The Inhibitory Effect And Mechanism Of Metformin On Activated Hepatic Stellate Cells In Liver Fibrosis

Background and AimsLiver fibrosis is a pathological disease due to acute or chronic injury to the liver.Hepatic stellate cell(HSC)palys a dominant role in the progress of liver fibrosis,and it acts as the primary effector cell in hepatic fibrosis.In normal liver,HSC is located in the space of Disse in quiescent state.With the stimulation of the etiological factors,HSC changed phenotype to activated type with the change in its function,which acts the same as myofibroblast cell.Activated HSC is proliferative,contractive,and can secret extracellular matrix(ECM)and cytokines.Pathological angiogenesis is widely mentioned in chronic liver diseases(CLDs),and is thought to have effect on both liver fibrosis and portal hypertension(PHT).HSC is considerded to be the liver-specific pericyte and participate in liver angiogenesis and sinusoidal remodeling.The pathological sinusoidal remodeling is featured by sinusoidal capillarization with the coverage of more contractile HSCs.Due to hyper-response to vasoconstrictor and diminished response to vasodilator,HSCs are contractile and reduce the caliber of sinusoids after contraction,thus causing a functional change in modulating hepatic tone,resulting in the increase of intra-hepatic vascular resistance(IHVR),and finally aggravate portal hypertension.HSC has an important role in sinusoidal remodeling and constitute a crossroad between inflammation,angiogenesis,and fibrosis.Besides,activated HSC playes a key role in mediating the augmented IHVR.So activated HSC is a promising therapeutic target in the treatment of CLDs.Metformin is a potent anti-hyperglyceminc agent with high safety,low side effect and price,and it is recommended as the first-line drug for patients with diabetes.Evidences from clinical and experimental animal models have demonstrated the protective activities of metformin beyond its antidiabetic effects.Previous studies showed that metformin could also have protective effect on CLDs.However,the mechanisms are still not well described.So further studies are needed to analyze the therapeutic effect of metformin in CLDs and its intrinsic mechanism.Platelet-derived growth factor(PDGF)signaling pathway is among the best characterized pathways in the activation of HSC.Reports show that PDGF induces activation of the downstream molecules extracellular signal-regulated kinase(ERK)and the Akt/mTOR in activated HSC,which are linked with HSC proliferation,migration and phenotype change.ERK and mTOR pathways are also associated with angiogenesis in previous studies.Activation of AMP-activated protein kinase(AMPK)inhibits the proliferation and migration of HSC induced by PDGF.The main effect of metformin is through activating AMPK,so we speculated that metformin can inhibit the pro-fibrogenic and pro-angiogenic response of activated HSC.In this study,we examined the effect of metformin on activated HSCs in vivo and in vitro.First,we used a fibrotic mouse model to determine whether metformin had effects on liver fibrosis.Metformin decreased the activation of HSCs,reduced the deposition of ECM,and inhibited pathogenic angiogenesis in CCl4-treated mice.The HSC cell line LX-2 and human umbilical vein endothelial cell(HUVEC)was used for in vitro studies.Metformin inhibited the activation,proliferation,motility,and contraction of activated HSCs,reduced the secretion of ECM,and decreased HSC-based angiogenesis.We also investigated the underlying mechanisms,with a focus on AMPK and the downstream Akt/mTOR and ERK signaling pathways.What's more,we treated liver cirrhotic rat with metformin,and metformin could decrease portal pressure in portal hypertension.These results showed that metformin could inhibit the pro-fibrogenic and pro-angiogenic effect of activated HSC in liver fibrosis in vivo and in vitro,decrease portal pressure in liver cirrhosis.Thus providing theoretical evidence for the use of metformin in the treatment of chronic liver diseases.Part1 The therapeutic effect of metformin on liver fibrotic miceAimTo investigate the therapeutic effect of metformin on carbon tetrachloride(CCl4)-induced liver fibrosis.MethodsWe used male C57BL/6 mice for in vivo study.Mice were randomly divided into 3 groups(a control group,a CCl4 group,and a metformin group).The liver fibrosis model was induced by intraperitoneal injection of CCl4 dissolved 1:1(v/v)in olive oil twice per week for 6 weeks,while the control mice were injected with olive oil alone.Mice in the metformin group were treated with metformin indrinking water at the same time.The level of fibrosis was detected by hematoxylin-eosin(HE)staining,Sirius Red(SR)staining,Western blot,RT-PCR and immunohistochemistry(IHC).We also tested the level of ALT,AST and TBIL in the serum.Results1.Mice developed marked liver fibrosis after intraperitoneal injection with CCl4 for 6 weeks,while metformin could attenuate this effect.This result was confirmed by HE staining and SR staining.Metformin could attenuate hepatocellular degeneration,decrease the accumulation of connective tissue,and reduce the infiltration of inflammatory cells.2.In CCl4-treated mice,the activities of serum ALT and TBIL were markedly up-regulated,while treatment with metformin could not reduce the levels of the injury to liver function.3.Mice exposed to CCl4 expressed more VEGF,indicating more intrahepatic angiogenesis than the control group.Treatment with metformin could significantly suppressed the expression of VEGF,indicating that merformin could inhibit angiogenesis in liver fibrosis.4.Mice exposed to CCl4 increased a-SMA at both the protein and mRNA levels,while co-treatment with metformin reduced this effect.This result indicated that the anti-fibrogenesis and anti-angiogenesis effect of metformin may be mediated by inhibiting the activation of HSCs.ConclusionMetformin decreased the activation of HSCs,reduced the deposition of ECM,and inhibited angiogenesis in CCl4-treated mice.Therefore,metformin attenuated CCl4-induced liver fibrosis in mice,the anti-fibrogenesis and anti-angiogenesis effect of metformin may be mediated by inhibiting the activation of HSCs.Part 2 Metformin attenuates pro-fibrogenic and pro-angiogenic response of activated hepatic stellate cells in vitro by activating AMP-activated protein kinaseAimTo investigate the inhibitory effect of metformin on activated hepatic stellate cell(HSC)and the possible signaling pathways involved.Methods1.The HSC cell line LX-2 and HUVEC were used for in vitro study.2.The effect of metformin on cell proliferation was detected by CCK8 assay.3.HSC was activated by PDGF-BB and pretreated with or without different concentrations of metformin.Expression of a-SMA,collagen type I and fibronectin was determined by Western blot.4.Cell motility was measured by the scratch test and Transwell assay.5.Collagen gel contraction assay was carried out to assess the effect of metformin on HSC contraction.6.As hypoxia is the most potent stimulus for VEGF expression,we further used CoCl2 to mimic a hypoxic environment.The HIF-la and VEGF expression in HSC was measured by western blot.7.The protein level in the supernatant was detected by ELISA assay in hypoxic condition.Tuber formation assay was performed to assess the effect of metformin on angiogenesis in vitro.8.We also analyzed the possible signaling pathways involved.The phosphorylation levels of AMPK,AKT,mTOR and ERK were measured by Western blot.And we used the indicated inhibitors and agonists to confirm the effect.Results1.Metformin inhibited the activation of HSC in a dose-dependent manner(IC50 = 50.01 mmol/L).2.PDGF-BB induced the activation of HSC,increased the secretion of ECM,and up-regulated the contractility and motility.Thus promoting the fibrogenic response of HSC in vitro.3.Metformin suppressed the expression of a-SMA,Collagen type I and Fibronectin,indicating that metformin could inhibit the activation of HSC and decreas the expression of ECM in vitro.4.Metformin decreased the migration and invasion of HSC,indicating that it can inhibited the motility of HSC,thus reduce the chemotaxis of HSC.5.Metformin inhibited the PDGF-BB induced contraction of HSC.6.Metformin decreased the expression of VEGF in HSC through inhibition of HIF-1? in both PDGF-BB and hypoxic conditions.7.Metformin down-regulated the secretion of VEGF by HSC and inhibited angiogenesis in hypoxic conditions in vitro.8.Metformin inhibited the fibrogenic response and pro-angiogenic response of activated HSC by PDGF-BB through inhibition of the Akt/mTOR and ERK pathways under the via the activation of AMPK.9.Metformin could decrease VEGF expression by activated HSC by down-regulating the mTOR/HIF-1? and ERK/HIF-1? pathways under hypoxic conditions via the activation of AMPK.ConclusionMetformin can inhibit the activation,proliferation,motility and contraction of HSC,reduce the secretion of ECM,attenuate HSC angiogenic properties,and decrease HSC-based angiogenesis.These effects of metformin are associated with inhibition of the Akt/mTOR and ERK pathways via the activation of AMPK.Therefore,metformin may be a novel therapeutic approach for the treatment of chronic liver diseases.Part 3 Effect of metformin on hepatic structure and portal hemodynamics in cirrhotic ratsAimTo investigate the effect of metformin on hepatic structure and portal hemodynamics in cirrhotic rats.MethodsWe used male Wistar rats for in vivo study.Rats were randomly divided into 3 groups(a control group,a CCl4 group,and a metformin group).The liver cirrhosis model was induced by intraperitoneal injection of CCl4 dissolved 1:1 in olive oil twice per week for 7 weeks,while the control mice were injected with olive oil alone.Rats in the metformin group were treated with metformin by gavage.After treatment for 2 weeks,the diameter of portal vein was measured by ultrasound.Portal vein pressure(PVP)was measured by directly puncture of the portal vein.All rats were sacrificed at the end of the study,and the weight of liver and spleen was measured.The level of fibrosis was detected by HE staining and Sirius Red staining.The structural change in the liver was observed by transmission electron microscope(TEM).The expression of a-SMA in liver tissue was measured by western blot.Results1.Rats developed marked liver cirrhosis after intraperitoneal injection with CCl4 for 7 weeks.2.Cirrhotic rats had lower body weight when compared with rats in the control group,while metformin could further decrease the body weight of cirrhotic rats.3.The weight of liver and spleen of cirrhotic rats were higher than rats in the control group.However,metformin could decrease the weight of liver and spleen,and spleen index in cirrhotic rats,indicating that metformin could attenuate the inflammatory response in spleen.4.Rats in the cirrhotic group had wider portal vein,while metformin could not change the diameter of portal vein in liver cirrhosis.5.The PVP was significantly higher in the cirrhotic rats than in the control group.Treatment with metformin could decrease PVP in the cirrhotic rats.6.Rats developed marked liver fibrosis after intraperitoneal injection with CCl4 for 7 weeks,while metformin could attenuate this effect.This result was confirmed by HE staining and Sirius Red staining.7.The expression of a-SMA at protein level was higher in the cirrhotic rats,and treatement with metformin can decrease the expression of a-SMA in cirrhotic liver tissure.8.Liver sinusoidal was covered with continuous basement membrane,and decreased open fenestrae and increased deposition of collagen were seen in the cirrhotic liver under TEM,while treatment with metformin could ameliorate this structural change.ConclusionMetformin could attenuate the structural change in cirrhotic rats,decrease the level of fibrosis,reduce the intra-hepatic vascular resistance,and further reduce portal pressure in cirrhotic rats,suggesting metformin is a novel therapeutic approach for the treatment of portal hypertension.