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AIMTo investigate the effect of metformin on activated hepatic stellate cells (HSCs) and the possible signaling pathways involved.METHODSA fibrotic mouse model was generated by intraperitoneal injection of carbon tetrachloride (CCl4) and subsequent treatment with or without metformin. The level of fibrosis was detected by hematoxylin-eosin staining, Sirius Red staining, and immunohistochemistry. The HSC cell line LX-2 was used for in vitro studies. The effect of metformin on cell proliferation (CCK8 assay), motility (scratch test and Transwell assay), contraction (collagen gel contraction assay), extracellular matrix (ECM) secretion (Western blot), and angiogenesis (ELISA and tube formation assay) was investigated. We also analyzed the possible signaling pathways involved by Western blot analysis.RESULTSMice developed marked liver fibrosis after intraperitoneal injection with CCl4 for 6 wk. Metformin decreased the activation of HSCs, reduced the deposition of ECM, and inhibited angiogenesis in CCl4-treated mice. Platelet-derived growth factor (PDGF) promoted the fibrogenic response of HSCs in vitro, while metformin inhibited the activation, proliferation, migration, and contraction of HSCs, and reduced the secretion of ECM. Metformin decreased the expression of vascular endothelial growth factor (VEGF) in HSCs through inhibition of hypoxia inducible factor (HIF)-1α in both PDGF-BB treatment and hypoxic conditions, and it down-regulated VEGF secretion by HSCs and inhibited HSC-based angiogenesis in hypoxic conditions in vitro. The inhibitory effects of metformin on activated HSCs were mediated by inhibiting the Akt/mammalian target of rapamycin (mTOR) and extracellular signal-regulated kinase (ERK) pathways via the activation of adenosine monophosphate-activated protein kinase (AMPK).CONCLUSIONMetformin attenuates the fibrogenic response of HSCs in vivo and in vitro, and may therefore be useful for the treatment of chronic liver diseases.

Aim To investigate the effect of carvedilol on liver fibrosis and hepatic sinusoidal capillarization in mice with carbon tetrachloride (CCl 4 )-induced fibrosis. Methods A liver fibrosis mouse model was induced by intraperitoneal CCl 4 injection for 8 weeks. The mice were divided into five experimental groups: the normal group, the oil group, the CCl 4 group, the CCl 4 +carvedilol (5 mg/kg/d) group, and the CCl 4 +carvedilol (10 mg/kg/d) group. The extent of liver fibrosis was evaluated by histopathological staining, and the changes in fenestrations of hepatic sinus endothelial cells were observed by scanning electron microscope (SEM). The expression of α-smooth muscle actin (α-SMA) and vascular endothelial markers was detected by immunohistochemistry and Western blot assays. The effect of carvedilol on cell apoptosis was studied via Terminal deoxynucleotidyl Transferase Mediated dUTP Nick End Labeling (TUNEL) assay, and the serum levels of matrix metalloproteinase-8 (MMP-8), vascular endothelial growth factor (VEGF), and angiopoietin-2 were detected through a Luminex assay. Results Liver fibrosis in CCl 4 -treated mice was attenuated by reduced accumulation of collagen and the reaction of inflammation with carvedilol treatment. Carvedilol reduced the activation of hepatic stellate cells (HSCs) and increased the number of apoptotic cells. The expression of α-SMA, CD31, CD34 and VWF (von Willebrand factor) was significantly decreased after carvedilol treatment. In addition, the number of fenestrae in the hepatic sinusoid showed notable differences between the groups, and the serum levels of MMP-8, VEGF and angiopoietin-2 were increased in the mice with liver fibrosis and reduced by carvedilol treatment. Conclusion The study demonstrated that carvedilol could prevent further development of liver fibrosis and hepatic sinusoidal capillarization in mice with CCl 4 -induced fibrosis.