多靶点酪氨酸激酶抑制剂阿西替尼治疗肝纤维化的作用与机制

Effects of multi-target tyrosine kinase inhibitor Acitinib on liver fibrosis

目的观察多靶点酪氨酸激酶抑制剂阿西替尼(Axitinib,AG-013736)对肝纤维化的治疗作用。方法用四氯化碳(carbon tetrachloride,CCL4)腹腔注射以诱导建立小鼠肝纤维化实验模型,于造模2周、4周时分别给予阿西替尼-羧甲基纤维素(CMC)溶液及CMC溶液灌胃处理,治疗1周、2周后处死小鼠,并取肝脏组织标本进行HE染色、Masson染色及α-SMA免疫组化检测;体外实验以不同浓度的阿西替尼CMC溶液分别干预人肝星状细胞系(LX2)及人正常肝细胞系(LO2),于48、72 h进行MTT检测,流式细胞术观察细胞凋亡,同时应用Western blotting检测凋亡蛋白的表达。结果HE和Masson染色显示CCL4腹腔注射可诱导小鼠肝纤维化,且处理4周组的肝纤维化程度重于2周组;造模后经阿西替尼溶液治疗后,肝组织胶原染色面积及α-SMA阳性表达低于造模4周组及CMC治疗对照组,差异有统计学意义(P<0.05);体外实验中,阿西替尼能够有效抑制肝星状细胞系LX2活性,促进其凋亡,同时,促凋亡蛋白Fas、Caspase-8以及Caspase-3蛋白表达增高,凋亡抑制基因Bcl-2的蛋白表达下降,而在对照肝细胞系LO2细胞中并无上述变化。结论阿西替尼通过诱导肝星状细胞凋亡发挥抗纤维化作用,且对正常肝细胞系无明显影响。

Objective To observe the therapeutic effects of Axitinib,a tyrosine kinase receptor inhibitor,on liver fibrosis.Methods In vivo,CCL4 was injected intraperitoneally to induce liver fibrosis in mice.After modeling,Axitinib-carboxymethyl cellulose(Axitinib-CMC)solution or CMC solution were administered by gavage.After 2 weeks of modeling,4 weeks of modeling,1 week of treatment and 2 weeks of treatment,the mice were killed and their liver tissues were stained by HE,Masson andα-SMA immunohistochemistry.In vitro,human hepatic stellate cell line(LX2)and human normal liver cell line(LO2)were intervened with different concentrations of Axitinib-CMC solution.MTT assay was performed 48 h and 72 h after the intervention.Flow cytometry was used to observe the apoptosis of the two cell lines.Western blotting was used to detect the expressions of Fas,Caspase-8,Caspase-3 and Bcl-2 proteins.Results HE and Masson staining results showed that CCL4 could induce liver fibrosis in the mice,and the degree of liver fibrosis was more severe in the 4-week than that in the 2-week treatment group.After treatment with Axitinib,the collagen staining area and the positive expression level ofα-SMA were significantly lower than those in 4-week group and CMC treatment control group(P<0.05).In vitro,Axitinib could effectively inhibit the viability of hepatic stellate cell line LX2 and promote its apoptosis.Meanwhile,the expressions of pro-apoptotic proteins Fas,Caspase-8 and Caspase-3 increased,while the expression of apoptosissuppressor gene Bcl-2 decreased.However,the above changes were not found in control hepatocyte line LO2.Conclusion Axitinib exerts an anti-fibrosis effect by inducing apoptosis of hepatic stellate cells,and has no significant effect on normal hepatocytes.