| Objective:Acanthoic acid (AA), (-)-primara-9(11),15-dien-19-oicacid, is a pimaradiene diterpene, isolated from the root bark of Acanthopanax koreamim Nakai (Araliaceae). In our previous research, we found the good protective effect of AA. In this study, we investigate protective effect of AA in carbon tetrachloride (CCl4)-induced mice and TGF-β stimulated hepatic stellate cells (HSCs).Methods:In vivo, CCl4-treated mice were intraperitoneally injected with 10% CCl4 in olive oil (2 ml/kg, twice a week for 4 weeks and then once a week for the next 4 weeks). In AA treated groups, mice were intragastricly ad ministrated with AA (20 mg/kg or 50 mg/kg) 3 times per week for 8 weeks. Mice were sacrificed at 8 weeks and then blood samples and the liver were collected to store at -80 ℃ until analyzed. Serum levels of aspartate a minotransferase (AST), alanine a rninotransferase (ALT) and tissue necrosis factor-α (TNF-α) levels were detected by using an Autodry Chemistry Analyzer. The pathological changes in liver tissues were evaluated by HE, immunohistochemistr, Masson stainings. We Determine the Protein levels of α-SMA, Collagen-I, MMP-13, TIMP-1, LXRs and NF-κB/IκB-α in liver tissue by Western Blotting.TGF-β (5 ng/ml) was added to activate HSC-T6 cells for 2 h, and then treated with AA (1,3, or 10 μmol/1) for 24 h before analysis. After a period of time, collecting cells, extracting tota protein and protein in the nucleus were analyzed by western blotting.GW3965 is systematica LXR agonists. Studies showed that LXR agonists can inhibit expression of any inflammatory regulator in mouse macrophage. Different concentrations (0.125,0.25,0.5,1,2 μM) of GW3965 was added to incubate the LX-2 cells for 18 h and we determined the protein levels of LXRs. Different concentrations (1,3,10,20 μmol/1) of AA and 2 μM GW3965 was added to incubate the LX-2 cells for 18 h and then activated by 1 μg/ml LPS for 30 mim. Finally, we determined the protein levels of LXRs, α-SMA, Collagen-I, TIMP-1, NF-κB/IκB-α.Results:In vivo, administration of AA reduced serum aminotransferase and tissue necrosis factor-α (TNF-α) levels evoked by CC14. Administration of AA reduced the expression of a-SMA and Collagen-I and regulated the ratio of MMP-13/TIMP-1. AA administration resulted in upregulating both LXRa and LXRfS and follow with inhibit NF-κB translocation.In vitro, AA with a dosage from 0.78 to 50μmol/l presented almost very lower cytotoxicity on HSC-T6 cell. AA significantly inhibited the increasing α-SMA expression stimulated by TGF-β,and increased expression of LXRβ.GW3965 is systematica LXR agonists.0.125-2 μM GW3965 significantly increased the expression of LXRs compared with that in normal group. The expression of α-SMA, Collagen-I and TIMP-1 were significantly decreased in 20 μM AA group compared with that in LPS group. Upon 1,3,10,20μM AA and 2 μM GW3965 administration, the IxB-a protein level significantly increased compared with LPS group. Following IκB-degradation, NF-κB was released from the physical restriction imposed by IκB-α and translocated to the nucleus.Conclusion:AA might ameliorate the liver fibrosis induced by carbon tetrachloride in mice via activating of LXRα and LXRβ, and inhibit activated HSCs via activating of LXRβ.0.125-2 μM GW3965 could significantly increase the expression of LXRs, andinhibited the expression of α-SMA, Collagen-I. AA might inhibited the expression of α-SMA, Collagen-I via activating of LXRa and LXRβ.and might inhibit the activated HSCs via modulating the nuclear translocation of NF-κB. AA has the same effect with GW3965 in activating LXRs, and was asystematica LXR agonists. Therefore, AA may be a potential candidate for the therapy of hepatic fibrosis. |
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