Anlotinib Attenuates Liver Fibrosis by Regulating the Transforming Growth Factor β1/Smad3 Signaling Pathway

Abstract

Background: Hepatic stellate cell hyperactivation is a central link in liver fibrosis development, transforming growth factor β1 (TGF-β1) is a key activator of HSCs.

Aims: This study investigated whether anlotinib attenuates CCl4 induced liver fibrosis in mice and explored its antifibrotic mechanism.

Methods: 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.

Results: 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.

Conclusions: 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.

Keywords: Anlotinib; HSC; Hepatic fibrosis; TGF-β1/Smad3 signaling pathway.