N1-acetyl Substituted Pyrrolidine Compounds A5Ameliorates Liver Cirrhosis Through Modulation Of Hepatic Stellate Cell Activity

Background:The formation of the central link of liver fibrosis is caused by activation and proliferation of hepatic stellate cells, inhibiting the activation and proliferation of hepatic stellate cells is an important way to treat liver fibrosis.Object:(2S,4R)-methyl l-acetyl-4-(N-(4-bromophenyl) sulfamoyloxy) pyrrolidine-2-carboxylate (CIP-A5) is the Nl-acetyl substituted pyrrolidine derivative which was designed against the structure of matrix metalloproteinase (MMP-2and MMP-9). CIP-A5has been considered as a candidate compound for treatment of liver cirrhosis. In this study, in rat hepatic stellate cells (HSC-T6) are cultured in vitro to observe the effect of CIP-A5on cell proliferation, apoptosis and fibrosis cytokine, to clarify the CIP-A5antifibrotic mechanism from the molecular level.Methods:(A) Gelatinase assay:Hydrolysis of succinylated gelatin by gelatinase was measured in the presence of compounds and inhibitory rate of compounds was valued. We used SDS-PAGE gelatin zymography to evaluate the effects of compounds on inhibition of MMP-2and MMP-9in the supernatants of HSC-T6cell culture.(B) The effect of CIP-A5on cell proliferation and apoptosis:HSC-T6cells were employed and the cell growth inhibition was checked by MTT assay and trypan blue staining. Cell surface of phosphatidylserine in apoptotic cells was quantitatively detected by using Annexin V/FITC and PI apoptosis detection kit. (C) The effect of CIP-A5on the expression of fibrosis cytokine:Western blot analysis was employed to evaluate the expression levels of fibrosis cytokines such as TIMP-1, TGF-β1, TNF-α, CTGF in HSC-T6cells.Results:The inhibition rates of substrate hydrolyzed by gelatinase were increased from28.4%to126.7%with increasing concentrations (5-30ng/ml) of CIP-A5exposed for30min. The activity of MMP-2, MMP-9in the supernatants of HSC-T6was increased in a dose-dependent manner by treatment with CIP-A5for24h. The inhibition rates were increased from38.6%to190.5%respectively for MMP-2and from22.4%to170.0%respectively for MMP-9. The up-regulated expression of MMP-9, pro-MMP-2, MMP-2was also valued using the assay of Western blot (P<0.05). Using Western blot assay, we also found that the expression levels of TGF-β1, TNF-α, CTGF in HSC-T6cells were significantly decreased exposure to CIP-A5.Conclusion:CIP-A5can play the role of liver fibrosis by inhibiting the hepatic stellate cell activation and proliferation induced by TGF-β, improving and enhancing gelatin enzyme activity, reducing the expression of fibrosis cytokines.