Anti-hepatic Fibrosis Effects And Mechanisms Of Two Active Ingredients From Plant

Liver fibrosis,characterized by abnormal depositions of fibrous connective tissue in livers,is a pathologic situation that may result in a series of advanced liver diseases.Transforming growth factor-β1（TGF-β）is a multifunctional cytokine involved in the regulation of cell growth and differentiation.HSCs were activated by TGF-βthrough promoting their proliferation and migration.Hepatocytes were also provoked by TGF-βthrough the EMT.Both activation of HSCs and EMT of hepatocytes were involved in the fibrogenic process in livers.Therefore,a method based on the determination of TGF-β-induced migration and proliferation of human HSCs LX-2,cytoskeletal changes of mouse hepatocytes AML-12,and detection of their marker proteins was established.Natural products,characterized by safety and eutherapeutic effects,have been increasingly attracted by a lot of attention.The provious researches have shown the ability of hepatoprotective activities of natural products.Therefore,the research focused on natural products and based on the above conditions to screen potential agents in vitro.It was found that SEPJ and limonin both inhibited TGF-β-induced EMT of hepatocytes and activation of HSCs in vitro.Further,we researched mechanisms of the two agents’anti-liver fibrosis activities in both vitro and vivo.SEPJ was a saponin extract from the rhizomes of P.japonicas.The analyzed characterized chemical compositions by LC-MS were Chikusetsusaponin V,Chikusetsusaponin IV,and Chikusetsusaponin IVa.The production of ROS was an important cellular event contributing to TGF-β-provoked EMT in hepatocytes and activation in HSCs.SEPJ inhibited TGF-β-induced accumulation of ROS in both LX-2 and AML-12 cells.Mechanistically,the levels of nuclear Nrf2 protein and upregulated the expression of Nrf2-responsive antioxidative enzymes such as HO-1,NQO1 and GCLC were increased by SEPJ in both types of cells.Furthermore,enhanced phosphorylation of Akt and GSK3βacted upstream of SEPJ-mediated activation of Nrf2.Knockdown of Nrf2 or inhibition of Akt diminished the protective activity of SEPJ against TGF-βin both LX-2 and AML-12 cells.These results indicated that the activation of Akt/GSK3β/Nrf2 signaling pathway was closely related to the activity of SEPJ in TGF-β-induced activation of LX-2 cells and EMT of AML-12 cells.The experiments in vivo revealed a dramatic mitigation by SEPJ of mouse liver fibrosis provoked by CCl₄.The results showed that SEPJ could improve the abnormal liver function indicators,expression of liver fibrosis marker protein and oxidative stress induced by CCl₄ in vivo.The pathological results further proved that SEPJ could improve the fibrosis hyperplasia and Hepatocytes necrosis induced by CCl₄in mice.Consistent with these findings in LX-2 and AML-12 cells,mouse hepatic Akt/GSK3β/Nrf2 axis was also potentiated by SEPJ.In conclusion,the activation of LX-2 HSCs and EMT of AML-12hepatocytes were mitigated by SEPJ and the CCl₄-provoked mouse liver fibrosis was also alleviated by SEPJ.Modulating the Akt/GSK3β/Nrf2 axis plays critical roles in its hepatoprotective effects.These findings support the consideration of SEPJ as a possible supportive reagent for controlling liver fibrosis clinically.Limonin is a natural tetracyclic triterpenoid compound which is generally enriched in seeds and peels of oranges and lemons.It was found that limonin repressed TGF-β-induced EMT in AML-12 hepatocytes and activation of LX-2 HSCs.In both kinds of cells,limonin suppressed TGF-β-induced phosphorylation and nuclear translocation of Smad2/3 and subsequent Smad2/3-dependent gene transcription.In addition,the transcription of Smad7 in both AML-12 and LX-2 cells were increased by limonin.Knockdown of Smad7 abrogated the suppressive effects of limonin on TGF-β-induced EMT in AML-12 cells and activation of LX-2 cells.Further studies revealed that limonin alleviated mouse liver fibrosis induced by CCl₄.Limonin could improve the abnormal liver function indicators and expression of liver fibrosis marker protein induced by CCl₄ in mice.Results of HE staining also showed that limonin could improve the conditions of CCl₄-induced liver fibrosis hyperplasia and necrosis of hepatocytes in mice.Consistent with these findings in vitro,limonin upregulated Smad7and suppressed phosphorylation and nuclear translocation of Smad2/3 in vivo.Transcription of Smad2/3-responsive genes was also inhibited by limonin.These 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 the transcription of Smad2/3-downstream genes is implicated in the hepatoprotective activity of limonin.This paper is designed to research active agents from natural products to control liver fibrosis by pharmacology and molecular biology research methods and to provide a theoretical basis for the prevention and treatment of liver fibrosis.