C-terminus modification of Streptomyces clavuligerus deacetoxycephalosporin C synthase improves catalysis with an expanded substrate specificity

Here we investigated the effect of pioglitazone, a peroxisome proliferator-activated receptor (PPAR)-gamma ligand, on early-phase hepatic fibrogenesis in vivo caused by acute carbon tetrachloride (CCl(4)) administration in the rat. Pioglitazone (1 mg/kg BW) prevented pericentral fibrosis and induction of alpha-smooth muscle actin (SMA) 72 h after CCl(4) administration (1 ml/kg BW). CCl(4) induction of alpha1(I)procollagen mRNA in the liver was blunted by pioglitazone to the levels almost 2/3 of CCl(4) alone. Pioglitazone also prevented CCl(4)-induced hepatic inflammation and necrosis, as well as increases in serum tumor necrosis factor-alpha levels. Further, pioglitazone inhibited the induction of alphaSMA and type I collagen in primary cultured hepatic stellate cells in a dose-dependent manner. In conclusion, pioglitazone inhibits both hepatic inflammation and activation of hepatic stellate cells, thereby ameliorating early-phase fibrogenesis in the liver following acute CCl(4).     

Leptin enhances alpha1(I) collagen gene expression in LX-2 human hepatic stellate cells through JAK-mediated H2O2-dependent MAPK pathways

Leptin, a liver profibrogenic cytokine, induces oxidative stress in hepatic stellate cells (HSCs), with increased formation of the oxidant H2O2, which signals through p38 and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways, stimulating tissue inhibitor of metalloproteinase-1 production. Since oxidative stress is a pathogenic mechanism of liver fibrosis and activation of collagen gene is a marker of fibrogenesis, we evaluated the effects of leptin on collagen I expression. We report here that, in LX-2 human HSCs, leptin enhances the levels of alpha1(I) collagen mRNA, promoter activity and protein. Janus kinase (JAK)1 and JAK2 were activated. H2O2 formation was increased; this was prevented by the JAK inhibitor AG490, suggesting a JAK-mediated process. ERK1/2 and p38 were activated, and the activation was blocked by catalase, consistent with an H2O2-dependent mechanism. AG490 and catalase also prevented leptin-stimulated alpha1(I) collagen mRNA expression. PD098059, an ERK1/2 inhibitor, abrogated ERK1/2 activation and suppressed alpha1(I) collagen promoter activity, resulting in mRNA down-regulation. The p38 inhibitor SB203580 and overexpression of dominant negative p38 mutants abrogated p38 activation and down-regulated the mRNA. While SB203580 had no effect on the promoter activity, it reduced the mRNA half-life from 24 to 4 h, contributing to the decreased mRNA level. We conclude that leptin stimulates collagen production through the H2O2-dependent and ERK1/2 and p38 pathways via activated JAK1 and JAK2. ERK1/2 stimulates alpha1(I) collagen promoter activity, whereas p38 stabilizes its mRNA. Accordingly, interference with leptin-induced oxidative stress by antioxidants provides an opportunity for the prevention of liver fibrosis.     

Antifibrogenic effect in vivo of low doses of insulin-like growth factor-I in cirrhotic rats.

Insulin-like growth factor-I (IGF-I) is produced mainly in the liver and it induces beneficial effects on the nutritional status, the liver function and oxidative hepatic damage in cirrhotic rats. The aim of this work was to analyze the effect of IGF-I on mechanisms of fibrogenesis in cirrhotic rats. Liver cirrhosis was induced by CCl(4) inhalation and phenobarbital in Wistar rats. Ten days after stopping CCl(4) administration (day 0), rats received either IGF-I (2 microg/100 g bw/day) (CI+IGF) or saline (CI) subcutaneously during 14 days. Animals were sacrificed on day 15. As control groups were used: healthy rats (CO) and healthy rats treated with IGF-I (CO+IGF). Liver histopathology, hydroxyproline content, prolyl hydroxylase activity, collagen I and III mRNA expression and the evolution of transformed Ito cells into myofibroblasts were assessed. Among the two control groups (CO+IGF), no differences were found in hydroxyproline content and these levels were lower than those found in the two cirrhotic groups. Compared with untreated cirrhotic rats, the CI+IGF-I animals showed a significant reduction in hydroxyproline content, prolyl hydroxylase activity and collagen alpha 1(I) and alpha1(III) mRNA expression. A higher number of transformed Ito cells (alpha-actin +) was observed in untreated cirrhotic animals as compared to CO and CI+IGF groups. In summary, treatment with IGF-I reduced all of the studied parameters of fibrogenesis. In conclusion, low doses of IGF-I induce in vivo an antifibrogenic effect in cirrhotic rats.     

Asiatic acid inhibits liver fibrosis by blocking TGF-beta/Smad signaling in vivo and in vitro

Liver fibrosis is a major cause of liver failure, but treatment remains ineffective. In the present study, we investigated the mechanisms and anti-hepatofibrotic activities of asiatic acid (AA) in a rat model of liver fibrosis induced by carbon tetrachloride (CCl(4)) and in vitro in TGF-beta1-stimulated rat hepatic stellate cell line (HSC-T6). Treatment with AA significantly attenuated CCl(4)-induced liver fibrosis and functional impairment in a dosage-dependent manner, including blockade of the activation of HSC as determined by inhibiting de novo alpha smooth muscle actin (a-SMA) and collagen matrix expression, and an increase in ALT and AST (all p<0.01). The hepatoprotective effects of AA on fibrosis were associated with upregulation of hepatic Smad7, an inhibitor of TGF-beta signaling, thereby blocking upregulation of TGF-beta1 and CTGF and the activation of TGF-beta/Smad signaling. The anti-fibrosis activity and mechanisms of AA were further detected in vitro in HSC-T6. Addition of AA significantly induced Smad7 expression by HSC-T6 cells, thereby inhibiting TGF-beta1-induced Smad2/3 activation, myofibroblast transformation, and collagen matrix expression in a dosage-dependent manner. In contrast, knockdown of Smad7 in HSC-T6 cells prevented AA-induced inhibition of HSC-T6 cell activation and fibrosis in response to TGF-beta1, revealing an essential role for Smad7 in AA-induced anti-fibrotic activities during liver fibrosis in vivo and in vitro. In conclusion, AA may be a novel therapeutic agent for liver fibrosis. Induction of Smad7-dependent inhibition of TGF-beta/Smad-mediated fibrogenesis may be a central mechanism by which AA protects liver from injury.     

Accelerated CCl4-induced liver fibrosis in Hjv-/- mice, associated with an oxidative burst and precocious profibrogenic gene expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl₄. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis.     

Aqueous extract of Anoectochilus formosanus attenuate hepatic fibrosis induced by carbon tetrachloride in rats.

The aim of this study was to investigate the effects of aqueous extract of Anoectochilus formosanus (AFE) on liver fibrogenesis in carbon tetrachloride (CCl4)-induced cirrhosis. Fibrosis was induced in rats by oral administration of CCl4 (20%, 0.5 ml/rat, p.o.) twice a week for 8 weeks. AFE (0.5 and 2.0 g/kg, p.o., daily for 8 weeks) was administered to rats simultaneously. AFE showed reducing actions on the elevated levels of GOT and GPT caused by CCl4. Liver fibrosis in rats induced by CCl4 led to the drop of serum albumin concentration; the AFE increased the albumin concentration. The CCl4-induced liver fibrosis markedly caused liver atrophy and splenomegalia, while AFE increased the liver weight, and decreased the spleen weight. The CCl4-induced liver fibrosis decreased the protein content, and increased collagen contents in rat's liver. AFE significantly increased the contents of protein and reduced the amount of collagen in the liver. In CCl4-treated rats, glutathione concentrations of liver were not affected. AFE significantly increased liver glutathione concentrations. All these results clearly demonstrate that AFE can reduce the liver fibrogensis in rats induced by CCl4.     

Role of nitric oxide in the regulation of fibrogenic factors in experimental liver fibrosis in mice

Previously, we have shown that an increased expression level of iNOS but a reduction in the expression of eNOS is associated with increased oxidative stress markers in CCl?-induced experimental liver fibrosis. The present study aimed to investigate the effect of L-arginine and 5-methylisothiourea hemisulfate (SMT) in the expression of profibrogenic factors in chronic liver injury. ICR mice were treated with CCl? with or without treatment of L-arginine, an NO donor, or SMT, an iNOS inhibitor. The expression of matrix metalloptroteinases (MMPs), tissue inhibitors of metalloproteinases (TIMPs), α-smooth muscle actin (α-SMA), tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR. The activity of the MMP-2 and MMP-9 were measured by zymography. Our results showed that CCl?-treated mice showed significant up-regulation of expression of pro-fibrogenic factors, TNF-α and COX-2. Treatment with L-arginine or SMT showed a significant reduction in CCl?-induced expression of these pro-fibrogenic factors, TNF-α and COX-2. In conclusion, both SMT and L-arginine effectively attenuated the progression of CCl?-induced liver fibrosis. SMT suppresses iNOS mediated NO production. However, L-arginine augments NO production. The similar effect of the two drugs on liver fibrosis indicates that there may be two distinct pathways of NOS mediated fibrogenesis in chronic liver injury by iNOS and eNOS. Our results suggest that eNOS-mediated liver fibrogenesis may play a more important role than that of iNOS in chronic liver injury. Taken together, these results support the contention that NO plays an active role in the progression of liver fibrosis and hepatocellular damage.     

Hrd1 participates in the regulation of collagen I synthesis in renal fibrosis

The production and accumulation of collagen-rich extracellular matrix are common hallmarks during the process of renal fibrogenesis. However, the mechanisms of the regulation of collagen synthesis in renal fibrosis are still unclear. Hrd1, an E3 ubiquitin ligase, plays important roles for protein folding in ER and transport to Golgi. Here, we examined the hypothesis that Hrd1 posttranslationally regulates collagen synthesis in renal interstitial fibrogenesis. Unilateral ureteral obstruction induced Hrd1 expression, predominantly in the renal interstitium and tubular epithelium of fibrotic kidneys. Transforming growth factor β1, as a key mediator in kidney fibrosis, significantly increased the expressions of Hrd1, α-smooth muscle actin, fibronectin as well as procollagen I and mature collagen I in dose-dependent manner in tubular epithelial cells, suggesting that collagen I maturation might be modulated during renal fibrosis. In cultured renal fibroblasts, Hrd1 knockdown decreased secreted collagen I ~60 % in the supernatant of NRK-49F cells. Conversely, Hrd1 overexpression increased secreted collagen I ~1.5-fold. Hrd1 overexpression significantly increased the expressions of both procollagen I and mature collagen I, ~2.2-fold and ~1.8-fold, respectively. However, Hrd1 knockdown markedly decreased the expression of mature collagen I ~80 %, while procollagen I expression only was decreased ~21 %. Moreover, short interfering RNA-induced knockdown of Sec23A blunted the increase in collagen I expression (both immature and mature form) by Hrd1 overexpression and returned collagen I expression toward control levels. These results indicate that Hrd1 plays an important role in the maturation of collagen I in renal fibrosis, and that Sec23A pathway is required for ER-to-Golgi procollagen trafficking to promote collagen synthesis.     

Modified synthetic siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibrogenesis in rats

BACKGROUND/AIMS: Fibrosis occurs in most chronic liver injuries and results from changes in the balance between synthesis and degradation of extracellular matrix (ECM) components. Matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs) are known to regulate the ECM turnover. We investigate the effect of modified synthetic small interfering RNA (siRNA) targeting TIMP-2 in rat model of liver fibrosis. METHODS: Rat hepatic fibrosis was induced by CCl4 for 8 weeks. After the 2-week CCl4 injection period, rats in the three siRNA groups simultaneously received a different dosage (0.05, 0.1 and 0.2 mg.kg(-1), respectively) of modified synthetic siRNA targeting TIMP-2 via the tail vein every 3 days for 6 weeks. The pathological changes in liver tissues were observed by light microscopy and transmission electron microscopy. Portal vein pressure and proliferating cell nuclear antigen were measured. Expression of TIMP-2, MMP-2, MT1-MMP, MMP-13, hepatocyte growth factor, collagen type I, collagen type III and alpha-SMA were evaluated by quantitative real-time polymerase chain reaction or Western blotting or gelatin zymography. RESULTS: Modified synthetic siRNA targeting TIMP-2 induced a dose-dependent inhibition of the TIMP-2 expression in the rat model of liver fibrosis with a similar trend in MMP-2 and MT1-MMP, but an increase in MMP-13. Rats administered siRNA targeting TIMP-2 showed promotion of ECM degradation, reduction in activated hepatic stellate cells and enhancement of hepatocyte regeneration. Furthermore, portal hypertension was also ameliorated after treatment with siRNA targeting TIMP-2. CONCLUSIONS: Knock-down of TIMP-2 expression attenuates CCl4-induced liver fibrosis and is a potential pharmacological target for gene therapy in liver fibrosis.     

Antagonistic regulation of transmembrane 4 L6 family member 5 attenuates fibrotic phenotypes in CCl(4) -treated mice

The development of liver fibrosis from chronic inflammation can involve epithelial-mesenchymal transition (EMT). Severe liver fibrosis can progress to cirrhosis, and further to hepatocellular carcinoma. Because the tetraspanin transmembrane 4 L6 family member 5 (TM4SF5) induces EMT and is highly expressed in hepatocellular carcinoma, it is of interest to investigate whether TM4SF5 expression is correlated with EMT processes during the development of fibrotic liver features. Using hepatic cells in vitro and a CCl(4) -mediated mouse liver in?vivo model, we examined whether TM4SF5 is expressed during liver fibrosis mediated by CCl(4) administration and whether treatment with anti-TM4SF5 reagent blocks the fibrotic liver features. Here, we found that TM4SF5 expression was induced by the transforming growth factor (TGF)β1 and epidermal growth factor signaling pathways in hepatocytes in vitro. In the CCl(4) -mediated mouse liver model, TM4SF5 was expressed during the liver fibrosis mediated by CCl(4) administration and correlated with α-smooth muscle actin expression, collagen I deposition, and TGFβ1 and epidermal growth factor receptor signaling activation in fibrotic septa regions. Interestingly, treatment with anti-TM4SF5 reagent blocked the TM4SF5-mediated liver fibrotic features: the formation of fibrotic septa with α-smooth muscle actin expression and collagen I deposition was attenuated by treatment with anti-TM4SF5 reagent. These results suggest that TM4SF5 expression mediated by TGFβ1 and growth factor can facilitate fibrotic processes during chronic liver injuries. TM4SF5 is thus a candidate target for prevention of liver fibrosis following chronic liver injury.     

15d-Deoxy-Delta12,14-prostaglandin J2 modulates collagen type I synthesis in human hepatic stellate cells by inducing oxidative stress

15 deoxy-Delta(12,14)-prostaglandin(2) (15d-PGJ(2)) is known to inhibit the proliferation of hepatic stellate cells (HSCs), major cellular components that cause hepatic fibrosis, in vitro. It also induces oxidative stress, which results in hepatic myofibroblast death. On the other hand, oxidative stress generally induces HSC proliferation and collagen synthesis in vitro, and liver fibrogenesis in vivo. In this study, we evaluated the effects of 15d-PGJ(2) at various concentrations on the viability and collagen synthesis of HSCs. 15d-PGJ(2) increased intracellular reactive oxygen species (ROS), and reduced the viability of human HSCs at concentrations 5 microM by inducing apoptotic cell death. In addition, the antioxidants alpha-tocopherol and N-acetylcysteine (NAC) blocked 15d-PGJ(2)-induced HSC death. Collagen I synthesis was increased 1.5-fold by 0.5 microM 15d-PGJ(2) treatment, but was reduced to 30% of the control level by 10 microM 15d-PGJ(2), and NAC pretreatment prevented these changes in collagen production by 15d-PGJ(2). We conclude that 15d-PGJ(2) may either induce or prevent hepatic fibrogenesis depending on its concentration.     

S-adenosylmethionine blocks collagen I production by preventing transforming growth factor-beta induction of the COL1A2 promoter

To study the anti-fibrogenic mechanisms of S-adenosylmethionine (AdoMet), transgenic mice harboring the -17 kb to +54 bp of the collagen alpha2 (I) promoter (COL1A2) cloned upstream from the beta-gal reporter gene were injected with carbon tetrachloride (CCl4) to induce fibrosis and coadministered either AdoMet or saline. Control groups received AdoMet or mineral oil. AdoMet lowered the pathology in CCl4-treated mice as shown by transaminase levels, hematoxylin and eosin, Masson's trichrome staining, and collagen I expression. beta-Galactosidase activity indicated activation of the COL1A2 promoter in stellate cells from CCl4-treated mice and repression of such activation by AdoMet. Lipid peroxidation, transforming growth factor-beta (TGFbeta) expression, and decreases in glutathione levels were prevented by AdoMet. Incubation of primary stellate cells with AdoMet down-regulated basal and TGFbeta-induced collagen I and alpha-smooth muscle actin proteins. AdoMet metabolites down-regulated collagen I protein and mRNA levels. AdoMet repressed basal and TGFbeta-induced reporter activity in stellate cells transfected with COL1A2 promoter deletion constructs. AdoMet blocked TGFbeta induction of the -378 bp region of the COL1A2 promoter and prevented the phosphorylation of extracellular signal-regulated kinase 1/2 and the binding of Sp1 to the TGFbeta-responsive element. These observations unveil a novel mechanism by which AdoMet could ameliorate liver fibrosis.     

Changes of the hepatic proteome in murine models for toxically induced fibrogenesis and sclerosing cholangitis

We investigated the changes in the hepatic proteome in murine models for toxic-induced fibrogenesis and sclerosing cholangitis. A comprehensive comparison of protein changes observed is made and the mechanistical basis of the expression changes is discussed. Hepatic fibrosis was induced by repetitive intraperitoneal CCl4 treatment of BALB/c mice or developed spontaneously in BALB/c-ATP-binding cassette, subfamily B, member 4 (Abcb4) knock out mice. Fibrosis was verified by a morphometric score and assessment of hydroxyproline content of liver tissue, respectively. The innovative difference in-gel electrophoresis (DIGE) technique was used to analyse protein expression levels of the mouse proteome. Results were confirmed by Western blotting and real-time RT-PCR. In CCl4-induced fibrosis 20 out of 40 and in BALB/c-Abcb4(-/-) mice 8 out of 28 differentially expressed proteins were identified utilizing DIGE. Only two proteins, selenium-binding protein (Sbp2) and carbonic anhydrase 3, have been unidirectionally expressed (i.e. down-regulated) in both models. Relevant differences in the pathogenesis of toxically induced liver fibrosis and sclerosing cholangitis exist. The only novel protein with regard to liver fibrosis depicting a unidirectional expression pattern in both animal models was Sbp2. An explicit protein function could not be clarified yet.     

Progression of hepatic stellate cell activation is associated with the level of oxidative stress rather than cytokines during CCl4-induced fibrogenesis

In order to identify a fibrogenic factor associated with the potential of hepatic stellate cells (HSC) activation that arises during the CCl4-induced fibrogenic process, the relationship between the activation of HSC and levels of several fibrogenic factors were investigated. After isolation of HSC from the liver at different stages of CCl4 intoxication, the activation of HSC was assessed by the expression of alpha-smooth muscle actin. Levels of cytokines and oxidative stress in liver homogenates and plasma were measured by enzyme linked immunosorbent assay and the colorimetric method. In primary culture, HSC isolated from a rat liver were gradually activated in a time-dependent manner according to CCl4 administration. The progression of HSC activation was closely correlated with parameters related to oxidative stress in liver homogenates rather than the tissue levels of several cytokines. Also, the levels of antioxidants and arginase activity were inversely correlated with HSC activation. In plasma, the levels of oxidative stress and cytokines in CCl4-treated rat livers were not associated with the activation of HSC found during the CCl4-induced fibrogenic process. The relationship between HSC activation and oxidative stress was also confirmed through several factor-treated HSC cultures. In conclusion, the activation of HSC was accelerated according to CCl4 administration, and the progression of HSC activation is absolutely related to the oxidative stress. These results show that enhanced oxidative stress is an important signal for activation of HSC in experimental liver fibrogenesis.     

大鼠肝纤维化过程中组织金属蛋白酶抑制因子I基因的原位表达

为了解组织金属蛋白酶抑制因子１（ＴＩＭＰ－１）基因在实验性肝纤维化形成中的作用,我们应用地高辛原位杂交技术对大鼠肝组织在四氯化碳（ＣＣｌ４）诱发肝纤维化形成过程中ＴＩＭＰ－１ｍＲ－ＮＡ的表达进行了研究。结果表明,ＣＣｌ４肝损伤早期（４周）肝组织中间质细胞（血管及窦内皮细胞及贮脂细胞）中显示ＴＩＭＰ－１的过度表达;ＣＣｌ４肝纤维化早期（８周）及肝纤维化晚期（１２周）,肝组织中间质细胞的ＴＩＭＰ－１表达持续维持在高水平。结果提示,肝间质细胞（内皮细胞及贮脂细胞）是肝内ＴＩＭＰ－１的主要来源细胞;ＴＩＭＰ－１的异常表达是大鼠肝纤维化过程中较早出现的分子变化,与肝纤维化发生有关;纤维化晚期持续高表达的ＴＩＭＰ－１通过抑制胶原酶而在肝纤维化持续进展中起重要作用。