Abstract  The effect of age and peroxidative stress on the concentration of a deoxyguanosine malondialdehyde adduct (dG-MDA) in rat tissues was investigated. Vitamin E deficiency had no effect on the dG-MDA content of liver DNA in rats fed a diet containing 10% corn oil. When 2% cod liver oil was added to this diet, the dG-MDA content of liver DNA doubled in the positive controls fed a high level of vitamin E (100 ppm dl-alpha-tocopherol), and there was a further increase when vitamin E was deleted. Neither iron nitrilotriacetate administration nor choline deficiency had any effect on the dG-MDA content of liver DNA. Carbon tetrachloride had a lowering effect. The failure of iron or carbon tetrachloride administration and of vitamin E deficiency to increase liver dG-MDA is consistent with their failure in previous experiments to affect the urinary excretion of dG-MDA. In contrast, these forms of peroxidative stress produce large increments in the urinary excretion of MDA adducts with lysine, reflecting increased formation and degradation of MDA-modified proteins. DNA appears to be protected from modification by MDA produced at extranuclear sites. The frequency of dG-MDA in different tissues of 4-month-old rats varied markedly: brain >> liver > kidneys and testes. Higher concentrations of dG-MDA were found in the liver and kidneys, but not the testes, of 25-month-old rats. The determinants of the concentration of dG-MDA in DNA merit further investigation.

Abstract  Pretreatment of rats with large doses of vitamin A (VA) potentiates the hepatotoxicity of CCl4. Because our previous studies indicate that VA treatment does not enhance CCl4 metabolism but does enhance CCl4-induced lipid peroxidation and activates liver Kupffer cells to release increased amounts of oxygen-centered free radicals, the current studies were designed to determine if VA treatment potentiates CCl4-induced liver injury through increased release of reactive oxygen species. Plasma clearance of colloidal carbon, an index of Kupffer cell phagocytic activity, was enhanced two- to threefold in rats treated for 7 days with VA (retinol, 250,000 IU/kg per day). Accordingly, VA treatment alone caused Kupffer cell activation. To determine if these activated Kupffer cells could potentiate hepatic injury through release of reactive oxygen species upon CCl4 challenge, polyethylene glycol coupled-superoxide dismutase (PEG-SOD, 10,000 IU/kg) or -catalase (PEG-CAT, 40,000 IU/kg) was given iv 2 hr after CCl4 (0.15 ml/kg, ip) to control or VA-pretreated rats to quench any released reactive oxygen. PEG-SOD and PEG-CAT effectively blocked VA potentiation of CCl4 liver injury as assessed at 24 hr by change in plasma ALT. Methylpalmitate (MP, 2 g/kg), an inhibitor of Kupffer cell phagocytosis and related oxygen burst, also blocked the potentiation of liver injury when given iv 24 hr before CCl4 to VA-pretreated rats. At the doses used, PEG-SOD or PEG-CAT did not influence CCl4 toxicity in control rats (at 0.15 or 2 ml CCl4/kg). Importantly, SOD, CAT, and MP blocked the enhanced lipid peroxidation induced by CCl4 in VA-pretreated rats. From these findings we conclude that the potentiation of CCl4 liver injury by VA pretreatment is mediated, at least in part, by active oxygen species released from Kupffer cells and possibly other macrophages that are activated by VA. Supporting this conclusion is the failure of VA pretreatment to increase the release of LDH from suspension of hepatocytes incubated with CCl4.

Abstract  This study investigated the role of cytochrome P-450 2E1 in enhanced microsomal lipid peroxidation in experimental alcoholic liver disease. We also examined the contribution of this isoform to the increased microsomal injury in alcoholic liver disease caused by carbon tetrachloride-induced or iron-induced oxidant stress. Adult male Wistar rats were intragastrically infused with a high-fat diet and ethanol or glucose for 16 wk; this resulted in hepatic lipid peroxidation and fibrogenesis in the ethanol-fed animals. Microsomes were isolated by differential centrifugation in the presence of 100 mumol/L deferoxamine, washed twice in buffer without deferoxamine and incubated in the absence or presence of ethanol (50 mmol/L), carbon tetrachloride (150 mumol/L), ferric citrate (50 mumol/L) or ferric citrate plus ethanol at 37 degrees C for 30 min in an NADPH-generating system. The basal rate of lipid peroxidation in microsomes isolated from ethanol-fed rats was increased by 52% compared with that in microsomes from controls. Carbon tetrachloride-induced and ferric citrate-induced lipid peroxidation were also accentuated in microsomes from ethanol-fed rats, by 76% and 108%, respectively. Ethanol added in vitro significantly reduced basal (-58%) and ferric citrate-induced (-48%) lipid peroxidation in microsomes from ethanol-fed rats, whereas it had an insignificant effect on that in control microsomes. In fact, this protective effect of ethanol on microsomes from ethanol-fed rats resulted in attenuation of the difference in the level of microsomal lipid peroxidation between the two groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract  OBJECTIVE: To observe the effect of acupuncture stimulation of "Taichong" (LR 3), "Qimen" (LR 14), etc. on hepatic platelet-derived growth factor (PDGF) signal pathway activity at the protein and mRNA levels in hepatic fibrosis rats.

METHODS: Forty-six SD rats were randomly divided into control (10 rats), model (12 rats), acupuncture (12 rats) and non-acupoint (12 rats) groups. Hepatic fibrosis model was established by intraperitoneal injection of mixture solution of 50% CCl4 and olive oil [1:1, 3 times on the 1st week (W), twice/W thereafter for 5 more weeks]. During modeling, acupuncture stimulation of "Taichong" (LR 3), "Qimen" (LR 14), "Ganshu" (BL 18) and "Zusanli" (ST 36) was conducted simultaneously. At the end of the experiments, all the rats were sacrificed for collecting their liver and blood samples, followed by separation of the hepatic stellate cells (HSCs). ELISA, Western blot and Real-time quantitative PCR techniques were used to detect the content of serum PDGF and expression levels of PDGF-beta receptor (PDGF-beta R), extracellular signal-regulated kinase (ERK1/2), c-jun N-terminal kinase (JNK) and P 38 genes and proteins of HSCs, respectively.

RESULTS: Compared to the control group, serum PDGF content, and expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein and P 38 protein of HSCs in the model group were upregulated significantly (P < 0.01, P < 0.05). In comparison with the model group, serum PDGF content, and the expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein of HSCs in the acupuncture group were down-regulated apparently (P < 0.05, P < 0.01). No significant differences were found between the acupuncture and non-acupoint groups in serum PDGF content and between the model group and non-acupoint group in the expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein, JNK protein and P 38 protein of HSCs, as well as between the model group and acupuncture group in the expression levels of JNK protein and P 38 protein of HSCs (P > 0.05).

CONCLUSION: Acupuncture intervention can effectively down-regulate serum PDGF content, and expression levels of PDGF-beta R mRNA and protein, ERK mRNA and protein of HSCs in liver fibrosis rats, which may contribute to its effect in improving liver fibrosis through down-regulating PDGF signal pathway activity.

Abstract  A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner.

Abstract  The dosage-efficacy/toxicity relationship of the 50% alcohol extracts of Polygonum multiflorum was comparatively investigated on either normal or CCl4-induced chronic liver injury rats, by determining the general condition, serum biochemical indices and liver histopathology, coupled with the factor analysis. The dosages were 10 and 20 g raw materials per kg body weight. Compared with the normal control group, the normal high dose group showed significant increases of the serum alanine transaminase (ALT), total bilirubin (TBIL), high mobility group box 1 (HMGB-1) and interleukin-1β (IL-1β) (P < 0.05 or P < 0.01), as well the frequent incidences of inflammatory cell infiltration, hepatic sinus enlargement and fiber stripes formation in histopathological sections. Compared with the model control group, the model low dose group showed significant declines of serum ALT, aspartate transaminase (AST) and total bile acid (TBA) (P < 0.05), as well the alleviation of vacuoles of hepatocytes, but no amelioration of the inflammatory cell infiltration and fibrous tissue hyperplasia; moreover, the model high dose group showed significant degeneration declines of serum HMGB-1, tumor necrosis factor-α (TNF-α) and IL-1β (P < 0.05, P < 0.01), as well the evident alleviation of vacuoles degeneration of hepatocytes, inflammatory cells infiltration and fibrosis degree. The factor analysis showed that the low dosage treatment had almost neither injuring effect on the normal rats nor protective effect on the model rats; while the high dosage treatment showed observable injuring effect on the normal rats, expressed by the significant increases of the factor-1 (HMGB-1, TNF-α and IL-1β as the main contributors) and factor-2 (TBIL, ALT and TBA as the main contributors) relative to the normal control group. The liver protective effect of the high dosage treatment could be observed with the significant reduction of the factor-1, indicating the effective alleviation of the expression of inflammatory cytokines. In conclusion, it could illustrated the phenomenon of symptom-based prescription theory of Polygonum multiflorum on rat livers: the high dosage of the herb had either an injuring effect on normal rats, or a therapeutic effect on the rats with chronic liver injury.

Abstract  Ethanol (alcohol)-mediated cell and tissue damage is partly caused by increased oxidative and nitrosative stress. The majority of reactive oxygen and nitrogen species (ROS/RNS) in alcohol-exposed cells/tissues are being produced through direct inhibition of the mitochondrial respiratory chain and induction/activation of ethanol-inducible cytochrome P450 2E1 (CYP2E1), inducible nitric oxide synthase (iNOS), NADPH-oxidase, and xanthine oxidase. We are particularly interested in studying the combined effects of activated CYP2E1, a pro-oxidant enzyme, and suppressed mitochondrial aldehyde dehydrogenase (ALDH2), an anti-oxidant defensive enzyme responsible for removal of toxic acetaldehyde and lipid peroxides, as we recently reviewed in Journal of Proteomics, on increased oxidative stress and their implications in our experimental models. In the past, we have developed a sensitive method of using biotin-N-maleimide as a specific probe to identify oxidatively-modified mitochondrial proteins and their inactivation in animal models of alcoholic fatty liver (AFLD). During this fiscal year, we continued our study to identify and characterize oxidatively-modified proteins in animal models of nonalcoholic acute liver disease to understand the mechanisms of mitochondrial dysfunction, apoptosis and tissue injury. In this effort, we identified and characterized oxidatively-modified cytosolic proteins in rat livers exposed to MDMA (3,4-methylenedioxymethamphetamine, ecstasy), which is often co-abused in alcoholic individuals. Our results published in Proteomics showed that many cytosolic proteins including anti-oxidant enzymes such as Cu-Zn-dependent cytosolic superoxide dismutase (SOD1) and peroxiredoxin were oxidized and inactivated after MDMA exposure. Consequently, we observed increased oxidative stress with elevated levels of lipid peroxides, stress-activated protein kinases such as JNK and p38 kinase, and phosphorylated (inactivated) Bcl-2 or Bcl-XL, all contributing to apoptosis of hepatocytes. As another model of nonalcoholic acute liver injury, we also studied the mechanism of hepatic damage caused by lipopolysaccharide (LPS). Our data published in Toxicol Letters revealed that LPS causes severe liver damage in mice deficient of peroxisomal proliferator-activated receptor alpha (PPAR-alpha) through activating the STAT-1 related inflammatory signaling pathways and increased oxidative/nitrosative stress. However, we observed a very low level of tissue damage in wild-type mice, strongly suggesting the protective role of PPAR-alpha in regulating STAT1 inflammatory signaling pathways and oxidative/nitrosative stress. In addition to oxidation, nitration of tyrosine (Tyr) residues of many proteins is also important in regulating the activities of cellular proteins. By using Cyp2e1-null mice, we have recently demonstrated that CYP2E1 is involved in promoting nitration and ubiquitin-dependent degradation of many proteins. To study the role of protein nitration in mitochondrial dysfunction and tissue damage, we have developed a method to purify nitrated proteins from mitochondrial fractions in liver tissues treated with a CYP2E1 substrate acetaminophen (APAP) or LPS. Increased protein nitration in mitochondria was observed within 1 and 2 h after treatment with APAP (or LPS) followed by disappearance of nitrated proteins at 4 h, possibly through ubiquitin-mediated degradation of nitrated proteins. Based on these results, nitrated proteins were purified by affinity purification with the antibody to nitrated proteins. Mass spectral analysis of the purified nitrated proteins revealed nitration of many mitochondrial proteins. Biochemical properties of nitrated mitochondrial proteins are being investigated to establish the effects of nitration of these proteins on their biological functions. We have recently reported the critical role of the activated JNK in promoting cell death by phosphorylating critical proteins including pro-apoptotic Bax and mitochondrial ALDH2. To better understand the role of JNK in regulating mitochondrial function and cell/tissue damage through protein phosphorylation, we have initiated a study to identify and characterize JNK-mediated phosphorylation of many mitochondrial proteins. To specifically activate JNK without activating other mitogen-activated protein kinases, we chose a model of carbon tetrachloride (CCL4)-induced liver injury. We observed that activated JNK translocates to mitochondria and that many mitochondrial proteins are phosphorylated in a time-dependent manner after CCL4 exposure. To further characterize the phosphorylated mitochondrial proteins, we have purified phosphorylated mitochondrial proteins by affinity purification. We plan to determine the identities of the phosphorylated proteins by mass-spectral analysis followed by biochemical characterizations of some selected phospho-proteins to further establish the role of phosphorylation of mitochondrial proteins in promoting mitochondrial dysfunction and tissue injury. Although many animal models exist for studying the mechanisms of alcoholic and nonalcoholic fatty liver diseases (AFLD and NAFLD, respectively) without or with inflammation, the roles of PPAR-alpha and CYP2E1 in these areas have not been fully characterized. PPAR-alpha is a ligand-activated transcription factor involved in controlling the expression of many genes in the fatty acid transport, inflammatory reactions, and fat metabolism. Moreover, the expressed level of PPAR-alpha in humans is much lower (less than one tenth) than that in rodents, suggesting Ppara-null mice can be used as a good model for studying the mechanisms of AFLD and NAFLD in human conditions. We hypothesized that Ppara-null mice are very sensitive to organ damage compared to WT mice while Cyp2e1-null mice are very resistant to tissue damage caused by ethanol and other potentially toxic agents or various diets. Based on this hypothesis, we studied the mechanism of NAFLD in WT mice and Ppara-null mice fed a high fat diet (HFD). Age- and gender-matched WT and Ppara-null mice were fed a liquid HFD (70% energy derived from fat) or a standard liquid diet (STD, 35% energy derived from fat) ad libitum for 3 weeks in a 2 x 2 design. Ppara-null mice fed a HFD exhibited the highest levels of hepatocyte ballooning, steatosis, inflammation, and ultimately NASH activity score among the 4 groups. Elevated levels of CYP2E1, tumor necrosis factor-alpha, and lipid peroxides (e.g., malondialdehyde) were observed in HFD-fed Ppara-null mice. Consequently, protein nitration and oxidation were also increased in Ppara-null mice fed a HFD compared to their WT counterparts. Increased oxidative stress and inflammation were associated with activation of JNK and p38 kinase, contributing to increased hepatocyte apoptosis in Ppara-null mice fed a HFD compared with WT mice. These results published in J of Nutrition demonstrate that inhibition of PPAR-alpha functions may increase susceptibility to nonalcoholic steatohepatitis (NASH) in the presence of a HFD. On the other hand, Cyp2e1-null mice fed a HFD were relatively resistant to the development of NAFLD. Based on our own data recently published and unpublished, we believe that Ppara-null or Cyp2e1-null mice are very useful in studying the mechanisms of AFLD and NAFLD treated with an ethanol-liquid diet alone or in combination with another potentially toxic agent including a HFD or nicotine, simulating human conditions. Upon establishment of AFLD and NAFLD in these mouse strains, we plan to perform translational research by evaluating the beneficial effects of various anti-oxidants including docosahexaenoic acid (DHA) against AFLD and NAFLD in Ppara-null or Cyp2e1-null mice compared to WT mice.

Abstract  AIM: To investigate the effects of a Cetraria islandica extract in an animal model of hepatopathy.

MATERIAL AND METHODS: The experiments were carried out on Wistar rats, treated intraperitoneally (excepting carbon tetrachloride which was administered orally by esogastric tube), at single daily dose, for 28 days and divided in eight groups of 6 rats each: Group 1: Saline solution (0.5ml/100g bw/day); Group 2: MgSO4 (5mg/kbw/day); Group 3: C. islandica (Ci) extract (21.56mg/kbw/day); Group 4: Ci+MgSO4; Group 5: carbon tetrachloride (CT).(0.1 ml/100g bw/day); Group 6: CT+MgSO4; Group 7: CT+Ci; Group 8: CT+Ci+MgSO4. At the end of the experiment blood samples were taken to assess the effects on the blood parameters,TGO, TGP, GGT, LDH, serum total billirubin, neutrophil phagocytic function (Nitroblue tetrazolium test) and serum complement activity. This parameters were determined with VITROS 750 XRC device, using Johnson&Johnson kits. The results were statistically processed with the help of "t-Student" test. p<0.05 was considered statistically significant.

RESULTS: In this carbon tetrachloride-induced hepatopathy animal model, C. islandica extract demonstrated hepatoprotective and immuno-stimulating effects (proved by normalization of glutamic-pyruvic transminase, glutamic-oxalocetic transminase, serum total billirubin, stimulation of neutrophils percentage and phagocytic functions, decreasing the carbon tetrachloride-induced basophilia and monocytosis, especially in association with magnesium).

CONCLUSIONS: Further studies are required to confirm the benefits of this association in hepatoprotective and immunomodulating therapies.

Abstract  OBJECTIVE: To study the effects of decreased leptin expression on liver fibrosis.

METHODS: The small interfering RNA, targeting leptin gene, was designed according to the secondary structure of leptin gene. The recombinant plasmids were encapsulated with lipofectamine and then injected into carbon tetrachloride (CCl4) induced rat liver fibrosis models. Leptin and I, III collage were detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT-PCR).

RESULTS: The mRNA and protein levels of leptin in the fibrotic liver transfected with leptin shRNA were significantly decreased compared with those in controls (P less than 0.01). The depositions of type I and type III collagens were also decreased (P less than 0.01).

CONCLUSION: Decreased leptin expression prevents liver fibrosis.

Abstract  It is well known that isolation and cultivation of primary hepatocytes cause major gene expression alterations. In the present genome-wide, time-resolved study of cultivated human and mouse hepatocytes, we made the observation that expression changes in culture strongly resemble alterations in liver diseases. Hepatocytes of both species were cultivated in collagen sandwich and in monolayer conditions. Genome-wide data were also obtained from human NAFLD, cirrhosis, HCC and hepatitis B virus-infected tissue as well as mouse livers after partial hepatectomy, CCl4 intoxication, obesity, HCC and LPS. A strong similarity between cultivation and disease-induced expression alterations was observed. For example, expression changes in hepatocytes induced by 1-day cultivation and 1-day CCl4 exposure in vivo correlated with R = 0.615 (p < 0.001). Interspecies comparison identified predominantly similar responses in human and mouse hepatocytes but also a set of genes that responded differently. Unsupervised clustering of altered genes identified three main clusters: (1) downregulated genes corresponding to mature liver functions, (2) upregulation of an inflammation/RNA processing cluster and (3) upregulated migration/cell cycle-associated genes. Gene regulatory network analysis highlights overrepresented and deregulated HNF4 and CAR (Cluster 1), Krüppel-like factors MafF and ELK1 (Cluster 2) as well as ETF (Cluster 3) among the interspecies conserved key regulators of expression changes. Interventions ameliorating but not abrogating cultivation-induced responses include removal of non-parenchymal cells, generation of the hepatocytes' own matrix in spheroids, supplementation with bile salts and siRNA-mediated suppression of key transcription factors. In conclusion, this study shows that gene regulatory network alterations of cultivated hepatocytes resemble those of inflammatory liver diseases and should therefore be considered and exploited as disease models.

Abstract  To explore the differential proteome pattern in mouse fibrosis liver in comparison to wild type. Mice were fed with carbon tetrachloride or olive oil vehicle for 15 weeks. Mouse livers from both groups were collected and submitted to MS platform for proteome screening. GO (Gene Ontology) biological process and KEGG (Kyoto Enyoolpedia of Genes and Genomes) pathway enrichment analysis were used to analyze differentially expressed proteins. As the results, we identified 17 382 and 20 486 unique peptides in control and carbon tetrachloride-induced groups, respectively. A total of 4 991 proteins (at least 1 unique peptide matched) were identified, of which 2 135 were differentially expressed (> or = 2 fold). In fibrosis mouse liver 1 264 proteins were up regulated and 871 proteins were down regulated. Proteins associated with DNA replication, cell cycle, ECM-receptor interaction, and splicesome were significantly increased in carbon tetrachloride-induced group. Proteins associated with small molecule metabolic process, protein transport, organonitrogen compound metabolic process, and tetrapyrrole biosynthetic processes were down regulated in carbon tetrachloride-induced mouse liver fibrosis tissue. Bioinformatics findings showed that fibrosis was closely related to the regulation of VEGF and T cell receptor signaling pathway, and further suggested that liver fibrosis was a complex signal transduction process that many biological processes such as liver metabolism, inflammation, and immune response are involved. Based this study, we can envision that protection of protein metabolism in liver parenchymal cells and blocking of inflammatory signaling transduction may be beneficial for liver fibrosis therapy.

Abstract  OBJECTIVE: To explore attenuation and mechanism of endoplasmic reticulum stress (ERS)-mediated hepatocyte apoptosis in rats with alcohol-induced liver injury by Qinggan Huoxue Recipe (QGHXR) and its disassembled formulas (Qinggan Recipe and Huoxue Recipe respectively).

METHODS: A rat model of chronic alcoholic liver injury was successfully established using a compound reagent of alcohol, corn oil, and pyrazol. The modeled rats were randomly divided into the model group, the QGHXR group, the Qinggan Recipe (QGR) group, and the Huoxue Recipe group (HXR). The CCl4 control group and the normal control group were also set up. There were ten rats in each group. All rats of modeled groups were gastrogavaged with alcohol compound reagent every morning. Rats in the QGHXR group (at the daily dose of 9. 5 g/kg, QGR group (at the daily dose of 3.0 g/kg), and HXR group (at the daily dose of 6.5 g/kg) were administered with corresponding medicines by gastrogavage every afternoon. Equal volume of normal saline was given to rats of the model group by gastrogavage. CCl4 was intraperitoneally injected at the dose of 0.3 mL/kg to rats in the CCl4 control group, once per week. Normal saline was given to rats in the normal control group by gastrogavage. The treatment was lasted for two weeks. Pathological changes of the liver were observed by histopathology. Serum total homocysteine (tHCY) level was detected by ELISA. The hepatocyte apoptosis rate was detected using flow cytometry. The gene and protein expressions of eukaryotic translation initiation factor 2 alpha (elF-2alpha), phosphorylation elF-2alpha (pelF-2alpha), glucose-regulated protein 78 (GRP78), and Caspase-3 in the liver were examined using Real-time PCR and Westen blot respectively.

RESULTS: Compared with the normal control group, typical pathological changes of chronic alcoholic liver injury such as steatosis, inflammation, and even fibrosis occurred in model rats. The hepatocyte apoptosis obviously increased, with the apoptosis rate reaching the five-fold of that in normal rats. Besides, early apoptosis dominated. The serum tHCY level significantly increased. The expressions of p-elF-2alpha, GRP78, and Caspase-3 protein obviously increased (P < 0.01). Expressions of GRP78 and Caspase-3 mRNA significantly increased (P < 0.05, P < 0.01). Compared with the model group, the degrees of the liver injury and the hepatocyte apoptosis in the QGHXR group, the QGR group, and the HXR group were significantly alleviated. The serum tHCY level was significantly lowered. The protein expressions of p-elF-2a, GRP78, and Caspase-3 obviously decreased (P < 0.01). mRNA expressions of GRP78 and Caspase-3 obviously decreased in the QGHXR group (P < 0.05, P < 0.01). Only GRP78 mRNA expression obviously decreased in the QGR group (P < 0.05).

CONCLUSION: QGHXR and its disassembled formulas could attenuate ERS-mediated hepatocyte apoptosis in alcohol-induced liver injury rats by lowering the serum tHCY level and expressions of ERS apoptosis correlated factors.

Abstract  Hepatic metallothionein (MT) expression, with various isoforms, and varying cellular localizations is a useful marker for clinico-pathogenesis of liver diseases. In acute liver toxicity caused by cadmium, carbon tetrachloride, or acetaminophen, MT plays a protective role, via the scavenging of radical species. In chronic hepatitis C patients, hepatic MT levels appear to be a biological factor associated with the severity of HCV infection, and are associated with a better response to IFN therapy. Transgenic mice that express HBsAg in the liver show hepatocellular damage, inflammation, regeneration, hyperplasia, and, eventually, neoplasia. The MT isoform, MT-1 help mitigate HBV-induced hepatitis. Analysis of MT gene expression in the livers of chronic hepatitis B patients is useful for understanding the features of distinct liver diseases and for judging disease progression. A profound down-regulation of isoform MT-1G in hepatocellular carcinoma was observed in 63% of tumors relative to the adjacent nonmalignant liver. MT has been implicated in the control of p53 folding with zinc exchange. Therefore, it appears MT may play a role in the pathogenesis of hepatocellular carcinoma. Overall MT is linked to a variety of liver diseases.

Abstract  The aim of this study is to investigate whether a Ca-deficient diet has an attenuating effect on carbon tetrachloride (CCl4)-induced hepatotoxicity. Four-week-old male ddY mice were fed a Ca-deficient diet for 4 weeks as a part of the experimental protocol. While hypocalcemia was observed, there was no significant change in body weight. The CCl4-exposed hypocalcemic mice exhibited a significant decrease in alanine aminotransferase and aspartate aminotransferase activities at both 6 h and 24 h even though markers of renal function remained unchanged. Moreover, lipid peroxidation was impaired and total antioxidant power was partially recovered in the liver. Studies conducted in parallel with the biochemical analysis revealed that hepatic histopathological damage was attenuated 24 h post CCl4 injection in hypocalcemic mice fed the Ca-deficient diet. Finally, this diet impaired CCl4-induced inflammatory responses. Although upregulation of Ca concentration is a known indicator of terminal progression to cell death in the liver, these results suggest that Ca is also involved in other phases of CCl4-induced hepatotoxicity, via regulation of oxidative stress and inflammatory responses.

Abstract  OBJECTIVE: To evaluate therapeutic and antioxidant effects of Uygur Herb Foeniculum Vulgare Mill (FVM) in hepatic fibrosis rats.

METHOD: Hepatic fibrosis model was built in rats by subcutaneous injection with 40% CCl4 olive oil mixture. At the same time the rats were given high lipoid-low protein animal feeds for 5 weeks. 94 male SD rats were randomly divided into six groups :blank control group (A-group), 8 rats were feed in normal; prevention model control group (B-group), 10 rats were given saline solution by intragastric administration during make of hepatic fibrosis model; FVM prevention group (C-group), 10 rats were given FVM by intragastric administration during make of hepatic fibrosis model; model control group (D-group), FVM treatment group (E-group); Fuzhenghuayu treatment group (F-group). 22 rats in each D, E, F-group were respectively given saline solution, FVM and Fuzhenghuayu by intragastric administration after hepatic fibrosis model were built. At the 5-th weekend, A, B, C- group rats were sacrificed. At the 6-th, 7-th, 8-th, 9-th weekend, 4-6 rats in D, E, F-group were sacrificed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) and 8 - hydroxy-2-deoxyguanosine (8-OHdG) were detected, liver tissue homogenate superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), malondialdehyde (MDA) were detected. Histopathologic changes were observed after H.E and Masson staining. The expression of alpha-smooth muscle actin(a-SMA) were detected by immunohistochemical staining. The data were analyzed by SPSS17.0 software.

RESULTS: The serum levels of ALT, AST, HA, and LN in the FVM prevention group were significantly reduced compared to the prevention model control group.(P less than 0.05). Rats in FVM treatment group appeared a marked lower serum levels of ALT, AST, HA compared to the model control group (P less than 0.05), and a distinguished lower Inflammation grade and fibrosis stage (P less than 0.05) when the liver section were assayed as well; Rats in FVM treatment group and FVM prevention group had a conspicuous lower content of MDA, 8-OHdG, fibre and a-SMA expression (P less than 0.05), a significantly higher level of SOD, GSH-Px compared to those of in the model control groups.

CONCLUSIONS: Foeniculum Vulgare Mill declines liver inflammation response ,and prevent the hepatic fibrosis progression,, this may be due to its effects of antioxidative results.

Abstract  OBJECTIVE: To investigate the role of Src family kinases (Fgr, Hck, Lyn) and the major protein kinase C substrate SSeCKS in non-alcoholic steatohepatitis (NASH) and determine the possible mechanism regulating differential expression.

METHODS: Kupffer cells were stimulated with CCL4 and effect on SSeCKS, Hck, Fgr, and Lyn expression was detected by real-time reverse transcription-PCR. Male Sprague-Dawley rats were used to create a NASH model by feeing a high fat diet. The modeled rats were divided into a model group and a normal group. After sacrifice, the extent of hepatic steatosis and inflammation was assessed, and the expression levels of SSeCKS and Hck, Fgr, Lyn were detected by immunohistochemical staining.

RESULTS: Expression of Lyn and Hck was decreased in the CCL4-stimulated Kupffer cells and the change in expression level was positively associated with levels of inflammatory stimuli (P < 0.01). The change in expression of SSeCKS in the CCL4-stimulated Kupffer cells was negatively correlated with inflammatory stimuli (P < 0.01). Fgr expression was very low in the unstimulated Kupffer cells and was not affected by the exposure to inflammatory stimuli. The number of inflammatory cells in the liver tissues of rars were negatively correlated with expression of Lyn, Hck and SSeCKS (P < 0.01), with low negative correlation for Lyn (r =-0.398, P < 0.01) and moderate negative correlation for Hck (r=-0.508, P < 0.01); the Lyn and Hck expression levels were highly positively correlated (r =0.942, P < 0.01).

CONCLUSION: Src family kinases (Lyn, Hck and Fgr) and SSeCKS are involved in development and progression of NASH, and their differential expression patterns are associated to a certain extent. The factors may represent potential targets of therapy for NASH-related inflammation.

Abstract  OBJECTIVE: To explore the effects and the molecular mechanisms of 4-phenylbutyric acid (PBA) on carbon tetraehloride (CCl4)-induced acute liver injury in mice.

METHODS: Sixty adult, healthy, male ICR mice were divided equally into the control group, PBA group, CCl4 12 h group, CCl4 24 h group, CCl4 48 h group, CCl4 72 h group, PBA+CCl4 12 h group, PBA+CCl4 24 h group, PBA+CCl4 48 h group, and PBA+CCl4 72 h group. The CCl4 groups and the PBA+CCl4 groups were intraperitoneally (i.p.) injected with CCl4 (300 mL/kg). In the PBA+CCl4 groups, the mice were i.p. injected with PBA (400 mg/kg). All mice were sacrificed to collect blood and liver specimens at different time points after the CCl4 treatment. Serum alanine aminotransferase (ALT) was detected. Histological examination was performed using hematoxylin-eosin staining and light microscopy, and apoptosis was detected using terminal transferase dUTP nick end labeling. The hepatic distribution of proliferating cell nuclear antigen (PCNA) was detected by immunohistochemistry. The hepatic protein expression of glucose-regulated protein (GRP78), C/EBP homologousprotein (CHOP), phosphorylated c-Jun N-terminal kinases (p-JNK), phosphorylated eukaryotic initiation factor 2a subunit (p-eIF2a), phosphorylated serine threonine kinase (p-akt), and nuclear factor-kappa B p65 (NF-kappa Bp65) were determined by western blot.

RESULTS: The serum ALT level in the PBA+CCl4 groups was reduced as compared with that in the CCl4 groups at the various time points examined.The liver-to-body weight ratio of two groups showed a significant difference only at the 48 h time point (P<0.01). PBA reduced the degree of hepatic necrosis and apoptosis caused by CCl4, and reduced the expression of hepatic GRP78 and other endoplasmic reticulum stress-related proteins (P<0.01). The protein levels of p-akt, NF-kappa Bp65 and PCNA was significantly decreased in the PBA+CCl4 groups (P<0.01).

CONCLUSION: The endoplasmic reticulum stress inhibitor PBA alleviated acute hepatic necrosis and apoptosis but restrained hepatic proliferation.

Abstract  OBJECTIVE: To compare the bidirectional effect of rhubarb total anthraquinone (TA) and total tannins (TT) on rats' liver.

METHODS: One hundred rats were randomly divided into 10 groups, i.e., the blank group, the model group, the blank + high dose TA group, the blank +low dose TA group, the blank + high dose TT group, the blank + low dose TT group, the model + high dose TA group, the model + low dose TA group, the model +high dose TT group, and the model + low dose TT group, 10 in each group. The carbon tetrachloride (CCI4) was used to prepare the acute liver injury rat model. TA and TT of rhubarb (at 5.40 g crude drugs/kg and 14.69 g crude drugs/kg) were intragastrically administrated to rats in all groups except the blank group and the model group, once daily for 6 successive days.The general state of rats, biochemical indices such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), laminin (LN), hyaluronic acid (HA), transforming growth factor beta1 (TGF-beta1), as well pathological results of rat liver tissues. Finally the protection laws of TA and TT for rats' liver were analyzed using factor analysis.

RESULTS: Compared with the blank control group, all biochemical indices increased in the blank group (P < 0.05, P < 0.01). HA also increased in the blank + high dose TA group; AST, ALT, and HA also increased in the blank +high dose TT group (P < 0.05). Compared with the model group, AST, ALT, ALP, HA, and TGF-beta1 significantly decreased in the model + low dose TA group, the model + high dose TA group, the model + low dose TT group (P < 0.05, P < 0.01). Serum AST, ALT, and ALP also decreased in the model + high dose TT group (P < 0.05, P < 0.01). Pathological results showed that mild swollen liver cells in the model + high dose TA group. Fatty degeneration and fragmental necrosis around the central veins occurred in the blank + high dose TA group. The pathological injury was inproved in the model +low dose TA group. Two common factors, liver fibrosis and liver cell injury, were extracted by using factor analysis. TA showed stronger improvement of the two common factors than TT.

CONCLUSIONS: Rhubarb TA and TT showed protective and harmful effects on rats' liver. At an equivalent dosage, TA had better liver protection than TT. High dose TT played a role in liver injury to some extent.

Abstract  This study is designed to explore the possible effects of Hemerocallis citrina baroni flavonids (HCBF) on liver fibrosis induced by CCl4 in rats. The liver fibrosis model was induced by CCl4, and HCBF were administered by gastric perfusion at 25 and 50 mg x kg(-1) qd for 50 days, while the contents of alanine transaminase (ALT), aspartate aminotransferase (AST), gamma glutamyl transpeptidase (GGT), alkaline phosphatase (ALP), superoxide dismutase (SOD), maleic dialdehyde (MDA) and transforming growth factor-β1 (TGF-β1) were measured and the contents of PINP were measured in liver tissue, and the expression of TGF-β1 were observed by immunohistochemisty and Western blot. The pathological changes of liver tissue were examined by HE. The results showed that HCBF (25, 50 mg x kg(-1)) improved the liver function significantly through reducing the level of ALT, AST, GGT and ALP (P < 0.05 or P < 0.01), and increasing the content of SOD (P < 0.01), while reducing the content of MDA (P < 0.05 or P < 0.01), the expression of TGF-β1 (P < 0.05) and the content of PINP (P < 0.05). The results suggest that HCBF (25, 50 mg x kg(-1)) may inhibit the liver injury induced by CCl4 by decreasing the oxidative stress.

Abstract  OBJECTIVE: To investigate the changes in Smad 2, 3, 4 and 7 of the transforming growth factor-beta 1 (TGF-b1)/Smad signaling pathways in carbon tetrachloride (CCL4)-induced hepatic fibrosis rats treated with TGF-b1 small interfering (si)RNA.

METHODS: Rats were randomly divided among five groups: non-fibrotic (normal); fibrosis-induced (model); fibrotic treated with 0.125 mg/kg TGF-b1 siRNA; fibrotic treated with 0.250 mg/kg TGF-b1 siRNA; and fibrotic treated with negative control TGF-b1 siRNA. The expression of Smad 2, 3, 4 and 7 was detected by real-time polymerase chain reaction (for mRNA), immunohistochemistry and Western blotting (for protein).

RESULTS: The mRNA and protein levels of Smad 2, 3 and 4 were significantly lower in the the fibrotic rats treated with either 0.250 mg/kg or 0.125 mg/kg TGF-b1 siRNA than in the fibrotic model or the negative control TGF-b1 siRNA rats (P less than 0.01). Moreover, the mRNA and protein expression levels of Smad 2, 3 and 4 were significantly lower in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05). Comparing the 0.250 mg/kg and 0.125 mg/kg TGF-b1 siRNA groups to the model group and the TGF-b1 siRNA negative control group showed significantly increased levels of mRNA and protein expression of Smad 7 (P less than 0.01). In addition, the expression levels of Smad 7 were significantly higher in the 0.250 mg/kg TGF-b1 siRNA group than in the 0.125 mg/kg group (P less than 0.05).

CONCLUSION: siRNA-mediated silencing of TGF-b1 in rats led to significantly reduced expression of Smad 2, 3 and 4, but significantly increased expression of Smad 7. TGF-b1 regulation of Smad signaling molecules may contribute to hepatic fibrosis in rats and represent a target of future therapeutic intervention.