Wu Ye-Ting, Li Qi-Zhe, Zhao Xue-Ke, Mu Mao, Zou Gao-Liang, Zhao Wei-Feng
Department of Infectious Diseases, The First Affiliated Hospital of Soochow University, No 188, Shizi Street, Suzhou, 215000, Jiangsu, China.
Department of Infectious Diseases, The Affiliated Hospital of Guizhou Medical University, Guiyang, Guizhou, China.
Dig Dis Sci. 2023 Nov;68(11):4186-4195. doi: 10.1007/s10620-023-08101-1. Epub 2023 Sep 8.
Hepatic stellate cell hyperactivation is a central link in liver fibrosis development, transforming growth factor β1 (TGF-β1) is a key activator of HSCs.
This study investigated whether anlotinib attenuates CCl4 induced liver fibrosis in mice and explored its antifibrotic mechanism.
We used the human hepatic stellate cell line LX-2 for in vitro assays and used TGF-β1 to induce hepatic fibrosis in LX-2 cells. We analyzed cytotoxicity using a cell-counting kit-8 and transwell chambers to detect the migratory ability of LX-2 cells. Western blotting was used to detect the protein levels of collagen type I, α-smooth muscle actin, and p-Smad3. In addition, mice with CCl4-induced hepatic fibrosis were used as in vivo models. Histopathological examination was performed using H&E staining, Masson's trichrome staining, and immunohistochemistry.
Anlotinib significantly reversed TGF-β1-induced protein levels of Col I, α-SMA and p-Smad3 and inhibits migratory and proliferative abilities in vitro using LX-2 cells. CCl4 cause F4 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 12 to 14 (Ishak), a mean ALT measurement of 130 U/L and a mean measurement AST value of 119 U/L in mice. However, the CCl4-induced changes were markedly attenuated by anlotinib treatment, which returned to F2 grade (Ishak) hepatic fibrosis, liver inflammatory scores ranged from 4 to 6 (Ishak), a mean ALT measurement of 40 U/L and a mean measurement AST value of 56 U/L in mice.
Our results suggest that anlotinib-mediated suppression of liver fibrosis is related to the inhibition of TGF-β1 signaling pathway. Hepatic stellate cell hyper activation is a central link in liver fibrosis development, transforming growth factor β1 is a key activator of HSCs. Anlotinib is a multi-targeted tyrosine kinase inhibitor that has similar targets to nintedanib, a clinically used anti-pulmonary fibrosis drug. Our study demonstrates an FDA-approved drug-anlotinib-that could prevent liver fibrosis and inflammation. Experiments in cell cultures and mice show that anlotinib can inhibit the activation of hepatic stellate cells by down-regulating the TGFβ1/smad3 pathway, thereby reversing liver fibrosis. In animal experiments, anlotinib showed protective effects on the CCl4-induced liver damage, including ameliorating liver inflammation, reversing liver fibrosis and reducing liver enzymes. This is a very good signal, anlotinib may be useful for halting or reversing the progression of liver fibrosis and could be employed in the development of novel therapeutic drugs for the management of chronic liver diseases.
肝星状细胞的过度活化是肝纤维化发展的中心环节，转化生长因子β1（TGF-β1）是肝星状细胞的关键激活因子。
本研究探讨安罗替尼是否能减轻四氯化碳诱导的小鼠肝纤维化，并探究其抗纤维化机制。
我们使用人肝星状细胞系LX-2进行体外实验，并用TGF-β1诱导LX-2细胞发生肝纤维化。使用细胞计数试剂盒-8分析细胞毒性，并使用Transwell小室检测LX-2细胞的迁移能力。采用蛋白质免疫印迹法检测Ⅰ型胶原蛋白、α-平滑肌肌动蛋白和p-Smad3的蛋白水平。此外，将四氯化碳诱导的肝纤维化小鼠作为体内模型。采用苏木精-伊红染色、Masson三色染色和免疫组织化学进行组织病理学检查。
安罗替尼显著逆转了TGF-β1诱导的LX-2细胞中Col I、α-SMA和p-Smad3的蛋白水平，并在体外抑制了其迁移和增殖能力。四氯化碳导致小鼠出现F4级（Ishak）肝纤维化，肝脏炎症评分为12至14分（Ishak），平均谷丙转氨酶测量值为130 U/L，平均谷草转氨酶测量值为119 U/L。然而，安罗替尼治疗显著减轻了四氯化碳诱导的变化，使小鼠恢复到F2级（Ishak）肝纤维化，肝脏炎症评分为4至6分（Ishak），平均谷丙转氨酶测量值为40 U/L，平均谷草转氨酶测量值为56 U/L。
我们的结果表明，安罗替尼介导的肝纤维化抑制与TGF-β1信号通路的抑制有关。肝星状细胞过度活化是肝纤维化发展的中心环节，转化生长因子β1是肝星状细胞的关键激活因子。安罗替尼是一种多靶点酪氨酸激酶抑制剂，其靶点与临床使用的抗肺纤维化药物尼达尼布相似。我们的研究证明了一种FDA批准的药物——安罗替尼——可以预防肝纤维化和炎症。细胞培养和小鼠实验表明，安罗替尼可以通过下调TGFβ1/smad3通路抑制肝星状细胞的活化，从而逆转肝纤维化。在动物实验中，安罗替尼对四氯化碳诱导的肝损伤显示出保护作用，包括减轻肝脏炎症、逆转肝纤维化和降低肝酶。这是一个非常好的信号，安罗替尼可能有助于阻止或逆转肝纤维化的进展，并可用于开发治疗慢性肝病的新型治疗药物。
