Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl_4-induced hepatic fibrosis

AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells. METHODS: We examined the inhibitory mechanism of silymarin on CCI4-induced hepatic cirrhosis in rats. At 4, 8, and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment. RESULTS: In the silymarin with CCU-treated group, increase of hepatic stellate cells and TGF-β1 production were lower than in the CCI4-treated group at early stages. Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCI4+silymarin-treated group decreased significantly. CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.     

Glycyrrhizic acid inhibits apoptosis and fibrosis in carbontetrachloride-induced rat liver injury

AIM:To investigate anti-apoptotic effects of glycyrrhizic acid(GA) against fibrosis in carbon tetrachloride(CCl4)-induced liver injury and its contributing factors.METHODS:Liver fibrosis was induced by administration of CCl4 for 8 wk.Pathological changes in the liver of rats were examined by hematoxylin-eosin staining.Collagen fibers were detected by Sirius red staining.Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of cleaved caspase-3,Bax,α-SMA,connective tissue growth factor(CTGF),matrix metalloproteinase(MMP) 2 and MMP9 proteins were evaluated by western blot analysis,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were estimated by real-time PCR.RESULTS:Treatment with GA significantly improved the pathological changes in the liver and markedly decreased the positive area of Sirius red compared with rats in the CCl4-treated group.TUNEL assay showed that GA significantly reduced the number of TUNEL-positive cells compared with the CCl4-treated group.The expression levels of cleaved caspase-3,Bax,α-SMA,CTGF,MMP2 and MMP9 proteins,and α-SMA m RNA,collagen type Ⅰ and Ⅲ m RNA were also significantly reduced by GA compared with the CCl4-treated group(P 0.05).CONCLUSION:GA treatment can ameliorate CCl4-induced liver fibrosis by inhibiting hepatocyte apoptosis and hepatic stellate cell activation.     

Effects of AT1 receptor antagonist, Iosartan, on rat hepatic fibrosis induced by CCl4

AIM To investigate effect of Iosartan, an AT1 receptor antagonist, on hepatic fibrosis induced by CCI4; and to determine whether or not AT1 receptors are expressed on hepatic stellate cells. M~THODS AND RESULTS Fifty male SpragueDawley rats, weighing (180 ± 20) g, were randomized into five groups (control group, model group, and three Iosartan treated groups ), in which all rats were given the subcutaneous injection of 40% CCl4 (every 3 days for 6 weeks) except for rats of control group. Rats of Iosartantreated groups were treated with Iosartan (20 mg/ kg, 10 mg/kg, 5 mg/kg, daily gavage). After 6 weeks liver tissue and serum samples of all rats were examined. Serum hyaluronic acid (HA), procollagen type Ⅲ (PC Ⅲ ) were detected by radioimmunoassays. van Giesion collagen staining was used to evaluate the extracellular matrix of rats with liver fibrosis. The expression of AT1 receptors, transforming growth factor-beta (TGFβ), and alpha-smooth muscle actin (α-SMA) in liver tissue were determined by immunohistochemical techniques. Compared with model group, serum ALT and AST of Iosartantreated groups were significantly reduced (t = 4.20, P < 0.01 and t = 4.57, P < 0.01 ). Serum HA and PC Ⅲ also had significant differences (t = 3.53, P<0.01 and t=2.20, P<0.05). The degree of fibrosis was improved by Iosartan and correlated with the expressions of AT1 receptors, TGF-β, and α-SMA in liver tissue. CONCLUSION AT1 receptor antagonist, Iosartan, could limit the progression of the hepatic fibrosis induced by CCl4. The mechanism may be related to the decrease in the expression of AT1 receptors and TGF-β, ameliorating the injury of hepatocytes; activation of local renin-angiotensin system might relate to hepatic fibrosis; and during progression of fibrosis, activated hepatic stellate cells might express AT1 receptors.     

ACEI attenuates the progression of CCl_4-induced rat hepatic fibrogenesis by inhibiting TGF-β1, PDGF-BB, NF-κB and MMP-2,9

AIM: Angiotensin II has pro-fibrotic function in the liver. Blockade of the renin-angiotensin-aldosterone-system (RMS) attenuates hepatic fibrosis. The aim of the present study was to determine the mechanism of angiotensin-converting enzyme inhibitor (ACEI) on the progression of rat hepatic fibrosis. METHODS: Forty male Wistar rats were divided into three groups. Model group (Mo): The rats were injected subcutaneously with 40% of CCl4 0.25 mL/100 g. Perindopril group (Pe): The rats were injected subcutaneously with 40% of CCl4. Perindopril, equivalent to 2 mg/(kg·d), was administrated. Control group (Nc): the rats were treated with olive oil only. After 4 and 6 wk, the rats were killed. The liver sections were stained with Masson. The protein expressions of AT1R, TGF-β1 and PDGF-BB were examined by Western blot. Nuclear factor κB (NF-κB) DNA binding activity was examined by EMSA (Electrophoretic gel mobility shift assay). Matrix metalloproteinase-2,9 (MMP-2,9) activity was assessed by zymography. Serum laminin (LN) and hyaluronic acid (HA) were measured using radioimmunoassays. RESULTS: Using Western blot, we clearly provided direct evidence for the expression of AT1R in liver. The expression was up-regulated when fibrogenesis occurred. Perindopril treatment significantly reduced mean fibrosis score, protein levels of AT1R, TGF-β1 and PDGF-BB, serum levels of HA and LN, and the activity of MMP-2,9. NPκB DNA binding activity markedly increased in model group, perindopril treatment considerably reduced NPκB DNA binding activity. CONCLUSION: Perindopril attenuates CCl4-induced hepatic fibrogenesis of rat by inhibiting TGF-β1, PDGF-BB, NF-κB and MMP-2,9.     

Expression and location of Smad2, 4 mRNAs during and after liver fibrogenesis of rats

AIM: To investigate the location alteration of Smad2 and Smad4 mRNAs in the liver during and after fibrogenesis in rats. METHODS: Eighty male Wistar rats weighing approximately 200 g each were used. The rat models of experimental hepatic fibrosis were established by injection with carbon tetrachloride (CCl4), normal rats and rats were injected with olive oil and served as control groups. In situ hybridization(ISH) was used to detect the Smad2 and Smad4 mRNA in liver. RESULTS: In situ hybridization showed Smad2 and Smad4 mRNA expressions in the cytoplasm of hepatic stellate cells (HSC), fibroblasts and myofibroblasts around the central vein and hepatic sinus during and after fibrogenesis. Expression of Smad2, 4 mRNA was higher than that in normal and control rats. CONCLUSION: In the process of and after hepatic fibrosis formation, HSC, fibroblasts and myofibroblasts are the major cells that express Smad2 and Smad4. The more serious the hepatic fibrosis is in the injured liver, the higher the level of Smad2 and Smad4 gene expression is during and after fibrogenesis respectively.     

Effects of oxymatrine on experimental hepatic fibrosis and its mechanism in vivo

AIM: Hepatic fibrogenesis has close relation with hepatic stellate cells (HSC)and tissue inhibitors of metalloproteinase (TIMP). Oxymatrine (OM) is a kind of Chinese herb that is found to have some effects on liver fibrosis. We aimed to determine the effects of OM on hepatic fibrosis and explore the possible mechanism. METHODS: Thirty-two rats were randomly divided into four groups; 16 were used to develop hepatic fibrosis by carbon tetrachloride (CCI4) and treated with or without OM, and 16 were used as controls. The expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) and α-smooth muscle actin (α-SMA) in the livers of rats was detected by immunohisto-chemical assay. Liver pathology was determined by H&E staining and reticulum staining. RESULTS: In CCl4-injured rats, the normal structure of lobules was destroyed, and pseudolobules were formed. Hyperplasia of fibers was observed surrounding the lobules. While the degree of fibrogenesis in liver tissues was significantly decreased in those rats with OM-treatment compared with those without OM treatment. The pseudolobules were surrounded by strong, multi-layer reticular fibers, which netted into pseudolobules in CCl4-injured rats, however, there was a significant decrease in reticular fibers in OM-treated rats. The expression of TIMP-1 in hepatic cells was weak in control groups, but strong in CCl4-injured groups, however, the expression of TIMP-1 was significantly inhibited by OM (F = 52.93, P<0.05). There was no significant change in the expression of α-SMA between CCl4-injured rats with or without OM treatment (F= 8.99, P>0.05). CONCLUSION: OM effectively inhibits CCl4-induced fibrogenesis in rat liver tissues, probably by reducing the expression level of TIMP-1.     

Effects of extract from Ginkgo biloba on carbon tetrachloride-induced liver injury in rats

AIM: To study the effects of extract from Ginkgo biloba (EGb) containing 22% flavonoid and 5% terpenoid on chronic liver injury and liver fibrosis of rats induced by carbon tetrachloride (CCl4). METHODS: All rats were randomly divided into control group, CCl4-treated group, colchicine-treated group and EGb-protected group. Chronic liver injury was induced in experimental groups by subcutaneous injection of CCl4 and fed with chows premixed with 79.5% corn powder, 20% lard and 0.5% cholesterol (v/v). EGb-protected group was treated with EGb (0.5 g/kg body weight per day) for 7 wk. At the end of wk 8, all the rats were killed. Liver function, liver fibrosis, oxidative stress and expression of transforming growth factorβ1 (TGF-β1), a-smooth muscle actin (α-SMA) and typeⅠcollagens in liver were determined. In addition, pathology changes of liver tissue were observed under light microscope. RESULTS: The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and albumin (Alb) in EGb-protected group were notably improved as compared with the CCL4-treated group (P < 0.01). The contents of serum hyaluronic acid (HA), typeⅢprocollagen (PCⅢ), typeⅣcollagen (CIV) and the expression of hepatic tissue TGF-β1,α-SMA and typeⅠcollagen in EGb-protected group were significantly lower than those in CCL4-treated groups (P < 0.05, P < 0.01). The degrees of liver fibrosis in EGb-protected groups were lower than those in CCL4-treated groups (6.58±1.25 vs 9.52±2.06, P < 0.05). Compared to the CCL4-treated group, the levels of plasma glutathoine peroxidase (Se-GSH-Px), superoxide dismutase (SOD) and malondialdehyde (MDA) were strikingly improved also in EGb-protected group (P < 0.05, P < 0.01). CONCLUSION: EGb resists oxidative stress and thereby reduces chronic liver injury and liver fibrosis in rats with liver injury induced by CCl4     

Alterations of mast cells and TGF-β1 on the silymarin treatment for CCl4-induced hepatic fibrosis

AIM: Silymarin is a potent antioxidant, antiinflammatory and anti-fibrogenic agent in the liver, which is mediated by alteration of hepatic Kupffer cell function, lipid peroxidation, and collagen production. Especially, in hepatic fibrogenesis, mast cells are expressed in chronic inflammatory conditions, and promote fibroblast growth and stimulate production of the extracellular matrix by hepatic stellate cells.METHODS: We examined the inhibitory mechanism of silymarin on CCl4-induced hepatic cirrhosis in rats. At 4, 8,and 12 wk, liver tissues were examined histopathologically for fibrotic changes produced by silymarin treatment.RESULTS: In the silymarin with CCl4-treated group,increase of hepatic stellate cells and TGF-β1 production were lower than in the CCl4-treated group at early stages.Additionally, at the late fibrogenic stage, expressions of TGF-β1 were weaker and especially not expressed in hepatocytes located in peripheral areas. Moreover, the number of mast cell in portal areas gradually increased and was dependent on the fibrogenic stage, but those of CCl4+silymarin-treated group decreased significantly.CONCLUSION: Anti-fibrotic and antiinflammatory effects of silymarin were associated with activation of hepatic stellate cells through the expression of TGF-β1 and stabilization of mast cells. These results suggest that silymarin prevent hepatic fibrosis through suppression of inflammation and hypoxia in the hepatic fibrogenesis.     

Antifibrotic effects of interleukin-10 on experimental hepatic fibrosis

BACKGROUND/AIMS: To study the effects of interleukin-10 on hepatic stellate cells and liver tissue in experimental rats hepatic fibrosis. METHODOLOGY: Rat hepatic fibrosis model induced by carbon tetrachloride was established. Liver tissues were harvested from the rats administered CCl4 with or without IL-10 treatment and the animals of the control group. The expression of TGF-beta1, MMP-2 and TIMP-1 in the liver tissues was measured by S-P immunohistochemistry. In addition, another model was established; HSCs in rats in each group were isolated. RT-PCR was employed to analyze TGF-beta1, MMP-2 and TIMP-1 mRNA expression in cells and immunocytochemistry was performed to detect protein expression of alpha-SMA, NF-kappaB, TGF-beta1, MMP-2 and TIMP-1 in HSCs. RESULTS: Rat hepatic fibrosis was developed successfully. The fibrosis changes were partially reversed by simultaneous administration of IL-10. The positive signals of TGF-beta1, MMP-2 and TIMP-1 were observed more frequently (P<0.05) in the CCl4-treated group compared to those in the IL-10-treated group and the control group. HSCs were successfully isolated. TGF-beta1, MMP-2 and TIMP-1 mRNA in HSCs increased obviously during the course of hepatic fibrosis, and their levels were decreased after the treatment with IL-10 (P<0.05). The immunocytochemistry positive levels for TGF-beta1, MMP-2, TIMP-1, alpha-SMA and NF-kappaB in the fibrogenesis group were increased significantly compared to the normal group (P<0.01). The positive signals decreased significantly (P<0.05) after the treatment with IL-10. CONCLUSIONS: The expression of TGF-beta1, MMP-2 and TIMP-1 increased in liver or in HSC of hepatic fibrosis rats and decreased after treatment with IL-10. The IL-10 could inhibit the activation of HSCs and make an antifibrogenic process come into effect in this way.     

Leptin augments inflammatory and profibrogenic responses in the murine liver induced by hepatotoxic chemicals

Lines of evidence suggested a possible link between leptin and hepatic fibrosis; however, whether leptin modulates the fibrogenesis in the liver remains unclear. The purpose of this study, therefore, was to evaluate the effect of leptin on inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. Male C57Bl/6 mice were given carbon tetrachloride (CCl(4)) (0.1 microL/g body weight [BW], intraperitoneally [IP]) and/or recombinant murine leptin (1 microg/g BW, IP) simultaneously, and sacrificed up to 72 hours later. Further, some mice were given thioacetamide (TAA; 200 microg/g BW, IP) and leptin 3 times per week for 4 weeks to evaluate the effect of leptin on chronic fibrogenic responses. A simultaneous injection of leptin enhanced acute CCl(4)-induced necroinflammatory and subsequent fibrotic changes in the hepatic lobules. The steady-state messenger RNA (mRNA) levels of alpha1(I) procollagen and heat shock protein 47 (HSP47) in the liver were potentiated when leptin was injected together with CCl(4). Expression of alpha smooth muscle actin (alpha-SMA) in the liver after CCl(4) treatment was also augmented markedly in combination with leptin. Further, leptin increased transforming growth factor beta1 (TGF-beta1) mRNA in the liver 24 hours after acute CCl(4) about 4-fold higher than CCl(4) alone. Moreover, leptin enhanced hepatic fibrosis and induction of alpha1(I) procollagen mRNA caused by chronic TAA administration. Collectively, these findings indicated that leptin augments both inflammatory and profibrogenic responses in the liver caused by hepatotoxic chemicals. It is postulated that the increase in systemic leptin levels enhances up-regulation of TGF-beta1, leading to activation of stellate cells, thereby augmenting the fibrogenic response in the liver.     

The epidermal growth factor receptor ligand amphiregulin participates in the development of mouse liver fibrosis

The hepatic wound-healing response to chronic noxious stimuli may lead to liver fibrosis, a condition characterized by excessive deposition of extracellular matrix. Fibrogenic cells, including hepatic stellate cells and myofibroblasts, are activated in response to a variety of cytokines, growth factors, and inflammatory mediators. The involvement of members of the epidermal growth factor family in this process has been suggested. Amphiregulin (AR) is an epidermal growth factor receptor (EGFR) ligand specifically induced upon liver injury. Here, we have addressed the in vivo role of AR in experimental liver fibrosis. To this end, liver fibrosis was induced in AR+/+ and AR-/- mice by chronic CCl(4) administration. Histological and molecular markers of hepatic fibrogenesis were measured. Additionally, the response of cultured human and mouse liver fibrogenic cells to AR was evaluated. We observed that AR was expressed in isolated Kupffer cells and liver fibrogenic cells in response to inflamatory stimuli and platelet-derived growth factor, respectively. We demonstrate that the expression of alpha-smooth muscle actin and collagen deposition were markedly reduced in AR-/- mice compared to AR+/+ animals. AR-/- mice also showed reduced expression of tissue inhibitor of metalloproteinases-1 and connective tissue growth factor, two genes that responded to AR treatment in cultured fibrogenic cells. AR also stimulated cell proliferation and exerted a potent antiapoptotic effect on isolated fibrogenic cells. CONCLUSION: These results indicate that among the different EGFR ligands, AR plays a specific role in liver fibrosis. AR may contribute to the expression of fibrogenic mediators, as well as to the growth and survival of fibrogenic cells. Additionally, our data lend further support to the role of the EGFR system in hepatic fibrogenesis.     

Taurine inhibits oxidative damage and prevents fibrosis in carbon tetrachloride-induced hepatic fibrosis

BACKGROUND/AIMS: The aim of the study was to examine the effects of taurine on hepatic fibrogenesis and in isolated hepatic stellate cells (HSC). METHODS: The rats of the hepatic damage (HD) group were administered carbon tetracholoride (CCl4) for 5 weeks and a subgroup received, in addition, a 2% taurine containing diet for 6 weeks (HDT). The HSC were isolated from normal rats and cultured for 4 days. RESULTS: The hepatic taurine concentration was decreased in the HD group. This loss and the hepatic histological damage and fibrosis (particularly in the pericentral region), were reduced following taurine treatment. Furthermore, the hepatic alpha-SMA, lipid hydroperoxide and 8-OHdG levels in serum and liver, as well as hepatic TGF-beta1 mRNA and hydroxyproline levels were significantly increased in the HD group, and most of these parameters were significantly reduced following taurine treatment. In contrast to the MAP-kinase and Akt expressions, which remained unchanged, the lipid hydroperoxide and hydroxyproline concentrations, as well as TGF-beta1 mRNA levels were significantly reduced by taurine in activated HSC. CONCLUSIONS: Oral taurine administration enhances hepatic taurine accumulation, reduces oxidative stress and prevents progression of hepatic fibrosis in CCl4-induced HD rats, as well as inhibits transformation of the HSC.     

TGF-beta/Smad signaling in the injured liver

TGF-beta, acting both directly and indirectly, represents a central mediator of fibrogenic remodeling processes in the liver. Besides hepatic stellate cells (HSCs), which are induced by TGF-beta to transdifferentiate to myofibroblasts and to produce extracellular matrix, hepatocytes are also strongly responsive for this cytokine, which induces apoptosis during fibrogenesis and provides growth control in regeneration processes. Based on this, TGF-beta-mediated hepatic responses to injury are the result of a complex interplay between the different liver cell types. In this review we summarize the knowledge about TGF-beta signal transduction in HSCs with special impact on Smad pathways. We further describe a molecular cross-talk between profibrogenic TGF-beta and antifibrogenic IFN-gamma signaling in liver cells. Finally, we introduce hepatocyte plasticity and epithelial-to-mesenchymal transition in the liver, which is well established in tumorigenesis, as a potential feature of fibrogenesis and highlight possible action points of TGF-beta in these contexts.     

Hepatic fibrogenesis requires sympathetic neurotransmitters

BACKGROUND AND AIMS: Hepatic stellate cells (HSC) are activated by liver injury to become proliferative fibrogenic myofibroblasts. This process may be regulated by the sympathetic nervous system (SNS) but the mechanisms involved are unclear. METHODS: We studied cultured HSC and intact mice with liver injury to test the hypothesis that HSC respond to and produce SNS neurotransmitters to promote fibrogenesis. RESULTS: HSC expressed adrenoceptors, catecholamine biosynthetic enzymes, released norepinephrine (NE), and were growth inhibited by alpha- and beta-adrenoceptor antagonists. HSC from dopamine beta-hydroxylase deficient (Dbh(-/-)) mice, which cannot make NE, grew poorly in culture and were rescued by NE. Inhibitor studies demonstrated that this effect was mediated via G protein coupled adrenoceptors, mitogen activated kinases, and phosphatidylinositol 3-kinase. Injury related fibrogenic responses were inhibited in Dbh(-/-) mice, as evidenced by reduced hepatic accumulation of alpha-smooth muscle actin(+ve) HSC and decreased induction of transforming growth factor beta1 (TGF-beta1) and collagen. Treatment with isoprenaline rescued HSC activation. HSC were also reduced in leptin deficient ob/ob mice which have reduced NE levels and are resistant to hepatic fibrosis. Treating ob/ob mice with NE induced HSC proliferation, upregulated hepatic TGF-beta1 and collagen, and increased liver fibrosis. CONCLUSIONS: HSC are hepatic neuroglia that produce and respond to SNS neurotransmitters to promote hepatic fibrosis.