Pathogenesis Of Hepatic Fibrosis Research Articles

A novel fused 1,2,4-triazine aryl derivative as antioxidant and nonselective antagonist of adenosine A2A receptors in ethanol-activated liver stellate cells

It has been detected that hepatic adenosine A2A receptors play an active role in the pathogenesis of hepatic fibrosis and suggest a novel therapeutic target in the treatment and prevention of hepatic cirrhosis. In this paper we examined if our new triazine derivative (IMT) can inhibit ethanol-induced activation of HSCs measured as increased α-SMA, collagen synthesis and enhanced oxidative stress in rat liver stellate cells. We also investigated its influence on cytokines (TGF-β, TNF-α) synthesis, MMP-2 and TIMP-1 production and ethanol-induced intracellular signal transduction. Moreover, with using of known adenosine A2A receptor agonist (CGS 21680), and antagonist (SCH 58261) we examined if this triazine derivative acts on adenosine receptors.We detected a strong antagonistic action of new triazine derivative (IMT) on ethanol-induced rat liver stellate cells activation, observed as a significant decrease in α-SMA, collagen synthesis, reactive oxygen species production, TGF-β, TNF-α, MMP-2 and TIMP-1 production as well as JNK, p38MAPK, NFκB, IκB, Smad3 phosphorylation. Moreover, IMT strongly inhibited activation of stellate cells by known selective agonist of adenosine A2A receptor (CGS 21680). When known A2A receptor antagonist (SCH 58261) was used together with IMT this effect was not spectacular. Additionally, only slight enhancement of inhibition was observed when cells were pretreated both IMT with SCH 58261, hence we suppose that IMT acts as nonselective antagonist of A2A receptors, and, besides its antioxidant activity, also by this way inhibited ethanol-induced stellate cell activation.

Molecular pathogenesis of hepatic fibrosis and current therapeutic approaches

The pathogenesis of hepatic fibrosis involves significant deposition of fibrilar collagen and other extracellular matrix proteins. It is a rather dynamic process of wound healing in response to a variety of persistent liver injury caused by factors such as ethanol intake, viral infection, drugs, toxins, cholestasis, and metabolic disorders. Liver fibrosis distorts the hepatic architecture, decreases the number of endothelial cell fenestrations and causes portal hypertension. Key events are the activation and transformation of quiescent hepatic stellate cells into myofibroblast-like cells with the subsequent up-regulation of proteins such as α-smooth muscle actin, interstitial collagens, matrix metalloproteinases, tissue inhibitor of metalloproteinases, and proteoglycans. Oxidative stress is a major contributing factor to the onset of liver fibrosis and it is typically associated with a decrease in the antioxidant defense. Currently, there is no effective therapy for advanced liver fibrosis. In its early stages, liver fibrosis is reversible upon cessation of the causative agent. In this review, we discuss some aspects on the etiology of liver fibrosis, the cells involved, the molecular pathogenesis, and the current therapeutic approaches.

The role of the renin–angiotensin system in liver fibrosis

Hepatic fibrosis, which is characterized by progressive inflammation and deposition of extracellular matrix components, is a common response to chronic liver disease. Hepatic fibrogenesis is a dynamic process that involves several liver cell types including hepatic stellate cells and Kupffer cells. In addition, recent evidence indicates that bile duct epithelial cells (i.e. cholangiocytes) also participate in the progression of biliary fibrosis that is observed during chronic cholestatic liver diseases, such as primary sclerosing cholangitis. To date, there are no effective treatments for hepatic fibrosis. Several recent studies have demonstrated that the renin-angiotensin system (RAS) plays a key role in hepatic fibrosis. Therapies targeting the RAS may represent a promising paradigm for the prevention and treatment of hepatic fibrosis in the setting of chronic liver disease. In this review, we provide a comprehensive update on the role of RAS in the pathogenesis of hepatic fibrosis in both animal models and human studies. We will discuss the profibrotic mechanisms activated by the RAS and the cell types involved. Studies that have utilized angiotensin receptor blockers (ARBs) and angiotensin-converting enzyme (ACE) inhibitors to modulate the RAS in order to ameliorate hepatic fibrosis will also be discussed. Although the cumulative evidence supports the potential for the use of ARBs and ACE inhibitors as treatment for hepatic fibrosis, extensive studies of the effectiveness of RAS therapeutics are necessary in patients with chronic liver disease.

Serum retinol binding protein 4 and clinical outcome in postoperative biliary atresia

Biliary atresia (BA), a chronic inflammatory disease of bile ducts, is characterized by the obliteration of bile flow. The aim of the present study was to investigate serum retinol binding protein 4 (RBP4) in postoperative BA patients and the association of RBP4 with clinical parameters and liver stiffness scores. A number of forty-eight BA patients post Kasai operation and 24 controls were enrolled. None of the patients had undergone liver transplantation. BA patients were classified into two groups according to their serum total bilirubin (TB) levels (non-jaundice, TB<2mg/dl vs. jaundice, TB≥2mg/dl) and liver stiffness (insignificant fibrosis, liver stiffness <7kPa vs. significant fibrosis, liver stiffness ≥7kPa). Serum RBP4 levels were determined by enzyme-linked immunosorbent assay (ELISA). Liver stiffness scores were measured by FibroScan. BA patients had lower RBP4 levels (14.9±1.0 vs. 18.7±1.0ng/ml, P=0.02), but higher liver stiffness than controls (29.5±3.3 vs. 5.0±0.5kPa, P<0.001). Serum RBP4 levels were significantly decreased in BA patients with jaundice, compared with those without jaundice (9.5±0.9 vs. 18.2±1.2ng/ml, P<0.001). Moreover, BA patients with significant liver fibrosis displayed lower serum RBP4 than those with insignificant fibrosis (14.1±1.2 vs. 21.2±1.4ng/ml, P=0.02). Further analysis showed that serum RBP4 was strongly correlated with liver stiffness and serum albumin (r=-0.72, P<0.001, and r=0.65, P<0.001, respectively). BA patients with portal hypertension (PH) had lower serum RBP4 than those without PH (12.8±1.2 vs. 19.2±1.7ng/ml, P=0.003). Serum RBP4 levels decreased in BA patients compared with normal participants, and its levels declined significantly in patients with more severe disease. RBP4 may play a role in the pathogenesis of hepatic fibrosis and serve as a possible biomarker reflecting disease severity in postoperative BA patients.

Immunohistochemical localization of transforming growth factor &amp;beta;-1 and its relationship with collagen expression in advanced liver fibrosis due to biliary atresia

Purpose:Biliary atresia (BA) is the most common indication of liver transplantation in children. Pathogenesis of hepatic fibrosis, which is a prominent feature of BA, remains obscure. The purpose of this work was to determine the cellular sources of transforming growth factor beta-1 (TGFβ1) and establish the relationship between TGFβ1-producing cells and extracellular matrix producing myofibroblasts (MFBs) in advanced BA.Methods:Trichrome staining and immunohistochemistry were carried out to determine the expression pattern of collagen and TGFβ1 protein in explant liver specimens from patients with BA. The intensities of portal and lobular TGFβ1 expressions were compared. Immunofluorescence technique was carried out to determine the relationship between α-smooth muscle actin (α-SMA)-positive-MFB and TGFβ1-positve cells.Results:Lobular TGFβ1 protein expression was significantly higher than portal (89 ± 6 versus 10 ± 1 arbitrary units, P ≤ 0.05), whereas no difference was noted in livers used as control (10 ± 1.6 versus 19 ± 5 arbitrary units, P = 0.11). TGFβ1 expression was more in the center of nodules versus MFB in surrounding fibrous septa. Contrary to TGFβ1 expression, α1-SMA was mostly expressed in the portal structures and the adjacent fibrous septa enacting lobulation of the parenchyma. The results obtained by coimmunofluorescence staining showed no colocalization of α-SMA and TGFβ1.Conclusions:TGFβ1 protein expression is mostly localized to hepatocytes in advanced BA. These findings suggest a paracrine mechanisms of TGFβ1-driven fibrogenesis in advanced BA.

Evolving challenges in hepatic fibrosis

Continued elucidation of the mechanisms of hepatic fibrosis has yielded a comprehensive and nuanced portrait of fibrosis progression and regression. The paradigm of hepatic stellate cell (HSC) activation remains the foundation for defining events in hepatic fibrosis and has been complemented by progress in a number of new areas. Cellular sources of extracellular matrix beyond HSCs have been identified. In addition, the role of chemokine, adipokine, neuroendocrine, angiogenic and NAPDH oxidase signaling in the pathogenesis of hepatic fibrosis has been uncovered, as has the contribution of extracellular matrix stiffness to fibrogenesis. There is also increased awareness of the contribution of innate immunity and greater understanding of the complexity of gene regulation in HSCs and myofibroblasts. Finally, both apoptosis and senescence have been recognized as orchestrated programs that eliminate fibrogenic cells during resolution of liver fibrosis. Ironically, the progress that has been made has highlighted the growing disparity between advances in the experimental setting and their translation into new diagnostic tools and treatments. As a result, focus is shifting towards overcoming key translational challenges in order to accelerate the development of new therapies for patients with chronic liver disease.

Changes in E-NTPDase 3 expression and extracellular nucleotide hydrolysis during the myofibroblast/lipocyte differentiation

Hepatic stellate cells (HSC) play a critical role in the development and maintenance of liver fibrosis. HSC are lipocytes that displayed the capacity to develop into myofibroblast-like cells. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate the concentration of extracellular nucleotides, signaling molecules that play a role in the pathogenesis of hepatic fibrosis. In the present study, we identified and compared the expressions of E-NTPDase family members in two different phenotypes of the mouse hepatic stellate cell line (GRX) and evaluated the nucleotide hydrolysis by these cells. We show that both phenotypes of GRX cell line expressed NTPDase 3 and 5. However, only activated cells expressed NTPDase 6. In quiescent-like cells, the hydrolysis of triphosphonucleosides was significantly higher, and was related to an increase in Entpd3 mRNA expression. The diphosphonucleosides were hydrolyzed at a similar rate by two phenotypes of GRX cells. We suggest that up-regulation of Entpd3 mRNA expression modulates the extracellular concentration of nucleotides/nucleosides and affect P2-receptor signaling differently in quiescent-like cells and may play a role in the regulation of HSC functions.

Role of small ubiquitin-related modifier-1 in the pathogenesis of hepatic fibrosis in rats

Role of small ubiquitin-related modifier-1 in the pathogenesis of hepatic fibrosis in rats

Long-term feeding of a synthetic diet rich in disaccharides induces hepatic fibrosis in nonalcoholic fatty liver disease in Zucker rats

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease, and is commonly observed in patients with obesity or type 2 diabetes. The pathogenesis of hepatic fibrosis is clinically important to the outcome of NAFLD, however, is not well understood. Since dietary habits are often considered to be responsible for NAFLD, we used a synthetic diet rich in disaccharides (12.1% calorie sucrose and lactose), which can be considered for human consumption. We examined the long-term (24 weeks) effect of this diet on the liver of Zucker (fa/fa) rats. The synthetic diet-fed Zucker (fa/fa) rats showed hepatic fibrosis during the development of NAFLD with no apparent infiltration of inflammatory cells. They showed significantly elevated hepatic mRNA levels of proinflammatory and profibrogenic cytokines. These findings suggest that excess long-term feeding of a diet similar to the synthetic diet used in our study leads to hepatic fibrosis during the development of NAFLD in patients with obesity or type 2 diabetes. Our results were different from NAFLD accompanied by infiltration of inflammatory cells. However, they also suggest that long-term feeding of this synthetic diet to Zucker (fa/fa) rats is useful for studying the hepatic fibrogenesis during the pathogenesis of NAFLD.

A Contradictory Role of A1 Adenosine Receptor in Carbon Tetrachloride- and Bile Duct Ligation-Induced Liver Fibrosis in Mice

Mice lacking A(1) adenosine receptors (A(1)AR) were thought to be protected from developing fatty liver; however, the contribution of A(1)AR to hepatic fibrosis has not been explored. Here we found that the expression of A(1)AR was decreased in fibrotic liver induced by chronic carbon tetrachloride (CCl(4)) but increased in that induced by bile duct ligation (BDL). Therefore, we examined whether A(1)AR contributes to hepatic fibrosis in CCl(4) and BDL animal models using A(1)AR knockout mice. Compared with wild-type (WT) mice, hepatic fibrosis resulting from chronic CCl(4) exposure was attenuated in A(1)AR(-/-) mice with markedly decreased collagen deposition and reduced hepatic stellate cell activation, whereas bile duct-ligated A(1)AR(-/-) mice displayed a significant increase in hepatic fibrosis. Hepatocyte damage was reduced in A(1)AR(-/-) mice after a single injection of CCl(4), with down-regulation of CYP2E1 and UCP2 gene expression in livers, which resulted in impaired liver sensitivity to CCl(4). However, BDL caused severe bile infarcts in livers of A(1)AR(-/-) mice, with significantly elevated levels of bile acid compared with those in WT mice. CCl(4) and BDL resulted in different expression patterns of genes involved in fibrogenesis in A(1)AR(-/-) mice. These results indicate that A(1)AR participates in the pathogenesis of hepatic fibrosis with a complex mechanism, and the effect of targeting adenosine and its receptors in the prevention of hepatic fibrosis should be cautiously evaluated.

Liver Fibrosis and Therapeutic Strategies: The Goal for Improving Metabolism

This review summarizes the current state of knowledge on non-alcoholic fatty liver disease (NAFLD) and the hepatitis C virus (HCV)-associated liver fibrosis, and provides insight into the role of dysmetabolism in hepatic fibrogenesis. Clinical relevance of drugs correcting these metabolic disturbances in the reversion of liver fibrosis will also be discussed. Liver fibrosis affects more than ten millions of people worldwide and may lead to cirrhosis, liver failure, and death. Recent epidemiological data indicate that the incidence of liver fibrosis is expected to triple during the next 10 to 15 years as a result of the HCV infection and NAFLD escalation. In accordance with the modern view of liver fibrogenesis, the pathways involved in the pathogenesis of hepatic fibrosis appear to be broadly similar regardless of the etiology. Some features of metabolic syndrome, including obesity, insulin resistance, and type 2 diabetes represent a strong risk factor in development and progression of hepatic fibrosis. However, whatever the cause, fibrosis culminates in cirrhosis and results in liver failure, thus, a potent anti-fibrotic therapy is urgently needed to reverse scarring and eliminate progression to cirrhosis.

Activity and expression of ecto-nucleotide pyrophosphate/phosphodiesterases in a hepatic stellate cell line

Nucleotides and nucleosides represent an important and ubiquitous class of molecules that interact with specific receptors, regulate a variety of activities within the liver, and play a role in the pathogenesis of hepatic fibrosis. Ecto-nucleotide pyrophosphatase/phosphodiesterases (E-NPPs) are ecto-enzymes that are located on the cell surface. NPP1, NPP2, and NPP3 (abbreviated as NPP1-3 hereafter) have been implicated in the hydrolysis of nucleotides; together with other ecto-nucleotidases, they control the events induced by extracellular nucleotides. We have identified and compared the expression of E-NPP family members in two different phenotypes of the mouse hepatic stellate cell line (GRX). In quiescent-like hepatic stellate cells (HSCs), E-NPP activity was significantly higher, NPP2 mRNA expression decreased and NPP3 mRNA increased. The differential NPP activity and expression in two phenotypes of GRX cells suggests that they are involved in the regulation of extracellular nucleotide metabolism in HSCs. However, the role of E-NPPs in the liver remains to be clarified.

Over-expression of C/EBP-α induces apoptosis in cultured rat hepatic stellate cells depending on p53 and peroxisome proliferator-activated receptor-γ

Hepatic stellate cells (HSCs) play a key role in the pathogenesis of hepatic fibrosis. In our previous studies, CCAAT enhancer binding protein-α (C/EBP-α) has been shown to be involved in the activation of HSCs and to have a repression effect on hepatic fibrosis in vivo. However, the mechanisms are largely unknown. In this study, we show that the infection of adenovirus vector expressing C/EBP-α gene (Ad-C/EBP-α) could induce HSCs apoptosis in a dose- and time-dependent manner by Annexin V/PI staining, caspase-3 activation assay, and flow cytometry. Also, over-expression of C/EBP-α resulted in the up-regulation of peroxisome proliferator-activated receptor-γ (PPAR-γ) and P53, while P53 expression was regulated by PPAR-γ. In addition, Fas, FasL, DR4, DR5, and TRAIL were studied. The results indicated that the death receptor pathway was mainly involved and regulated by PPAR-γ and p53 in the process of apoptosis triggered by C/EBP-α in HSCs.

Role of micro-RNAs in the pathogenesis of hepatic fibrosis

Role of micro-RNAs in the pathogenesis of hepatic fibrosis

Targeted inhibition of platelet-derived growth factor receptor-β subunit in hepatic stellate cells ameliorates hepatic fibrosis in rats

The activation of hepatic stellate cells (HSCs) is the key event of the pathogenesis of hepatic fibrosis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs, and PDGF receptor-beta subunit (PDGFR-beta) is required for the proliferation of HSCs induced by PDGF. In this study, a high gene-silencing-efficacy PDGFR-beta small interference RNA (siRNA) was synthesized that could suppress the PDGFR-beta expression and inhibit the activation and proliferation but could not induce the apoptosis of HSCs in vitro. To avoid the side effect of nonspecific interference of PDGFR-beta, we constructed an HSCs-specific short hairpin RNA (shRNA) expression plasmid in which PDGFR-beta shRNA was driven by a glial fibrillary acidic protein (GFAP) promoter. The double-staining immunofluorescence examination indicated that GFAP promoter could target the transgene expression into HSCs in carbon tetrachloride induced acute injured rat's liver and bile duct ligation (BDL)-induced chronic injured rat's liver. Furthermore, HSCs-specific PDGFR-beta shRNA could relieve liver injury and hepatic fibrosis in the rat's model induced by BDL. This study demonstrates that PDGFR-beta siRNA may be presented as an antifibrogenic agent. The application of HSCs-specific RNA interference induced by the GFAP promoter might supply a new powerful tool for cell-specific gene therapy of hepatic fibrogenesis.

Ecto‐5′‐nucleotidase (CD73) ‐mediated extracellular adenosine production plays a critical role in hepatic fibrosis

Adenosine is a potent endogenous regulator of tissue repair that is released from injured cells and tissues. Hepatic fibrosis results from chronic hepatic injury, and we have previously reported that endogenously generated adenosine, acting at A(2A) receptors, plays a role in toxin-induced hepatic fibrosis. Adenosine may form intracellularly and then be transported to the extracellular space or it may form extracellularly from adenine nucleotides released from injured cells. Because ecto-5'-nucleotidase (CD73) catalyzes the terminal step in extracellular adenosine formation from AMP, we determined whether CD73 plays a role in the development of hepatic fibrosis. Mice were treated overnight with PBS, CCl(4), ethanol, or thioacetamide (TAA); their livers were harvested, and slices were incubated in medium for 20 h before adenosine concentration in the supernatant was measured by HPLC. Hepatic fibrosis was induced by CCl(4) or TAA treatment in CD73 knockout (CD73KO and C57BL/6 background) and C57BL/6 control mice [wild-type (WT)] mice and quantified by digital analysis of picrosirius red stained slides and hydroxyproline content. mRNA expression was quantified by real-time polymerase chain reaction, and protein was quantified by Western blot or enzyme-linked immunosorbent assay. Livers from WT mice treated with CCl(4), ethanol, and TAA released 2- to 3-fold higher levels of adenosine than livers from comparably treated CD73KO mice. CD73KO mice were protected from fibrosis with significantly less collagen content in the livers of CD73KO than WT mice after treatment with either CCl(4) or TAA. There were far fewer alpha-smooth muscle actin positive hepatic stellate cells in CCl(4)-treated KO mice than that in WT mice. After CCl(4) treatment, the mRNA level of A(1), A(2A), A(2B), and A(3) adenosine receptors, tumor necrosis factor-alpha, interleukin (IL) -1beta, IL-13r alpha1, matrix metalloproteinase (MMP)-2, MMP-14, tissue inhibitor of metalloproteinase (TIMP) -1, and TIMP-2, and IL-13 level increased markedly in both CD73KO and WT mice, but Col1 alpha1, Col3 alpha1, and transforming growth factor-beta1 mRNA increased much more in WT mice than that in KO mice. Moreover, IL-13r alpha2, MMP-13 mRNA, and MMP-13 protein were higher in KO mice than that in WT mice. These results indicate that adenosine, formed extracellularly from adenine nucleotides, plays a major role in the pathogenesis of hepatic fibrosis and that inhibition of adenosine production or blockade of adenosine receptors may help prevent hepatic fibrosis.

Curcumin: potential for hepatic fibrosis therapy?

The beneficial antioxidative, anti-inflammatory and antitumorigenic effects of curcumin have been well documented in relation to cancer and other chronic diseases. Recent evidence suggests that it may be of therapeutic interest in chronic liver disease. Hepatic fibrosis (scarring) occurs in advanced liver disease, where normal hepatic tissue is replaced with collagen-rich extracellular matrix and, if left untreated, results in cirrhosis. Curcumin inhibits liver cirrhosis in a rodent model and exerts multiple biological effects in hepatic stellate cells (HSCs), which play a central role in the pathogenesis of hepatic fibrosis. In response to liver injury, these cells proliferate producing pro-inflammatory mediators and extracellular matrix. Curcumin induces apoptosis and suppresses proliferation in HSCs. In addition, it inhibits extracellular matrix formation by enhancing HSC matrix metalloproteinase expression via PPARgamma and suppressing connective tissue growth factor (CTGF) expression. In this issue, Chen and co-workers propose that curcumin suppresses CTGF expression in HSC by inhibiting ERK and NF-kappaB activation. These studies suggest that curcumin modulates several intracellular signalling pathways in HSC and may be of future interest in hepatic fibrosis therapy.

Elevation of serum stem‐cell factor in postoperative biliary atresia

Biliary atresia (BA) is one of the most common causes of neonatal cholestasis. Stem-cell factor (SCF) has been implicated in the development of fibrosis in various diseases. The objective of the present study was to examine the significant role of SCF in BA. Fifty-seven pediatric patients with BA after Kasai operation and 30 healthy children were recruited. The mean ages of BA patients and controls were 6.1 +/- 0.6 years and 6.1 +/- 0.7 years, respectively. The patients were categorized into two groups according to their serum levels of total bilirubin (TBil < 2 mg/dL, no jaundice vs TBil > or = 2 mg/dL, persistent jaundice) and alanine aminotransferase (ALT < 100 vs ALT > or = 100 U/L). The serum SCF levels were determined on commercially available enzyme-linked immunosorbent assay. The mean serum SCF level of the BA children was higher than that of normal controls (748.3 +/- 17.9 pg/mL vs 582.2 +/- 17.3 pg/mL; P < 0.001). Subsequent analysis demonstrated that the BA patients with serum ALT > or = 100 U/L had significantly greater levels of serum SCF compared to those with serum ALT < 100 U/L (796.5 +/- 22.6 pg/mL vs 694.7 +/- 25.0 pg/mL, respectively; P = 0.002). In addition, serum SCF levels were significantly elevated in the patients with portal hypertension (PH) compared with those without PH (810.0 +/- 18.8 pg/mL vs 634.1 +/- 20.1 pg/mL, P < 0.001). The current study showed that BA patients had higher serum SCF levels compared with controls. The significant elevation in SCF levels is associated with the presence of PH and the degree of hepatic injury. These findings suggest that SCF may play a part in the pathogenesis of hepatic fibrosis in BA patients after Kasai procedure.

Differential effects of TGF-β on connective tissue growth factor (CTGF/CCN2) expression in hepatic stellate cells and hepatocytes

Connective tissue growth factor (CTGF/CCN2) has been implicated in the pathogenesis of hepatic fibrosis and suggested as a downstream mediator of the fibrogenic master cytokine TGF-beta. We investigated the effect of TGF-beta1 on CTGF/CCN2 expression in cultured rat hepatic stellate cells and hepatocytes by means of Western and Northern blotting, immunocytochemistry, reporter gene analysis, and metabolic labelling. We found that the expression of CTGF/CCN2 in hepatic stellate cells is (i) only marginally (if at all) stimulated by TGF-beta and by a constitutively active type I TGF-beta receptor, (ii) independent from Smad2/3 phosphorylation, (iii) not reduced by TGF-beta1 antagonists or ALK5-receptor inhibitors and (iv) not upregulated during transdifferentiation to myofibroblasts in culture. However, expression and secretion of CTGF/CCN2 in cultured hepatocytes increased spontaneously during culture and was strongly stimulated by TGF-beta1. In bile-duct ligated and CCl(4)-treated rat livers, a strong CTGF/CCN2 expression in hepatocytes was noticed. Endothelin-1 stimulated CTGF/CCN2 expression in stellate cells but not in hepatocytes. Pathway specific signalling inhibitors point to the involvement of non-Smad signalling cascades but their contribution to CTGF/CCN2 regulation is different in both cell types. The results do not reveal a relevant interrelation between TGF-beta function and CTGF/CCN2 expression in hepatic stellate cells, which is in contrast to hepatocytes.

De novo synthesis of glutathione is a prerequisite for curcumin to inhibit hepatic stellate cell (HSC) activation

On liver injury, quiescent hepatic stellate cells (HSC), the most relevant cell type for hepatic fibrogenesis, become active, characterized by enhanced cell growth and overproduction of extracellular matrix (ECM). Oxidative stress facilitates HSC activation and the pathogenesis of hepatic fibrosis. Glutathione (GSH) is the most important intracellular antioxidant. We previously showed that curcumin, the yellow pigment in curry from turmeric, significantly inhibited HSC activation. The aim of this study is to elucidate the underlying mechanisms. It is hypothesized that curcumin might inhibit HSC activation mainly by its antioxidant capacity. Results from this study demonstrate that curcumin dose and time dependently attenuates oxidative stress in passaged HSC demonstrated by scavenging reactive oxygen species and reducing lipid peroxidation. Curcumin elevates the level of cellular GSH and induces de novo synthesis of GSH in HSC by stimulating the activity and gene expression of glutamate-cysteine ligase (GCL), a key rate-limiting enzyme in GSH synthesis. Depletion of cellular GSH by the inhibition of GCL activity using L-buthionine sulfoximine evidently eliminates the inhibitory effects of curcumin on HSC activation. Taken together, our results demonstrate, for the first time, that the antioxidant property of curcumin mainly results from increasing the level of cellular GSH by inducing the activity and gene expression of GCL in activated HSC in vitro. De novo synthesis of GSH is a prerequisite for curcumin to inhibit HSC activation. These results provide novel insights into the mechanisms of curcumin as an antifibrogenic candidate in the prevention and treatment of hepatic fibrosis.