C23 ameliorates carbon tetrachloride-induced liver fibrosis in mice

BACKGROUND C23,an oligo-peptide derived from cold-inducible RNA-binding protein(CIRP),has been reported to inhibit tissue inflammation,apoptosis and fibrosis by binding to the CIRP receptor;however,there are few reports on its role in liver fibrosis and the underlying mechanism is unknown.AIM To explore whether C23 plays a significant role in carbon tetrachloride(CCl4)-induced liver fibrosis.METHODS CCl4 was injected for 6 weeks to induce liver fibrosis and C23 was used beginning in the second week.Masson and Sirius red staining were used to examine changes in fiber levels.Inflammatory factors in the liver were detected and changes inα-smooth muscle actin(α-SMA)and collagen I expression were detected via immu-nohistochemical staining to evaluate the activation of hematopoietic stellate cells(HSCs).Western blotting was used to detect the activation status of the trans-forming growth factor-beta(TGF-β)/Smad3 axis after C23 treatment.RESULTS CCl4 successfully induced liver fibrosis in mice,while tumor necrosis factor-alpha(TNF-α),IL(interleukin)-1β,and IL-6 levels increased significantly and the IL-10 level decreased significantly.Interestingly,C23 inhibited this process.On the other hand,C23 significantly inhibited the activation of HSCs induced by CCl4,which inhibited the expression ofα-SMA and the synthesis of collagen I.In terms of mechanism,C23 can block Smad3 phosphorylation significantly and inhibits INTRODUCTION At present there is no specific and effective drug for treating liver fibrosis caused by acute or chronic injury.Although preclinical research has made breakthroughs,their suitability as clinical treatments is still unknown.The activation of hepatic stellate cells(HSCs)caused by chronic inflammation is a key process in the development of liver fibrosis and activated HSCs expressα-smooth muscle actin(α-SMA)and transdifferentiate into myofibroblasts with proliferation,migration and secretion abilities,synthesizing the extracellular matrix to deposit in the hepatocyte space and subse-quently forming liver fibrosis[1].Although therapeutic strategies have improved due to past few efforts there is no ideal treatment for hepatic fibrosis[2].Extracellular cold inducible RNA binding protein(CIRP)has been shown to play a role in various acute and chronic inflammatory diseases by promoting tissue inflammation and apoptosis and inducing fibrosis through its receptor Toll-like receptor 4(TLR4)[3].C23 is a recognized competitive inhibitor of CIRP that can competitively bind to CIRP receptors and reduce tissue damage in inflammatory diseases[4].C23 has been shown to significantly reduce serum tumor necrosis factor-alpha(TNF-α),IL(interleukin)-6 and IL-1βlevels.In addition,it can reduce tissue TLR4,TNF-α,IL-6 and IL-1βlevels and inhibit the colocalization of CIRP and TLR4,which plays a significant role in systemic inflammation[5].Re-search has shown that CIRP induces the inflammatory phenotype of lung fibroblasts in a TLR4-dependent manner[6].On the other hand,CIRP is associated with markers of fibrosis andα-SMA is significantly positively correlated with CIRP.Cirp-/-mice exhibit attenuated expression ofα-SMA and collagen(COL1A1 and COL3A1),decreased hydroxyproline content,decreased histological fibrosis scores,and improved pulmonary hypertension[7].C23 inhibited the release of TNF-α,the degradation of IκB and the nuclear translocation of NF-κB in CIRP-stimulated macrophages in a dose-dependent manner and C23 treatment significantly increased the serum levels of lactic dehydrogenase,alanine ami-notransferase,IL-6,TNF-αand IL-1βin septic CLP mice[8].Based on previous research we hypothesized that C23 might alleviate liver fibrosis by inhibiting acute and chronic inflammation.As a selective hepatotoxic chemical carbon tetrachloride(CCl4).can induce inflammation and activate HSCs,promoting liver fibrosis.This study reveals the role and mechanism of C23 in CCl4-induced liver fibrosis in mice.at room temperature for 30 minutes.The gray value of each group was calculated after chemiluminescence.

Effect of Mongolian Vinegar Soaked Licorice on Liver Fibrosis Induced by Carbon Tetrachloride Combined with High-fat Diet in Rats

[Objectives]To determine the improvement effect of vinegar soaked licorice on liver fibrosis induced by carbon tetrachloride(CCl_(4))combined with high-fat diet in rats.[Methods]Subcutaneous injection of 40%-60%CCl_(4)olive oil solution(0.3 mL/100 g)combined with a high-fat diet was used for 5 weeks to establish the rat model with liver fibrosis.After the modeling,the rats were divided into a low dose(0.8 g/kg),a medium dose(2.5 g/kg),a high dose(5 g/kg)group,a colchicine(1.5 mg/kg)positive group,and a vinegar group(2 mL/kg).The serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels in the rats were measured automatically.The serum hyaluronic acid(HA)was detected by radioimmunoassay,and the serum laminin(LN)and procollagen type III peptide(PIIIP)levels were measured by enzyme-linked immunoassay.Liver histopathological morphological changes were observed by HE and Masson staining,and expressions of cytochrome CYP2E1 and transcription factor Nrf2 were detected by immunohistochemistry.[Results]The rat liver fibrosis model was established successfully at the 6~(th)week.Compared with the model group,the levels of ALT,AST,HA,LA,PIIIP,CYP2E1 and Nrf2 of all the examined indexes in the dosing group were decreased(P<0.05).As shown in the pictures of liver pathological tissue sections,the liver fibrosis was significantly alleviated in the positive group and the 3 administration groups.[Conclusions]Vinegar soaked licorice can significantly improve the liver fibrosis induced by carbon tetrachloride combined with high-fat diet in rats,and the effect of the high-dose group was similar to that of the positive group.

Caspase-12 mediates carbon tetrachloride-induced hepatocyte apoptosis in mice

AIM: To investigate the role of caspase-12 and its downstream targets in carbon tetrachloride (CCl4)-induced hepatocyte apoptosis.

Impaired balance of T helper 17/T regulatory cells in carbon tetrachloride-induced liver fibrosis in mice

AIM: To investigate the effect of T helper (Th) 17/T regulatory (Treg) cells on hepatic fibrosis in mice and its possible mechanism.

Dechlorination of carbon tetrachloride by the catalyzed Fe-Cu process

The dectrochemical reduction characteristics of carbon tetrachlofide （CT） were investigated using cyclic voltammetry in this study. In addition, the difference in reduction mechanisms of CT between Master Builders＇ iron and the catalyzed Fe-Cu process was discussed. The results showed that CT was reduced directly on the surface of copper rather than by atomic hydrogen produced at the cathode in the catalyzed Fe-Cu process. The reduction was realized largely by atomic hydrogen in Master Builders＇ iron. The entire CT in 350 ml aqueous solution with 320 mg/L was reduced to trichloromethane and dichloromethane in 2.25 h when 100 g of scrap iron with Fe/Cu ratio of 10：1 （w/w） were used. Moreover, the reduction rate slowed with time. CT could be reduced at acidic, neutral and alkaline pH from solution by Fe-Cu bimetallic media, but the mechanisms were different. The degradation rate was not significantly influenced by pH in the catalyzed Fe-Cu process; in Master Builders＇ iron it clearly increased with decreasing pH. The kinetics of the reductions followed pseudo-first order in both cases. Furthermore, the reductions under acidic conditions proceeded faster than that under the neutral and alkaline conditions. The catalyzed Fe-Cu process was superior to Master Builders＇ iron in treating CT-containing water and this advantage was particularly noticeable under alkaline conditions. The reduction was investigated in the cathode （Cu） and anode （Fe） compartments respectively, the results showed that the direct reduction pathway played an important role in the reduction by the catalyzed Fe-Cu process. The catalyzed Fe-Cu process is of practical value.

Effects of Haobie Yangyin Ruanjian Decoction on hepatic fibrosis induced by carbon tetrachloride in rats

AIM:To explore the anti-fibrotic effect of Haobie Yangyin Ruanjian Decoction(HYRD)on CCl4-induced hepatic fibrosis in rats and its modulation on the transforming growth factor(TGF)β-Smad signaling pathway.METHODS:Fifty-six healthy Wistar rats were randomly divided into five groups:normal control group(n=6),CCl4-induced hepatic fibrosis group(n=14) and three treatment groups(the treated rats received HYRD via oral administration at daily dosages of 8.2,2.5 and 0.82 g/kg,respectively)of HYRD(n=12,respectively).Experimental hepatic fibrosis was induced by subcutaneous injection of carbon tetrachloride solution(CCl4 dissolved in peanut oil,4:6,V/V)with 0.5 mL/100 g body weight for the first time,and then 0.3 mL/100 g body weight twice a week for 8 wk.In the former 2 wk,rats were raised by feedstuffⅠ(80% corn meal,20%lard,0.5%cholesterol).After 2 wk,they were raised by feedstuffⅡ(corn meal and 0.5% cholesterol).Except for the control group,30%alcohol solution was given orally to each rat every other day from the beginning,1 mL for each rat.Liver function parameters and hepatic hydroxyproline content were detected by chromatometry.Serum levels of hyaluronic acid(HA),typeⅣcollagen(CIV),typeⅢprecollagen(PCⅢ)and laminin(LN)were assayed with radioimmunoassay.Deposition of collagen was observed with hematoxylin-eosin staining and collagen staining.Gene expression of TGFβ1 and Smad3 were detected with real-time reverse transcriptase-polymerase chain reaction and Western blotting,respectively.RESULTS:The serum levels of alanine transaminase and aspartate transaminase were increased in the model group compared with the control group(P<0.01),and they were decreased in the three treatment groups compared with the model group.The serum levels of total protein and albumin were decreased in the model group and increased in the three treatment groups.The hepatic hydroxyproline content and serum levels of PCⅢ,HA,LN and CIV were markedly increased in the model group compared with the control group,and decreased in the treatment groups.The gene expression of TGFβ1 and Smad3 was enhanced in the model group compared with the control group,and HYRD could down regulate their expression.CONCLUSION:HYRD can inhibit hepatic fibrosis induced by CCl4 in rats,which is probably associated with its down-regulation on fibrogenic signal transduction of TGFβ-Smad pathway.

Restrictive model of compensated carbon tetrachloride-induced cirrhosis in rats

AIM： To develop a simplified and quick protocol to induce cirrhosis and standardize models of partial liver resection in rats. METHODS： In Fischer F344 rats two modified protocols of phenobarbital-carbon tetrachloride （CCl4） （dilution 50%） gavage to induce cirrhosis （frequency adjusted according to weight, but each subsequent dose was systematically administered） were tested, i.e. the rapid and slow protocols. Prothrombin time （PT） and total bilirubin （TB） were also evaluated. Animals from the rapid group underwent 15% hepatectomy and animals from the slow group underwent 70% hepatectomy. RESULTS： Rapid protocol： This corresponded to 1 gavage/4 d over 6 wk （mortality 30%）. Mean PT was 35.2 ±2.8 s （normal： ld.5 s）, and mean TB was 1.8 ± 0.2 mg/dL （normal： 0.1 mg/dL）. Slow protocol： This cop responded to 1 gavage/6 d over 9 wk （mortality 10%）. Mean PT was 11.8 ± 0.2 s （normal： 14.5 s）, and mean TB was 0.4 ± 0.04 mg/dL （normal： 0.1 mg/dL）. Pathological analyses were performed in both protocols which showed persistent cirrhosis at 3 mo. Rat mortality in the rapid garage group who underwent 15% hepatectomy and in the slow garage group who underwent 70% hepatectomy was 50% and 70%, respectively, CONCLUSION： Our modified model is a simplified method to induce cirrhosis which is rapid （6 to 9 wk）, efficient and stable up to 3 mo. Using this method, ＂Child Pugh A＂ or ＂Child Pugh BC＂ cirrhotic rats were obtained. Our models of cirrhosis and hepatectomy can be used in various situations focusing on postoperative survival.

Protective effects of Foeniculum vulgare root bark extract against carbon tetrachloride-induced hepatic fibrosis in mice

AIM To investigate the protective effects of Foeniculum vulgare root bark (FVRB), a traditional Uyghur medicine, against carbon tetrachloride (CCl4)-induced hepatic fibrosis in mice. METHODS Mice were randomly divided into eight groups (n = 20 each). Except for the normal control group, mice in the rest groups were intraperitoneally injected (i.p.) with 0.1% CCl4-olive oil mixture at 10 mL/kg twice a week to induce liver fibrosis. After 4 wk, mice were treated concurrently with the 70% ethanol extract of FVRB (88, 176, 352 and 704 mg/kg, respectively) daily by oral gavage for 4 wk to evaluate its protective effects. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), triglyceride (TG), hexadecenoic acid (HA), laminin (LN), glutathione (GSH), superoxide dismutase (SOD), and malondialdehyde (MDA) in liver tissues were measured. Hematoxylin-eosin (H and E) staining and Masson trichrome (MT) staining were performed to assess histopathological changes in the liver. The expression of transforming growth factor beta 1 (TGF-beta 1), matrix metalloprotein 9 (MMP-9) and metallopeptidase inhibitor 1 (TIMP-1) was detected by immunohistochemical analysis. Additionally, TGF-beta 1 and alpha-smooth muscle actin (alpha-SMA) protein expression was measured by Western blot. RESULTS A significant reduction in serum levels of AST, ALT, TG, HA and LN was observed in the FVRB-treated groups, suggesting that FVRB displayed hepatoprotective effects. Also, the depletion of GSH, SOD, and MDA accumulation in liver tissues was suppressed by FVRB. The expression of TGF-beta 1, MMP-9 and TIMP-1 determined by immunohistochemistry was markedly reduced in a dose-dependent manner by FVRB treatment. Furthermore, protective effects of FVRB against CCl4-induced liver injury were confirmed by histopathological studies. Protein expression of TGF-beta 1 and alpha-SMA detected by Western blot was decreased by FVRB treatment. CONCLUSION Our results indicate that FVRB may be a promising agent against hepatic fibrosis and its possible mechanisms are inhibiting lipid peroxidation and reducing collagen formation in liver tissue of liver fibrosis mice.

In vitro and in vivo protective effects of proteoglycan isolated from mycelia of Ganoderma lucidum on carbon tetrachlorideinduced liver injury

AIM： To investigate the possible mechanism of the protective effects of a bioactive fraction, Ganoderma lucidum proteoglycan （GLPG）isolated from Ganoderma luddum mycelia, against carbon tetrachloride-induced liver injury. METHODS： A liver injury model was induced by carbon tetrachloride. Cytotoxicity was measured by MTY assay. The activities of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） were determined with an automatic multifunction-biochemical analyzer and the levels of superoxide dismutase （SOD） and TNF-α were determined following the instructions of SOD kit and TNF radioimmunoassay kit. Uver sections were stained with hematoxylin and eosin （H＆E） for histological evaluation and examined under light microscope. RESULTS： We found that GLPG can alleviate the L-02 liver cells injury induced by carbon tetrachloride （CCh） through the measurements of ALT and AST activities and the administration of GLPG to L-02 cells did not display any toxicity. Furthermore, histological analysis of mice liver injury induced by CCh with or without GLPG pretreatment indicated that GLPG can significantly suppress the toxicity induced by CCh in mice liver. We also found that GLPG reduced TNF-α level induced by CCh in the plasma of mice, whereas increased SOD activity in the rat serum. CONCLUSION： GLPG has hepatic protective activity against CCl4 induced injury both in vitro and in vivo. The possible antihepatotoxic mechanisms may be related to the suppression of TNF-α level and the free radical scavenging activity.

(Z)-5-(4-methoxybenzylidene)thiazolidine-2,4-dione protects rats from carbon tetrachloride-induced liver injury and fibrogenesis

AIM： To evaluate the hepatoprotective roles of （Z）- 5-（4-methoxybenzylidene）thiazolidine-2,4-dione （SKLB010） against carbon tetrachloride （CCI4）-induced acute and chronic liver injury and its underlying mecha- nisms of action.

Adsorption characteristics of carbon tetrachloride from aqueous solution onto polyacrylonitrile-based activated carbon fiber

The isotherm,mechanism and kinetics of carbon tetrachloride(CT) adsorption by polyacrylonitrile-based activated carbon fiber(PAN-ACF) were investigated in batch reactors and a continuous flow reactor,and the regeneration of PAN-ACF was also studied.Freundlich and Dubinin-Radushkevich(D-R) adsorption equations can well describe the adsorption isotherm.CT is mainly adsorbed on the exterior surface of PAN-ACF with low boundary layer effect and rate-controlling step of intra-particle diffusion.The adsorption dynamics in the batch reactor well fits with the pseudo-first-order model,and the breakthrough curves in the continuous flow reactor can be well described by the Yoon-Nelson model.The ACF can be recycled through thermal regeneration,whereas the adsorption capacity decreases from 7.87 to 4.98 mg/g after the fourth regeneration.78%-94%of CT can be removed from the wastewater of a fluorine chemical plant on a pilot scale,which confirms the efficacy of ACF under industrial conditions.The results indicate that PAN-ACF is applicable to CT removal from wastewater.

Hepatoprotective effect of Cichorium intybus L.,a traditional Uighur medicine,against carbon tetrachloride-induced hepatic fibrosis in rats

AIM: To investigate the hepatoprotective effect of a Cichorium intybus L. extract (CIE) on CCl4-induced hepatic fibrosis in rats.

Filtrate of fermented mycelia from Antrodia camphorata reduces liver fibrosis induced by carbon tetrachloride in rats

AIM： To investigate the effects of filtrate of fermented mycelia from Antrodia camphorata （FMAC） on liver fibrosis induced by carbon tetrachloride （CCI4） in rats. METHODS： Forty Wistar rats were divided randomly into control group and model group. All model rats were given 200 mL/L CCI4 （2 mL/Kg, po） twice a week for 8 wk. Four weeks after CCh treatment, thirty model rats were further divided randomly into 3 subgroups： CCh and two FMAC subgroups. Rats in CCI4 and 2 FMAC subgroups were treated with FMAC 0, 0.5 and 1.0 g/kg, daily via gastrogavage beginning at the fitch week and the end of the eighth week. Spleen weight, blood synthetic markers （albumin and prothrombin time） and hepatic malondialdehyde （MDA） and hydroxyproline （HP） concentrations were determined. Expression of collagen I, tissue inhibitor of metalloproteinases （TIMP）-1 and transforming growth factor β1 （TGF-β1） mRNA were detected by RTPCR. Histochemical staining of Masson＇s trichrome was performed. RESULTS： CCI4 caused liver fibrosis, featuring increased prothrombin time, hepatic MDA and HP contents, and spleen weight and decreased plasma albumin level. Compared with CCh subgroup, FMAC subgroup （1 g/kg） significantly decreased the prothrombin time （36.7±7.2 and 25.1±10.2 in CCh and FMAC groups, respectively, P〈 0.05） and increased plasma albumin concentration （22.7± 1.0 and 30.7±2.5 in CCk and FMAC groups, respectively, P 〈 0.05）. Spleen weight was significantly lower in rats treated with CCh and FMAC （1 g/kg） compared to CCh treated rats only （2.7±0.1 and 2.4±0.2 in CCk and FMAC groups, respectively, P〈0.05）. The amounts of hepatic MDA and HP in CCI4± FAMC （1 g/kg） subgroup were also lower thanthose in CCh subgroup （MDA： 3.9±0.1 and 2.4±0.6 in CCh and CCI4 ＋ FMAC groups, respectively, P〈 0.01; HP： 1730.7±258.0 and 1311.5±238.8 in CCI4 and CCI4＋FMAC groups, respectively, P〈0.01）. Histologic examinations showed that CCI4＋FMAC subgroups had thinner or less fibrotic septa than CCh group. RT-PCR analysis indicated that FMAC （1 g/kg） reduced mRNA levels of collagen I, TIMP-1 and TGF-β1 （collagen I： 5.63±2.08 and 1.78±0.48 in CCh and CCI4＋FMAC groups, respectively, P〈0.01; TIMP-1： 1.70±0.82 and 0.34±0.02 in CCh and CCI4 ＋ FMAC groups, respectively, P〈0.01; TGF-β1：38.03±11.9 and 4.26±2.17 in CCh and CCI4＋FMAC groups, respectively, P〈0.01） in the CCI4-treated liver. CONCLUSION： It demonstrates that FMAC can retard the progression of liver fibrosis induced by CCh in rats.

Ameliorative effect of Ganoderma lucidum on carbon tetrachloride-induced liver fibrosis in rats

AIM： To investigate the effects of Reishi mushroom, Ganoderma lucidum extract （GLE）, on liver fibrosis induced by carbon tetrachloride （CCl4） in rats. METHODS： Rat hepatic fibrosis was induced by CCl4. Forty Wistar rats were divided randomly into 4 groups： control, CCl4, and two GLE groups. Except for rats in control group, all rats were administered orally with CCl4 （20%, 0.2 mL/100 g body weight） twice a week for 8 weeks. Rats in GLE groups were treated daily with GLE （1 600 or 600 mg/kg） via gastrogavage throughout the whole experimental period. Liver function parameters, such as ALT, AST, albumin, and albumin/globulin （A/G） ratio, spleen weight and hepatic amounts of protein, malondiladehyde （MDA） and hydroxyproline （HP） were determined. Histochemical staining of Sirius red was performed. Expression of transforming growth factor β1 （TGF-β1）, methionine adenosyltransferase （MAT1） 1A and MAT2A mRNA were detected by using RT-PCR. RESULTS： CCl4 caused liver fibrosis, featuring increase in plasma transaminases, hepatic MDA and HP contents, and spleen weight; and decrease in plasma albumin, A/G ratio and hepatic protein level. Compared with CCl4 group, GLE （600, 1 600 mg/kg） treatment significantly increased plasma albumin level and A/G ratio （P〈0.05） and reduced the hepatic HP content （P〈0.01）. GLE （1 600 mg/kg） treatment markedly decreased the activities of transaminases （P〈 0.05）, spleen weight （P〈 0.05） and hepatic MDA content （P〈0.05）; but increased hepatic protein level （P〈0.05）. Liver histology in the GLE （1 600 mg/kg）-treated rats was also improved （P〈0.01）. RT-PCR analysis showed that GLE treatment decreased the expression of TGF-β1 （P〈 0.05-0.001） and changed the expression of MAT1A （P〈0.05-0.01） and MAT2A （P〈 0.05-0.001）. CONCLUSION： Oral administration of GLE significantly reduces CCl4-induced hepatic fibrosis in rats, probably by exerting a protective effect against hepatocellular necrosis by its free-radical scavenging ability.

A novel polyherbal formulation containing thymoquinone attenuates carbon tetrachloride-induced hepatorenal injury in a rat model

Objective:To evaluate a novel polyherbal formulation(BSVT)containing the standardized extracts from the leaves of Boerhavia diffusa,Solidago virgaurea,Vitex negundo,and thymoquinone in CCl4 induced hepatorenal toxicity in rats.Methods:A total of 36 rats were divided into six groups including normal control,CCl4(2 mL/kg,i.p.),CCl4(2 mL/kg,i.p.)+Cystone?(750 mg/kg p.o.),CCl4(2 mL/kg,i.p.)+BSVT(25 mg/kg,p.o.),CCl4(2 mL/kg,i.p.)+BSVT(50 mg/kg,p.o.),and CCl4(2 mL/kg,i.p.)+BSVT(100 mg/kg,p.o.).All treatments were given for four weeks.Serum levels of aspartate transaminase,alanine transaminase,alkaline phosphatase,cholesterol,total protein,serum urea,blood urea nitrogen and creatinine were assessed.Superoxide dismutase,malondialdehyde,and glutathione peroxidase were evaluated in tissue homogenate.The histopathological study of liver and kidney tissues was also done.Results:Aspartate transaminase,alanine transaminase,alkaline phosphatase,cholesterol,serum urea,blood urea nitrogen and creatinine were significantly elevated(P<0.001)while total protein was considerably reduced in the CCl4 group as compared to the normal control(P<0.001),which indicated hepatorenal toxicity.In addition,superoxide dismutase and glutathione peroxidase activities were significantly decreased(P<0.001)while malondialdehyde levels were increased markedly(P<0.001).Treatment with BSVT formulation recovered these parameters towards a normal level in a dose-dependent manner.Conclusions:BSVT formulation ameliorates the hepatorenal toxicity in a dose-dependent manner.Furthermore,clinical studies are required to confirm its efficacy.

Mechanism underlying carbon tetrachloride-inhibited protein synthesis in liver

AIM: To study the mechanism underlying carbon tetrachloride (CCl4)-induced alterations of protein synthesis in liver. METHODS: Male Sprague-Dawley rats were given CCl4 (1 mL/100 g body weight) and 3H-leucine incorporation. Malondialdehyde (MDA) level in the liver, in vitro response of hepatocyte nuclei nucleotide triphosphatase (NTPase) to free radicals, and nuclear export of total mRNA with 3'-poly A+ were measured respectively. Survival response of HepG2 cells to CCl4 treatment was assessed by methyl thiazolyl tetrazolium. Km and Vmax values of nuclear envelope NTPase activity in liver of rats treated with CCl4 were assayed by a double-reciprocal plot. RESULTS: The protein synthesis was inhibited while the MDA level was signif icantly increased in liver of rats treated with CCl4. In addition, CCl4 decreased the NTPase binding capacity of nuclear envelope (Km value) in cultured HepG2 cells. Moreover, in vitro ferrous radicals from Fenton's system suppressed the NTPase activity of liver nuclear envelope in a dose-dependent manner. Down-regulation of the nuclear envelope NTPase activity indicated a lower energy provision for nucleocytoplasmic transport of mRNA molecules, an evidence in CCl4-treated HepG2 cells correspondingly supported by the nuclear sequestration of poly (A)+ mRNA molecules in morphological hybridization research. CONCLUSION: Inhibition of mRNA transport, suggestive of decreased NTPase activity of the nuclear envelope, may be involved in carbon tetrachloride-inhibited protein synthesis in liver.

Management of carbon tetrachloride-induced acute liver injury in rats by syngeneic hepatocyte transplantation in spleen and peritoneal cavity

AIM:Acute hepatitis may seldom have a fulminant course. In the treatment of this medical emergency,potential liver support measure must provide immediate and sufficient assistance to the hepatic function.The goal of our study was to study the adequacy of hepatocyte transplantation (HCTx)in two different anatomical sites,splenic parenchyma and peritoneal cavity,in a rat model of reversible acute hepatitis induced by carbon tetrachloride(CCl_4). METHODS:After CCl_4 intoxication,84 male Wistar rats used as recipients were divided in to four experimental groups accordingly to their treatment:Group A(n=-24):intrasplenic transplantation of 10×10~6 isolated hepatocytes,Group B(n=24): intraperitoneal transplantation of 20×10~6 isolated hepatocytes attached on plastic microcarriers,Group C(n=-18):intrasplenic injection of 1 mL normal saline(sham-operated controls), Group D(n=-18):intraperitoneal injection of 2.5 mL normal saline(sham-operated controls).Survival,liver function tests (LFT)and histology were studied in all four groups,on d 2, 5 and 10 post-HCTx. RESULTS:The ten-day survival(and mean survival)in the 4 groups was 72.2%(8.1±3.1),33.3%(5.4±3.4),0% (3.1±1.3)and 33.3%(5.4±3.6)in groups A,B,C,D, respectively(P_(AB0<0.05,P_(AC)<0.05,P_(BD)=NS).In the final survivors,LFT(except alkaline phosphatase)and hepatic histology returned to normal,independently of their previous therapy.Viable hepatocytes were identified within splenic parenchyma(in group A on d 2)and both in the native liver and the fatty tissue of abdominal wall(in group B on d 5). CONCLUSION:A significantly better survival of the intrasplenically transplanted animals has been demonstrated. Intraperitoneal hepatocytes failed to promptly engraft.A different timing between liver injury and intraperitoneal HCTx may give better results and merits further investigation.

Negundoside,an irridiod glycoside from leaves of Vitex negundo,protects human liver cells against calcium-mediated toxicity induced by carbon tetrachloride

AIM： To evaluate the protective effect of 2′-p-hydroxy benzoylmussaenosidic acid [negundoside （NG）, against carbon tetrachloride （CCl4）-induced toxicity in HUH-7 cel Is.METHODS： CCI4 is a well characterized hepatotoxin, and inducer of cytochrome P450 2E1 （CYP2E1）-mediated oxidative stress. In addition, lipid peroxidation and accumulation of intracellular calcium are important steps in the pathway involved in CCl4 toxicity. Liver cells （HUH-7） were treated with CCI4, and the mechanism of the cytoprotective effect of NG was assessed. Silymarin, a known hepatoprotective drug, was used as control. RESULTS： NG protected HUH-7 cells against CCl4 toxicity and loss of viability without modulating CYP2E1 activity. Prevention of CCl4 toxicity was associated with a reduction in oxidative damage as reflected by decreased generation of reactive oxygen species （ROS）, a decrease in lipid peroxidation and accumulation of intracellular Ca^2＋ levels and maintenance of intracellular glutathione homeostasis. Decreased mitochondrial membrane potential （MMP）, induction of caspases mediated DNA fragmentation and cell cycle arrest, as a result of CCl4 treatment, were also blocked by NG. The protection afforded by NG seemed to be mediated by activation of cyclic adenosine monophosphate （cAMP） synthesis and inhibition of phospholipases （cPLA2）. CONCLUSION： NG exerts a protective effect on CYP2E1-dependent CCl4 toxicity via inhibition of lipid peroxidation, followed by an improved intracellular calcium homeostasis and inhibition of Ca^2＋-dependent proteases.

Protective Effect of Extract Powder of Turmeric on Carbon Tetrachloride Induced Acute Hepatic Injury in Mice

To explore the protective effect of extract powder of turmeric on carbon tetrachloride （CCl4）-induced acute hepatic injury, the mice were administrated with extract powder of turmeric with different doses （50, 100, 200 mg/kg） for 7 d. Then the mice were treated with 0.12% CCl4 by intraperitoneal injection. The levels of ALT, AST in serum and activities of SOD, CAT, MDA, GSH-Px in liver tissue were detected and the liver lesions were examined. The results showed that the activities of ALT, AST and the level of MDA in extract powder of turmeric group were signif- icantly decreased, and the activities of SOD, CAT, GSH-Px were significantly increased, and liver pathology were improved compared with the injured group （P〈 0.05 or P〈0.01）. It indicated that the extract powder of turmeric had significant protective effect against CCl4 induced acute hepatic injury in mice.

Liver Nuclear Activation of Carbon Tetrachloride or Bromotrichloromethane to Trichloromethyl and Trichloromethylperoxyl Free Radicals.Their Reactions With Lipids and Proteins

The formation of·CCl3 radicals in liver nuclei was suggested by spin trapping of them with N-t-butyl-α-phenylnitrone followed by GC/MS detection of the resulting adduct. Comparison of its formation in microsomal biotransformation of CCl4 was made. In aerobic nuclear activation mixtures containing NADPH and CCl4, significant decrease in the arachidonic acid content of nuclear lipids was observed (27. 8%, compared to control), the intensity of this decrease was lower than that occurring in the corresponding microsomal incubation mixtures (29.1%). Significant decreases in arachidonic acid content of nuclear and endoplasmic reticulum lipids were also observed in animals at 6 hours of poisoning with the haloalkane. During aerobic nuclear metabolism of CCl4 or CBrCl3, cholesterol oxidation products were detected: a ketocholesterol, an epoxide like structure and 7-ketocholesterol. Nuclear protein carbonyl formation was not promoted during nuclear CCl4 biotransformation. NADPH by itself may lead to protein carbonyl formation during prolonged periods of incubation. CBrCl3 in contrast, led to decreased protein carbonyl formation. No increase in nuclear protein carbonyl formation was observed in CCl4 intoxicated animals during periods of time between 1 to 6 hours after treatment. The results indicate that during nuclear biotransformation of CCl4 or CBrCl3 reactive free radicals, PUFA degradation, reactive aldehydes and cholesterol oxidation products are formed, nearby DNA and regulatory proteins.