Biotinylated Oligo-α-(1 → 4)-d-galactosamines and Their N-Acetylated Derivatives: α-Stereoselective Synthesis and Immunology Application.

Using 3-O-benzoyl-4,6-O-di-tert-butylsilylidene-2-azido-2-deoxy-selenogalactoside, biotinylated oligo-α-(1 → 4)-d-galactosamines comprising from two to six GalN units were prepared for the first time together with their N-acetylated derivatives. The combination of blocking groups used herein provided stereocontrol for the α-stereospecific glycosylation, to show also high efficiency of phenyl 2-azido-2-deoxy-selenogalactosides as glycosyl donors. The obtained glycoconjugates are related to fragments of exopolysaccharide galactosaminogalactan (GG) found in Aspergillus fumigatus, which is the most important airborne human fungal pathogen in industrialized countries. The synthesized glycoconjugates were arrayed on streptavidin-coated plates and used to investigate the GG epitopes recognized by mouse monoclonal antibodies against GG and by human antibodies in the sera of patients with aspergillosis. The obtained data showed that the oligo-α-(1 → 4)-d-galactosamines and their N-acetylated derivatives allowed the first precise analysis of the specificity of the antibody responses to this extremely complex fungal polysaccharide.

Luzindole attenuates LPS/d-galactosamine-induced acute hepatitis in mice.

Melatonin is a well-documented hormone that plays central roles in the regulation of sleep-wake cycles. There is cumulative evidence to suggest that melatonin is also a pleiotropic regulator of inflammation, and luzindole has been widely used as a melatonin receptor antagonist. This study investigated the potential effects of luzindole on LPS/d-galactosamine (d-GalN)-induced acute hepatitis. The results indicated that treatment with luzindole alleviated histological damage in the liver, reduced the level of transaminases in plasma and improved the survival of LPS/d-GalN-exposed mice. Treatment with luzindole also suppressed the production of the pro-inflammatory cytokines TNF-α and IL-6 in LPS/d-GalN-exposed mice. In addition, treatment with luzindole inhibited the activation of caspase-3, -8 and -9, and suppressed the cleavage of caspase-3 and poly(ADP-ribose) polymerase. Therefore, treatment with luzindole attenuates LPS/d-GalN-induced acute liver injury, suggesting that luzindole might have potential value for the intervention of inflammation-based hepatic disorders.

Protective effects of ginsenoside Rg1 against lipopolysaccharide/d-galactosamine-induced acute liver injury in mice through inhibiting toll-like receptor 4 signaling pathway.

Acute liver injury (ALI) is a dramatic liver disease characterized by large areas of inflammation in the liver. This study aimed to investigate the protective effects of ginsenoside Rg1 (Rg1), a biologically active component in Panax ginseng, on lipopolysaccharide/d-galactosamine (LPS/D-GalN)-induced ALI in mice, and meanwhile explore the molecular mechanism in vivo and in vitro. Mice were pretreated with Rg1 for three days prior to LPS (40 µg/kg)/D-GalN (700 mg/kg) administration. The results showed that Rg1 improved the survival rate and reduced the liver to body weight ratios in mice. Rg1 also reduced the production of oxidative markers such as MDA and MPO induced by LPS/D-GalN. In addition, Rg1 significantly decreased the production of inflammatory cytokines including TNF-α, IL-6, IL-1ß, Mip-2, Mcp-1, iNOS, and increased the activity of anti-inflammatory cytokine IL-10. Moreover, Rg1 inhibited the protein expression of TLR4 and its downstream genes including NF-κB and MAPKs, which are involved in inflammatory response. Rg1 dramatically reduced oxidative stress by regulating the expression of efflux transporters Mrp2 and various enzymes including GCLC, GCLM, HO-1 and NQO1. However, the changes in these genes and protein induced by Rg1 were abrogated by TLR4 antagonist TAK-242 in vitro. In conclusion, Rg1 had hepatoprotective effect on LPS/D-GalN-induced ALI in mice. The protection may be associated with the inhibition of TLR4. These findings suggest that Rg1 may be a promising agent for prevention against ALI.

Oxyresveratrol prevents lipopolysaccharide/d-galactosamine-induced acute liver injury in mice.

Oxyresveratrol (Oxy) is a natural polyhydroxystilbene abundant in mulberry that has anti-inflammation and anti-oxidant activities. We evaluated the protective effect of Oxy in the context of the lipopolysaccharide and d-galactosamine (LPS/d-GalN) induced acute liver injury. Oxy restricted the development of histopathological changes, markedly reduced the activity of alanine transaminase (ALT) and aspartate transaminase (AST), which are indicators of impaired liver function. Oxy significantly regulated the contents of oxidative stress related enzymes and products, and inhibited expressions of inflammatory mediators and cytokines. Oxy treatment diminished the Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling pathway in liver, activated the Kelch-like ECH-associated protein 1(Keap1)-nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, and increased expressions of heme oxygenase 1 (HO-1) and quinine oxidoreductase 1(NQO1). Pretreatment with Oxy decreased LPS/d-GalN stimulated hepatocyte apoptosis by efficaciously raising the B-cell lymphoma 2 (Bcl-2)/Bcl-2 associated X (Bax) ratio, inhibiting the expression and activation of caspases, and activating the phosphoinoside-3-kinase (PI3K)-Akt pathway. Our results demonstrate the hepatoprotective efficacy of Oxy. The protection is mainly due to the prevention of TLR4/NF-κB pathway activation, induced activation of Keap1-Nrf2 signaling pathway, and decreased hepatocyte apoptosis. Oxy warrants further study as a potential therapeutic agent candidate for the management of acute liver injury.

Toll-Like Receptor Ligand-Induced Liver Injury in D-Galactosamine-Sensitized Mice: Differences between TLR7/8 and TLR9 Ligands, Cytokine Patterns, and Cross-Tolerance Induction by TLR2 Ligand Pretreatment.

Administration of Toll-like receptor ligands (TLRLs) is known to cause liver injury in D-galN-sensitized mice. In the present study, we aimed to complement preceding reports on the TLRL/D-galN system by analyzing comparisons among TLRLs, mouse strain dependence, effects on serum levels of cytokines, and effects of sequential administrations of different TLRLs. In a preliminary set of analyses, we first confirmed that liver failure can be induced by diverse TLRLs, including LTA and R848 in combination with D-galN. Analysis using TLR4-deficient mice excluded potential confounding effects of endogenous TLR4Ls that include those referred to as DAMPs in CpG DNA/D-galN hepatotoxicity. Subsequently, we showed that LTA pretreatment could prevent mortality in both CpG DNA/D-galN- and R848/D-galN-treated mice compared to without pretreatment. Incidentally, we observed that without the LTA pretreatment, CpG DNA/D-galN showed relatively higher liver-specific toxicity whereas R848/D-galN showed more symptoms of multiple organ failure. These findings suggest that, in D-galN-sensitized mice, different TLRLs not only show similarity in the ability to induce hepatic injury but also exhibit distinctive abilities in inducing systemic inflammation and multiple organ failure. These findings also suggest the potential usefulness of cross-tolerance induction using LTA in the prevention of organ failure in TLRL-mediated acute inflammation.

Preventive effects of interleukin-6 in lipopolysaccharide/d-galactosamine induced acute liver injury via regulating inflammatory response in hepatic macrophages.

Lipopolysaccharide/d-Galactosamine (LPS/d-Gal)-induced acute liver injury is characterized by significant inflammatory responses including TNF-α and interleukin-6 (IL-6) and is a widely applied experimental model for inflammation research. TNF-α is critical in the progression of LPS/d-Gal-induced liver injury. However, the role of IL-6 in this model is still unknown. In the present study, we aim to elucidate the involvement of IL-6 in the pathogenesis of acute liver injury induced by LPS/d-Gal in mice and its underlying mechanism. To induce acute liver injury, LPS (50µg/kg body weight) and d-Gal (400mg/kg body weight) were injected intraperitoneally in the C57BL/6 mice. The vehicle (saline) or a single dose of recombinant IL-6 (200µg/kg body weight) was administered 2h prior to LPS/d-Gal injection. Mice were sacrificed 2h and 6h after LPS/d-Gal injection. The results indicated that IL-6 treatment could protect mice from LPS/d-Gal-induced tissue damage, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) elevation, as well as hepatocyte apoptosis and inflammation. Furthermore, in vitro study showed that IL-6 treatment could significantly suppress LPS-triggered expression of proinflammatory cytokines and chemokines, TNF-α, RANTES and MCP-1 in macrophages while promoting the expression of M2 markers, such as Arg-1 and Mrc-1 in macrophages. Taken together, these findings revealed a novel and unexpected role of IL-6 in ameliorating LPS/d-Gal-induced acute liver injury via regulating inflammatory responses in hepatic macrophages.

CQMUH-011, a novel adamantane sulfonamide compound, inhibits lipopolysaccharide- and D-galactosamine-induced fulminant hepatic failure in mice.

CQMUH-011, a novel adamantane sulfonamide compound, was shown to suppress macrophage activation and proliferation in our previous study. However, it is unknown whether CQMUH-011 has anti-inflammatory and hepatoprotective properties. In this study, we investigated the potential effects and mechanisms of CQMUH-011 on lipopolysaccharide (LPS)-induced RAW264.7 cell activation in vitro and LPS- and D-galactosamine (D-GalN)-induced fulminant hepatic failure (FHF) in vivo. The results showed that in RAW264.7 cells challenged by LPS, CQMUH-011 inhibited cell proliferation and induced cell cycle arrest and apoptosis. Furthermore, CQMUH-011 reduced tumor necrosis factor (TNF)-α and interleukin (IL)-1ß production and down-regulated the overexpression of toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB induced by LPS in RAW264.7 cells. In vivo, CQMUH-011 reduced serum levels of aspartic aminotransferase and alanine transaminase and improved the mortality and hepatic pathological damage induced by LPS/D-GalN in mice. Moreover, CQMUH-011 significantly inhibited the serum levels of proinflammatory mediators, including TNF-α, IL-6, IL-1ß, nitric oxide (NO), and prostaglandin E2 (PGE2), and down-regulated the protein expression of TLR4, p38 mitogen-activated protein kinases, NF-κB, NF-κB inhibitor α (IκBα), IκB kinase ß (IKKß), cyclooxygenase-2 (COX-2) and inducible NO synthases (iNOS) induced by LPS/D-GalN in mice. In conclusion, these results demonstrated that CQMUH-011 has a notable anti-inflammatory effect and protects mice from LPS/D-GalN-induced FHF and that the molecular mechanisms might be related to the inhibition of the TLR4/NF-κB signaling pathway activation, the subsequent decrease in proinflammatory mediator production, and the inhibition of macrophage activation.

Antioxidation, anti-inflammation and anti-apoptosis by paeonol in LPS/d-GalN-induced acute liver failure in mice.

To evaluate the hepatoprotective effects and potential mechanisms of paeonol (Pae) against acute liver failure (ALF) induced by lipopolysaccharide (LPS)/d-galactosamine (d-GalN) in mice, we examined anti-oxidative, anti-inflammatory and anti-apoptotic activities of Pae. We found that Pae pretreatment markedly reduced the activities of alanine transaminase and aspartate transaminase as well as the histopathological changes induced by LPS/d-GalN. Catalase, glutathione and superoxide dismutase activities increased and reactive oxygen species activity decreased after Pae treatment compared with LPS/d-GalN treatment. Pretreatment with Pae also significantly inhibited the expression levels of iNOS, nitric oxide (NO), COX-2 and prostaglandin E2 (PGE2). In addition, Pae administration prevented the phosphorylated expression of IκB kinase, inhibitor kappa B in the nuclear factor-kappa B (NF-κB) signaling pathway, and suppressed the phosphorylated expression of extracellular signal-regulated kinase (ERK), c-jun-N-terminal kinase and p38 in the MAPK signaling pathway. Pretreatment with Pae also inhibited hepatocyte apoptosis by reducing the expression of caspases 3, 8, 9, and Bax, and increasing Bcl-2. In total, protective effects of Pae against LPS/d-GalN-induced ALF in mice are attributed to its antioxidative effect, inflammatory suppression in NF-κB and MARK signaling pathways, and inhibition of hepatocyte apoptosis inhibition. Therefore, Pae can be a potential therapeutic agent in attenuating LPS/d-GalN-induced ALF in the future.

Interleukin-23 mediates the pathogenesis of LPS/GalN-induced liver injury in mice.

BACKGROUND: Interleukin-23 (IL-23) is required for T helper 17 (Th17) cell responses and IL-17 production in hepatitis B virus infection. A previous study showed that the IL-23/IL-17 axis aggravates immune injury in patients with chronic hepatitis B virus infection. However, the role of IL-23 in acute liver injury remains unclear. OBJECTIVE: The purpose of this study was to determine the role of the inflammatory cytokine IL-23 in lipopolysaccharide/d-galactosamine (LPS/GalN)-induced acute liver injury in mice. METHODS: Serum IL-23 from patients with chronic hepatitis B virus (CHB), acute-on-chronic liver failure (ACLF) and healthy individuals who served as healthy controls (HCs) was measured by ELISA. An IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody was administered intravenously at the time of challenge with LPS (10µg/kg) and GalN (400mg/kg) in C57BL/6 mice. Hepatic pathology and the expression of Th17-related cytokines, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and the stabilization factor Csf3 were assessed in liver tissue. RESULTS: Serum IL-23 was significantly upregulated in ACLF patients compared with CHB patients and HCs (P<0.05 for both). Serum IL-23 was significantly upregulated in the non-survival group compared with the survival group of ACLF patients, which was consistent with LPS/GalN-induced acute hepatic injury in mice (P<0.05 for both). Moreover, after treatment, serum IL-23 was downregulated in the survival group of ACLF patients (P<0.001). Compared with LPS/GalN mice, mice treated with either an IL-23p19 neutralizing antibody or an IL-23p40 neutralizing antibody showed less severe liver tissue histopathology and significant reductions in the expression of Th17-related inflammatory cytokine, including IL-17 and TNF-α; neutrophil chemoattractants, including Cxcl1, Cxcl2, Cxcl9, and Cxcl10; and stabilization factors Csf3 within the liver tissue compared with LPS/GalN mice (P<0.05 for all). CONCLUSION: High serum IL-23 was associated with mortality in ACLF patients and LPS/GalN-induced acute liver injury in mice. IL-23 neutralizing antibodies attenuated liver injury by reducing the expression of Th17-related inflammatory cytokines, neutrophil chemoattractants and stabilization factors within the liver tissue, which indicated that IL-23 likely functions upstream of Th17-related cytokine and chemokine expression to recruit inflammatory cells into the liver.

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.

The angiotensin-converting enzyme inhibitor captopril rescues mice from endotoxin-induced lethal hepatitis.

The renin-angiotensin system is classically regarded as a crucial regulator of circulatory homeostasis, but recent studies also revealed its pro-inflammatory roles. The beneficial effects of the angiotensin-converting enzyme inhibitor (ACEI) in severe inflammatory injury in the lung and heart have been previously reported, but its potential effects on lethal hepatitis were unknown. In this study, a mouse model with LPS/d-galactosamine (GalN)-induced fulminant hepatitis were used to test the protective potential of captopril, a representative ACEI. The results indicated that treatment with captopril significantly decreased the plasma level of alanine aminotransferase and aspartate aminotransferase, alleviated the histopathological damage of the liver tissue and improve the survival rate of LPS/GalN-challenged mice. These effects were accompanied by reduced mRNA levels of TNF-α and IL-6 in the liver, and decreased protein level of TNF-α and IL-6 in the plasma. In addition, the activation of caspases 3, 8 and 9, and the presence of TUNEL-positive apoptotic cells, were also suppressed by captopril treatment. The above evidence suggested that the renin-angiotensin system might be involved in the development of LPS/GalN-induced fulminant hepatitis and ACEI might have potential value in lethal hepatitis.

Tamoxifen Attenuates Lipopolysaccharide/Galactosamine-induced Acute Liver Failure by Antagonizing Hepatic Inflammation and Apoptosis.

Bacterial lipopolysaccharide (LPS)-induced acute liver failure (ALF) is a common severe clinical syndrome in intensive care unit. No other methods are available for its prevention apart from supportive treatment and liver transplantation. Tamoxifen (TAM) was reported to attenuate ALF induced by excessive acetaminophen, while its effect on LPS-induced ALF remained unknown. For this, in the present study, we comprehensively assessed whether TAM can attenuate ALF induced by LPS/galactosamine (GaIN). Mice were given TAM once a day for three times. Twelve hours after the last treatment, mice were given LPS/GaIN (intraperitoneally [i.p.]). Survival, plasma transaminases, and histopathology were examined. Serum TNF-α and IL-1ß were analyzed by ELISA. Hepatic apoptosis was analyzed by TUNEL and caspase-3 Western blotting, respectively. Compared to the model group, ALF induced by LPS/GaIN was alleviated remarkably following TAM administration, as evidenced by the improvement of survival (87.5% vs. 37.5%), hepatic swell, moderate transaminases, slightly increased serum TNF-α, IL-1ß (P < 0.05), and moderate histopathology. In respect of apoptosis, severe hepatocellular apoptosis was reduced notably by TAM treatment confirmed by less TUNEL-positive hepatocytes and decreased caspase-3 cleavage. The results demonstrated that TAM could attenuate LPS/GaIN-induced ALF effectively, probably due to hepatic inflammation and apoptosis antagonism. Furthermore, it was the first report about the effect of TAM on LPS/GaIN-induced ALF.

Biochanin A protects lipopolysaccharide/D-galactosamine-induced acute liver injury in mice by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

Biochanin A, an isoflavone existed in red clover and peanuts, has been reported to possess a wide spectrum of pharmacological activities, such as anti-inflammatory and antioxidant effects. However, the protective effects and mechanism of biochanin A on liver injury have not been reported. In this study, acute liver injury was induced by intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (D-GalN). Biochanin A was administrated 1h prior to LPS/D-GalN challenge. Serum ALT, AST, IL-1ß, and TNF-α levels, hepatic malondialdehyde (MDA), GPx, SOD, and Catalase contents, tissue histology, IL-1ß, TNF-α, NLRP3, and Nrf2 expression were detected. The results showed that serum ALT, AST, IL-1ß, and TNF-α levels and hepatic MDA content increased after LPS/GalN treatment. These changes were attenuated by biochanin A. Meanwhile, biochanin A dose-dependently up-regulated the expression of Nrf2 and HO-1. Biochanin A also inhibited hepatic IL-1ß and TNF-α expression in a dose-dependent manner. Biochanin A did not inhibit LPS/D-GalN-induced hepatic NLRP3, ASC, and caspase-1 expression. However, the interaction of NLRP3 with ASC and caspase-1 were inhibited by biochanin A. In addition, LPS/D-GalN-induced up-regulation of thioredoxin-interacting protein (TXNIP) and interaction between TXNIP and NLRP3 were also inhibited by biochanin A. In conclusion, biochanin A protected against LPS/GalN-induced liver injury by activating the Nrf2 pathway and inhibiting NLRP3 inflammasome activation.

Preparation and evaluation of luteolin-phospholipid complex as an effective drug delivery tool against GalN/LPS induced liver damage.

CONTEXT: The phyto-phospholipid complexation technique is a promising approach to improve the bioavailability and efficacy of flavonoids. OBJECTIVE: The objective of this study was to improve the bioavailability and efficacy of luteolin by phospholipid complexation against inflammatory liver damage. MATERIALS AND METHODS: The phospholipid complex of luteolin (LPC) was prepared by solvent evaporation accompanied by freeze drying. The physicochemical properties of LPC were investigated by means of spectroscopy, differential scanning calorimetry (DSC) and X-ray diffraction (XRD). Pharmacokinetic parameters in rats were determined and the hepatoprotective potential was assessed against D-galactosamine and lipopolysaccharide (GalN/LPS) induced hepatic damage. RESULTS: LPC showed drug loading of 74.14% and average particle size 147.4 nm. The results of FTIR, thermal and diffraction studies confirmed the formation of complex. The aqueous/n-octanol solubility showed improvements. LPC showed an increase in relative in vivo bioavailability to 535.31% of pure luteolin. The histological and biochemical changes induced by GalN/LPS were significantly ameliorated by LPC. DISCUSSION: Hepatoprotective effect of LPC was more profound than luteolin with a particle size suitable for passive targeting of inflammatory sites. CONCLUSION: LPC was successfully formulated under optimized conditions and is an efficient drug delivery system for oral administration of luteolin with enhanced bioavailability and hepatoprotective potential.

The presence of a galactosamine substituent on the arabinogalactan of Mycobacterium tuberculosis abrogates full maturation of human peripheral blood monocyte-derived dendritic cells and increases secretion of IL-10.

Slow-growing and pathogenic Mycobacterium spp. are characterized by the presence of galactosamine (GalN) that modifies the interior branched arabinosyl residues of the arabinogalactan (AG) that is a major heteropolysaccharide cell wall component. The availability of null mutants of the polyprenyl-phospho-N-acetylgalactosaminyl synthase (Rv3631, PpgS) and the (N-acetyl-) galactosaminyl transferase (Rv3779) of Mycobacterium tuberculosis (Mtb) has provided a means to elucidate the role of the GalN substituent of AG in terms of host-pathogen interactions. Comparisons of treating human peripheral blood monocyte-derived dendritic cells (hPMC-DCs) with wild-type, Rv3631 and Rv3779 mutant strains of Mtb revealed increased expression of DC maturation markers, decreased affinity for a soluble DC-SIGN probe, reduced IL-10 secretion and increased TLR-2-mediated NF-κB activation among GalN-deficient Mtb strains compared to GalN-producing strains. Analysis of surface expression of a panel of defined or putative DC-SIGN ligands on both WT strains or either Rv3631 or Rv3779 mutant did not show significant differences suggesting that the role of the GalN substituent of AG may be to modulate access of the bacilli to immunologically-relevant receptor domains on DCs or contribute to higher ordered pathogen associated molecular pattern (PAMP)/pattern recognition receptor (PRR) interactions rather than the GalN-AG components having a direct immunological effect per se.

Emodin ameliorated lipopolysaccharide-induced fulminant hepatic failure by blockade of TLR4/MD2 complex expression in D-galactosamine-sensitized mice.

Emodin has been reported to possess anti-inflammatory and anti-oxidant activities. The aim of this study was to explore the effect and mechanism of emodin on lipopolysaccharide (LPS)-induced fulminant hepatic failure (FHF) in D-galactosamine (D-GalN)-sensitized mice. Our results showed that pretreatment with emodin inhibited the elevation of plasma aminotransferases, alleviated the hepatic histopathological abnormalities and improved the survival rate of LPS/D-GalN-primed mice. Moreover, emodin markedly attenuated the increased serum and hepatic tumor necrosis factor-α (TNF-α) production, and activated hepatic p38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signal pathways in LPS/D-GalN-challenged mice. Furthermore, using an in vitro experiment, we found that emodin dose-dependently suppressed TNF-α production, dampened AP-1 and NF-κB activation, and blocked toll-like receptor (TLR) 4/myeloid differentiation factor (MD) 2 complex expression in LPS-elicited RAW264.7 mouse macrophage cells. Taken together, these data suggested that emodin could effectively prevent LPS-induced FHF, which might be mediated by inhibition of TNF-α production, deactivation of MAPKs and NF-κB, and blockade of TLR4/MD2 complex expression.

Thymoquinone attenuates liver fibrosis via PI3K and TLR4 signaling pathways in activated hepatic stellate cells.

Thymoquinone (TQ) is the major active compound derived from the medicinal Nigella sativa. In the present study, we investigated the anti-fibrotic mechanism of TQ in lipopolysaccharide (LPS)-activated rat hepatic stellate cells line, T-HSC/Cl-6. T-HSC/Cl-6 cells were treated with TQ (3.125, 6.25 and 12.5µM) prior to LPS (1µg/ml). Our data demonstrated that TQ effectively decreased activated T-HSC/Cl-6 cell viability. TQ significantly attenuated the expression of CD14 and Toll-like receptor 4 (TLR4). TQ also significantly inhibited phosphatidylinositol 3-kinase (PI3K) and serine/threonine kinase-protein kinase B (Akt) phosphorylation. The expression of α-SMA and collagen-I were significantly decreased by TQ. Furthermore, TQ decreased X linked inhibitor of apoptosis (XIAP) and cellular FLIP (c-FLIPL) expression, which are related with the regulation of apoptosis. Furthermore, TQ significantly increased the survival against LPS challenge in d-galactosamine (d-GlaN)-sensitized mice, and decreased the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which were in line with in vitro results. Our data demonstrated that TQ attenuates liver fibrosis partially via blocking TLR4 expression and PI3K phosphorylation on the activated HSCs. Therefore, TQ may be a potential candidate for the therapy of hepatic fibrosis.

The protective role of the transmembrane thioredoxin-related protein TMX in inflammatory liver injury.

AIMS: Accumulating evidence indicates that oxidative stress is associated with inflammation, and the cellular redox status can determine the sensitivity and the final outcome in response to inflammatory stimuli. To control the redox balance, mammalian cells contain a variety of oxidoreductases belonging to the thioredoxin superfamily. The large number of these enzymes suggests a complex mechanism of redox regulation in mammals, but the precise function of each family member awaits further investigations. RESULTS: We generated mice deficient in transmembrane thioredoxin-related protein (TMX), a transmembrane oxidoreductase in the endoplasmic reticulum (ER). When exposed to lipopolysaccharide (LPS) and d-(+)-galactosamine (GalN) to induce inflammatory liver injury, mutant mice were highly susceptible to the toxicants and developed severe liver damage. LPS-induced production of inflammatory mediators was equivalent in both wild-type and TMX(-/-) mice, whereas neutralization of the proinflammatory cytokine tumor necrosis factor-α suppressed the toxic effects of LPS/GalN in the mutant mice. Liver transcriptional profiles revealed enhanced activation of the p53-signaling pathway in the TMX(-/-) mice after LPS/GalN treatment. Furthermore, TMX deficiency also caused increased sensitivity to thioacetamide, which exerts its hepatotoxicity through the generation of reactive oxygen species. INNOVATION: The present study is the first to address the role of the oxidoreductase TMX in inflammatory liver injury. The phenotype of mice deficient in TMX suggests a functional link between redox regulation in the ER and susceptibility to oxidative tissue damage. CONCLUSION: We conclude that TMX plays a major role in host defense under the type of inflammatory conditions associated with oxidative stress.

The anti-inflammatory effects of ethyl acetate on Lipopolysaccharide/D-galactosamine challenged mice and Lipopolysaccharide activated RAW264.7 cells.

Ethyl acetate (EA) is an ordinary organic compound in fruits, wine and cosmetics, and used as a solvent frequently. With the recent observation in our experiment, we suspected that EA could affect immune function, in particular macrophage activity. In this paper, we tested EA's protect effect against death in Lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced endotoxic shock model in mice. And also found EA decreased the LPS-induced mRNA expression of mediators of inflammation including cyclooxygenase 2 (COX2), inducible NOS (iNOS), and tumor necrosis factor α (TNF α) in RAW264.7 cells. Consequently, EA decreased the production of, TNF α and the inflammatory agent nitric oxide (NO) in RAW264.7 cells treated with LPS. Other pro-inflammatory cytokines such as IL-1h and IL-6 were similarly decreased by EA treatment of RAW264.7 cells. The potential mechanism may associate with NF-κB activity as we shown. Taken together, these results suggest that EA has anti-inflammatory properties.