Curcumin ameliorates intrahepatic angiogenesis and capillarization of the sinusoids in carbon tetrachloride-induced rat liver fibrosis

Neoangiogenesis and the development of an abnormal angio-architecture in the liver are strongly linked with progressive fibrogenesis. This study aimed to evaluate the ability of curcumin to protect liver fibrosis-associated angiogenesis and capillarization of the sinusoids in experimental rats. Liver fibrosis was induced by intraperitoneal injection of carbon tetrachloride (CCl₄) with or without curcumin for 6 weeks. The results suggest that curcumin treatment markedly attenuated CCl₄-induced liver fibrosis, as assessed by histology and hydroxyproline content, and inhibited hepatic stellate cell activation. Curcumin ameliorated hepatic angiogenesis, as assessed by measuring microvessel density using Von Willebrand factor staining and by examining the expression of the endothelial cell markers CD31 and vascular endothelial growth factor receptor (VEGFR)-2 in the livers. Pathologic remodeling of liver sinusoidal capillarization, as assessed by electron-microscopic analysis of Disse's space and by evaluation of the levels of basement membrane protein expression, was also attenuated by curcumin administration. The intrahepatic gene or protein expression of hypoxia-inducible factor-1α, VEGFR-1, placental growth factor, and cyclooxygenase-2 decreased with treatment with curcumin in fibrotic rats. In conclusion, curcumin ameliorates hepatic angiogenesis and sinusoidal capillarization in CCl₄-induced rat liver fibrosis through suppressing multiple proangiogenic factors.     

Oxymatrine attenuates CCl4-induced hepatic fibrosis via modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways

Oxymatrine (OMT) is able to effectively protect against hepatic fibrosis because of its anti-inflammatory property, while the underlying mechanism remains incompletely understood. In this study, forty rats were randomly divided into five groups: control group, model group (carbon tetrachloride, CCl₄) and three OMT treatment groups (30, 60, 120 mg/kg). After CCl₄ alone, the fibrosis score was 20.2 ± 0.8, and the level of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hydroxyproline content, and collagen I expression was elevated, but OMT blunted these parameters. Treatment with OMT prevented CCl₄-induced increases in expression of pro-inflammatory and pro-fibrotic cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-α, meanwhile OMT promoted the expression of anti-inflammatory and anti-fibrotic factors such as interleukin (IL)-10 and bone morphogenetic protein and activin membrane-bound inhibitor (Bambi). Moreover, lipopolysaccharides (LPS) and high mobility group box-1 (HMGB1), which activates Toll-like receptor 4 (TLR4) and modulate hepatic fibrogenesis through hepatic stellate cells (HSCs) or Kupffer cells, were significantly decreased by OMT treatment. These results were further supported by in vitro data. First, OMT suppressed the expression of TLR4 and its downstream pro-inflammatory cytokines, lowered the level of HMGB1, TGF-β1 in macrophages. Then, OMT promoted Bambi expression and thereby inhibited activation of HSCs mediated by transforming growth factor (TGF)-β1. In conclusion, this study showed that OMT could effectively attenuate the CCl₄-induced hepatic fibrosis, and this effect may be due to modulation of TLR4-dependent inflammatory and TGF-β1 signaling pathways.     

Chlorogenic acid reduces liver inflammation and fibrosis through inhibition of toll-like receptor 4 signaling pathway

Chlorogenic acid (CGA) is a type of polyphenol with anti-inflammatory, antioxidant activities. Our previous studies showed CGA could efficiently inhibit carbon tetrachloride (CCl₄)-induced liver fibrosis in rats. However, the specific underlying mechanism remains unclear. The aim of this study is to investigate the effects of CGA on liver inflammation and fibrosis induced by CCl₄ and whether they are related to inhibition of toll-like receptor 4 (TLR4) signaling pathway. Male Sprague-Dawley (SD) rats were administrated CCl₄ together with or without CGA for 8 weeks. Histopathological and biochemical analyses were carried out. The mRNA and protein expression levels of proinflammatory and profibrotic mediators were detected by RT-PCR and Western blot, respectively. The levels of serum proinflammatory cytokines were detected by ELISA. CGA significantly attenuated CCl₄-induced liver damage and symptoms of liver fibrosis, accompanied by reduced serum transaminase levels, collagen I and α-smooth muscle actin (α-SMA) expression. As compared with the CCl₄-treated group, the expression levels of TLR4, myeloid differentiation factor 88 (MyD88), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were reduced in the treatment group of CCl₄ and CGA, whereas bone morphogenetic protein and activin membrane-bound inhibitor (Bambi) expression was increased. CGA also suppressed CCl₄ induced nuclear factor-κB (NF-κB) activation. Moreover, the hepatic mRNA expression and serum levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were significantly increased in CCl₄-treated rats and attenuated by co-treatment with CGA. Our data indicate that CGA can efficiently inhibit CCl₄-induced liver fibrosis in rats and the protective effect may be due to the inhibition of TLR4/MyD88/NF-κB signaling pathway.     

Liver fibrosis in mice induced by carbon tetrachloride and its reversion by luteolin

Hepatic fibrosis is effusive wound healing process in which excessive connective tissue builds up in the liver. Because specific treatments to stop progressive fibrosis of the liver are not available, we have investigated the effects of luteolin on carbon tetrachloride (CCl₄)-induced hepatic fibrosis. Male Balb/C mice were treated with CCl₄ (0.4 ml/kg) intraperitoneally (i.p.), twice a week for 6 weeks. Luteolin was administered i.p. once daily for next 2 weeks, in doses of 10, 25, and 50 mg/kg of body weight. The CCl₄ control group has been observed for spontaneous reversion of fibrosis. CCl₄-intoxication increased serum aminotransferase and alkaline phosphatase levels and disturbed hepatic antioxidative status. Most of these parameters were spontaneously normalized in the CCl₄ control group, although the progression of liver fibrosis was observed histologically. Luteolin treatment has increased hepatic matrix metalloproteinase-9 levels and metallothionein (MT) I/II expression, eliminated fibrinous deposits and restored architecture of the liver in a dose-dependent manner. Concomitantly, the expression of glial fibrillary acidic protein and α-smooth muscle actin indicated deactivation of hepatic stellate cells. Our results suggest the therapeutic effects of luteolin on CCl₄-induced liver fibrosis by promoting extracellular matrix degradation in the fibrotic liver tissue and the strong enhancement of hepatic regenerative capability, with MTs as a critical mediator of liver regeneration.     

Effects of standardized bilberry fruit extract (Mirtoselect®) on resolution of CCl4-induced liver fibrosis in mice

Bilberry (Vaccinium myrtillus L) has been traditionally used in the treatment of various liver disorders. The aim of this study was to investigate the effects of bilberry fruit extract (BE) on carbon tetrachloride (CCl₄)-induced hepatic fibrosis. Male Balb/C mice were treated with CCl₄ dissolved in olive oil (20% v/v, 2 ml/kg) intraperitoneally (i.p.), twice a week for 7 weeks. BE (1, 5, and 10 mg/kg) was given to mice for next 15 days, 72 h after the last dose of CCl₄. The CCl₄ administration increased oxidative stress as well as the expression of tumor necrosis factor-α (TNF-α) and transforming growth factor-β1 (TGF-β1) in the liver. Furthermore, increased α-smooth muscle actin (α-SMA) expression and hydroxyproline levels indicated activation of hepatic stellate cells (HSCs) and enhanced collagen production. BE 10 mg/kg markedly attenuated oxidative stress, decreased TNF-α, TGF-β1, and α-SMA expression, and eliminated hepatic collagen deposits. These results indicate that BE, in a dose dependent manner, induces the resolution of liver fibrosis by decreasing oxidative stress and inactivating HSCs via down-regulation of fibrogenic cytokines, TGF-β1 and TNF-α.     

Oligo-peptide I-C-F-6 inhibits hepatic stellate cell activation and ameliorates CCl4-induced liver fibrosis by suppressing NF-κB signaling and Wnt/β-catenin signaling

Oligo-peptide I-C-F-6 is a Carapax trionycis extract component that has an effect on hepatic fibrosis, however, its mechanism of action is still unclear. This study investigated whether oligo-peptide I-C-F-6 could inhibit liver fibrosis by suppressing NF-κB and Wnt/β-catenin signaling, which are important in liver fibrosis. HSC-T6 cells were treated with oligo-peptide I-C-F-6, and rats were divided randomly into five groups: control (saline), CCl₄, CCl₄ plus oligo-peptide I-C-F-6 (0.12 and 0.24 mg/kg), and CCl₄ plus colchicine (0.11 mg/kg). Here, we demonstrated that oligo-peptide I-C-F-6 ameliorated liver injury, inflammation, and hepatic fibrogenesis induced by CCl₄. Oligo-peptide I-C-F-6 also inhibited the activation of hepatic stellate cells (HSCs) in vivo and in vitro, as evaluated by the expression of transforming growth factor-β1 (TGF-β1) and α-smooth muscle actin (α-SMA), which is a specific marker of HSC activation. Moreover, oligo-peptide I-C-F-6 significantly reduced the expression and distribution of β-catenin, P-AKT, phospho (P)-GSK-3β, nuclear factor κB (NF-κB) P65, phospho-P65, and IκB kinase α/β (IKK-α/β) levels; additionally, IκB-α level was elevated both in vivo and in vitro. Together, these results indicate that oligo-peptide I-C-F-6 has hepatoprotective and anti-fibrotic effects in animal models of liver fibrosis, the mechanism of which may be related to modulating NF-κB and Wnt/β-catenin signaling.     

Isochlorogenic acid A attenuates progression of liver fibrosis through regulating HMGB1/TLR4/NF-κB signaling pathways

OBJECTIVE Liver fibrosis is a chronic damage process related to the further progression of hepatic cirrhosis and has yet no truly effective treatment is available. This study aimed to investigate the effects of isochlorogenic acid A(ICQA) on liver fibrosis induced by carbon tetrachloride(CCl4) and clarify the underlying mechanism. METHODS Rats were treated with CCl4 for eight weeks in order to induce liver fibrosis and simultaneously orally administered with ICQA(10, 20 and 40 mg·kg~(-1)). RESULTS ICQA had significant protective effect on liver injury, inflammation, and fibrosis in rats. Meanwhile, ICQA prevented the activation of hepatic stellate cells(HSC) as indicated by inhibiting the overexpression of a-smooth muscle actin(a-SMA). In addition, reduced fibrosis was found to be associated with decreased protein expression of high-mobility group box 1(HMGB1) and toll like receptor(TLR)4. Moreover, ICQA supressed the cytoplasmic translocation of HMGB1 in rat liver. Further investigations indicated that ICQA treatment significantly attenuated nuclear translocation of the nuclear factor-κB(NF-κB) p65 and inhibited degradation of IkB a expression in the liver of rats with liver fibrosis. CONCLUSION ICQA has hepatoprotective and anti-fibrotic effects in rats with liver fibrosis through modulating the HMGB1/TLR4/NF-κB signaling pathways.     

Therapeutic effects of AdipoRon on liver inflammation and fibrosis induced by CCl4 in mice

ObjectiveThe present work aimed to investigate the effects of AdipoRon against acute hepatitis and liver fibrosis induced by carbon tetrachloride (CCl₄) in mice.MethodsC57BL/6 mice were randomly divided into five groups: control, model, AdipoRon groups (three different dosages), CCl₄ was administered to induce acute hepatitis or liver fibrosis except for control group. The liver function, inflammatory and fibrotic profiles were evaluated by histology, immunohistochemistry and expression analysis, respectively.ResultsAdipoRon pretreatment effectively attenuated oxidative stress and hepatocellular damage in acute CCl₄ intoxication, demonstrated by marked reduction in peroxidation indexes [hepatic malonaldehyde (MDA), total nitric oxide synthase (tNOS), inducible nitric oxide synthase (iNOS)] and serum transaminases [alanine aminotransferase (ALT), aspartate transaminase (AST)]. Moreover, AdipoRon attenuated the severity of fibrosis induced by sustaining CCl₄ challenge, with the alleviation of fibrous deposit and architecture distortion. The levels of canonical fibrosis markers (aminotransferases, hydroxyproline, hyaluronic acid, laminin) were also dose-dependently modulated by AdipoRon. Immunochemistry and expression analysis showed AdipoRon restrained the proinflammatory and profibrotic cytokines (TNF-α, TGF-β1, α-SMA, COL1A1), which somehow, ascribed the anti-fibrotic action to inhibiting hepatic stellate cells (HSCs) activation and quenching specific inflammation-fibrogenesis pathways.ConclusionsAdipoRon demonstrates a remedial capacity against hepatitis and fibrosis induced by CCl₄, potentially by inflammation restraint and HSC deactivation, which might pave the way for its therapeutical application in hepatic fibrosis.     

Oridonin ameliorates carbon tetrachloride-induced liver fibrosis in mice through inhibition of the NLRP3 inflammasome

Liver fibrosis is characterized by the activation of hepatic stellate cells (HSCs) and accumulation of the extracellular matrix. There are limitations in the current therapies for liver fibrosis. Recently, oridonin was shown to induce apoptosis in HSCs. Thus, we aimed to determine the roles of oridonin in chronic liver injury and fibrosis. Liver fibrosis was induced by CCl₄ in mice injected intraperitoneally with oridonin for 6 weeks. The administration of oridonin significantly attenuated liver injury and reduced ALT levels. In addition, Sirius Red staining and the expression of α-smooth muscle actin (α-SMA) were significantly reduced by oridonin in murine livers with fibrosis. The expression of NLRP3, caspase-1, and IL-1β was downregulated with the oridonin treatment. Furthermore, the expression of F4/80 in liver tissues was also decreased by oridonin treatment. These results demonstrate that oridonin ameliorates chronic liver injury and fibrosis. Mechanically, oridonin may inhibit the activity of the NLRP3 inflammasome and inflammation in the liver. These results highlight the potential of oridonin as a therapeutic agent for liver fibrosis.     

Nrf2 pathway activation contributes to anti-fibrosis effects of ginsenoside Rg1 in a rat model of alcohol- and CCl4-induced hepatic fibrosis

Aim:

To investigate the anti-fibrosis effects of ginsenoside Rg1 on alcohol- and CCl₄-induced hepatic fibrosis in rats and to explore the mechanisms of the effects.

Methods:

Rats were given 6% alcohol in water and injected with CCl₄ (2 mL/kg, sc) twice a week for 8 weeks. Rg1 (10, 20 and 40 mg/kg per day, po) was administered in the last 2 weeks. Hepatic fibrosis was determined by measuring serum biochemical parameters, HE staining, Masson''s trichromic staining, and hydroxyproline and α-SMA immunohistochemical staining of liver tissues. The activities of antioxidant enzymes, lipid peroxidation, and Nrf2 signaling pathway-related proteins (Nrf2, Ho-1 and Nqo1) in liver tissues were analyzed. Cultured hepatic stellate cells (HSCs) of rats were prepared for in vitro studies.

Results:

In the alcohol- and CCl₄-treated rats, Rg1 administration dose-dependently suppressed the marked increases of serum ALT, AST, LDH and ALP levels, inhibited liver inflammation and HSC activation and reduced liver fibrosis scores. Rg1 significantly increased the activities of antioxidant enzymes (SOD, GSH-Px and CAT) and reduced MDA levels in liver tissues. Furthermore, Rg1 significantly increased the expression and nuclear translocation of Nrf2 that regulated the expression of many antioxidant enzymes. Treatment of the cultured HSCs with Rg1 (1 μmol/L) induced Nrf2 translocation, and suppressed CCl₄-induced cell proliferation, reversed CCl_4- induced changes in MDA, GPX, PCIII and HA contents in the supernatant fluid and α-SMA expression in the cells. Knockdown of Nrf2 gene diminished these actions of Rg1 in CCl₄-treated HSCs in vitro.

Conclusion:

Rg1 exerts protective effects in a rat model of alcohol- and CCl₄-induced hepatic fibrosis via promoting the nuclear translocation of Nrf2 and expression of antioxidant enzymes.     

Design of a highly potent GLP-1R and GCGR dual-agonist for recovering hepatic fibrosis

Currently, there is still no effective curative treatment for the development of late-stage liver fibrosis. Here, we have illustrated that TB001, a dual glucagon-like peptide-1 receptor/glucagon receptor (GLP-1R/GCGR) agonist with higher affinity towards GCGR, could retard the progression of liver fibrosis in various rodent models, with remarkable potency, selectivity, extended half-life and low toxicity. Four types of liver fibrosis animal models which were induced by CCl₄, α-naphthyl-isothiocyanate (ANIT), bile duct ligation (BDL) and Schistosoma japonicum were used in our study. We found that TB001 treatment dose-dependently significantly attenuated liver injury and collagen accumulation in these animal models. In addition to decreased levels of extracellular matrix (ECM) accumulation during hepatic injury, activation of hepatic stellate cells was also inhibited via suppression of TGF-β expression as well as downstream Smad signaling pathways particularly in CCl₄-and S. japonicum-induced liver fibrosis. Moreover, TB001 attenuated liver fibrosis through blocking downstream activation of pro-inflammatory nuclear factor kappa B/NF-kappa-B inhibitor alpha (NFκB/IKBα) pathways as well as c-Jun N-terminal kinase (JNK)-dependent induction of hepatocyte apoptosis. Furthermore, GLP-1R and/or GCGR knock-down results represented GCGR played an important role in ameliorating CCl₄-induced hepatic fibrosis. Therefore, TB001 can be used as a promising therapeutic candidate for the treatment of multiple causes of hepatic fibrosis demonstrated by our extensive pre-clinical evaluation of TB001.     

Hepatoprotective effects of zingerone on carbon tetrachloride- and dimethylnitrosamine-induced liver injuries in rats

In this study, we investigated the hepatoprotective and anti-fibrotic effects of zingerone, one of the active components of ginger, against carbon tetrachloride (CCl₄)- and dimethylnitrosamine (DMN)-induced liver injuries in rats, respectively. Oral administration of zingerone (10 mg/kg) reduced CCl₄-induced abnormalities in liver histology, serum alanine aminotransferase and aspartate aminotransferase levels, and liver malondialdehyde levels. Zingerone treatment attenuated CCl₄-induced increases in inflammatory mediators, including tumor necrosis factor-α, interleukin-1β, cyclooxygenase-2, and inducible nitric oxide synthase mRNA levels. Western blot analysis showed that zingerone suppressed activation of nuclear factor-kappa B (NF-κB) p65 and phosphorylation of extracellular signal-regulated kinase, c-Jun NH₂-terminal kinase, and p38 mitogen-activated protein kinases (MAPKs). Liver fibrosis induced by DMN (10 mg/kg, intraperitoneally) was ameliorated by administration of zingerone (10 and 20 mg/kg, orally). Zingerone treatment reduced DMN-induced elevation of hydroxyproline content and hepatic stellate cell activation. In conclusion, zingerone showed antioxidative and anti-inflammatory effects in CCl₄-intoxicated rats by inhibiting oxidative stress and NF-κB activation via blockade of the activation of upstream MAPKs. Moreover, zingerone had hepatoprotective and anti-fibrotic effects against DMN-induced liver injury suggesting its usefulness in the prevention of liver inflammation and the development of hepatic fibrosis.     

Hsian-tsao (Mesona procumbens Heml.) prevents against rat liver fibrosis induced by CCl4 via inhibition of hepatic stellate cells activation

In this study, the protective effect of extract of Hsian-tsao (Mesona procumbens) (EHT) against liver fibrogenesis in carbon tetrachloride (CCl₄)-injured rats was evaluated. The inhibitory effect of oleanolic acid (OA) and ursolic acid (UA), which are the active compounds in EHT, on the activation of hepatic stellate cells (HSC) was also determined. The results showed that EHT at a dosage of 1.2 g/kg of b.w. significantly reduced the liver injury induced by CCl₄ in rats. It also decreased the activity of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and the deposition of collagen in the liver. Oral administration of EHT reduced the levels of alpha-smooth muscle actin (α-SMA) and the activity of metalloproteinases (MMPs) in rats injured by treatment with CCl₄. In addition, we performed experiments with the rat hepatic stellate cell line HSC-T6 in which we induced the expression of MMP-2 and α-SMA with phorbol-12-myristate-13-acetate (PMA). Treating these cells with OA (20 μM) or UA (10 μM) caused a decrease in the levels of both proteins. Taken together, our data indicate that EHT can efficiently inhibit CCl₄-induced liver fibrosis in rats. EHT may therefore be a useful functional food for preventing liver fibrosis.     

Evodiamine ameliorates liver fibrosis in rats via TGF-β1/Smad signaling pathway

Liver fibrosis is considered to be a result of chronic liver pathological changes, and hepatic stellate cells (HSCs) play an important role during this process. Evodiamine, an indole alkaloid derived from Evodia rutaecarpa, exhibits pharmacological activities. This study focused on the effects of evodiamine on carbon tetrachloride (CCl₄)-induced liver fibrosis in rats and HSCs in vitro via the TGF-β1/Smad signaling pathway. A liver fibrosis rat model was established by the intraperitoneal injection of CCl₄ (3 ml/kg, 30% in olive oil). Evodiamine (15 and 25 mg/kg) was administered orally for 8 weeks. HSCs were treated with different evodiamine concentrations. The results indicated that evodiamine could improve the histopathological abnormalities in liver tissues and decrease the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), hydroxyproline, and total bilirubin (TBIL). Concentrations of IL-6, tumor necrosis factor-α (TNF-α), collagen-I (COL-I), and collagen-III (COL-III) were reduced by evodiamine. Western blotting and real-time PCR showed that protein expression of transforming growth factor-β (TGF-β1), p-Smad 2/3 (phosphorylation of Smad 2/3), and smooth muscle alpha-actin (α-SMA) as well as mRNA expression of TGF-β1 and α-SMA in liver tissues were downregulated by evodiamine. The cell proliferation, production of hydroxyproline, and the protein expression of TGF-β1, p-Smad 2/3, and α-SMA in HSCs were dose-dependently reduced by evodiamine. Collectively, evodiamine had an antifibrosis effect in CCl₄-induced liver fibrosis, and reduced HSCs proliferation and collagen metabolism in vitro. The major mechanism was downregulation of relative expression of TGF-β1, p-Smad 2/3, and α-SMA.     

TRPM7 channel regulates PDGF-BB-induced proliferation of hepatic stellate cells via PI3K and ERK pathways

TRPM7, a non-selective cation channel of the TRP channel superfamily, is implicated in diverse physiological and pathological processes including cell proliferation. Recently, TRPM7 has been reported in hepatic stellate cells (HSCs). Here, we investigated the contribution role of TRPM7 in activated HSC-T6 cell (a rat hepatic stellate cell line) proliferation. TRPM7 mRNA and protein were measured by RT-PCR and Western blot in rat model of liver fibrosis in vivo and PDGF-BB-activated HSC-T6 cells in vitro. Both mRNA and protein of TRPM7 were dramatically increased in CCl₄-treated rat livers. Stimulation of HSC-T6 cells with PDGF-BB resulted in a time-dependent increase of TRPM7 mRNA and protein. However, PDGF-BB-induced HSC-T6 cell proliferation was inhibited by non-specific TRPM7 blocker 2-aminoethoxydiphenyl borate (2-APB) or synthetic siRNA targeting TRPM7, and this was accompanied by downregulation of cell cycle proteins, cyclin D1, PCNA and CDK4. Blockade of TRPM7 channels also attenuated PDGF-BB induced expression of myofibroblast markers as measured by the induction of α-SMA and Col1α1. Furthermore, the phosphorylation of ERK and AKT, associated with cell proliferation, decreased in TRPM7 deficient HSC-T6 cells. These observations suggested that TRPM7 channels contribute to perpetuated fibroblast activation and proliferation of PDGF-BB induced HSC-T6 cells via the activation of ERK and PI3K pathways. Therefore, TRPM7 may constitute a useful target for the treatment of liver fibrosis.     

Gingerol protects against experimental liver fibrosis in rats via suppression of pro-inflammatory and profibrogenic mediators

6-Gingerol (Gin) is known to possess hepatoprotective effects. Liver fibrosis is a major health concern that results in significant morbidity and mortality. There is no FDA-approved medication for liver fibrosis. The present work aimed at exploring the beneficial effects of Gin against liver fibrosis in rats. Experimental fibrosis was induced by challenging animals with CCl₄ for 6 weeks. Gin significantly ameliorated the increase in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, albumin, total cholesterol (TC) and triglyceride (TG) concentrations, and liver index. These effects were confirmed by light and electron microscopic examinations. The antifibrotic effects were confirmed by examining Masson trichrome-stained liver sections which indicated reduced collagen deposition in Gin-treated animals. Further, Gin administration hampered alpha-smooth muscle actin (α-SMA) expression and significantly reduced hepatic content of transforming growth factor-beta (TGF-β). Also, Gin elicited profound antioxidant actions as indicated by preventing reduced glutathione (GSH) depletion and lipid peroxide accumulation. The observed antifibrotic activities involved decreased production of nuclear factor κB (NF-κB), tumor necrosis factor alpha (TNF-α), expression of toll-like receptor 4 (TLR4), intercellular adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM). Involvement of Gin anti-inflammatory activity was verified by the decreased expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in livers of animals treated with Gin. Thus, it can be concluded that Gin protects against CCl₄-induced liver fibrosis in rats. This can be ascribed, at least partly, to its antioxidant, anti-inflammatory effects as well as the inhibition of NF-κB/TLR-4 expression.     

Curcumin inhibits collagen synthesis and hepatic stellate cell activation in-vivo and in-vitro

We previously demonstrated that curcumin, a well-known antioxidant, inhibits collagen deposition in carbon tetrachloride-induced liver injury in rats. The major effector cells responsibleforcollagensynthesis in the liver are activated hepatic stellate cells. In this study,we investigated the inhibitory effects of curcumin on the collagen synthesis and activation of rat hepatic stellate cells in-vitro, and on hepatic stellate cell activation in-vivo. The effects of curcumin on the production of collagen and smooth muscle alpha-actin proteins and of alpha1(I) collagen mRNA were studied in-vivo and in-vitro. The effect of curcumin on DNA synthesis was also determined in-vitro. In-vivo, treatment with curcumin reduced collagen deposition and smooth muscle alpha-actin-positive areas and lowered mRNA levels of type I collagen in the liver. In-vitro, curcumin at a concentration of 5 microg mL(-1) reduced DNA synthesis, and downregulated smooth muscle alpha-actin and type I collagen expression, and alpha1(I) collagen mRNA expression. We concluded that curcumin inhibits collagen synthesis and hepatic stellate cell activation in-vivo and in-vitro, and thus may prove a valuable anti-fibrogenic agent.     

β-Arrestin2 deficiency attenuates oxidative stress in mouse hepatic fibrosis through modulation of NOX4

Hepatic fibrosis is a disease characterized by excessive deposition of extracellular matrix (ECM) in the liver. Activation of hepatic stellate cells (HSCs) is responsible for most of ECM production. Oxidative stress and reactive oxygen species (ROS) may be important factors leading to liver fibrosis. NADPH oxidase 4 (NOX4) is the main source of ROS in hepatic fibrosis, but the mechanism by which NOX4 regulates oxidative stress is not fully understood. β-Arrestin2 is a multifunctional scaffold protein that regulates receptor endocytosis, signaling and trafficking. In this study, we investigated whether β-arrestin2 regulated oxidative stress in hepatic fibrosis. Both β-arrestin2 knockout (Arrb2 KO) mice and wild-type mice were intraperitoneally injected with carbon tetrachloride (CCl₄) to induce hepatic fibrosis. Arrb2 KO mice showed significantly attenuated liver fibrosis, decreased ROS levels and NOX4 expression, and reduced collagen levels in their livers. In vitro, NOX4 knockdown significantly inhibited ROS production, and decreased expression of alpha-smooth muscle actin in angiotensin II-stimulated human HSC cell line LX-2. Through overexpression or depletion of β-arrestin2 in LX-2 cells, we revealed that decreased β-arrestin2 inhibited ROS levels and NOX4 expression, and reduced collagen production; it also inhibited activation of ERK and JNK signaling pathways. These results demonstrate that β-arrestin2 deficiency protects against liver fibrosis by downregulating ROS production through NOX4. This effect appears to be mediated by ERK and JNK signaling pathways. Thus, targeted inhibition of β-arrestin2 might reduce oxidative stress and inhibit the progression of liver fibrosis.     

Smad2 increases the apoptosis of activated human hepatic stellate cells induced by TRAIL

The activation of hepatic stellate cells (HSCs) plays a critical role in the development of liver fibrosis. The induction of apoptosis in activated HSCs during the recovery phase of hepatic fibrosis represents a potential anti-fibrotic therapy. We have previously shown that Smad2 protects against hepatic fibrogenesis; however, the role of Smad2 in the regulation of activated HSC apoptosis remains unknown. We hypothesized that Smad2 regulates the apoptosis of activated HSCs, leading to the resolution of liver fibrosis. To test this hypothesis, the livers of rats were harvested at 0 and 4 weeks after hepatic fibrosis was established by CCl₄ injection. Furthermore, TGF-β1-activated HSCs were treated with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) following the silencing or overexpression of Smad2. Both the phosphorylation of Smad2 and TRAIL were detected in fibrotic liver tissues. The results of TUNEL and α-SMA double-staining showed an increase in the apoptosis of activated HSCs during the spontaneous recovery phase. The knockdown of Smad2 reduced TRAIL-induced apoptosis in TGF-β1-activated human LX-2 cells and resulted in an increased expression of α-SMA and collagen I (Col. I). In contrast, the overexpression of Smad2 increased TRAIL-induced HSC apoptosis and reduced the expression of α-SMA and Col. I. The mechanisms underlying these findings were associated with the Smad2-mediated down-regulation of X-linked inhibitor of apoptosis protein (XIAP), resulting in enhanced caspase-3 activity and apoptosis. In conclusion, Smad2 enhances TRAIL-induced apoptosis in activated HSCs, which facilitates the resolution of hepatic fibrosis.