Curcumin downregulates Smad pathways and reduces hepatic stellate cells activation in experimental fibrosis

Introduction and ObjectivesCurcumin, a polyphenol, is a natural compound that has been widely studied as a hepatoprotector; however, only a few studies have examined its ability to reduce fibrosis in previously established cirrhosis. The objective of this study was to investigate whether curcumin could reduce carbon tetrachloride (CCl₄)-induced fibrosis and if so, to determine the action mechanisms involved in the reduction process.Materials and MethodsCCl₄ was administered to male Wistar rats (400 mg/kg, three times a week, i. p.) for 12 weeks; curcumin (100 mg/kg body weight twice per day, p. o.) was administered from week 9–12 of CCl₄ treatment. Biochemical markers of hepatic injury and oxidative stress were evaluated. Hematoxylin and eosin, Masson’s trichrome stains, transmission electron microscopy; immunohistochemistry, and zymography assays were carried out. Moreover, Smad3 and α-SMA mRNA and protein levels were studied. Western blotting by TGF-β, CTGF, Col-I, MMP-13, NF-κB, IL-1, IL-10, Smad7, pSmad3, and pJNK proteins was developed.Results and ConclusionsCurcumin reduced liver damage, oxidative stress, fibrosis, and restored normal activity of MMP-9 and MMP-2. Besides, curcumin restored NF-κB, IL-1, IL-10, TGF-β, CTGF, Col-I, MMP-13, and Smad7 protein levels. On the other hand, curcumin decreased JNK and Smad3 phosphorylation. Furthermore, curcumin treatment decreased α-SMA and Smad3 protein and mRNA levels. Curcumin normalized GSH, and NF-κB, JNK-Smad3, and TGF-β-Smad3 pathways, leading to a decrement in activated hepatic stellate cells, thereby producing its antifibrotic effects.     

Suppression of macrophage infiltration inhibits activation of hepatic stellate cells and liver fibrogenesis in rats

BACKGROUND & AIMS: Monocytes/macrophages infiltrate into injured livers. We tried to clarify their roles in inflammation and subsequent fibrogenesis by inhibiting their infiltration with a mutated form (7ND; 7 amino acids at the N-terminal were deleted) of monocyte chemoattractant protein 1, which may function as a dominant-negative mutant. METHODS: Rats were injected via the tail vein with an adenovirus expressing either human 7ND (Ad7ND), a truncated type II transforming growth factor beta receptor (AdTbeta-TR), which works as a dominant-negative receptor, bacterial beta-galactosidase (AdLacZ), or saline. Seven days later, the rats were treated with dimethylnitrosamine for 1-21 days. RESULTS: Within 24 hours after a single dimethylnitrosamine injection, macrophages were observed in livers. With a 3-day dimethylnitrosamine treatment, activated hepatic stellate cells were detectable in livers in AdLacZ-, AdTbeta-TR-, and saline-injected rats. In contrast, in the Ad7ND-treated rats, infiltration of macrophages was markedly reduced, and activated hepatic stellate cells were not detectable. After a 3-week dimethylnitrosamine treatment, fibrogenesis was almost completely inhibited, and activated hepatic stellate cells were hardly seen in livers in both Ad7ND- and AdTbeta-TR-treated rats. CONCLUSIONS: Our results show that blockade of macrophage infiltration inhibits activation of hepatic stellate cells and leads to suppression of liver fibrogenesis. The presence of activated hepatic stellate cells in the initial phase after injury and its absence at a later phase in the AdTbeta-TR-treated livers indicate that transforming growth factor beta is not an activating factor for hepatic stellate cells, and this suggests that transforming growth factor beta is required for the survival of activated hepatic stellate cells. Our study suggests that infiltrated macrophages may themselves produce an activating factor for hepatic stellate cells.     

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells

AIM:To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis.METHODS:Male Sprague-Dawley rats underwent common bile duct ligation(BDL) for 14 d and were treated with Gardenia jasminoides by gavage.The ef-fects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells(LX-2) in vitro.RESULTS:Treatment with Gardenia jasminoides decreased serum alanine aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,146.6 ± 15 U/L vs 77 ± 6.5 U/L,P = 0.0007) and aspartate aminotransferase(BDL vs BDL + 100 mg/kg Gardenia jasminoides,188 ± 35.2 U/L vs 128 ± 19 U/L,P = 0.005) as well as hydroxyproline(BDL vs BDL + 100 mg/kg Gardenia jasminoides,438 ± 40.2 μg/g vs 228 ± 10.3 μg/g liver tissue,P = 0.004) after BDL.Furthermore,Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor β1(TGF-β1),collagen type?Ⅰ?(Col?Ⅰ) and α-smooth muscle actin(α-SMA).Gardenia jasminoides significantly suppressed the upregulation of TGF-β1,Col?Ⅰand α-SMA in LX-2 exposed to recombinant TGF-β1.Moreover,Gardenia jasminoides inhibited TGF-β1-induced Smad2 phosphorylation in LX-2 cells.CONCLUSION:Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.     

Role of hepatic stellate cell/hepatocyte interaction and activation of hepatic stellate cells in the early phase of liver regeneration in the rat

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are known to play a role in hepatic regeneration. We investigated hepatocyte/HSC interaction and HSC activation at various times after 70% partial hepatectomy (PHx) in the rat. METHODS: The hepatic microcirculation was studied using intravital fluorescence microscopy (IVFM). Desmin and alpha-SMA within liver tissue were detected by immunohistochemistry. In isolated parenchymal liver cells (PLCs) and HSCs, double immunostaining was used to identify activated HSC. RESULTS: Using IVFM, hepatocyte-clusters were often seen in vivo at 3 days after PHx (PHx3). Distance between HSC fell from 61.7+/-2.1 microm in controls to 36.1+/-1.4 microm (P<0.001) while the HSC/hepatocyte ratio rose (0.71+/-0.01 to 1.08+/-0.03; P<0.001). In >80% of in vivo microscopic fields in the PHx3 group, clusters of HSCs were observed especially near hepatocyte-clusters. At PHx1 and PHx3, >20% of cells in the PLC-fraction were HSCs which adhered to hepatocytes. At PHx3, in addition to desmin staining, isolated HSCs were also positive for BrdU and alpha-SMA, and formed clusters. HSCs in the HSC-fraction were only positive for desmin which indicated that adherence to hepatocytes is required for HSC activation. CONCLUSIONS: Our data suggest that HSCs are activated by adhering to hepatocytes in the early phase of liver regeneration.     

Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin

BACKGROUND & AIMS: Obesity is one of the risk factors for liver fibrosis, in which plasma adiponectin, an adipocytokine, levels are decreased. Hepatic stellate cells play central roles in liver fibrosis. When they are activated, they undergo transformation to myofibroblast-like cells. Adiponectin suppresses the proliferation and migration of vascular smooth muscle cells, whose characteristics are similar to those of hepatic stellate cells. Adiponectin could have biological significances in liver fibrosis. METHODS: The role of adiponectin on liver fibrosis induced by the administration of carbon tetrachloride twice a week for 12 weeks was tested by using adiponectin-knockout mice and an adenovirus-mediated adiponectin-expression system. We also investigated the effect of adiponectin in activated hepatic stellate cells. RESULTS: When mice were administered carbon tetrachloride (300 microL/kg body weight) twice a week for 12 weeks, knockout mice showed extensive liver fibrosis with an enhanced expression of transforming growth factor-beta 1 and connective tissue growth factor compared with wild-type mice (P < 0.05). Injection of adenovirus producing adiponectin (AdADN) before carbon tetrachloride (1000 microL/kg body weight) treatment prevented liver fibrosis in wild-type mice (P < 0.001). Injection of AdADN at 6 weeks attenuated liver fibrosis even though carbon tetrachloride was given for an additional 6 weeks (total of 12 weeks). In cultured hepatic stellate cells, adiponectin suppressed platelet-derived growth factor-induced proliferation and migration and attenuated the effect of transforming growth factor-beta 1 on the gene expression of transforming growth factor-beta 1 and connective tissue growth factor and on nuclear translocation of Smad2. CONCLUSIONS: The findings indicate that adiponectin attenuates liver fibrosis and could be a novel approach in its prevention.     

Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells

BACKGROUND & AIMS: Tumor desmoplasia is one of the representative histopathologic findings in ductal pancreatic adenocarcinoma. The aims of this study were to examine the cellular and molecular mechanisms of fibrogenesis associated with pancreatic adenocarcinomas. METHODS: Immunostainings were performed with human pancreatic adenocarcinomas (n = 27) and tumors induced in nude mice (n = 36) by subcutaneously injecting MiaPaCa2, Panc1, and SW850 with and without pancreatic stellate cells. Matrix-producing cells were isolated from pancreatic adenocarcinomas and compared with pancreatic stellate cells isolated from tissue of chronic pancreatitis. Paracrine stimulation of pancreatic stellate cells by carcinoma cells was studied regarding matrix synthesis (collagen and c-fibronectin on protein and messenger RNA level) and cell proliferation (bromodeoxyuridine incorporation). RESULTS: High numbers of desmin and alpha-smooth muscle actin-positive cells were detected in 26 of 27 pancreatic adenocarcinomas. Intense fibronectin and collagen stainings were associated with these cells. By using cytofilament stainings, gene expression profiling, and morphological examinations, the matrix-producing cells obtained by the outgrowth method from pancreatic adenocarcinomas were identified as pancreatic stellate cells. Supernatants of MiaPaCa2, Panc1, and SW850 cells stimulated proliferation and collagen type I and c-fibronectin synthesis of cultured pancreatic stellate cells. Preincubation of the carcinoma cell supernatants with neutralizing antibodies against fibroblast growth factor 2, transforming growth factor beta 1, and platelet-derived growth factor significantly reduced the stimulatory effects. Subcutaneous injection of carcinoma cells and pancreatic stellate cells induced fast-growing subcutaneous fibrotic tumors in nude mice. Morphometric analysis of carcinoma cells (cytokeratin stainings) showed a high density of carcinoma cells in these tumors. CONCLUSIONS: Pancreatic stellate cells strongly support tumor growth in the nude mouse model. The increased deposition of connective tissue in pancreatic carcinoma is the result of a paracrine stimulation of pancreatic stellate cells by carcinoma cells.     

Natural killer cells ameliorate liver fibrosis by killing activated stellate cells in NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent manners

BACKGROUND & AIMS: Viral hepatitis infection, which is a major cause of liver fibrosis, is associated with activation of innate immunity. However, the role of innate immunity in liver fibrosis remains obscure. METHODS: Liver fibrosis was induced either by feeding mice with the 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet or by injecting them with carbon tetrachloride. The Toll-like receptor 3 ligand, polyinosinic-polycytidylic acid, was used to activate innate immunity cells and mediators, including natural killer cells and interferon gamma. RESULTS: In the mouse model of DDC-induced liver fibrosis, natural killer cell activation by polyinosinic-polycytidylic acid induced cell death to activated hepatic stellate cells and attenuated the severity of liver fibrosis. Polyinosinic-polycytidylic acid treatment also ameliorated liver fibrosis induced by carbon tetrachloride. The observed protective effect of polyinosinic-polycytidylic acid on liver fibrosis was diminished through either depletion of natural killer cells or by disruption of the interferon gamma gene. Expression of retinoic acid early inducible 1, the NKG2D ligand, was undetectable on quiescent hepatic stellate cells, whereas high levels were found on activated hepatic stellate cells, which correlated with the resistance and susceptibility of quiescent hepatic stellate cells and activated hepatic stellate cells to natural killer cell lysis, respectively. Moreover, treatment with polyinosinic-polycytidylic acid or interferon gamma enhanced the cytotoxicity of natural killer cells against activated hepatic stellate cells and increased the expression of NKG2D and tumor necrosis factor-related apoptosis-inducing ligand on liver natural killer cells. Blocking NKG2D or tumor necrosis factor-related apoptosis-inducing ligand with neutralizing antibodies markedly diminished the cytotoxicity of polyinosinic-polycytidylic acid-activated natural killer cells against activated hepatic stellate cells. CONCLUSIONS: Our findings suggest that natural killer cells kill activated hepatic stellate cells via retinoic acid early inducible 1/NKG2D-dependent and tumor necrosis factor-related apoptosis-inducing ligand-dependent mechanisms, thereby ameliorating liver fibrosis.     

The nuclear receptor SHP mediates inhibition of hepatic stellate cells by FXR and protects against liver fibrosis

BACKGROUND AND AIMS: The farnesoid X receptor (FXR) is an endogenous sensor for bile acids and inhibits bile acid synthesis by inducing small heterodimer partner (SHP) gene expression. The aim of this study was to investigate whether FXR is expressed by and modulates function of hepatic stellate cells (HSCs). METHODS: The antifibrotic activity of FXR ligand was tested in 2 rodent models: the porcine serum and bile duct ligation (BDL). RESULTS: Twelve-week administration of 1-10 mg/kg 6-ethyl chenodeoxycholic acid (6-ECDCA), a synthetic FXR ligand, to porcine serum-treated rats prevented liver fibrosis development and reduced liver expression of alpha1(I) collagen, TGF-beta1 and alpha-SMA mRNA by approximately 90%. Therapeutic administration of 6-ECDCA, 3 mg/kg, to BDL rats reduced liver fibrosis and alpha1(I) collagen, transforming growth factor (TGF)-beta1, alpha-SMA, and tissue metalloproteinase inhibitor (TIMP)-1 and 2 messenger RNA (mRNA) by 70%-80%. No protection was observed in BDL rats treated with CDCA, 3 mg/kg, and ursodeoxycholic acid, 15 mg/kg. FXR expression was detected in HSCs. Exposure of HSCs to FXR ligands caused a 3-fold increase of SHP, reduced alpha1(I)collagen and TGF-beta1 by approximately 60%-70% and abrogates alpha1(I) collagen mRNA up-regulation induced by thrombin and TGF-beta1. By retrovirus infection and small interference RNA, we generated SHP overexpressing and SHP-deficient HSC-T6. Using these cell lines, we demonstrated that SHP binds JunD and inhibits DNA binding of adaptor protein (AP)-1 induced by thrombin. CONCLUSIONS: By demonstrating that an FXR-SHP regulatory cascade promotes resolution of liver fibrosis, this study establish that FXR ligands might represent a novel therapeutic option to treat liver fibrosis.     

Tetramethylpyrazine reduces glucose and insulin-induced activation of hepatic stellate cells by inhibiting insulin receptor-mediated PI3K/AKT and ERK pathways

Hepatic stellate cell (HSC) activation is the central event during liver fibrogenesis. Metabolic syndrome characterized by hyperglycemia and hyperinsulinemia contributes to nonalcoholic steatohepatitis-associated liver fibrosis. This study was to investigate the effects of tetramethylpyrazine (TMP) on HSC activation induced by glucose and insulin (Glu/Ins) and the underlying mechanisms. Results showed that Glu/Ins significantly stimulated proliferation, invasion, adhesion, and extracellular matrix (ECM) production in HSCs. TMP inhibited HSC proliferation, invasion and adhesion, and reduced the expression of marker genes related to HSC activation in Glu/Ins-activated HSCs. Mechanistic evidence revealed that TMP reduced insulin receptor (InsR) expression and blocked the downstream phosphatidylinositol-3-kinase (PI3K)/AKT and extracellular signal-regulated kinase (ERK) cascades, which was required for TMP attenuation of HSC activation. Moreover, TMP modulated the genes relevant to ECM homeostasis favoring ECM degradation. It could be concluded that TMP inhibited Glu/Ins-stimulated HSC activation and ECM production by inhibiting InsR-mediated PI3K/AKT and ERK pathways.     

纤维化大鼠肝星状细胞IL-10/IL-10R的表达及IL-10干预的影响

目的研究实验性肝纤维化大鼠肝星状细胞白介素10(IL-10)/白介素10受体(IL-10R)表达及IL-10干预对其表达的影响。方法60只清洁级SD大鼠,随机分为正常对照(A组)、肝纤维化(B组)和IL-10干预组(C组)。B组和C组以CCl4腹腔内注射构建肝纤维化模型,C组并予IL-10腹腔内注射干预造模。于第7周和第11周分别随机选取一批大鼠,分离肝星状细胞,抽提总RNA,以半定量RT-PCR法检测各组HSC中IL-10和IL-10R mRNA表达情况。结果病理组织学证实肝纤维化模型构建成功,原代HSC分离培养成功,IL-10干预可以减轻其肝纤维化程度。肝纤维化时IL-10和IL-10R的mRNA表达均较正常组有所增强,但随着肝纤维化进展,IL-10的表达逐渐减弱。IL-10干预可以使二者的表达上调,以第11周时变化最为显著。结论大鼠肝HSC可表达IL-10及IL-10R,IL-10可作用于HSC而发挥抗肝纤维化作用。     

Curcumin diminishes the impacts of hyperglycemia on the activation of hepatic stellate cells by suppressing membrane translocation and gene expression of glucose transporter-2

Diabetes is featured by elevated levels of blood glucose, i.e. hyperglycemia, which might be a risk factor for hepatic fibrogenesis in patients with non-alcoholic steatohepatitis. Hepatic stellate cells (HSCs) are the major effectors during hepatic fibrogenesis. This study was designed to evaluate impacts of high levels of glucose on HSC activation, assess roles of the phytochemical curcumin in attenuating the glucose impacts, and elucidate underlying mechanisms. In this report, levels of intracellular glucose were measured. Contents and gene expression of glucose transporter-2 (GLUT2) in cell fractions were examined. Levels of cellular glutathione and oxidative stress were analyzed. We observed that high levels of glucose induced cell proliferation, type I collagen production and expression of genes relevant to HSC activation, and elevated intracellular glucose levels in cultured HSCs. Curcumin eliminated the stimulatory impacts. Curcumin abrogated the membrane translocation of GLUT2 by interrupting the p38 MAPK signaling pathway. In addition, curcumin suppressed glut2 expression by stimulating the activity of peroxisome proliferator-activated receptor-gamma (PPARγ) and de novo synthesis of glutathione. In conclusion, hyperglycemia stimulated HSC activation in vitro by increasing intracellular glucose, which was eliminated by curcumin by blocking the membrane translocation of GLUT2 and suppressing glut2 expression. The latter was mediated by activating PPARγ and attenuating oxidative stress. Our results presented evidence to impacts of hyperglycemia on stimulating HSC activation and hepatic fibrogenesis, and provided novel insights into the mechanisms by which curcumin eliminated the hyperglycemia-caused HSC activation and potential therapeutic strategies for treatment of diabetes-associated hepatic fibrogenesis.