胞活化在肝癌肿瘤微环境中的作用机制

Sorafenib inhibition of hepatic stellate cell activation in the microenvironment of hepatocellular carcinoma

目的研究索拉菲尼对肝星状细胞活性及活化的影响及其在肝脏肿瘤微环境中的作用机制。方法通过LX2细胞和HepG2细胞共培养，观察LX2对HepG2细胞增殖的影响。通过MTT检测观察索拉菲尼对LX2增殖的影响。用不同浓度索拉菲尼干预LX2细胞，细胞组化检测LX2细胞α—SMA的表达；ELISA检测LX2上清液PDGF-BB和TGF-β1浓度变化；蛋白印迹检测LX2细胞ERKl、ERK2、Akt信号通路的表达。将不同浓度索拉菲尼干预的LX2细胞和HepG2细胞共培养24h，观察对HepG2细胞侵袭能力影响。结果LX2和HepG2细胞共培养后实验组HepG2细胞明显较对照组多。经不同浓度索拉菲尼干预LX2细胞12h、24h、36h和48h后，实验组细胞活性弱于对照组，具有浓度及时间依赖性。对照组α—SMA的表达强于实验组，具有浓度和时间依赖性。随着索拉菲尼药物浓度的升高及同一浓度下时间的延长，上清中PDGF-BB和TGF-β1的浓度降低。实验组与对照组中ERKl、ERK2、AKT的表达基本相同，但各自磷酸化状态的表达随着药物浓度的升高呈下降趋势。随着药物浓度的升高LX2诱导HepG2侵袭的能力逐渐减弱。结论索拉菲尼可通过抑制α—SMA的表达、抑制肝星状细胞PDGF信号通路及下调上清液中的PDGF-BB和TGF-β1的表达，从而抑制HSC的活性及活化，继而抑制HepG2的增殖及侵袭。

Objective To investigate the effects and mechanisms of sorafenib on hepatic stellate cell viability and activation in the microenvironment of liver tumor. Methods The effects of LX2 cells on HepG2 cell proliferation were observed by coculture of LX2 and HepG2 cells. MTT assay was used to observe the effects of sorafenib on LX2 proliferation, and expression of a-smooth muscle actin （α- SMA） was measured immunocytochemically in LX2 cells treated with different concentrations of sor- afenib. Changes in PDGF-BB and TGF-βI concentrations were detected in LX2 supernatant using ELISA. Expression of ERK1, ERK2, and AKT signaling pathways were measured using Western blot. Furthermore, LX2 cells were cocultured with HepG2 cells for 24 hours to observe their effects on the invasive ability of HepG2 cells. Result After coculture of LX2 and HepG2 cells, HepG2 cells increased in the experimental group more than those of in the control group. After treatment with va- rious concentrations of sorafenib for 12, 24, 36 or 48 hours, the viability of treated LX2 cells was lower than the controls in a concentration- and time-dependent manner. As sorafenib concentration and time of exposure increased, α-SMA expression became weaker in treated cells. PDGF-BB and TGF-β1 concentrations decreased with higher sorafenib concentrations and with longer exposure under the same concentration. ERK1, ERK2 and Akt expression was identical between treated and control groups, but their phosphorylated expression decreased with increased concentrations of sorafenib. The invasive ability of HepG2 cells induced by LX2 gradually decreased as sorafenib concentrations increased. Conclusions Sorafenib suppressed α-SMA expression, inhibited PDGF-dependent signaling pathways in HSCs, downregulated PDGF-BB and TGF-β1 expression in the supernatant of HSCs, and restrainedthe viability and activation of HSCs. Sorafenib treatment therefore resulted in suppressed proliferation and invasion of HepG2 cells.