Limonin relieves TGF-β-induced hepatocyte EMT and hepatic stellate cell activation in vitro and CCl4-induced liver fibrosis in mice via upregulating Smad7 and subsequent suppression of TGF-β/Smad cascade

Liver fibrosis is a pathological process as a result of intrahepatic deposition of excessive ECM. EMT of hepatocytes and activation of HSCs both play important roles in the etiology of liver fibrosis. Here, we found that limonin repressed TGF-β-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs. Limonin suppressed TGF-β-provoked Smad2/3 C-terminal phosphorylation and subsequent nuclear translocation. However, limonin exerted few effects on Smad2/3 phosphorylation atlinker region. Mechanistically, limonin increased Smad7 in both AML-12 and LX-2 cells. Knockdown of Smad7 abrogated inhibitory effects of limonin on TGF-β-induced changes in both two cells. Further studies revealed that limonin upregulated Smad7 and declined C-terminal phosphorylation and nuclear translocation of Smad2/3 to alleviate mouse CCl4-induced liver fibrosis. Our findings indicated that limonin inhibits TGF-β-induced EMT of hepatocytes and activation of HSCs in vitro and CCl4-induced liver fibrosis in mice. Upregulated Smad7 which suppresses Smad2/3-dependent gene transcription is implicated in the hepatoprotective activity of limonin.