Gan Shen Fu Fang ameliorates liver fibrosis in vitro and in vivo by inhibiting the inflammatory response and extracellular signal-regulated kinase phosphorylation.

BACKGROUND: Liver fibrosis is a common health problem worldwide and there is still a lack of effective medicines. The Chinese herbal medicine, Gan Shen Fu Fang (GSFF) is composed of salvianolic acid B and diammonium glycyrrhizinate. In this study, we observed the effects of GSFF on liver fibrosis in vivo and in vitro in an attempt to provide some hope for the treatment. AIM: To observe the effects of GSFF on liver fibrosis in vivo and in vitro and investigate the mechanism from the perspective of the inflammatory response and extracellular signal-regulated kinase (ERK) phosphorylation. METHODS: Common bile duct-ligated rats were used for in vivo experiments. Hepatic stellate cells-T6 (HSC-T6) cells were used for in vitro experiments. Hematoxylin and eosin staining and Masson staining, biochemical assays, hydroxyproline (Hyp) assays, enzyme-linked immunoasorbent assay and western blotting were performed to evaluate the degree of liver fibrosis, liver function, the inflammatory response and ERK phosphorylation. The CCK8 assay, immunofluorescence and western blotting were applied to test the effect of GSFF on HSC-T6 cell activation and determine whether GSFF had an effect on ERK phosphorylation in HSC-T6 cells. RESULTS: GSFF improved liver function and inhibited liver fibrosis in common bile duct-ligated rats after 3 wk of treatment, as demonstrated by histological changes, hydroxyproline assays and collagen I concentrations. GSFF alleviated inflammatory cell infiltration and reduced the synthesis of pro-inflammatory cytokines [tumor necrosis factor-α (TNF-α) and interlukin-1ß] and NF-κB. In addition, GSFF decreased ERK phosphorylation. In vitro, GSFF inhibited the viability of HSC-T6 cells with and without transforming growth factor ß1 (TGF-ß1) stimulation and decreased the synthesis of collagen I. GSFF had the greatest effect at a concentration of 0.5 µmol/L. GSFF inhibited the expression of α-smooth muscle actin (α-SMA), a marker of HSC activation, in HSC-T6 cells. Consistent with the in vivo results, GSFF also inhibited the phosphorylation of ERK and downregulated the expression of NF-κB. CONCLUSION: GSFF inhibited liver fibrosis progression in vivo and HSC-T6 cell activation in vitro. These effects may be related to an alleviated inflammatory response and downregulated ERK phosphorylation.

HBOA ameliorates CCl4-incuded liver fibrosis through inhibiting TGF-ß1/Smads, NF-κB and ERK signaling pathways.

An ingredient was isolated from Acanthus ilicifolius and identified as 4-hydroxy-2(3H)-benzoxazolone (HBOA). Its protective effects and underlying mechanism on liver fibrosis were investigated. Briefly, rats were intragastrically administrated with 50% CCl4 twice a week for 12 weeks to induce liver fibrosis. Meanwhile, the animals were treated with various medicines from weeks 8 to 12. Then the histological change, serum biochemical index, inflammatory factors and hepatocyte apoptosis were detected. Moreover, the TGF-ß1/Smads, NF-κB and ERK signaling pathways were also detected to illustrate the underlying mechanism. The results showed that HBOA significantly ameliorated CCl4-induced liver injury and collagen accumulation in rats, as evidenced by the histopathologic improvement. Moreover, HBOA markedly decreased hepatocyte apoptosis by regulating the expression levels of caspase-3, -9 and -12, as well as the Bcl-2 family. The mechanism study showed that HBOA significantly decreased the expressions of α-smooth muscle actin (α-SMA) and collagen and inhibited the generation of excessive extracellular matrix (ECM) components by restoring the balance between matrix metalloproteinases (MMPs) and its inhibitor (TIMPs). HBOA markedly alleviated oxidative stress and inflammatory cytokines through inhibiting the NF-κB pathway. In addition, HBOA significantly down-regulated the levels of TGF-ß1, Smad2/3, Smad4 and up-regulated the level of Smad7, inhibiting the TGF-ß1/Smads signaling pathway. Moreover, HBOA significantly blocked the ERK signaling pathway, leading to the inactivation of hepatic stellate cells. This study suggests that HBOA exerts a protective effect against liver fibrosis via modulating the TGF-ß1/Smads, NF-κB and ERK signaling pathways, which will be developed as a potential agent for the treatment of liver fibrosis.

Protective Effect of Ganshuang Granules () on Liver Cirrhosis by Suppressing Regulatory T Cells in Mouse Model.

OBJECTIVE: To investigate the potential antifibrotic mechanisms of Chinese medicine Ganshuang Granules (, GSG) and to provide clinical therapeutic evidence of its effects. METHODS: A cirrhotic mouse model was established by intraperitoneally injecting a mixture of CCl4 (40%) and oil (60%) at 0.2 mL per 100 g of body weight twice a week for 12 weeks. After 12-week modeling, GSG was intragastric administrated to the mice for 2 weeks, and the mice were divided into low-, medium- and high-dose groups at doses of 1, 2 and 4 g/(kg·day), respectively. Liver morphology changes were observed using Masson's trichrome staining and B-ultrasound. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and hyaluronic acid (HA) in serum were detected using an automatic biochemistry analyzer. The expressions of desmin, smooth muscle actin (SMA) and Foxp3 in liver were detected by immunoflfluorescence. The regulatory T cell (Treg) frequency was determined through flflow cytometry analysis. Collagen-I, SMA, IL-6, tumor necrosis factor α (TNF-α), interleukin (IL)-1ß and transforming growth factor ß1 (TGF-ß1) expression levels were measured using quantitative polymerase chain reaction (qPCR). RESULTS: Masson's staining result showed fewer pseudolobule structures and fibrous connective tissue in the GSG-treatment groups than in the spontaneous recovery group. Ultrasonography showed that GSG treatment reduced the number of punctate hyperechoic lesions in mice cirrhotic livers. The serum ALT, AST, HA levels were significantly ameliorated by GSG treatment (ALT: F=8.104, P=0.000; AST: F=7.078, P=0.002; and HA: F=7.621, P=0.001). The expression levels of collagen-I and SMA in the cirrhotic livers were also attenuated by GSG treatment (collagen-I: F=3.938, P=0.011; SMA: F=4.115, P=0.009). Tregs, which were elevated in the fibrotic livers, were suppressed by GSG treatment (F=8.268, P=0.001). The expressions of IL-6, TNF-α and IL-1ß increased, and TGF-ß levels decreased in the cirrhotic livers after GSG treatment (IL-6: F=5.457, P=0.004; TNF-α: F=6.023, P=0.002; IL-1ß: F=6.658, P=0.001; and TGF-ß1: F=11.239, P=0.000). CONCLUSIONS: GSG promoted the resolution/regression of cirrhosis and restored liver functions in part by suppressing Treg cell differentiation, which may be mediated by hepatic stellate cells.

Yiguanjian decoction enhances fetal liver stem/progenitor cell-mediated repair of liver cirrhosis through regulation of macrophage activation state.

AIM: To investigate whether Yiguanjian decoction (YGJ) has an anti-liver cirrhotic effect and whether it regulates hepatic stem cell differentiation. METHODS: A rat model of liver cirrhosis was established via subcutaneous injection of carbon tetrachloride (CCl4) for 8 wk. From the beginning of the ninth week, the rats received 2-acetylaminofluorene (2-AAF) by oral gavage and a DLK-1+ fetal liver stem/progenitor cell (FLSPC) transplant or an FLSPC transplant in combination with YGJ treatment for 4 wk. In vitro, lipopolysaccharide (LPS)-activated macrophages were co-cultured with WB-F344 cells, and the differentiation of WB-F344 cells was observed in the presence and absence of YGJ treatment. RESULTS: FLSPC transplantation improved liver function and histopathology, and inhibited the activation of the non-canonical Wnt signaling pathway, while activating the canonical Wnt signaling pathway. YGJ enhanced the therapeutic effects of FLSPCs and also promoted the liver regeneration differentiation of FLSPCs into hepatocytes. In vitro, LPS-activated macrophages promoted the differentiation of WB-F344 cells into myofibroblasts, and the canonical Wnt signaling was inhibited while the non-canonical Wnt signaling was activated in WB-F344 cells. YGJ suppressed the activation of macrophages and then inhibited non-canonical Wnt signaling and promoted canonical Wnt signaling. CONCLUSION: YGJ enhances FLSPC-mediated repair of liver cirrhosis through regulation of macrophage activation state, and YGJ in combination with stem cell transplantation may be a suitable treatment for end-stage liver cirrhosis.

Yu Gan Long reduces rat liver fibrosis by blocking TGF-ß1/Smad pathway and modulating the immunity.

Yu Gan Long (YGL) is a Chinese traditional herbal medicine that has been used in the treatment of liver fibrosis for many years in clinical practice. However, its anti-hepatofibrotic mechanism has not been studied yet. In this study, the effect and mechanism of YGL in reducing liver fibrosis was demonstrated in vivo. Our results showed that liver fibrosis biomarkers collagen IV (Col IV), type III precollagen (PCIII), hyaluronuc acid (HA) and laminin (LN), were increased after CCl4 treatment and decreased by YGL. Among the liver fibrosis indicators, α-smooth muscle actin (α-SMA) was decreased by YGL in the CCl4-treated rats, while MMP2 and MMP9 was upregulated followed by TIMP1 downregulation. Proteins involved in liver fibrosis such as p-Smad2, p-Smad3 and Smad4 were down-regulated, while Smad7 protein was up-regulated by YGL after CCl4-induced liver damage. YGL also suppressed the increase of TGF-ß1, TNF-α, IL-1ß, IL-6, IL-4 and IL-17 A induced by CCl4 treatment, while promoted IFN-γ expression. Finally, the transcription factors ROR-γt and GATA3 were decreased, while T-bet was increased after YGL treatment. These results suggested that YGL attenuated CCl4-induced hepatic fibrosis by accelerating the extracellular matrix degradation, blocking the TGF-ß1/Smad signaling pathway and modulating the balance among IL-4, IL-17 A and IFN-γ, demonstrating YGL protective effect and its potential mechanisms in treating liver fibrosis.

An ω-3-enriched diet alone does not attenuate CCl4-induced hepatic fibrosis.

Exposure to the halogenated hydrocarbon carbon tetrachloride (CCl4) leads to hepatic lipid peroxidation, inflammation and fibrosis. Dietary supplementation of ω-3 fatty acids has been increasingly advocated as being generally anti-inflammatory, though its effect in models of liver fibrosis is mixed. This raises the question of whether diets high in ω-3 fatty acids will result in a greater sensitivity or resistance to liver fibrosis as a result of environmental toxicants like CCl4. In this study, we fed CCl4-treated mice a high ω-3 diet (using a mix of docosahexaenoic acid and eicosapentaenoic acid ethyl esters). We also co-administered an inhibitor of soluble epoxide hydrolase, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), which has been shown to boost anti-inflammatory epoxy fatty acids that are produced from both ω-3 and ω-6 dietary lipids. We showed that soluble epoxide inhibitors reduced CCl4-induced liver fibrosis. Three major results were obtained. First, the ω-3-enriched diet did not attenuate CCl4-induced liver fibrosis as judged by collagen deposition and collagen mRNA expression. Second, the ω-3-enriched diet raised hepatic tissue levels of several inflammatory lipoxygenase metabolites and prostaglandins, including PGE2. Third, treatment with TPPU in drinking water in conjunction with the ω-3-enriched diet resulted in a reduction in liver fibrosis compared to all other groups. Taken together, these results indicate that dietary ω-3 supplementation alone did not attenuate CCl4-induced liver fibrosis. Additionally, oxylipin signaling molecules may play role in the CCl4-induced liver fibrosis in the high ω-3 diet groups.

Artesunate alleviates hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats.

The current study was performed in order to explore the effect of artesunate (Art) on experimental hepatic fibrosis and the potential mechanism involved. Art, a water-soluble hemisuccinate derivative of artemisinin extracted from the Chinese herb Artemisia Annua, is a safe and effective antimalarial drug. Hepatic fibrosis was induced in SD rats by multiple pathogenic factors. Rats were treated concurrently with Art (28.8 mg/kg) given daily by oral gavage for 6 or 8 weeks to evaluate its protective effects. Our data demonstrated that Art treatment obviously attenuated hepatic fibrosis, characterized by less inflammatory infiltration and accumulation of extracellular matrix (ECM). Art remarkably decreased endotoxin, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels as well. Art significantly downregulated protein and mRNA expression of α-smooth muscle actin (α-SMA), toll-like receptors 4 (TLR4), myeloid differentiation factor 88 (MyD88) and transforming growth factor beta 1 (TGF-ß1). Art also significantly inhibited the nuclear transcription factor kappa B p65 (NF-κB p65) translocation into the nucleus. In addition, there were no remarkable differences between the N group and the NA group. In conclusion, we found that Art could alleviate hepatic fibrosis induced by multiple pathogenic factors and inflammation through the inhibition of LPS/TLR4/NF-κB signaling pathway in rats, suggesting that Art may be a potential candidate for the therapy of hepatic fibrosis.

Protective effect of theaflavin-enriched black tea extracts against dimethylnitrosamine-induced liver fibrosis in rats.

Liver cirrhosis is responsible for hepatic fibrosis resulting in high mortality and is also a risk factor for developing hepatocellular carcinoma (HCC), which is the fifth most common cancer in men and the seventh in women globally. Several studies have found effective anti-cancer activities of theaflavins, the major black tea polyphenols. The objective of this study was to investigate the protective effects of theaflavin-enriched black tea extracts (TF-BTE) on hepatic fibrosis induced by dimethylnitrosamine (DMN) administration in Sprague-Dawley (SD) rats. Treatment of SD rats with DMN (10 mg per kg bw) for 4 weeks produced inflammation and remarkable liver fibrosis assessed by serum biochemistry and histopathological examination. Fibrotic status and the activation of hepatic stellate cells were improved by oral administration of 40% theaflavins in black tea extracts (40% TF-BTE) as evidenced by histopathological examination. Oral administration of 40% TF-BTE at a low dose of 50 mg per kg bw per day and a high dose of 100 mg per kg bw per day attenuated the DMN-induced elevation of serum GOT (glutamate oxaloacetate transaminase) and GPT (glutamic pyruvic transaminase) levels and reduced necrosis, bile duct proliferation, and inflammation. Western blot analyses revealed that TF-BTE inhibited the expression of liver alpha-smooth muscle actin (α-SMA) and transforming growth factor-ß1 (TGF-ß1) protein. The histochemical examination showed the inhibitory effect of TF-BTE on the p-Smad3 expression. Overall, these data demonstrated that TF-BTE exhibited hepatoprotective effects on experimental fibrosis, potentially by inhibiting the TGF-ß1/Smad signaling.

Hepatoprotective Effects of Traditional Chinese Medicine on Liver Fibrosis from Ethanol Administration following Partial Hepatectomy.

The aim of this study was to establish the effective hepatoprotective properties of traditional Chinese medicines (TCMs) in fibrotic rat liver regeneration after partial hepatectomy (PHx). Fibrosis was induced in rats by ethanol (EtOH, 5 ml/kg) administration for 6, 24, 72, and 168 h. The rats were then fed four TCMs (1 g/kg/day, Codonopsis pilosula (CP), Salvia miltorrhiza Bunge (SMB), Bupleurum kasi (BK), and Elephantopus scaber L (ESL)) to Spraque-Dawley rats for 6, 24, 72 and 168 h, respectively. Surgical 70% cirrhotic fibrosis PHx was then conducted at 6, 24, 72, and 168 h. The effects on liver regeneration were examined to estimate and measure hepatocyte growth factor (HGF), focal adhesion kinase (FAK), Cyclin D1, Cyclin E, and retinoblastoma protein (pRb) protein expression using Western blotting analysis. Cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, tissue inhibitors of metalloproteinase (TIMP)-1, TIMP-2 and TIMP-3 mRNA by Reverse transcription polymerase chain reaction (RT-PCR) were analyzed in cirrhotic fibrosis rats. Transforming growth factor-ß1 (TGF-ß1), Cyclin D1, Cyclin E, pRb and E2F mRNA expression levels were determined in fibrotic rats following PHx using RT-PCR. We found elevated glutamyl oxaloacetic transaminase (GOT), glutamyl pyrubic transaminase (GPT), alkaline phosphatase (ALP), gammaglutamyl transpeptidase (γ-GT), glutathione (GSH), nonprotein sulfhydryl (NPSH) and total bilirubin in serum after 6 h EtOH administration. These levels were progressively decreased over 168 h. Total protein and albumin were reduced in serum after 6 h administration and then progressively increased. In contrast, tissues disorder histology and morphology were determined in liver sections. After rats were fed TCMs we found that SMB extraction not only induced HGF, FAK, Cyclin D1, and pRb protein expression and Cyclin D1 mRNA increases, but also reduced MMP-2 and MMP-9 after 24 and 72 h post injury. In the cell cycle S phase the Cyclin E protein expression was increased by ESL. CP induced TIMP-1, TIMP-2 and TIMP-3 mRNA increases in fibrotic rats. We detected liver regeneration in fibrotic rats. We also found that the liver regeneration index increased from 6 to 168 h post PHx. After 168 h fibrotic liver regeneration rats exhibited reduced TGF-ß1 mRNA expression and enhanced Cyclin D1, Cyclin E, pRb and E2F mRNA expression. TCMs play a crucial role in the early mediating process in fibrotic rat liver regeneration after PHx.

Garcinia Cambogia attenuates diet-induced adiposity but exacerbates hepatic collagen accumulation and inflammation.

AIM: To investigate long-term effects of Garcinia Cambogia (GC), weight-loss supplement, on adiposity and non-alcoholic fatty liver disease in obese mice. METHODS: Obesity-prone C57BL/6J mice were fed a high-fat diet (HFD, 45 kcal% fat) with or without GC (1%, w/w) for 16 wk. The HFD contained 45 kcal% fat, 20 kcal% protein and 35 kcal% carbohydrate. They were given free access to food and distilled water, and food consumption and body weight were measured daily and weekly, respectively. Data were expressed as the mean ± SE. Statistical analyses were performed using the statistical package for the social science software program. Student's t test was used to assess the differences between the groups. Statistical significance was considered at P < 0.05. RESULTS: There were no significant changes in body weight and food intake between the groups. However, the supplementation of GC significantly lowered visceral fat accumulation and adipocyte size via inhibition of fatty acid synthase activity and its mRNA expression in visceral adipose tissue, along with enhanced enzymatic activity and gene expression involved in adipose fatty acid ß-oxidation. Moreover, GC supplementation resulted in significant reductions in glucose intolerance and the plasma resistin level in the HFD-fed mice. However, we first demonstrated that it increased hepatic collagen accumulation, lipid peroxidation and mRNA levels of genes related to oxidative stress (superoxide dismutase and glutathione peroxidase) and inflammatory responses (tumor necrosis factor-α and monocyte chemoattractant protein-1) as well as plasma alanine transaminase and aspartate transaminase levels, although HFD-induced hepatic steatosis was not altered. CONCLUSION: GC protects against HFD-induced obesity by modulating adipose fatty acid synthesis and ß-oxidation but induces hepatic fibrosis, inflammation and oxidative stress.

Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice.

Toll-like receptor 4 (TLR4) and its coreceptor, myeloid differentiation factor-2 (MD-2), are key in recognition of lipopolysaccharide (LPS) and activation of proinflammatory pathways. Here we tested the hypothesis that TLR4 and its coreceptor MD-2 play a central role in nonalcoholic steatohepatitis (NASH) and liver fibrosis in nonalcoholic fatty liver disease. Mice of control genotypes and those deficient in MD-2 or TLR4 [knockout (KO)] received methionine choline-deficient (MCD) or methionine choline-supplemented (MCS) diet. In mice of control genotypes, MCD diet resulted in NASH, liver triglycerides accumulation, and increased thiobarbituric acid reactive substances, a marker of lipid peroxidation, compared with MCS diet. These features of NASH were significantly attenuated in MD-2 KO and TLR4 KO mice. Serum alanine aminotransferase, an indicator of liver injury, was increased in MCD diet-fed genotype controls but was attenuated in MD-2 KO and TLR4 KO mice. Inflammatory activation, indicated by serum TNF-α and nictoinamide adenine dinucleotide phosphate oxidase complex mRNA expression and activation, was significantly lower in MCD diet-fed MD-2 KO and TLR4 KO compared with corresponding genotype control mice. Markers of liver fibrosis [collagen by Sirius red and α-smooth muscle actin (SMA) staining, procollagen-I, transforming growth factor-ß1, α-SMA, matrix metalloproteinase-2, and tissue inhibitor of matrix metalloproteinase-1 mRNA] were attenuated in MD-2 and TLR4 KO compared with their control genotype counterparts. In conclusion, our results demonstrate a novel, critical role for LPS recognition complex, including MD-2 and TLR4, through NADPH activation in liver steatosis, and fibrosis in a NASH model in mice.

Effects of dahuangzhechong pills on cytokines and mitogen activated protein kinase activation in rats with hepatic fibrosis.

UNLABELLED: RELEVANCE TO ETHNOPHARMACOLOGY: Dahuangzhechong pill (DHZCP), a well-known and canonical Chinese medicine formula from "The Synopsis of Prescriptions of the Golden Chamber", is officially approved and recommended by Chinese association of integrative medicine for the prevention and treatment of hepatic fibrosis in China. AIM OF THE STUDY: To test the hypothesis that therapeutic effects of DHZCP on hepatic fibrosis are conferred by regulating cytokine profile through a mitogen activated protein kinase (MAPK) pathway. MATERIALS AND METHODS: Hepatic fibrosis is inducted by carbon tetrachloride (CCl(4)) in rats which then were randomly divided into six groups: hepatic fibrosis model group, high dose DHZCP group, low dose DHZCP group, Fufang Biejia Ruangan Pian (FBRP) group, Colchicine group and control group. Pathological, immunohistochemical, multiplex immunoassay and protein expression studies (Western blotting) are performed. RESULTS: DHZCP significantly decreases the levels of alanine aminotransferase, aspartate aminotransferase, hyaluronic acid, laminin, type IV collagen and procollagen III, and reverses hepatic fibrosis in rat model. DHZCP also could reduce the expression of α-smooth muscle actin, and lower the serum level of tumor necrosis factor alpha (TNF-α) and interleukin 13 (IL-13). The expressions of phosphorylated p38 MAPK and extracellular signal-regulated kinase (ERK) are down-regulated, while no significant changes are found in phosphorylation of c-Jun N-terminal kinase (JNK). CONCLUSIONS: DHZCP can alleviate hepatic fibrosis induced by CCl(4). The anti-fibrotic effects of DHZCP are conferred by decreasing the secretion of TNF-α and IL-13 through down-regulating p38 and ERK phosphorylation.

The biological response modifier AM3 attenuates the inflammatory cell response and hepatic fibrosis in rats with biliary cirrhosis.

BACKGROUND: An inflammatory immune system response ensues in the liver and in the systemic circulation in cirrhosis, where it contributes to hepatic fibrosis and peripheral vasodilation. Modulation of the inflammatory response without increasing susceptibility to infection is a therapeutic target in cirrhosis. AM3 is a low-toxicity biological response modifier with regulatory effects on innate and adaptative immunity, and the ability to normalise the production of tumour necrosis factor alpha (TNFalpha). AIMS: This was an experimental study to investigate the effects of oral AM3 on the systemic and hepatic inflammatory response, liver fibrosis and on the haemodynamic abnormalities of portal hypertension in rats with biliary cirrhosis. DESIGN: Bile-duct ligated rats received a 3-week oral course of AM3 or placebo. RESULTS: In cirrhotic rats, AM3 blunted the inflammatory switch of circulating and intrahepatic monocytes and T-cells to TNFalpha and interferon gamma (IFNgamma) production, respectively. AM3 modified the intrahepatic polarisation pattern of the regulatory cytokines, decreasing the mRNA expression of transforming growth factor beta1 (TGFbeta1), interleukin 4 (IL4), and IFNgamma, and increasing that of IL10. Total and IFNgamma-producing natural killer (NK) cells were lowered by AM3 in the peripheral blood and liver of cirrhotic rats. The immunomodulatory effects of AM3 led to reduced hepatic fibrogenesis in cirrhotic rats, as shown by decreased area of liver fibrosis, hydroxyproline content and mRNA expression of procollagen alpha1(I). Besides, AM3 lowered portal pressure and systemic hyperaemia. CONCLUSIONS: The biological response modifier AM3 reverses the concurrent inflammatory immune system activation in peripheral blood and liver of experimental established cirrhosis, which results in reductions of hepatic fibrosis, portal pressure and peripheral vasodilation.

Reduction of periductal fibrosis in liver fluke-infected hamsters after long-term curcumin treatment.

Chronic infection with the liver fluke, Opisthorchis viverrini, induces advanced periductal fibrosis and is a relative risk factor for cholangiocarcinoma in Southeastern Asia. We examined the reducing effect of curcumin on hepatobiliary fibrosis using O. viverrini-infected hamsters supplemented with dietary 1% curcumin (w/w) as an animal model. The expression profile of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs), cytokines, and collagens was assessed in relation to liver fibrosis. Histopathological studies revealed that curcumin had no effect on fibrosis at the short-term infection (21 days and 1 month); however, peribiliary fibrosis was significantly reduced after the long-term curcumin treatment for 3 months, compared to the untreated group. Expression of alpha-smooth muscle actin was associated with the reduction of liver fibrosis. A decrease in hepatic hydroxyproline level and mRNA expression of collagen I and III supported the reduction of fibrosis. The expression of TIMP-1, TIMP-2, and tumor necrosis factor-alpha genes was also decreased after curcumin treatment. In contrast, curcumin increased mRNA expression of MMP-13, MMP-7 (at 6 months), interleukin-1 beta, and transforming growth factor beta, implying that increased MMPs activity contributes to extracellular matrix degradation. These results suggest that curcumin reduces periductal fibrosis after long-term treatment by tissue resorption via inhibition of TIMPs expression and enhancement of MMPs expression mediated by cytokines. In conclusion, curcumin may serve as a promising nutraceutical agent exerting antifibrotic effect in O. viverrini-infected patients and contribute to cholangiocarcinoma prevention.

[Changes in the hemodynamics of rats with immunological liver fibrosis].

OBJECTIVE: To observe the changes in the hemodynamics of rats with immunological liver fibrosis and explore the pathogenesis of "blood stasis" in liver fibrosis. METHODS: Rat models of liver fibrosis were established by multiple intraperitoneal injections of pig serum. The hematocrit, blood viscosity at the shear rate of 150/s, 30/s, 5/s, and 1/s, serum markers for liver fibrosis, and serum transaminase levels were measured in the control and model rats. RESULTS: The hematocrit, blood viscosity at different shear rates, hyaluronic acid (HA), laminin (LN), procollagen type III (PCIII), type IV collagen (CIV), glutamic-pyruvic transaminase (ALT) and glutamic-oxaloacetic transaminase (AST) increased significantly in the rats with experimental liver fibrosis appeared as compared with those in the control rats. Positive correlations were noted between blood viscosity at different shear rates and serum concentrations of the fibrosis markers (HA, LN, PCIII, and CIV) in the model rats. CONCLUSION: The changes in the hemodynamics in rats with immunological liver fibrosis suggests the role of "blood stasis" in the pathogenesis of liver fibrosis and provide experimental evidence for therapies to "activate the blood circulation and dissipate blood stasis" for treatment of liver fibrosis.

Interferon-beta reduces the mouse liver fibrosis induced by repeated administration of concanavalin A via the direct and indirect effects.

Type I interferons (IFNs), IFN-alpha and IFN-beta, are widely used for treating chronic hepatitis C. Although retrospective studies have suggested that type I IFNs have direct antifibrotic effects, little is known about these mechanisms. The present study was designed to clarify the preventive mechanisms of type I IFNs in the progression of fibrosis for the establishment of a more effective therapy. A murine fibrosis model comprising immunological reactions was induced by the administration of concanavalin A (0.3 mg/body) into mice once a week for 4 weeks. Liver injury and the degree of fibrosis were determined by measuring the serum alanine aminotransferase activities and liver hydroxyproline contents with or without IFN-beta pretreatment. IFN-beta suppressed the hepatocellular injury and increased the hydroxyproline content induced by repeated concanavalin A injections, but had no effect on established fibrosis. Furthermore, IFN-beta reduced the expressions of transforming growth factor-beta, basic fibroblast growth factor, collagen type I A2 and tissue inhibitor of metalloproteinase 1 messenger RNAs, which are related to the progression of liver fibrosis. The IFN-beta reduced the liver injury and fibrosis induced by immunological reactions. These data suggest that type I IFNs suppress the progression of cirrhosis through inhibition of repeated hepatocellular injury and/or factors that promote the liver fibrosis induced by hepatitis virus infection.

Dalteparin sodium prevents liver injury due to lipopolysaccharide in rat through suppression of tumor necrosis factor-alpha production by Kupffer cells.

BACKGROUND: Sensitization of Kupffer cells (KC) to lipopolysaccharide (LPS) and overproduction of tumor necrosis factor (TNF)-alpha play important roles in the pathogenesis of alcoholic liver damage and sepsis-associated organ injury. Therefore, suppression of TNF-alpha should prove useful for treatment of LPS-induced liver injury. Recently, heparin has been reported to diminish TNF-alpha production from macrophages in response to LPS. Dalteparin sodium (DS) is a low-molecular-weight heparin with a mean molecular weight of 5,000. DS elicits an antithrombotic effect through a mechanism depending on anti-factor Xa activity but not on the antithrombin activity. DS is thus suitable for treatment of disseminated intravascular coagulation because it has a much smaller prohemorrhagic property. In this study, we evaluated whether DS could prevent LPS-induced liver injury. METHODS: Female Wistar rats were administered DS (50 IU/kg intraperitoneally) followed by challenge with LPS (5 mg/kg intravenously) 2 hr later. Livers and sera were collected 24 hr later. KC from rats were isolated and cultured in RPMI 1640 supplemented with 10% fetal bovine serum. After the addition of LPS (10 microg/ml) to the culture media, intracellular Ca2+ was measured by using a fluorescent indicator, fura-2. RESULTS: LPS (5 mg/kg intravenously) caused focal necrosis and neutrophil infiltration in the control liver. The histological changes and increased alanine aminotransferase levels caused by LPS injection were diminished by treatment with DS. LPS increased intracellular Ca2+ of KC in control rats from the basal level (26 +/- 6 nmol/liter) to 280 +/- 18 nmol/liter. This increase was blunted by DS (126 +/- 28 nmol/liter). The DS treatment decreased the LPS-induced TNF-alpha production by KC from 911 +/- 78 pg/ml to 309 +/- 45 pg/ml (p < 0.05). CONCLUSIONS: These results indicate that DS reduces the LPS-induced liver injury through suppression of TNF-alpha production.

Effect of branched-chain amino acids on the composition and cytolytic activity of liver-associated lymphocytes in rats.

BACKGROUND: Although branched-chain aminoacids (BCAA) are reported to be effective in prolongation of the mean survival time of patients with liver cirrhosis, it is not clear whether BCAA could influence the immune function in those patients. METHODS: Branched-chain amino acids were given as a supplement to carbon tetrachloride-induced cirrhotic rats, and an aminogram of the liver and kinetics of liver-associated lymphocytes (LAL) were then analysed. RESULTS: Liver cirrhosis was established at the 12th week, and glutathione S-transferase placental form (GST-P)-positive lesions, which are known to be pre-neoplastic lesions, occupied 1.72+/-0.84% of the liver at the 16th week in the controls. At this time the LAL showed an increase in the number of CD5-, CD8- and CD18-positive cells and augmentation of lectin-dependent cellular cytotoxicity (LDCC) activity. Furthermore, supplementation of BCAA increased the number of LAL, especially CD8-positive cells and natural killer cells, and augmented LDCC activity of LAL at the 16th week. The number of LAL was positively correlated with the valine concentration in the plasma and liver, and the area of GST-P-positive lesions tended to be decreased in the BCAA group. CONCLUSION: These findings suggest that BCAA administration has stimulatory effects on the local immune systems of the liver, which may have a potential to inhibit hepatocarcinogenesis. Moreover, among all amino acids valine might be an important amino acid for enhancing the immune function of LAL.

N-acetylcysteine improves cytotoxic activity of cirrhotic rat liver-associated mononuclear cells.

Liver cirrhosis, which is associated with decreased plasma and hepatic glutathione (GSH), has been reported to cause the suppression of NK activity in peripheral blood mononuclear cells. Since low GSH levels in lymphocytes are known to alter lymphocyte function, we examined the correlation between intracellular GSH levels and the cytotoxic activity of liver-associated mononuclear cells (liver MNC). We show here that rat liver contains a highly active population of NK cells (CD3- NKR-P1 + cells) that kill Yac-1 in vitro and that the cytotoxic activity of this NK population is directly proportional to liver MNC GSH. This proportionality is independent of the methods used to alter GSH level. Thus, in vitro treatment of liver MNC with buthionine sulfoximine to lower GSH levels lowers the cytotoxic activity. MNC from cirrhotic liver, in which implanted tumor cells grow faster, have both low GSH levels and low cytotoxicity, and supplementation of cirrhotic liver MNC with N-acetylcysteine raises GSH levels and increases cytotoxicity. These findings suggest a physiologic mechanism, i.e. decreased GSH, may be causally associated with the increased incidence of hepatoma in cirrhotic individuals and the increased growth of hepatoma cells in cirrhotic animals. Thus, we suggest that the GSH is important to the optimal functioning of the hepatic immunity that protects against hepatoma development.