Cytokine blockade inhibits hepatic tissue inhibitor of metalloproteinase‐1 expression and up‐regulates matrix metalloproteinase‐9 in toxic liver injury

Abstract: Background: Tissue inhibitor of metalloproteinases (TIMP)‐1, the most important endogenous inhibitor of matrix metalloproteinases, plays a pivotal role in the pathogenesis of liver fibrosis and may represent an effective therapeutic target in the design of antifibrotic strategies for chronic liver diseases. Methods: Intraperitoneal application of a single dose of either tumor necrosis factor (TNF)‐α or interleukin (IL)‐1β in mice led to an enhanced expression of hepatic TIMP‐1 after 4–16 h. Male Sprague–Dawley rats were treated with carbon tetrachloride (CCl₄) in the presence and absence of specific TNF‐α and IL‐1β inhibitors. Results: Real‐time PCR revealed a significant increase of TIMP‐1 mRNA in total rat liver 24 h after CCl₄ injection. Repetitive injection of both, etanercept and anakinra, before and after CCl₄ injection effectively inactivated TNF‐α and IL‐1β. Anticytokine pretreatment reduced the increase of TIMP‐1 expression after a single CCl₄ injection by 50% and 75%, respectively. In contrast to CCl₄‐treated rats with and without TNF‐α blockade, IL‐1β inactivation caused a sevenfold increase in matrix metalloproteinases‐9 mRNA levels. Conclusions: In conclusion, TIMP‐1 expression is up‐regulated in the early phase of toxic liver injury by proinflammatory cytokines such as TNF‐α and IL‐1β in rodents. Pharmacological inactivation of these cytokines significantly reduces TIMP‐1 gene expression. Our data provide a potential new antifibrotic approach.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

β‐Glucuronidase inhibitor tectorigenin isolated from the flower of Pueraria thunbergiana protects carbon tetrachloride‐induced liver injury

Background/Aim: To understand the relationship between the fluctuation in serum β‐glucuronidase and hepatotoxicity, an inhibitor of β‐glucuronidase was isolated from the flowers of Pueraria thunbergiana and its hepatoprotective activity was measured. Method: Tectorigenin was isolated from the flowers of pueria thunbergiana as an inhibitor of β‐glucuronidase, and serum ALT, AST and biological parameters as markers for its hepatoprotective activity were measured on CCl₄‐induced liver injury in mice. The relationship between serum β‐glucuronidase and hepatoprotective activities in mice was measured. Results: When tectorigenin at a dose of 100 mg/kg was intraperitoneally administered on CCl₄‐induced liver injury in mice, it significantly inhibited the increase of plasma ALT, AST and LDH activities. The inhibitory effect of tectorigenin is much more potent than that of dimethyl diphenyl bicarboxylate (DDB), which has been used as a commercial hepatoprotective agent. When tectoridin transformed to tectorigenin by intestinal bacteria was orally administered to mice, it showed hepatoprotective activity. However, when tectoridin was intraperitoneally administrated to mice, it did not exhibit hepatoprotective activity. Moreover, orally administered tectoridin not only inhibited β‐glucuronidase but also increased GSH content and GST activity on CCl₄‐induced hepatotoxicity of mice. Conclusion: We insist that an inhibitor of β‐glucuronidase tectorigenin may be hepatoprotective and tectoridin should be a prodrug transformed to tectorigenin.     

Serum levels of matrix metalloproteinase‐9, tissue inhibitors of metalloproteinase‐1 and their ratio are associated with impaired lung function in the elderly: A population‐based study

Background and objective: Matrix metalloproteinases (MMP) and their inhibitors, tissue inhibitors of metalloproteinases (TIMP), regulate homeostasis and turnover of the extra cellular matrix. The aim of this study was to investigate the associations of serum MMP‐9 and TIMP‐1 with lung function. Methods: Spirometry was performed in a population‐based sample of 888 subjects aged 70 years. Serum MMP‐9 and TIMP‐1 concentrations were measured by ELISA. Results: Lower FEV₁ values were associated with higher serum levels of MMP‐9 (P = 0.001) and TIMP‐1 (P < 0.001), and a higher ratio of MMP‐9 to TIMP‐1 (P = 0.02). These associations were significant after adjustment for gender, weight, height, BMI, current smoking, pack years of smoking and the time for which samples were frozen. After stratification for gender, the associations between FEV₁ and MMP‐9, TIMP‐1, and their ratio, were significant in men but not in women. Conclusions: Lower FEV₁ was significantly but weakly associated with higher serum levels of MMP‐9, TIMP‐1 and a higher MMP‐9/TIMP‐1 ratio. This association was stronger in men than in women, suggesting a possible role for extracellular matrix remodelling in the development of impaired lung function. These associations may also partly explain the association between low FEV₁ and cardiovascular disease.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone on carbon tetrachloride‐induced hepatic cirrhosis in rats

Aim: The present study was undertaken to evaluate the effects of 1‐O‐hexyl‐2,3,5‐trimethylhydroquinone (HTHQ), a synthesized vitamin E derivative, on carbon tetrachloride (CCl₄)‐induced cirrhosis. Methods: Rats were treated with hypodermic injections of CCl₄ twice a week to induce the hepatic cirrhosis, and given drinking water containing HTHQ or solvent. Primary cultures of rat hepatocytes were performed to evaluate the effects of HTHQ on the expression of inducible nitric oxide synthase (iNOS). Results: Masson's staining of rat livers showed fibrosis around pseudo‐lobules in the CCl₄ group, the lesions being reduced in the CCl₄ HTHQ group. Increases in liver tissue hydroxyproline and α₁(I) collagen, α‐smooth muscle actin and iNOS induced by CCl₄, were also markedly diminished by HTHQ. Furthermore, both HTHQ and vitamin E attenuated interleukin‐1β‐induced iNOS protein expression in cultured hepatocytes, the potency of HTHQ being 10‐times higher than that of vitamin E. Conclusion: HTHQ may inhibit development of hepatic cirrhosis in rats, more potently than vitamin E, by inhibiting the iNOS expression in hepatocytes. Because vitamin E has a radical scavenging action, roles of NO and peroxynitrite will be discussed in the effects of HTHQ on the fibrosis.     

Conditional knockout of heparin‐binding epidermal growth factor‐like growth factor in the liver accelerates carbon tetrachloride‐induced liver injury in mice

Aim: We previously demonstrated that heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF) is induced in response to several liver injuries. Because the HB‐EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver‐specific HB‐EGF conditional knockout mice using the interferon‐inducible Mx‐1 promoter driven cre recombinase transgene and investigated its role during acute liver injury. Methods: We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl₄) in HB‐EGF KO mice and wild‐type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB‐EGF‐dependent anti‐apoptosis and wound‐healing process of the liver in vitro. Results: HB‐EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl₄ injection. We also demonstrated that HB‐EGF treatment inhibited tumor necrosis factor‐α‐induced apoptosis of AML12 mouse hepatocytes and promoted the wound‐healing response of these cells. Conclusion: This study showed that HB‐EGF plays a protective role during acute liver injury.     

多西环素对乳腺癌移植瘤小鼠MMP-2及TIMP-2 mRNA表达的影响

目的 研究多西环素在小鼠乳腺癌肺转移模型中对基质金属蛋白酶-2(MMP-2)及金属蛋白酶组织抑制因子-2(TIMP-2)mRNA表达的影响.方法 30只TAⅡ近交系小鼠建立肺转移性乳腺癌BCML-TAⅡ99模型,随机分为对照组及给药组,每组15只,于可触及肿物后次日,给药组腹腔注射盐酸多西环素0.1 ml(10 mg/ml),对照组给予等量生理盐水.7 w后处死动物,剥离肿瘤称重,部分新鲜组织采用逆转录多聚酶链反应(RT-PCR)方法 检测肿瘤组织MMP-2及TIMP-2 mRNA表达水平.结果 多西环素给药组小鼠肿瘤生长大小及转移率分别为(2.718±0.337)g和13.3%均低于对照组[(4.313±0.468)g,53.3%,P＜0.01,P＜0.05].与对照组比较,多西环素治疗组MMP-2 mRNA为0.637±0.088,明显低于对照组 (1.035±0.097,P＜0.05);TIMP-2 mRNA为0.778±0.148,明显高于对照组 (0.484±0.125,P＜0.05).结论 多西环素通过调节MMP-2及TIMP-2 mRNA的表达水平,对小鼠乳腺癌肺转移模型中肿瘤生长及转移发挥抑制作用.     

Identification of paraxanthine as the most potent caffeine‐derived inhibitor of connective tissue growth factor expression in liver parenchymal cells

Background: Recently, we identified hepatocytes as the major cellular source of profibrogenic connective tissue growth factor (CTGF/CCN2) in the liver. Based on reports of a hepatoprotective effect of coffee consumption, we were the first to provide evidence that caffeine suppresses transforming growth factor (TGF)‐β dependent and ‐independent CTGF expression in hepatocytes in vitro and in vivo, thus suggesting this xanthine‐alkaloid as a potential therapeutic agent. Aim: This study aims at comparing the inhibitory capacities of caffeine and its three demethylated derivates paraxanthine, theophylline and theobromine on CTGF expression in hepatocytes and hepatic stellate cells (HSC). Results: Our data suggest paraxanthine as the most important pharmacological repressor of hepatocellular CTGF expression among the caffeine‐derived metabolic methylxanthines with an inhibitory dosage (ID)₅₀ of 1.15 mM, i.e. 3.84‐fold lower than what is observed for caffeine. In addition, paraxanthine displayed the least cell toxicity as proven by the water‐soluble tetrazolium‐1 cell vitality assay. However, caffeine or any of the metabolites did not inhibit CTGF expression in HSC. At the toxicological threshold concentration of 1 mM for paraxanthine, we observed an inhibition of hepatocellular CTGF synthesis by 44%, which was strongly reverted in the presence of the specific competitive cyclic adenosine monophosphate inhibitor Rp‐adenosine 3′,5‐cyclic monophosphorothioate triethylammonium salt. Furthermore, CTGF protein expression induced by various concentrations of TGF‐β (0.13–1 ng/ml) is still reduced by, on average, 27%/45% in the presence of paraxanthine (1.25 mM/2.5 mM). Conclusion: Our data provide an evidence‐based suggestion of the caffeine‐derived primary metabolite paraxanthine as a potentially powerful antifibrotic drug by its inhibitory effect on (hepatocellular) CTGF synthesis.     

Polymorphisms in the transforming growth factor‐β1 gene and the risk of asthma: A meta‐analysis

Background and objective: Polymorphisms in the transforming growth factor‐β1 (TGF‐β1) gene have been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. A meta‐analysis was performed to investigate the association between polymorphisms in the TGF‐β1 gene and asthma susceptibility. Methods: Searches were performed of Medline (Ovid), PubMed, the Chinese Biological Medicine Database (CBM), the Chinese Journals Full‐text Database (CNKI), the Cochrane Library Database and the Web of Science, covering all papers published up to 30 April 2009. Statistical analysis was performed using Revman4.2.8 and STATA10.0 software. Results: Two polymorphisms (?509C/T and 915G/C(G25C)) were investigated in 14 studies, involving 2979 asthma patients and 4941 control subjects. The results showed that individuals carrying the ?509T allele (TT+TC) had a 36% increased risk of asthma, when compared with homozygotes (?509CC) (OR 1.36, 95% CI: 1.12–1.65). However, there was no significant association with risk of asthma in carriers of the 915C allele (GC+CC) compared with 915GG homozygotes (OR 1.05, 95% CI: 0.65–1.70). In a subgroup analysis by ethnicity, the risk of asthma associated with the ?509T allele was significantly elevated among Asians (OR 1.50, 95% CI: 1.04–2.17) but not Caucasians (OR 1.16, 95% CI: 1.00–1.36). In a subgroup analysis by age, the ?509T allele was associated with a significantly elevated risk of asthma among adults (OR 1.45, 95% CI: 1.09–1.92) but not children (OR 1.19, 95% CI: 0.96–1.46). Conclusions: This meta‐analysis suggested that the ?509C/T polymorphism in the TGF‐β1 gene may be a risk factor for asthma. To further evaluate gene–gene and gene–environment interactions between polymorphisms in the TGF‐β1 gene and asthma susceptibility, more studies involving thousands of patients are required.     

The −509C/T polymorphism of transforming growth factor‐β1 is associated with increased risk for development of chronic idiopathic neutropenia

Objective: Impaired granulopoiesis in chronic idiopathic neutropenia (CIN) has been associated with an inflammatory bone marrow (BM) microenvironment consisting of pro‐inflammatory and pro‐apoptotic mediators, such as tumor necrosis factor (TNF)‐α, transforming growth factor (TGF)‐β1, and Fas‐Ligand (Fas‐L). In this study, we evaluated the frequency of TNF‐α, TGF‐β1 and Fas‐L gene polymorphisms in CIN patients and explored their role in excessive cytokine production and their association with CIN development. Methods: The TNF‐α?308G/A, TGF‐β1 ?509C/T, +869T/C, +915G/C, and Fas‐L ?844T/C polymorphisms were studied in 57 CIN patients, and 100 healthy controls from Crete, a well‐defined area with genetically homogeneous population, using a polymerase chain reaction‐based restriction fragment length polymorphism assay. Results: The mutant genotype C/T or T/T of TGF‐β1 ?509C/T polymorphism was more common in CIN patients than in controls (P = 0.033). Compared to wild‐type genotype, the TT genotype was associated with increased risk for CIN development (OR: 5.7; 95% CI: 1.18–27.26; P = 0.033). Compared to controls, patients with CT and TT genotypes displayed increased TGF‐β1 levels in serum (P < 0.0001 and P = 0.0002, respectively) and BM (P < 0.0001 and P = 0.0002, respectively). No significant difference was found between patients and controls in the frequency of TNF‐α?308G/A, TGF‐β1 +869T/C and +915G/C and Fas‐L ‐844T/C polymorphisms. Conclusions: The TGF‐β1 ?509C/T polymorphism is associated with increased risk for CIN and contributes to the pathophysiology of the disorder by inducing TGF‐β1 overproduction. This is the first study providing evidence that genetic factors may predispose to CIN and may have a role in the pathophysiology of the disorder.     

Endoglin in human liver disease and murine models of liver fibrosis—A protective factor against liver fibrosis

Background & Aims

Liver fibrosis is the outcome of chronic liver injury. Transforming growth factor‐β (TGF ‐β) is a major profibrogenic cytokine modulating hepatic stellate cell (HSC ) activation and extracellular matrix homeostasis. This study analyses the effect of Endoglin (Eng), a TGF ‐β type III auxiliary receptor, on fibrogenesis in two models of liver injury by HSC ‐specific endoglin deletion.

Methods

Eng expression was measured in human and murine samples of liver injury. After generating GFAP^Cre(+)Eng^ΔHSC mice, the impact of Endoglin deletion on chronic liver fibrosis was analysed. For in vitro analysis, Eng^flox/flox HSC s were infected with Cre‐expressing virus to deplete Endoglin and fibrogenic responses were analysed.

Results

Endoglin is upregulated in human liver injury. The receptor is expressed in liver tissues and mesenchymal liver cells with much higher abundance of the L‐Eng splice variant. Comparing GFAP^{C re(?)}Eng^f/f to GFAP^{C re(+)}Eng^ΔHSC mice in toxic liver injury, livers of GFAP^{C re(+)}Eng^ΔHSC mice showed 39.9% (P  < .01) higher Hydroxyproline content compared to GFAP^{C re(?)}Eng^f/f littermates. Sirius Red staining underlined these findings, showing 58.8% (P  < .05) more Collagen deposition in livers of GFAP^{C re(+)}Eng^ΔHSC mice. Similar results were obtained in mice subjected to cholestatic injury.

Conclusion

Endoglin isoforms are differentially upregulated in liver samples of patients with chronic and acute liver injury. Endoglin deficiency in HSC significantly aggravates fibrosis in response to injury in two different murine models of liver fibrosis and increases α‐SMA and fibronectin expression in vitro. This suggests that Endoglin protects against fibrotic injury, likely through modulation of TGF ‐β signalling.