Hepatic collagenolytic activity in rats after carbon tetrachloride poisoning.
ABSTRACT: 1. Collagenolytic activity towards acid-soluble collagen labelled with [(14)C]-proline was assayed in rat liver with and without carbon tetrachloride poisoning. The products of enzymic digestion were found to be free amino acids and peptides. 2. The hepatic collagenolytic activity increased under conditions of single-dose and subacute carbon tetrachloride poisoning, and correlated with hydroxyproline content. The highest activity was found during recovery from subacute poisoning. 3. Under the same experimental conditions, hepatic acid-proteinase activity changed independently of the collagenolytic activity and also of hepatic hydroxyproline content. 4. The increased collagenolytic activity during carbon tetrachloride poisoning was found mainly in the supernatant fraction. 5. The ratio of the collagenolytic activity to hepatic hydroxyproline content increased during recovery from single-dose and subacute poisoning, and decreased during subacute poisoning.
Project description:1. The collagen hydroxyproline in rat liver was composed of 3.5% neutral-soluble collagen, 4.9% acid-soluble collagen and 91.6% insoluble collagen. In labelling studies with [(14)C]proline in vitro, the specific radioactivities of neutral-soluble, acid-soluble and insoluble collagens in rat liver were found to be 233000, 69000 and 830d.p.m./mumol of hydroxyproline respectively after 1h. 2. During subacute carbon tetrachloride poisoning the hepatic content of insoluble collagen markedly increased, whereas those of soluble collagens did not change. During recovery from subacute poisoning hepatic contents of soluble collagens were markedly decreased. 3. After 8 weeks of carbon tetrachloride poisoning the specific radioactivities of hepatic soluble collagens increased, while that of insoluble collagen decreased. During recovery from subacute poisoning, the specific radioactivities of soluble collagens decreased to the normal range and that of insoluble collagen further decreased. 4. Hepatic collagenolytic activity solubilizing insoluble collagen, which differs from mammalian collagenase, decreased under the conditions of the subacute poisoning and also during recovery from subacute poisoning.
Project description:The effect of administration of carbon tetrachloride and dimethylnitrosamine in vivo on hepatic microsomal function related to drug metabolism was measured. It was found that the capacity of isolated microsomes to demethylate dimethylaniline was diminished during the first hour after carbon tetrachloride poisoning and during the second hour after dimethylnitrosamine poisoning. Thereafter the microsomes from carbon tetrachloride-poisoned livers showed a continuous decline in activity so that at 24hr. there was little residual capacity to undertake demethylation. Microsomes from dimethylnitrosamine-poisoned animals were not different from controls at 24hr. During the first 3hr. there was a transient rise in the accumulation of the N-oxide intermediate in carbon tetrachloride-poisoned livers, with a subsequent fall to below control values. In dimethylnitrosamine poisoning there was a parallel decrease in N-oxide accumulation with decreased demethylation. In the latter part of the first 24hr. the ratio of N-oxide accumulation to demethylation was increased in both instances. At 2hr. after poisoning with either compound there was no evidence of altered NADPH(2)-dependent neotetrazolium reduction or lipid peroxidation. NADPH(2)-dependent azo-dye cleavage was decreased. There was no difference in microsomal cytochrome b(5) content, but there was a decrease in the amount of cytochrome P-450. This latter change was correlated with the decreased capacity for NADPH(2)-dependent oxidative demethylation. It is suggested that dimethylnitrosamine is associated with a defect in microsomal NADPH(2)-dependent electron transport at the level of cytochrome P-450. In addition to affecting cytochrome P-450, carbon tetrachloride is associated with a second severe block involving the release of formaldehyde from the N-oxide intermediate.
Project description:1. Blood and liver concentrations of carbon tetrachloride were measured, at intervals after an oral dose, in rats given stock and protein-free diets. The values did not correlate with the resistance to poisoning found in the rats on protein-free diets. 2. The metabolism of carbon tetrachloride to carbon dioxide in vivo and in liver microsomal preparations was depressed in animals given protein-free diets. 3. Rats given a single dose of DDT [1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane] were highly sensitive to carbon tetrachloride poisoning. The livers of such animals had an increased microsomal protein content and greatly increased microsomal activity in the demethylation of Pyramidon (aminopyrine) and in the conversion of (14)CCl(4) into (14)CO(2). 4. The incorporation of [(14)C]leucine into protein by liver slices was depressed by carbon tetrachloride. This effect was decreased by addition of SKF525A (2-diethylaminoethyl 2,2-diphenyl-2-propylacetate) and in slices from rats given protein-free diets. It is suggested that the toxicity of carbon tetrachloride is closely linked to its metabolism.
Project description:Effective and well-tolerated anti-fibrotic drugs are currently lacking. Therefore, this study was carried out to investigate the potential anti-fibrotic effects of imatinib, nilotinib and silymarin on established hepatic fibrosis in the carbon tetrachloride (CCl(4)) rat model. Male Wistar rats received intraperitoneal injections of CCl(4) twice weekly for 8weeks, as well as daily intraperitoneal treatments of imatinib (10 and 20mg/kg), nilotinib (10 and 20mg/kg) and silymarin (100mg/kg) during the last 4weeks of CCl(4)-intoxication. At the end of the study, hepatic damage was evaluated by analysis of liver function tests and hepatic oxidative stress parameters. Hepatic fibrosis was evaluated by histopathology and morphometry, as well as collagen and 4-hydroxyproline contents. Nilotinib (20mg/kg) was the most effective treatment to counteract CCl(4)-induced hepatic injury as indicated by liver function tests and histopathology. Nilotinib (10mg/kg), nilotinib (20mg/kg) and silymarin (100mg/kg) treatments reduced the mean score of hepatic fibrosis by 31%, 68% and 47%, respectively, and hepatic collagen content by 47%, 49% and 18%, respectively in CCl(4)-treated rats. Hepatic morphometric evaluation and 4-hydroxyproline content revealed that CCl(4)-induced fibrosis was ameliorated significantly by nilotinib (20mg/kg) and imatinib (20mg/kg). Unlike nilotinib, imatinib (20mg/kg) showed some sort of hepatic injury evidenced by elevation of serum aminotransferases and total bilirubin levels, and hepatic total nitrate/nitrite content, as well as characteristic anisonucleosis visualized with the hematoxylin-eosin staining. In conclusion, this study provides the evidence that nilotinib exerts anti-fibrotic activity and suggests that it may be valuable in the treatment of hepatic fibrosis in humans.
Project description:Collagenolytic cathepsin activity was detected in lysed rabbit peritoneal polymorphonuclear leucocytes. The pH optimum was around 3, and activity was greatly enhanced by the presence of cysteine and EDTA. Digestion of polymeric collagen resulted in the release of alpha, beta, and gamma-chains. Collagenolytic cathepsin activity was associated mainly with the granule fraction isolated from homogenates by differential centrifugation. The granule fraction was further fractionated by isopycnic density-gradient centrifugation, and the collagenolytic cathepsin activity was shown to be associated with the azurophil and tertiary granules, both lysosome-like organelles.
Project description:The ubiquitous cross-linking enzyme tissue transglutaminase (TG2) has been implicated in irreversible collagen stabilization in liver fibrosis, although functional evidence is lacking. We studied the contribution of TG2 to hepatic fibrotic matrix stability, as well as liver fibrosis progression and regression in TG2-deficient mice.Advanced liver fibrosis was induced by carbon tetrachloride or thioacetamide in TG2(-/-) mice and their wild-type littermates to study fibrosis progression and its spontaneous regression for up to 36 weeks. Pattern and extent of fibrosis were analyzed by histology and hepatic hydroxyproline quantification. Dynamic changes in hepatic matrix cross-linking were assessed by stepwise collagen extraction. Expression of 7 TGs and fibrosis-related genes was determined by quantitative reverse-transcription polymerase chain reaction.Transglutaminase activity was increased in fibrosis, and the level of TG2 messenger RNA correlated with the expression of fibrosis-related genes. Biochemical analysis revealed progressive collagen stabilization, with an up to 6-fold increase in the highly cross-linked, pepsin-insoluble fraction (26%). In TG2(-/-) mice, hepatic TG activity was significantly decreased, but chronic administration of carbon tetrachloride or thioacetamide led to a comparable extent and pattern of liver fibrosis, as in wild-type mice. In TG2(-/-) mice, the composition of hepatic collagen fractions and levels of fibrosis-related transcripts were unchanged, and fibrosis reversal was not facilitated.TG2 and TG activity are up-regulated during hepatic fibrosis progression, but do not contribute to fibrogenesis or stabilization of the collagen matrix. TG2 deletion does not promote regression of liver fibrosis. TG2-independent collagen cross-linking is a remarkable feature of progressing hepatic fibrosis and represents an important therapeutic target for liver fibrosis.
Project description:Hawthorn (HAW) is a herbal preparation extracted from <i>Crataegus oxyacantha</i>. HAW has cardioprotective, antioxidants, anti-inflammatory, and anti-hypotensive effects. HAW's effect on hepatic fibrosis remains, however, unknown. This study evaluated the impact of HAW on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats and elucidated its mechanisms. HAW reduced liver index and the serum liver enzyme markers and reduced liver damage, and fibrosis as confirmed by histopathological scoring of hematoxylin-eosin staining. Collagen deposition was reduced in HAW group compared to CCl4 group as confirmed by Masson staining, hydroxyproline content, and both mRNA and protein levels of alpha-smooth muscle actin, collagen 1 and 3. HAW also down regulated the gene expressions of inflammatory markers including interleukin-IL-1?, tumor necrosis factor-?, transforming growth factor-? 1, nuclear factor kappa-B, and cyclooxygenase-2 and decreased the myeloperoxidase activity. The effects of HAW was also associated with decreased levels of hepatic oxidative stress markers (malondialdehyde and P.Carbonyl) and with increased activity of superoxide dismutase. Those effects are possibly mediated by blocking the pro-oxidant machinery and down regulating the inflammatory and profibrotic responses. Finally, chlorogenic acid, epicatechin, rutin, vitexin quercetin, and iso quercetin were identified as the major species of polyphenols of the HAW herbal preparation used here. Therefore, HAW's potent protecting effects against liver fibrosis predicts a significant beneficial application.
Project description:1. Ribosomes and microsomes isolated from the livers of rats that had received carbon tetrachloride 1hr. previously had decreased endogenous capacity to incorporate amino acid. 2. The capacity of the isolated structures to respond to a synthetic messenger, polyuridylic acid, and to incorporate phenylalanine was investigated. 3. It was found that ribosomes from carbon tetrachloride-treated animals, prepared with detergent and at high ionic strength, could be restored to the same specific activity as control particles with polyuridylic acid but that these particles required more Mg(2+) in the incubation mixture. 4. Microsomes could also be stimulated to control activities with polyuridylic acid, but had a narrow optimum range of Mg(2+) concentration. 5. Microsomes prepared from poisoned animals could be preprogrammed with polyuridylic acid to a significantly greater degree than could control particles, and this response was greater with increasing Mg(2+) concentrations. These data suggested that in carbon tetrachloride poisoning the messenger-ribosome interaction had been altered. 6. Attempts to deprogramme particles from control and treated animals resulted in decreased endogenous activity of both particles and a decreased capacity for the treated particles to be restored with the synthetic messenger. 7. It is suggested that two effects are present in carbon tetrachloride poisoning, namely an alteration of the messenger-ribosome interaction and an increased lability of the ribosome, as either separate or related events.
Project description:1. An enzyme system present in a rat liver lysosome-rich fraction was found to liberate soluble hydroxyproline-containing products from insoluble collagen, with maximum activity at pH3.45. It was concluded that a form of cathepsin D was involved since synthetic substrates specific for trypsin were not hydrolysed. Collagenolysis was enhanced by thiol compounds and inhibited by Cu(2+) ions and the anti-inflammatory drugs phenylbutazone and ibufenac. 2. The possibility that behaviour of collagen and collagenolysis were modified by various substances, either by destruction of intramolecular and intermolecular bonds in tropocollagen or by electrostatic interactions, is discussed. Insoluble collagen was found to bind electrostatically to chondromucoprotein. This interaction was inhibited by some anti-inflammatory drugs. 3. Possible roles of the lysosomal collagenolytic enzyme system in experimental lathyrism in rats given penicillamine, and in erosion of cartilage in rheumatoid arthritis, are considered. 4. Collagenolysis in vivo, which may depend on complex interrelationships between collagen, chondromucoprotein and metal ions, is discussed in relation to possible effects, both harmful and beneficial, of anti-inflammatory drugs used in rheumatoid arthritis.
Project description:Connective tissue growth factor (CTGF) has been recognized as a central mediator and promising therapeutic target in hepatic fibrosis. In this study, we generated a novel virus-like particle (VLP) CTGF vaccine by inserting the 138-159 amino acid (aa) fragment of CTGF into the central c/e1 epitope of C-terminus truncated hepatitis B virus core antigen (HBc, aa 1-149) using a prokaryotic expression system. Immunization of BALB/c mice with the VLP vaccine efficiently elicited the production of anti-CTGF neutralizing antibodies. Vaccination with this CTGF vaccine significantly protected BALB/c mice from carbon tetrachloride (CCl4)-induced hepatic fibrosis, as indicated by decreased hepatic hydroxyproline content and lower fibrotic score. CCl4 intoxication-induced hepatic stellate cell activation was inhibited by the vaccination, as indicated by decreased ?-smooth muscle actin expression and Smad2 phosphorylation. Vaccination against CTGF also attenuated the over-expression of some profibrogenic factors, such as CTGF, transforming growth factor-?1, platelet-derived growth factor-B and tissue inhibitor of metalloproteinase-1 in the fibrotic mouse livers, decreased hepatocyte apoptosis and accelerated hepatocyte proliferation in the fibrotic mouse livers. Our results clearly indicate that vaccination against CTGF inhibits fibrogenesis, alleviates hepatocyte apoptosis and facilitate hepatic regeneration. We suggest that the vaccine should be developed into an effective therapeutic measure for hepatic fibrosis.