D-galactosamine Treatment Research Articles

Protective Mechanisms of Juncus effusus and Carbonized Juncus effusus against D-Galactosamine-Induced Acute Liver Injury in Mice.

This study investigated the hepatoprotective effects of Juncus effusus (J. effusus) and Carbonized J. effusus against liver injury caused by D-galactosamine (D-GalN) in mice. J. effusus and Carbonized J. effusus were administered by gavage once daily starting seven days before the D-GalN treatment. The results of the study indicated that J. effusus and Carbonized J. effusus suppressed the D-GalN-induced generation of serum alanine transaminase (ALT), aspartate aminotransferase (AST), hepatic malondialdehyde (MDA) and tumor necrosis factor-alpha (TNF-α) was observed. The values of superoxide dismutase (SOD) exhibited an increase. In addition, J. effusus and Carbonized J. effusus promoted the protein expression of nuclear factor erythroid 2-related factor 2 (Nrf2), NADPH quinone oxidoreductase-1 (NQO-1), heme oxygenase-1 (HO-1) as well as the mRNA expression of Nrf2, HO-1, NQO-1 and Glutamate cysteine ligase catalytic subunit (GCLC). The compressed Carbonized J. effusus demonstrated the optimum impact. These results suggest that J. effusus and Carbonized J. effusus protect against D-GalN-induced acute liver injury through the activation of the Nrf2 pathway.

Omeprazole Increases Survival Through the Inhibition of Inflammatory Mediaters in Two Rat Sepsis Models.

ABSTRACTBackground:Omeprazole (OMZ) is a proton pump inhibitor that is used to reduce gastric acid secretion, but little is known about its possible liver protective effects. This study investigated whether OMZ has beneficial effects in rat septic models of LPS-induced liver injury after D-galactosamine (GalN) treatment and 70% hepatectomy (PH), and to determine the mechanisms of OMZ in an in vitro model of liver injury.Methods:In the in vivo models, the effects of OMZ were examined 1 h before treatments in both models on survival, nuclear factor (NF)-κB activation, histopathological analysis, and proinflammatory mediator expression in the liver and serum. In the in vitro model, primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of OMZ. The influence of OMZ on nitric oxide (NO) product and inducible NO synthase (iNOS) induction and on the associated signaling pathway was analyzed.Results:OMZ increased survival and decreased tumor necrosis factor-alpha, iNOS, cytokine-induced neutrophil chemoattractant 1, IL-6, and IL-1β mRNA expression, and increased IL-10 mRNA expression in the livers of both GaIN/LPS- and PH/LPS-treated rats. Necrosis and apoptosis were inhibited by OMZ in GaIN/LPS rats, but OMZ had no effects on necrosis in PH/LPS rats. OMZ inhibited iNOS induction partially through suppression of NF-κB signaling in hepatocytes.Conclusions:OMZ inhibited the induction of several inflammatory mediators, resulting in the prevention of LPS-induced liver injury after GalN liver failure and PH, although OMZ showed different doses and mechanisms in the two models.

Possible Association Between DHEA and PKCε in Hepatic Encephalopathy Amelioration: A Pilot Study.

Objective: Hepatic encephalopathy (HE) is a neuropsychiatric syndrome caused by liver failure and by an impaired neurotransmission and neurological function caused by hyperammonemia (HA). HE, in turn, decreases the phosphorylation of protein kinase C epsilon (PKCε), contributing to the impairment of neuronal functions. Dehydroepiandrosterone (DHEA) exerts a neuroprotective effect by increasing the GABAergic tone through GABAA receptor stimulation. Therefore, we investigated the protective effect of DHEA in an animal model of HE, and the possible modulation of PKCε expression in different brain area.Methods: Fulminant hepatic failure was induced in 18 male, Sprague–Dawley rats by i.p. administration of 3 g/kg D-galactosamine, and after 30 min, a group of animals received a subcutaneous injection of 25 mg/kg (DHEA) repeated twice a day (3 days). Exploratory behavior and general activity were evaluated 24 h and 48 h after the treatments by the open field test. Then, brain cortex and cerebellum were used for immunoblotting analysis of PKCε level.Results: DHEA administration showed a significant improvement of locomotor activity both 24 and 48 h after D-galactosamine treatment (****p < 0.0001) but did not ameliorate liver parenchymal degeneration. Western blot analysis revealed a reduced immunoreactivity of PKCε (*p < 0.05) following D-galactosamine treatment in rat cortex and cerebellum. After the addition of DHEA, PKCε increased in the cortex in comparison with the D-galactosamine-treated (***p < 0.001) and control group (*p < 0.05), but decreased in the cerebellum (*p < 0.05) with respect to the control group. PKCε decreased after treatment with NH4Cl alone and in combination with DHEA in both cerebellum and cortex (****p < 0.0001). MTS assay demonstrated the synergistic neurotoxic action of NH4Cl and glutamate pretreatment in cerebellum and cortex along with an increased cell survival after DHEA pretreatment, which was significant only in the cerebellum (*p < 0.05).Conclusion: An association between the DHEA-mediated increase of PKCε expression and the improvement of comatose symptoms was observed. PKCε activation and expression in the brain could inhibit GABA-ergic tone counteracting HE symptoms. In addition, DHEA seemed to ameliorate the symptoms of HE and to increase the expression of PKCε in cortex and cerebellum.

Forsythia Fruit Prevents Fulminant Hepatitis in Mice and Ameliorates Inflammation in Murine Macrophages.

Forsythia Fruit (FF), the fruit of Forsythia suspensa, has been used since ancient times as an herbal medication in East Asia to treat inflammation, gonorrhea, and pharyngitis. However, the efficacy of FF against liver damage due to inflammation has not been studied. Here, we explored the protective effects of FF in a mouse hepatitis model induced by lipopolysaccharide (LPS)/D-galactosamine (GalN) treatment. We measured inflammatory cytokine and aminotransferase levels in mouse blood and analyzed the effects of FF on inflammatory gene and protein expression levels in liver tissue. Our results show that FF treatment effectively lowers inflammatory cytokine and serum aminotransferase levels in mice and inhibits the expression of hepatic cytokine mRNA and inflammatory proteins. Furthermore, treatment with FF activated the antioxidant pathway HO-1/Nrf-2 and suppressed severe histological alteration in the livers of LPS/D-GalN-treated mice. Further investigation of the effects of FF on inflammatory reactions in LPS-stimulated macrophages showed that pretreatment with FF inhibits inflammatory mediator secretion and activation of inflammatory mechanisms both in a mouse macrophage RAW 264.7 cells and in primary peritoneal macrophages. These results show that FF has potential worth as a candidate for the treatment of fulminant inflammatory reactions and subsequent liver injury.

Verapamil inhibits early acute liver failure through suppressing the NLRP3 inflammasome pathway.

Acute liver failure (ALF) is a rare disease characterized by the sudden onset of serious hepatic injury, as manifested by a profound liver dysfunction and hepatic encephalopathy in patients without prior liver disease. In this paper, we aim to investigate whether verapamil, an antagonist of TXNIP, inhibits early ALF through suppressing the NLRP3 inflammasome pathway. Firstly, an ALF mouse model was induced by lipopolysaccharide (LPS)/D‐galactosamine (GalN) treatment. The optimal concentration of verapamil in treating early ALF mice was determined followed by investigation on its mechanism in LPS/GalN‐induced liver injury. Western blot analysis and co‐immunoprecipitation were performed to determine the activation of the TXNIP/NLRP3 inflammasome pathway. Subsequently, overexpression of NLRP3 in mouse liver was induced by transfection with AAV‐NRLP3 in vivo and in vitro to identity whether verapamil inhibited early ALF through suppressing the activation of NLRP3 inflammasome. We found that ALF was induced by LPS/GalN in mice but was alleviated by verapamil through a mechanism that correlated with suppression of the NLRP3 inflammasome pathway. Oxidative stress and inflammatory response were induced by intraperitoneal injection of LPS/GalN, but alleviated with injection of verapamil. Overexpression of NLRP3 via AAV in mouse liver in vivo and in vitro reduced the therapeutic effect of verapamil on LPS/GalN‐induced ALF. Taken together, the TXNIP antagonist verapamil could inhibit activation of the NLRP3 inflammasome, inflammatory responses and oxidative stress to alleviate LPS/GalN‐induced ALF.

Ratio of Creatine Kinase to Alanine Aminotransferase as a Biomarker of Acute Liver Injury in Dystrophinopathy.

Objective To investigate the ratios of creatine kinase (CK) to aminotransferases as biomarkers of acute liver injury in dystrophinopathy. Methods C57 and mdx (dystrophic) mice were treated with a hepatotoxic reagent D-galactosamine (D-GalN). The degrees of liver and muscle injury were assessed using histological examinations. To examine whether serum CK-adjusted aminotransferase levels could indicate liver status in dystrophic mice, the CK/alanine aminotransferase (ALT) and CK/aspartate aminotransferase (AST) ratios were analyzed. Furthermore, we enrolled 658 male patients with dystrophinopathy and 378 male patients without muscle and liver injury as control, whose serum ALT, AST, and CK levels were examined. Results Animal experiments indicated that D-GalN treatment could induce acute liver injury but not muscle injury. Additionally, D-GalN decreased the CK/ALT and CK/AST ratios in both C57 mice and mdx mice (P < 0.001). However, there was an overlap of the CK/AST ratio between dystrophic mice with and without acute liver injury. In patients with dystrophinopathy, CK-adjusted ALT diminished the variability associated with age, genotype, clinical phenotype, and motor function (P > 0.05). Conclusions CK/ALT is a potential biomarker for the differential evaluation of acute liver injury in dystrophic mice, which highlights the value to further evaluate the practice of CK/ALT in dystrophinopathy patients.

Andrographolide ameliorates d-galactosamine/lipopolysaccharide-induced acute liver injury by activating Nrf2 signaling pathway.

Andrographolide (ADH), a diterpenoid lactone extracted from Andrographis paniculata, has been found to have anti-inflammatory and anti-oxidative effects. However, its protective effects and mechanisms on liver injury have not been investigated clearly. This study takes an attempt to reveal the protective effects and mechanism of ADH on lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute liver injury in mice. The mice liver injury model was induced by LPS (60 mg/kg) and D-GalN (800 mg/kg), and ADH was given 1 h after LPS and D-GalN treatment. Hepatic tissue histology was measured by H&E staining. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were detected by detection kits. The levels of TNF-α and IL-1β were detected by ELISA. Moreover, malondialdehyde (MDA) and reactive oxygen species (ROS) contents were also detected. Meanwhile, the expression of Nrf2, HO-1, and NF-κB were detected by western blot analysis. The results showed that ADH treatment improved liver histology and decreased the levels of ALT, AST, MPO, IL-1β, TNF-α, as well as MDA and ROS levels of hepatic tissues in a dose-dependent manner. ADH also inhibited LPS/D-GalN-induced NF-κB activation. The expression of Nrf2 and HO-1 were increased by treatment of ADH. In conclusion, ADH protected against LPS/D-GalN-induced liver injury by inhibiting NF-κB and activating Nrf2 signaling pathway.

Protective effects of morin on lipopolysaccharide/d-galactosamine-induced acute liver injury by inhibiting TLR4/NF-κB and activating Nrf2/HO-1 signaling pathways

Morin, a bioactive flavonoid extracted from the bark of Moraceae plants and many medicinal herbs, has anti-inflammatory and antioxidative effects. In this research, we explored the protective effects of morin against lipopolysaccharide (LPS) and d-galactosamine (D-GalN) induced acute liver injury in mice. Mice were given an intraperitoneal injection of morin before LPS and D-GalN treatment and the HepG2 cells were only given morin to investigate its effects. The results showed that morin markedly inhibited the production of serum alanine transaminase (ALT), aspartate aminotransferase (AST), interleukin-6 (IL-6), tumor necrosis factor (TNF-α) and hepatic TNF-α, IL-6, and myeloperoxidase (MPO) induced by LPS/D-GalN. In order to evaluate morin effect in the future, we investigated the expression of nuclear factor E2 related factor 2 (Nrf2), nuclear factor-kappaB (NF-κB), toll like receptor 4 (TLR4) on liver injury. Taken together, these results suggested that morin could exert the anti-inflammatory and anti-oxidative effects against LPS/D-GalN-induced acute liver injury by activating Nrf2 signal pathways and inhibiting NF-κB activation.

Mangiferin alleviates lipopolysaccharide and D-galactosamine-induced acute liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation

Mangiferin, a glucosylxanthone from Mangifera indica, has been reported to have anti-inflammatory effects. However, the protective effects and mechanisms of mangiferin on liver injury remain unclear. This study aimed to determine the protective effects and mechanisms of mangiferin on lipopolysaccharide (LPS) and D-galactosamine (D-GalN)-induced acute liver injury. Mangiferin was given 1h after LPS and D-GalN treatment. The results showed that mangiferin inhibited the levels of serum ALT, AST, IL-1β, TNF-α, MCP-1, and RANTES, as well as hepatic malondialdehyde (MDA) and ROS levels. Moreover, mangiferin significantly inhibited IL-1β and TNF-α production in LPS-stimulated primary hepatocytes. Mangiferin was found to up-regulate the expression of Nrf2 and HO-1 in a dose-dependent manner. Furthermore, mangiferin inhibited LPS/d-GalN-induced hepatic NLRP3, ASC, caspase-1, IL-1β and TNF-α expression. In conclusion, mangiferin protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

Antidiabetic drug metformin is effective on the metabolism of asymmetric dimethylarginine in experimental liver injury

AimsWe aimed to investigate the pharmacological efficiency of metformin on asymmetric dimethylarginine (ADMA) metabolism in inflammation caused by the lipopolysaccharide (LPS)/d-galactosamine (d-GalN) treatment. MethodsAdult Sprague-Dawley rats were injected LPS/d-GalN intraperitoneally. One half of the animals was injected metformin (250mgkg−1 body mass for one week) prior to LPS/d-GalN treatment. Six hours after the LPS/d-GalN injection, livers were removed, and used for the measurements of dimethylarginine dimethylaminohydrolase (DDAH) and myeloperoxidase (MPO) activities, glutathione (GSH), ADMA and arginine levels. Liver tissues were examined histopathologically. The Kruskal–Wallis (posthoc Mann–Whitney U) test was used for the statistics.LPS/d-GalN injections caused liver injury as evidenced by the activities of aminotransferases and arginase. GSH level and DDAH activity were decreased in the liver. Metformin pretreatment alleviated the activity of serum enzymes, and attenuated histopathological lesions caused by LPS/d-GalN injections. LPS/d-GalN-induced inflammation, as confirmed by the increased MPO activity, created an asymmetrical distribution of arginine and ADMA between the tissue and plasma. Metformin decreased tissue ADMA level while it restored the DDAH activity and GSH. ConclusionOur findings showed that metformin administration for one week has a potency to protect liver through regulating ADMA metabolism in LPS/d-GalN-induced injury.

결명자 추출물이 D-Galactosamine 유발 간 손상에 미치는 영향

This study was performed to evaluate the biological activity and protective effect of Cassia tora ethanol extracts against D-galactosamine induced hepatotoxicity in rats. Male Sprague-Dawley rats were grouped into normal group, D-galactosamine treated group(control), D-galactosamine plus 0.25% Cassia tora extracts treated group and D-galactosamine plus 0.5% Cassia tora extracts treated group. Normal and control group were fed control diet and Cassia tora extracts treated groups were fed experimental diets containing 0.25% or 0.5% Cassia tora ethanol extracts for 5 weeks. Body weight gain and liver weight of rats were not significantly different between groups. Cholesterol and triglyceride concentrations in serum and liver were significantly lower in rats treated only with D-galactosamine compared to normal group, and improved in Cassia tora extracts supplemented rats. D-galactosamine treatment significantly increased serum aspartate transaminase, alanine transaminase, gamma glutamyl transferase and alkaline phosphatase, however, the activities of aspartate transaminase and alanine transaminase were significantly decreased in Cassia tora extracts supplemented rats when compared with D-galactosamine treated control group. Cassia tora extracts significantly suppressed the D-galactosamine-induced elevation of liver thiobarbituric acid reactive substances(TBARS) contents. Superoxide dismutase activity was decreased by D-galactosamine treatment, however by the supplementation of Cassia tora ethanol extracts, significantly increased in dose-dependent manner. Glutathione peroxidase activity in rats fed diets containing Cassia tora extracts was decreased compared to control. Based on these results, we concluded that Cassia tora ethanol extracts may prevents the D-galactosamine-induced hepatotoxicity probably via an antioxidant mechanism.

Protective Effects of HV-P411 Complex Against D-Galactosamine-Induced Hepatotoxicity in Rats

This study examined the hepatoprotective effect of the HV-P411 complex, an herbal extract mixture from the seeds of Vitis vinifera, Schisandra chinensis and Taraxacum officinale, against D-galactosamine (D-GalN)-induced hepatitis. Hepatotoxicity was induced by D-GalN (700 mg/kg, i.p.), and the HV-P411 complex was administered orally 48, 24, and 2 h before and 6 h after D-GalN injection. Increases in serum aminotransferase activity and lipid peroxidation and a decrease in hepatic glutathione content were attenuated by the HV-P411 complex 24 h after D-GalN treatment. The HV-P411 complex attenuated the increases in serum tumor necrosis factor-α, interleukin (IL)-6 level and cyclooxygenase-2 protein production and their mRNA expressions, while increases in serum IL-10 level and heme oxygenase-1 protein production and their mRNA expressions were augmented by the HV-P411 complex. The increased translocation of nuclear factor-κB and c-Jun phosphorylation were attenuated by treatment with the HV-P411 complex. Our results suggest that the HV-P411 complex prevents D-GalN-induced hepatotoxicity via antioxidative and anti-inflammatory activities.

Protective effects of Mg-CUD against D-galactosamine-induced hepatotoxicity in rats

This study examined the protective effects of magnesium chenoursodeoxycholic acid (Mg-CUD), a magnesium trihydrate salt of chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), against D-galactosamine (D-GalN)-induced liver injury. Hepatotoxicity was induced by intraperitoneal injection of D-GalN (700 mg/kg) and Mg-CUD (15.625, 31.25 and 62.5 mg/kg) was administered orally once a day for 2 weeks and 6 h after D-GalN injection. Significant increases in the level of serum alanine aminotransferase activity and lipid peroxidation were attenuated by Mg-CUD 24 h after D-GalN treatment. Hepatic glutathione/oxidized glutathione ratio was decreased, and this decrease was attenuated by Mg-CUD. Mg-CUD attenuated the increase in the levels of serum tumor necrosis factor (TNF)-α and interleukin (IL)-6, while it augmented the increase in serum IL-10 level and heme oxygenase (HO)-1 protein expression. Mg-CUD attenuated increased levels of TNF-α, IL-6, and IL-1β mRNA expression. Increased levels of IL-10 and HO-1 mRNA expression were augmented by Mg-CUD. The increased nuclear level of nuclear factor-κB (NF-κB) and decreased cytosolic level of Inhibitory κB-α protein were attenuated by Mg-CUD. Nuclear phosphorylated c-Jun (p-c-Jun) level showed a significant increase and this increase was attenuated by Mg-CUD. Our results suggest that Mg-CUD ameliorates D-GalN-induced acute hepatitis and that this protection is likely due to its anti-oxidative and anti-inflammatory activities, and inhibition of NF-κB nuclear translocation and nuclear p-c-Jun expression.

MyD88-dependent pro-inflammatory cytokine response contributes to lethal toxicity of staphylococcal enterotoxin B in mice

An elevated pro-inflammatory cytokine response is the primary cause of death by toxic shock after exposure to staphylococcal enterotoxin B (SEB). Identifying an intracellular signal mediator that predominantly controls the pro-inflammatory response is important for developing a therapeutic strategy. We examined the role of the signaling adaptor MyD88 in cell culture and in a mouse model of toxic shock. Our results indicated that elevated tumor necrosis factor-α, interferon-γ, interleukin (IL)-1α/β and IL-6 production from mouse spleen cells treated with SEB alone or in combination with lipopolysaccharide (LPS) was regulated by MyD88. Elevated levels of MyD88 protein in spleen cells, as well as in CD11c(+) or Mac3(+) cells, and activation of nuclear factor-κB in spleen cells were observed in mice treated with SEB. An SEB-dose dependent lethality was observed in LPS-potentiated and in D-galactosamine-sensitized mice. D-Galactosamine treatment of spleen cells had no effect in cytokine induction but rather increased the sensitivity to toxic shock in mice. Our results demonstrated an impaired pro-inflammatory cytokine production by spleen cells of MyD88(-/-) mice in response to SEB or SEB plus LPS. Most importantly, MyD88(-/-) mice were resistant to SEB-induced death. These results demonstrate that MyD88-dependent pro-inflammatory signaling is responsible for SEB intoxication. In addition, our studies also demonstrated that LPS potentiation, in comparison to D-galactosamine sensitization, contributes to a stronger SEB-induced lethality. This is due to the pro-inflammatory cytokine response elicited by MyD88 after exposure to SEB and LPS. These findings offer an important insight upon SEB intoxication and subsequent therapy targeting MyD88.

Staphylococcal enterotoxin A induction of pro‐inflammatory cytokines and lethality in mice is primarily dependent on MyD88

Staphylococcal enterotoxin (SE) -induced toxic shock is triggered by inflammatory cytokine signal amplification after SE binding to major histocompatibility complex class II molecules on antigen-presenting cells and T-cell receptors. Identifying host cellular elements contributing to this pro-inflammatory signal amplification is critical for developing a strategy for therapeutic intervention. Myeloid differentiation primary-response protein 88 (MyD88) is an intracellular signalling adaptor protein primarily known for mediating pro-inflammatory cytokine responses. We investigated the role of MyD88 in staphylococcal enterotoxin A (SEA) -treated cell cultures and mouse models of toxic shock. Our results demonstrated that elevated levels of tumour necrosis factor-alpha, interferon-gamma, interleukin-1alpha/beta (IL-1alpha/beta), IL-2 and IL-6 production correlated with up-regulation of MyD88 after treatment of spleen cells and mice with SEA alone or in combination with lipopolysaccharide (LPS). The SEA-induced lethality was also observed in (LPS-independent) D-galactosamine-sensitized mice. While LPS potentiated SEA-induced cytokine responses, D-galactosamine treatment had no additive effect. Most importantly, our results demonstrated that MyD88(-/-) mice were resistant to SEA-induced toxic shock and had reduced pro-inflammatory cytokine responses. These results suggest that SEA-induced lethality is primarily dependent on MyD88. Our findings offer an important insight on potential therapeutic treatment of SEA-induced toxic shock targeting MyD88.

Quantitative Analyses and Transcriptomic Profiling of Circulating Messenger Rnas As Biomarkers of Rat Liver Injury

Serum aminotransferases have been the clinical standard for evaluating liver injury for the past 50-60 years. These tissue enzymes lack specificity, also tracking injury to other tissues. New technologies assessing tissue-specific messenger RNA (mRNA) release into blood should provide greater specificity and permit indirect assessment of gene expression status of injured tissue. To evaluate the potential of circulating mRNAs as biomarkers of liver injury, rats were treated either with hepatotoxic doses of D-(+)-galactosamine (DGAL) or acetaminophen (APAP) or a myotoxic dose of bupivacaine HCl (BPVC). Plasma, serum, and liver samples were obtained from each rat. Serum alanine aminotransferase and aspartate aminotransferase were increased by all three compounds, whereas circulating liver-specific mRNAs were only increased by the hepatotoxicants. With APAP, liver-specific mRNAs were significantly increased in plasma at doses that had no effect on serum aminotransferases or liver histopathology. Characterization of the circulating mRNAs by sucrose density gradient centrifugation revealed that the liver-specific mRNAs were associated with both necrotic debris and microvesicles. DGAL treatment also induced a shift in the size of plasma microvesicles, consistent with active release of microvesicles following liver injury. Finally, gene expression microarray analysis of the plasma following DGAL and APAP treatment revealed chemical-specific profiles. The comparative analysis of circulating liver mRNAs with traditional serum transaminases and histopathology indicated that the circulating liver mRNAs were more specific and more sensitive biomarkers of liver injury. Further, the possibility of identifying chemical-specific transcriptional profiles from circulating mRNAs could open a range of possibilities for identifying the etiology of drug/chemical-induced liver injury.

Hepatocyte Transplantation and the Differentiation Fate of Host Oval Cells in Acute Severe Hepatic Injury

Oval cells and hepatocytes rarely proliferate simultaneously. This study aimed to determine the impacts of hepatocyte transplantation on the response and fate of oval cells that are activated to proliferate in acute severe hepatic injury. Retrorsine + D-galactosamine (R+D-gal) treatment was used to induce acute hepatic injury and to elicit extensive activation of oval cells in male dipeptidyl peptidase IV-deficient F344 rats. These rats were then randomized to receive wild-type hepatocyte transplantation or vehicle intraportally. The kinetics of oval cell response and their differentiation fate were analyzed. Results showed that oval cells were activated early and differentiated into hepatocytes in R+D-gal-treated rats without hepatocyte transplantation. With hepatocyte transplantation, the oval cells were recruited later and continued to proliferate in parallel with the massive proliferation of transplanted hepatocytes. They formed ductules and differentiated into biliary cells. When hepatocytes were transplanted at the day when oval cells were at their peak response, the numerous activated oval cells ceased to differentiate into hepatocytes and remained in ductular form. The ductular oval cells were capable of differentiating into hepatocytes again when the donor hepatocytes were inhibited to proliferate. We conclude that hepatocyte transplantation changes the mechanism of liver reconstitution and affects the differentiation fate of host oval cells in acute severe hepatic injury.

Impaired Hepatocyte Regeneration in Acute Severe Hepatic Injury Enhances Effective Repopulation by Transplanted Hepatocytes

Efficient repopulation by transplanted hepatocytes in the severely injured liver is essential for their clinical application in the treatment of acute hepatic failure. We studied here whether and how the transplanted hepatocytes are able to efficiently repopulate the toxin-induced acute injured liver. Male dipeptidyl peptidase IV-deficient F344 rats were randomized to receive retrorsine plus D-galactosamine (R+D-gal) treatment or D-galactosamine-alone (D-gal) to induce acute hepatic injury, and retrorsine-alone. In these models, retrorsine was used to inhibit the proliferation of endogenous hepatocytes while D-galactosamine induced acute hepatocyte damage. Wild-type hepatocytes (1 x 10(7)/ml) were transplanted intraportally 24 h after D-galactosamine or saline injection. The kinetics of proliferation and repopulation of transplanted cells and the kinetics of cytokine response, hepatic stellate cell (HSC) activation, and matrix metalloproteinase (MMP2) expression were analyzed. We observed that early entry of transplanted hepatocytes into the hepatic plates and massive repopulation of the liver by transplanted hepatocytes occurred in acute hepatic injury induced by R+D-gal treatment but not by D-gal-alone or retrorsine-alone. The expressions of transforming growth factor-alpha and hepatocyte growth factor genes in the R+D-gal injured liver were significantly upregulated and prolonged up to 4 weeks after hepatocyte transplantation. The expression kinetics were parallel with the efficient proliferation and repopulation of transplanted hepatocytes. HSC was activated rapidly, markedly, and prolongedly up to 4 weeks after hepatocyte transplantation, when the expression of HGF gene and repopulation of transplanted hepatocytes were reduced afterward. Furthermore, the expression kinetics of MMP2 and its specific distribution in the host areas surrounding the expanding clusters of transplanted hepatocytes are consistent with those of activated HSC. Impaired hepatocyte regeneration after acute severe hepatic injury may initiate serial compensatory repair mechanisms that facilitate the extensive repopulation by transplanted hepatocytes that enter early the hepatic plates.

Bioimaging nitric oxide in activated macrophages in vitro and hepatic inflammation in vivo based on a copper–naphthoimidazol coordination compound

Nitric oxide (NO) serves as a messenger for cellular signaling and physiological reactions such as inflammatory responses in vivo. Fluorescent bioimaging of nitric oxide is a very useful tool in NO functional research. Although many encouraging results have been achieved in the field of NO fluorescent detection, there is rarely satisfying result in inflammatory NO imaging in vivo. Here we report that fluorescent 5′-chloro-2-(2′-hydroxyphenyl)-1H-naphtho[2,3- d]imidazol can coordinate with Cu(II) to form a non-fluorescent coordination compound, which is able to directly and quickly image NO in cellular system or in vivo inflammation system with a turn-on fluorescence, based on a redox action of Cu(II). It was used to image NO produced by inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS) activated murine macrophages. More importantly, it could image the NO production in an acute severe hepatic injury (ASHI) model of BALB/c mice induced by integrative LPS and d-galactosamine (GalN) treatment. The results prove that the 5′-chloro-2-(2′-hydroxyphenyl)-1H-naphtho[2,3- d]imidazol coordinated with cupric ions can serve as an excellent NO bioimaging agent in different biological systems especially in inflammation related systems, and it may be valuable for diagnostic and pathological studies of NO related diseases.

Modulation of Spontaneous and Lipopolysaccharide-Induced Nitric Oxide Production and Apoptosis by D-Galactosamine in Rat Hepatocyte Culture: The Significance of Combinations of Different Methods

ABSTRACTApoptotic markers and signals produced by xenobiotics as hepatotoxic D-galactosamine (D-GalN) and lipopolysaccharide (LPS) are extensively investigated in vivo. The contribution of various cells and factors as nitric oxide (NO) in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia was reported. Therefore, the aim of the present work was to study the in vitro effect of D-GalN on nonstimulated or LPS-treated rat hepatocytes in culture and the potential involvement of NO in this process. Our results showed that the spontaneous and LPS-induced NO production was completely blocked by D-GalN during 0 to 24 hours. However, D-GalN slightly enhanced NO production during 24 to 48 hours. D-GalN was more potent to induce hepatocyte apoptosis and necrosis during 24 to 48 than 0 to 24 hours as evidenced morphologically (Annexin V/propidium iodide staining) and biochemically (caspase-3-like activity, alanine-aminotransferase leakage, MTT test). Interestingly, D-GalN treatment suppressed mitochondrial cytochrome C release throughout the study. LPS addition to D-GalN considerably aggravated apoptotic/necrotic markers only during 0 to 24 hours. Surprisingly, a share of apoptotic cells was distinctly lower after LPS + GalN treatment than after LPS alone during 0 to 24 hours, while 24- to 48-hour incubation produced massive apoptotic/necrotic hepatocytes. It may be concluded that there is a significant modulation of NO production by D-GalN. Because the role of NO is only partly decisive in the apoptotic/necrotic events, and considering the fraction of the cells completing apoptosis while others that turn toward necrosis (aponecrosis), caution should be exercised in apoptosis data interpretation and combinations of different test methods should be applied.