Taurine inhibits deposition of extracellular matrix in experimental liver fibrosis in rats]

OBJECTIVE: To study the effect of taurine on liver fibrosis and its mechanism. METHODS: Fibrosis was induced by the administration of carbon tertrachloride(CCl4) in rats. Some of the animals were treated with taurine. The rats were killed after 12 weeks of CCl4 treatment. Depositions of type I, III and IV collages, laminin and hyaluronic acid were studied in liver sections by immunohistochemical technique using specific antibody. The hepatic contents of type I, III procollage and tissue inhibitor of metalloproteinase-1(TIMP-1) mRNA were determined by Northern blot hybridization. RESULTS: A significant elevations of hepatic collagen I, III, IV, laminin and hyaluronic acid were observed after 12 weeks of liver injury in animals without taurine treatment, and a definite increase in the amounts of hepatic type I, III procollagen and TIMP-1 mRNA was noted. Taurine prevented increases in type I, III procollagen mRNA expression as well as the accumulation of the collagens, laminin and hyaluronic acid in the liver. CONCLUSION: The data indicate that taurine has a protective effect in CCl4-induced hepatic fibrosis. The results suggest taurine might be of potential value in clinical practice.     

Rat liver fibrosis regresses better with pegylated interferon alpha2b and ursodeoxycholic acid treatments than spontaneous recovery.

BACKGROUND/AIMS: Fibrosis and cirrhosis are common complications of chronic liver diseases. An imbalance between fibrogenesis and fibrolysis results in scarring of the liver parenchyma. We aimed to investigate the possible antifibrotic effectiveness of a newly modified interferon molecule peginterferon alpha2b (PEG-IFNalpha2b) which has better antiviral activity, and ursodeoxycholic acid (UDCA). METHODOLOGY: Liver fibrosis was established on 60 male Sprague Dawley rats with CCl4 in 12 weeks. After cessation of CCl4 Group I was left for spontaneous recovery. Group II was treated with PEG-IFN 1.5 microg/kg/week, Group III with UDCA 25 mg/kg/day and Group IV with combination of both drugs. All rats were killed at week 16. Histopathologic fibrosis scores, tissue hydroxyproline, TIMP-1 and MMP-13 levels were determined. Hepatic stellate cell apoptosis was detected by dual staining with TUNEL technique and anti-alpha smooth muscle actin. RESULTS: Fibrosis scores were lower in Group II, III and IV than Group I (p<0.05 for group I vs. II and III; p<0.01 for group I vs. IV). Tissue hydroxyproline levels were significantly decreased in Group II, III and IV when compared to Group I (p<0.05 for group I vs. II, p<0.01 for group I vs. III and IV). Lower liver TIMP-1 and higher MMP-13 levels were measured in Group II, III, and Group IV than Group I (p<0.01 for TIMP-1 and p<0.01, for MMP). Activated HSC apoptosis was significantly increased in Group II, III and IV when compared to Group I (p<0.01, for all). There was significantly higher apoptosis in Group II than Group III and IV (p<0.01). CONCLUSION: Treatment with both PEG-IFNalpha2b and UDCA improved CCl4 induced rat liver fibrosis. Significantly higher effects were obtained using these agents in combination.     

The serum concentrations of the aminoterminal propeptide of procollagen type III and the hepatic content of mRNA for the alpha 1 chain of procollagen type III in carbon tetrachloride-induced rat liver fibrogenesis.

Serum concentrations of the aminoterminal propeptide of procollagen type III (PIIIP) are elevated in fibrogenic diseases of the liver, but the mechanism of elevation is not fully understood. To investigate the mechanism, we compared serum concentrations of PIIIP with total liver content of mRNA for the pro alpha 1 (III) chain, in rats with carbon tetrachloride (CCl4)-induced liver fibrosis. Adult male rats received CCl4 in mineral oil twice weekly for 8 weeks and were compared with age-matched controls. Serum concentrations of PIIIP were measured by a specific radioimmunoassay; molecular sizes of PIIIP in serum were also determined. Pro alpha 1 (III) mRNA content in the liver was quantitated by RNA slot-blot hybridization and chemical measurement of total hepatic RNA content. Total collagen content of the liver was estimated by hydroxyproline measurement. All CCl4-treated animals had septal fibrosis after 4 weeks, and evidence of cirrhosis (regenerative nodules, ascites) was seen after 7 weeks of treatment. Serum concentrations of PIIIP and pro alpha 1 (III) mRNA content in the liver were correlated well until cirrhosis has established. They increased simultaneously after 3 weeks of treatment, 1 week before any elevation of hepatic hydroxyproline could be detected. After cirrhosis has established, pro alpha 1 (III) mRNA content in the liver decreased markedly, but serum PIIIP levels continued to be elevated. Hepatic hydroxyproline plateaued after 5 weeks. The molecular sizes of serum PIIIP indicate the release of intact native procollagen peptide during the development of cirrhosis. In conclusion, at least in CCl4-induced liver fibrosis in the rats, serum PIIIP levels can be used as a fibrogenic marker for the period progressing to cirrhosis. But the use of the serum PIIIP levels in cirrhosis seems to be limited by factors other than liver fibrogenesis.     

Dynamic changes of capillarization and peri-sinusoid fibrosis in alcoholic liver diseases

AIM:To investigate the dynamic changes of capillarization and peri-sinusoid fibrosis in an alcoholic liver disease model induced by a new method.METHODS:Male SD rats were randomly divided into 6 groups,namely normal,4d,2w,4w,9w and 11w groups.The animals were fed with a mixture of alcohol for designated days and then decollated,and their livers were harvested to examine the pathological changes of hepatocytes, hepatic stellate cells,sinusoidal endothelial cells,sinusoid, perisinusoid.The generation of three kinds of extra cellular matrix was also observed.RESULTS:The injury of hepatocytes became severer as modeling going on.Under electronic microscope,fatty vesicles and swollen mitochondria in hepatocytes,activated hepatic stellate cells with fibrils could been seen near or around it.Fenestrae of sinusoidal endothelial cells were decreased or disappeared,sinusoidal basement was formed.Under light microscopy typical peri-sinusoid fibrosis, griddinglike fibrosis, broaden portal areas, hepatocyte‘s fatty and balloon denaturation,iron sediment,dot necrosis,congregated lymphatic cells and leukocytes were observed.Type I collagen showed an increasing trend as modeling going on,slightly recovered when modeling stopped for 2 weeks. Meanwhile,type IV collagen decreased rapidly when modeling began and recovered after modeling stopped for 2 weeks.Laminin increased as soon as modeling began and did not recover when modeling stopped for 2 weeks.CONCLUSION:The pathological changes of the model were similar to that of human ALD,but mild in degree.It had typical peri-sinusoid fibrosis, however,capillarization seemed to be instable.It may be related with the reduction of type IV collagen in the basement of sinusoid during modeling.     

Measurement of carboxy terminal peptide of type I procollagen in sera of patients with viral hepatitis and liver cirrhosis

In the present investigation, a radioimmunoassay for carboxy terminal peptide of human type I procollagen (type 1 C-peptide) was developed. Its clinical implication for serodiagnosis of hepatic fibrosis in 85 patients with viral hepatitis, 45 patients with post-hepatitic liver cirrhosis and 37 patients with alcoholic liver diseases was evaluated in comparison with that of the previously established amino terminal peptide (type III N-peptide) assay. Anti-sera against type I procollagen was obtained by immunization of rabbit with purified type I procollagen from culture medium of IMR-90. The serum level of type I C-peptide in normal subjects was found to be 42 ng/ml (s.d. = 19). Type I C-peptide levels in patients with acute hepatitis were within normal range, while in chronic hepatitis, the mean type I C-peptide level increased as the grade of fibrosis advanced from grade I to III. However, there was no statistically significant difference between the mean type I C-peptide level of grade III and that of liver cirrhosis. Increments of type I C-peptide levels were also observed in alcoholic liver fibrosis (fatty liver with fibrosis and liver cirrhosis). On the other hand, type III N-peptide assay appeared to reflect not only the degree of hepatic fibrosis, but also the degree of hepatic inflammation, giving the high levels in acute viral hepatitis. Collectively, the results indicate the usefulness of type I C-peptide assay for monitoring hepatic fibrosis in viral hepatitis as well as in alcoholic liver disease.     

慢性乙型肝炎肝纤维化分期与血清肝纤维化标志物的相关性分析

目的 探讨血清透明质酸、Ⅲ型前胶原、层粘蛋白、Ⅳ型胶原等血清肝纤维化标志物与慢性肝炎肝组织炎症活动度及纤维化程度的相关性。方法 278例慢性肝炎患者经肝脏活栓后常规病理检查,肝活检前同时采血检测血清透明质酸、Ⅲ型前胶原、层粘蛋白、Ⅳ型胶原,结果应用x^2检验及t检验进行统计学处理。结果 肝组织纤维化程度与炎症活动度呈正相关关系,透明质酸可反映中度以上慢性肝炎炎症活动度及纤维化程度,且呈正相关;肝脏存在纤维化时层粘蛋白水平升高,与纤维化程度正相关;Ⅲ型前胶原、Ⅳ型胶原水平升高与炎症活动度有关。结论 血清透明质酸、Ⅲ型前胶原、层粘蛋白、Ⅳ型胶原可不同程度反映肝纤维纤维化程度,可作为血清肝纤维化检测指标,透明质酸更可反映肝硬化发展趋势。     

Cell types involved in collagen and fibronectin production in normal and fibrotic human liver

Three collagen types (I, III and IV) and fibronectin were localized in normal and alcoholic human liver by light and electron microscopy using the indirect immunoperoxidase technique. In normal liver, most of the bundles of collagen fibers stained for type pro-III collagen while only a few reacted for type I. Basement membranes stained for type IV collagen which formed discontinuous discrete deposits in sinusoids. Only fibronectin appeared as an almost continuous layer in the space of Disse. At the intracellular level, hepatocytes were found to contain little type I collagen and large amounts of fibronectin. Fat-storing cells strongly stained for type IV collagen and expressed low amounts of types I and III collagen and fibronectin. Endothelial cells contained low amounts of all the components. Alcoholic livers were studied at three stages: steatosis, fibrosis and cirrhosis. Qualitative and quantitative differences were observed in extracellular and intracellular distributions of matrix proteins. Increased amounts of all components were usually found in fibrotic and cirrhotic livers compared to normal liver. In two fibrotic livers which contained numerous bundles of collagen in the sinusoids, fat-storing cells stained more intensely for type III collagen. In a cryptogenic fibrotic liver, abundant type IV collagen was observed in hepatocytes. These results suggest that hepatocytes, fat-storing cells and endothelial cells are engaged in production of extracellular matrix components in normal human liver. In fibrosis, hepatocytes which normally did not synthesize types III and IV collagen may produce these collagens.     

Vascular endothelial growth factor and receptor interaction is a prerequisite for murine hepatic fibrogenesis

BACKGROUND: It has been shown that expression of the potent angiogenic factor, vascular endothelial growth factor (VEGF), and its receptors, flt-1 (VEGFR-1) and KDR/Flk-1 (VEGFR-2), increased during the development of liver fibrosis. AIMS: To elucidate the in vivo role of interaction between VEGF and its receptors in liver fibrogenesis. METHODS: A model of CCl(4) induced hepatic fibrosis was used to assess the role of VEGFR-1 and VEGFR-2 by means of specific neutralising monoclonal antibodies (R-1mAb and R-2mAb, respectively). R-1mAb and R-2mAb were administered after two weeks of treatment with CCl(4), and indices of fibrosis were assessed at eight weeks. RESULTS: Hepatic VEGF mRNA expression significantly increased during the development of liver fibrosis. Both R-1mAb and R-2mAb treatments significantly attenuated the development of fibrosis associated with suppression of neovascularisation in the liver. Hepatic hydroxyproline and serum fibrosis markers were also suppressed. Furthermore, the number of alpha-smooth muscle actin positive cells and alpha1(I)-procollagen mRNA expression were significantly suppressed by R-1mAb and R-2mAb treatment. The inhibitory effect of R-2mAb was more potent than that of R-1mAb, and combination treatment with both mAbs almost completely attenuated fibrosis development. Our in vitro study showed that VEGF treatment significantly stimulated proliferation of both activated hepatic stellate cells (HSC) and sinusoidal endothelial cells (SEC). VEGF also significantly increased alpha1(I)-procollagen mRNA expression in activated HSC. CONCLUSIONS: These results suggest that the interaction of VEGF and its receptor, which reflected the combined effects of both on HSC and SEC, was a prerequisite for liver fibrosis development.     

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.     

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     

Types of collagen synthesized by normal rat liver hepatocytes in primary culture

Collagen formation is an important function of liver parenchymal cells that may be relevant to the pathogenesis of hepatic fibrosis. The types of collagen synthesized by cultured normal rat liver hepatocytes were examined. Cells isolated from adult rats by enzymatic dispersion of the liver were established in primary monolayer culture. Cells were then incubated with radiolabeled proline for 20 hr in the presence of ascorbate and the lathrogen beta-aminopropionitrile. Collagen secreted into the cell media was assessed separately from that in the cell layer. The greater proportion of newly synthesized collagen was associated with the cell layer. Collagen types were identified by ion exchange chromatography and by polyacrylamide gel electrophoresis. Types I, III, IV, and V collagen were present in both media and cell layer. Types III and V were the predominant types found. Very little Type I collagen was synthesized by these cultured normal hepatocytes. The percentages of Types I, III, IV, and V collagens, combining media and cell layer, were 6, 38, 19, and 36, respectively.     

Expression of collagen XVIII mRNA in rat liver fibrosis]

OBJECTIVE: To measure and study quantitatively the collagen XVIII mRNA in normal and fibrotic rat livers. METHODS: We used ribonuclease protection assay to investigate the collagen XVIII mRNA expression in rat liver fibrosis induced by complete bile duct occlusion (BDO). The expression level of procollagen 1 (XVIII) mRNA was compared with that of procollagen 1 (I) and tissue inhibitor of metalloproteinase 1 (TIMP1). RESULTS: mRNA levels of procollagen and TIMP 1 increased 20- and 4-fold in BDO rat livers, respectively. In contrast, hepatic procollagen 1 mRNA level increased only 1.8-fold in fibrotic rat livers. CONCLUSION: C XVIII mRNA is upregulated slightly in liver fibrosis, which is probably correlated with the fact that CXVIII is mainly expressed by hepatocytes.     

Cyclin E1 controls proliferation of hepatic stellate cells and is essential for liver fibrogenesis in mice

Liver fibrogenesis is associated with the transition of quiescent hepatocytes and hepatic stellate cells (HSCs) into the cell cycle. Exit from quiescence is controlled by E-type cyclins (cyclin E1 [CcnE1] and cyclin E2 [CcnE2]). Thus, the aim of the current study was to investigate the contribution of E-type cyclins for liver fibrosis in man and mice. Expression of CcnE1, but not of its homolog, CcnE2, was induced in fibrotic and cirrhotic livers from human patients with different etiologies and in murine wild-type (WT) livers after periodical administration of the profibrotic toxin, CCl(4) . To further evaluate the potential function of E-type cyclins for liver fibrogenesis, we repetitively treated constitutive CcnE1(-/-) and CcnE2(-/-) knock-out mice with CCl(4) to induce liver fibrosis. Interestingly, CcnE1(-/-) mice were protected against CCl(4) -mediated liver fibrogenesis, as evidenced by reduced collagen type I α1 expression and the lack of septum formation. In contrast, CcnE2(-/-) mice showed accelerated fibrogenesis after CCl(4) treatment. We isolated primary HSCs from WT, CcnE1(-/-) , and CcnE2(-/-) mice and analyzed their activation, proliferation, and survival in vitro. CcnE1 expression in WT HSCs was maximal when they started to proliferate, but decreased after the cells transdifferentiated into myofibroblasts. CcnE1(-/-) HSCs showed dramatically impaired survival, cell-cycle arrest, and strongly reduced expression of alpha smooth muscle actin, indicating deficient HSC activation. In contrast, CcnE2-deficient HSCs expressed an elevated level of CcnE1 and showed enhanced cell-cycle activity and proliferation, compared to WT cells. Conclusions: CcnE1 and CcnE2 have antagonistic roles in liver fibrosis. CcnE1 is indispensable for the activation, proliferation, and survival of HSCs and thus promotes the synthesis of extracellular matrix and liver fibrogenesis. (HEPATOLOGY 2012;56:1140-1149).