Injection Of d-GalN Research Articles

Endotoxin tolerance ameliorates lipopolysaccharide/D-galactosamine-induced acute liver failure by negative regulation of the NF-κB/NLRP3 and activation of Nrf2/HO-1 via Sitr1

Acute liver failure (ALF) is a potentially fatal disorder characterized by extensive hepatocyte necrosis and rapid decline in liver function. Numerous factors, including oxidative stress, cell death, and inflammatory responses, are associated with its pathogenesis. Endotoxin tolerance (ET) refers to the phenomenon in which the body or cells exhibit low or no response to high-dose lipopolysaccharide (LPS) stimulation after pre-stimulation with low-dose LPS. However, the specific mechanism through which ET regulates LPS/D-galactosamine (D-GalN)-induced ALF remains unclear. An ALF mouse model was established by intraperitoneal injection of D-GalN (400 mg/kg) and LPS (10 mg/kg). A low dose of LPS (0.1 mg/kg/d) was continuously administered to mice for 5 d before modeling to assess the protective effect of ET. The data from this study showed that ET alleviated the inflammatory response in mice with LPS/D-GalN-induced ALF. ET inhibited LPS-induced oxidative damage and pyroptosis in macrophages in vitro. RNA sequencing analysis showed that the NF-κB/NLRP3 pathway was linked to the anti-inflammatory and antioxidative effects of ET. Furthermore, using western blot, RT-qPCR, and immunofluorescence, we verified that ET inhibited the NF-κB/NLRP3 pathway and triggered the Nrf2/HO-1 signaling pathway to attenuate oxidative stress and cell pyroptosis. Sirt1 knockdown reversed this protective effect. In summary, our research elucidates that ET prevents ALF advancement by upregulating Sirt1 levels, triggering the Nrf2/HO-1 signaling axis, and suppressing the NF-κB/NLRP3 signaling cascade to inhibit oxidative stress and cell pyroptosis. Our results provide a mechanistic explanation for the protective effect of ET against ALF.

The effects of rhein on D-GalN/LPS-induced acute liver injury in mice: Results from gut microbiome-metabolomics and host transcriptome analysis.

Rhubarb is an important traditional Chinese medicine, and rhein is one of its most important active ingredients. Studies have found that rhein can improve ulcerative colitis by regulating gut microbes, but there are few reports on its effects on liver diseases. Therefore, this study aims to investigate these effects and underlying mechanisms. Mice were given rhein (100 mg/kg), with both a normal control group and a model group receiving the same amount of normal saline for one week. Acute liver injury was induced in mice by intraperitoneal injection of D-GalN (800 mg/kg)/LPS (10 ug/kg). Samples (blood, liver, and stool) were then collected and assessed for histological lesions and used for 16S rRNA gene sequencing, high-performance liquid chromatography-mass spectrometry (LC-MS) and RNA-seq analysis. The levels of ALT and AST in the Model group were abnormal higher compared to the normal control group, and the levels of ALT and AST were significantly relieved in the rhein group. Hepatic HE staining showed that the degree of liver injury in the rhein group was lighter than that in the model group, and microbiological results showed that norank_o:Clostridia_UCG-014, Lachnoclostridium, and Roseburia were more abundant in the model group compared to the normal control group. Notably, the rhein treatment group showed reshaped disturbance of intestinal microbial community by D-GalN/LPS and these mice also had higher levels of Verrucomicrobia, Akkermansiaceae and Bacteroidetes. Additionally, There were multiple metabolites that were significantly different between the normal control group and the model group, such as L-α-amino acid, ofloxacin-N-oxide, 1-hydroxy-1,3-diphenylpropan-2-one,and L-4-hydroxyglutamate semialdehyde, but that returned to normal levels after rhein treatment. The gene expression level in the model group also changed significantly, various genes such as Cxcl2, S100a9, Tnf, Ereg, and IL-10 were up-regulated, while Mfsd2a and Bhlhe41 were down-regulated, which were recovered after rhein treatment. Overall, our results show that rhein alleviated D-GalN/LPS-induced acute liver injury in mice. It may help modulate gut microbiota in mice, thereby changing metabolism in the intestine. Meanwhile, rhein also may help regulate genes expression level to alleviate D-GalN/LPS-induced acute liver injury.

Convallatoxin inhibits IL-1β production by suppressing zinc finger protein 91 (ZFP91)-mediated pro-IL-1β ubiquitination and caspase-8 inflammasome activity.

ZFP91 positively regulates IL-1β production in macrophages and may be a potential therapeutic target to treat inflammatory-related diseases. We investigated whether this process is modulated by convallatoxin, which is a cardiac glycoside isolated from the traditional Chinese medicinal plant Adonis amurensis Regel et Radde. In vitro, the mechanisms by which convallatoxin inhibits ZFP91-regulated IL-1β expression were investigated using molecular docking, western blotting, RT-PCR, ELISA, immunofluorescence and immunoprecipitation assays.In vivo, mice liver injury was induced by an intraperitoneal injection of D-GalN and LPS, colitis was induced by oral administration of dextran sulfate sodium (DSS) in drinking water and peritonitis was induced by an intraperitoneal injection of alum. We confirmed that convallatoxin inhibited the release of IL-1β by down-regulating ZFP91. Importantly, we found that convallatoxin significantly reduced K63-linked polyubiquitination of pro-IL-1β regulated by ZFP91 and decreased the efficacy of pro-IL-1β cleavage. Moreover, convallatoxin suppressed ZFP91-mediated activation of the non-canonical cysteine-requiring aspartate protease-8 (caspase-8) inflammasome and MAPK signalling pathways in macrophages. Furthermore, we showed that ZFP91 promoted the assembly of the caspase-8 inflammasome complex, whereas convallatoxin treatment reversed this result. Mice in vivo studies further demonstrated that convallatoxin ameliorated D-GalN/LPS-induced liver injury, DSS-induced colitis and alum-induced peritonitis by down-regulating ZFP91. We show for the first time that convallatoxin-mediated inhibition of ZFP91 is an important regulatory event that prevents inappropriate inflammatory responses to maintain immune homeostasis. This mechanism provides new insight for the development of convallatoxin as a novel anti-inflammatory drug targeting ZFP91. This article is part of a themed issue on Inflammation, Repair and Ageing. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v179.9/issuetoc.

The Qingchangligan Formula Alleviates Acute Liver Failure by Regulating Galactose Metabolism and Gut Microbiota.

The Qingchangligan formula (QCLGF) is a traditional Chinese medicine that has significant clinical potential for patients with acute liver failure (ALF). However, the experimental evidence of the effect of QCLGF on ALF and the associated mechanisms remain elusive. We aimed to evaluate the function of QCLGF in ALF and the underlying mechanism. ALF was induced in rats by intraperitoneal injection of D-GalN (1100 mg/kg). The Qingchangligan formula was administered to the rats (6.725 g/kg · d) for 5 days, and the model group and the control group were given the same amount of physiological saline. Then 16S rRNA gene sequencing, high performance gas chromatography-mass spectrometry (GC-MS), and RNA-seq analysis were performed on the samples. The levels of ALT and AST in the ALF rats were abnormal (5322.08 ± 566.27 U/L and 7655.95 ± 1238.08 U/L, respectively) compared with the normal control (98.98 ± 6.90 U/L and 99.63 ± 10.94 U/L, respectively). The levels of ALT and AST in the QCLGF rats (2997.67 ± 469.24 U/L and 4158.40 ± 596.07 U/L, respectively) were closer the normal control group. Liver HE staining showed that the degree of liver damage in the QCLGF rats was lighter than that in the ALF rats. The overall structure of the gut microbiota after ALF was significantly altered, including Proteobacteria, Blautia, Romboutsia, Parabacteroides, UCG-008, Parasutterella, Ruminococcus, norank_f:Lachnospiraceae, the Eubacterium_xylanophilum_group, Oscillibacter, and Eisenbergiella. QCLGF balanced the structure and abundance of intestinal flora. The levels of D(+)galactose, isopropyl beta-D-1-thiogalactopyranoside and D-mannitol were lighter in the plasma of the ALF rats than in the normal control rats, but there were significantly elevated levels of those metabolites in the QCLGF rats. The gene expression changed significantly in the ALF rats. QCLGF regulated the expression of THBS1 and the KEGG pathways of carbohydrate metabolism, lipid metabolism, signal transduction, the immune system, and infectious disease: bacterial. QCLGF may alleviating intestinal flora disorder, regulating galactose metabolism and downregulating the expression of THBS1 to alleviate D-GalN induced acute liver failure.

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Liver disease is among the most serious ailments and can be classified as acute or chronic hepatitis, hepatosis, and cirrhosis. Besides expensive and ineffectual modern therapeutic agents like steroids and chemotherapy, South East Asian countries like India and China have an edge in treating hepatic disorders employing their native botanicals. The present study was designed to evaluate the protective effects of Arogyavardhani on D-galactosamine (d-GalN)-induced fulminant hepatic failure. Rats were given an intraperitoneal injection of d-GalN (270 mg/kg). Arogyavardhani (10 mg/kg and 50 mg/kg) was administered orally for 14 days continuously, and on the last day at 1hour before the d-GalN injection. Rats were sacrificed 24 hours after the d-GalN. Silymarin (100 mg/kg body weight) was given orally as a standard hepatoprotective drug. The liver injury was assessed biochemically, investigating biochemical parameters like ALT, AST, ALP, Bilirubin, Total Protein, and Albumin. The survival rates after the application of Arogyavardhani at 24 h were also observed. D-galactosamine administration induced a significant increase (P≤0.01) in total bilirubin associated with a marked elevation in the activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) as compared to control rats. The pretreatment of Arogyavardhani attenuated these changes in a dose-dependent manner. The survival rate of the OC groups was significantly higher than that of the d-GalN group. Our results suggest that Arogyavardhani has hepatoprotective effects against D-galactosamine induced hepatic damage. Therefore, Arogyavardhani may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.

Hepatoprotective and Antioxidant Capacity of Mallotus repandus Ethyl Acetate Stem Extract against d-Galactosamine-Induced Hepatotoxicity in Rats.

Mallotus repandus (M. repandus) is traditionally used to treat muscle pain, itching, fever, rheumatic arthritis, and a variety of liver disorders. The aim of the present work was to evaluate the hepatoprotective activity and the antioxidant potential of the ethyl acetate stem extract of M. repandus (ESMR) against d-galactosamine (d-GalN)-induced hepatopathy, along with a possible mechanism of action in rats. In vivo hepatoprotective activity of ESMR was examined using d-galactosamine (d-GalN)-induced hepatotoxicity in Sprague–Dawley rats. For this purpose, levels of serum diagnostic markers, activity of hepatic antioxidant enzymes, and liver histo-architecture were employed to assess the protective efficacy of ESMR. Furthermore, the total phenolic, flavonoid, and tannin contents were quantitated, and the antioxidant capacity of the extract was evaluated using different methods such as 2,2′-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO), hydrogen peroxide (H2O2), and hydroxyl radical (OH•) scavenging assays. Intraperitoneal d-GalN injection triggered hepatotoxicity, as shown by the noticeable increase in the serum hepatic marker enzymes, bilirubin content, γ-glutamyl transferase (GGT), total cholesterol (TC), triglycerides (TGs), and malondialdehyde (MDA), whereas glutathione, superoxide dismutase, and catalase levels were significantly lower compared with that of the control. Pretreatment with ESMR reduced the hepatic enzyme levels along with bilirubin, GGT, and MDA compared to the d-GalN-intoxicated group. These results were supported by histopathological studies, where d-galactosamine caused coagulative necrosis, hemorrhage, and inflammation. However, pretreatment with ESMR ameliorated the histo-architectural changes and brought them back to normal. Results also revealed that the total polyphenolic, flavonoid, and tannin content, and total antioxidant capacity of ESMR were 136.30 ± 0.78 mg GAE/g mg, 38.72 ± 0.85 mg QE/g, 75.88 ± 0.54 mg TAE/g, and 123.16 ± 0.24 mg AAE/g, respectively. In addition, ESMR inhibited free radicals with IC50 values of 94.47 ± 0.51, 127.33 ± 0.36, 164.12 ± 0.45, and 254.14 ± 0.35 μg/mL in DPPH, NO, H2O2, and OH• free radical scavenging assays, respectively. These findings highlight the protective role of ESMR against hepatic injury induced by d-GalN, which may be attributed to its higher antioxidant properties, thereby scientifically justifying its traditional use.

Ameliorative effect of ethanolic extract of roots of Tetracera akara (Burm. f.) Merr. on D-galactosamine induced hepatotoxicity in Wistar rats by downregulation of inflammatory mediators like TNFα, COX-2 and iNOS

Tetracera akara, a climbing shrub locally called Nennalvalli or Pattuvalli, is an ethnomedicinal plant used by Kani tribe of Kerala to treat chronic liver disorders and inflammatory conditions. The present study was aimed to evaluate the hepatoprotective activity of ethanolic extract of roots of Tetracera akara root on D-Galactosamine induced hepatotoxicity in Wistar rats. Hepatotoxicity was induced in Wistar rats by intraperitoneal injection of D-GalN (400 mg/kg in saline) in Wistar rats. Ethanolic extract of T. akara root (TA ETH) was administered to the experimental rats in varying doses of (50, 150 and 300 mg/kg/day), p. o. for 7 days. The hepatoprotective effect was evaluated by the estimation of biochemical markers of hepatic injury, anti-oxidant status of the liver by estimating hepatic catalase, superoxide dismutase, glutathione and malondialdehyde, gene and protein expression level of inflammatory marker genes and histopathological evaluation of experimental animals. Administration of TA ETH (150 and 300 mg/kg) significantly (P ≤0.05) restored the levels of serum bilirubin, protein and other hepatic enzymes almost comparable to the standard drug Silymarin-treated groups. The levels of antioxidant enzymes like SOD and CAT were elevated and lipid peroxidation was inhibited as evident from the reduced levels of MDA. The gene expression studies by quantitative PCR method showed that TA ETH significantly (P ≤0.05) downregulated pro inflammatory cytokines, inflammatory COX-2 genes and upregulated IL 10 gene levels in D-GalN induced liver tissue, which was further confirmed in protein estimation by ELISA method. The histopathological observations were in correlation with the biochemical findings showing the presence of normal hepatic architecture, which further evidenced the hepatoprotective effect of TA ETH. Ethanolic extract of the root of T. akara possesses significant hepatoprotective activity mainly by scavenging reactive free radicals, boosting the endogenous antioxidant system in liver and inhibiting pro-inflammatory mediator like TNF α, COX-2, iNOS and promoting the anti-inflammatory IL 10, thus substantiating the tribal claim.

Up‐regulation of microRNA‐30b/30d cluster represses hepatocyte apoptosis in mice with fulminant hepatic failure by inhibiting CEACAM1

Recently, impacts of microRNAs have been unraveled in human diseases, and we aimed to confirm the role of miR-30b/30d in fulminant hepatic failure (FHF). Expression of miR-30b/30d and CEACAM1 in serum of FHF patients and healthy people was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Mice FHF models were established by injection of D-Galn and lipopolysaccharide, and were treated with miR-30b/30d mimics. Oxidative stress, liver injury, and inflammatory reaction in mouse liver tissues were measured using oxidative stress-related factor kits, hematoxylin-eosin staining and enzyme-linked immunosorbent assay, respectively. Moreover, cell cycle distribution and apoptosis of hepatocytes of mice were determined by flow cytometry, and the target relation between miR-30b/30d and CEACAM1 was confirmed by bioinformatic method and dual luciferase reporter gene assay. MiR-30b/30d expression was positively, and CEACAM1 expression was negatively related to prognosis of FHF patients. Up-regulation of miR-30b/30d attenuated oxidative stress, liver injury, and inflammatory reaction, and improved survival rate of FHF mice. Furthermore, elevated miR-30b/30d ameliorated apoptosis and cell cycle arrest of hepatocytes of FHF mice. CEACAM1 was a target gene of miR-30b/30d. This study highlights that up-regulated miR-30b/30d attenuates the progression of FHF by targeting CEACAM1, which may be helpful to FHF treatment.

M1/M2-macrophage Polarization-based Hepatotoxicity in d-galactosamine-induced Acute Liver Injury in Rats.

d-galactosamine (d-GalN) is a well-known hepatotoxic agent that causes liver injury. Conversely, hepatic macrophages play a crucial role in maintaining liver tissue integrity. Macrophage functions were investigated in hepatic lesions induced by a single intraperitoneal injection of d-GalN (800 mg/kg body weight [BW]) in 6-week-old F344 rats. Blood and liver samples were examined at 8 hr and on 1, 2, 3, and 5 days postsingle injection (PSI). Hepatic lesions consisting of degeneration/sporadic foci of coagulation necrosis, inflammatory cell reaction, and reparative fibrosis were seen on PSI days 1 and 2, reflected by significantly increased serum levels of aspartate transaminase and alanine transaminase and upregulation of CD68 M1 (tumor necrosis factor-α, interleukin [IL]-6, and interferon-γ) and CD163 M2 (transforming growth factor-β1, IL-10, monocyte chemoattractant protein-1, and IL-4) macrophage-related factors. Double immunofluorescence staining on PSI day 2 demonstrated that 82% of hepatic macrophages expressed of CD163/CD68 simultaneously; 65-75% of MHC class II macrophages showed co-expression of CD163 or CD68 and 95% CD204-expressing macrophages reacted to CD163 or CD68. These findings showed that both M1- and M2-macrophages contributed to the development of hepatic lesions induced by d-GalN and provided information about macrophage activation, indicating the importance of analysis of macrophage phenotypes for hepatotoxicity based on M1/M2-polarization.

Qingchangligan formula attenuates the inflammatory response to protect the liver from acute failure induced by d-galactosamine/lipopolysaccharide in mice

Ethnopharmacological relevanceThe Qingchangligan formula, a traditional Chinese medicine comprising five herbs, is useful for treatment of patients with liver failure; however, its protective and regulatory mechanisms remain elusive. Aim of the studyTo test the hypothesis that the Qingchangligan formula protects mice against acute liver failure by inhibiting liver inflammation. Materials and methodsAcute liver failure (ALF) was induced by intraperitoneal injection of D-GalN (700mg/kg) plus LPS (10μg/kg). The Qingchangligan formula was administered to mice in three doses of 50mg/kg (on day 1, day 2, and day 3) prior to D-GalN/LPS injection by intragastric administration. The mice in different groups were sacrificed at 6h after D-GalN/LPS injection, and liver samples and blood were collected for analysis. ResultsAdministration of the Qingchangligan formula not only ameliorated liver injury, as evidenced by reduced transaminase levels and well-preserved liver architecture, but also decreased the lethality in ALF mice. Moreover, in the ALF model, pretreatment with the Qingchangligan formula alleviated liver inflammation and decreased hepatocyte apoptosis. Further demonstrating the protective effects of the Qingchangligan formula, we found that pretreatment with the Qingchangligan formula reduced the expression of inflammatory cytokines by decreasing the expression of components of the mitogen-activated protein kinase (MAPK) pathway and promoting autophagy in vitro and in vivo. ConclusionsOur findings demonstrated that the Qingchangligan formula exerts a protective effect against the pathophysiology of ALF, especially in regulating liver inflammation, and provide a rationale for using the Qingchangligan formula as a potential therapeutic strategy to ameliorate ALF.

Genistein modulates the expression of NF-κB and MAPK (p-38 and ERK1/2), thereby attenuating d-Galactosamine induced fulminant hepatic failure in Wistar rats

Genistein is an isoflavanoid abundantly found in soy. It has been found to play an important role in the prevention of various chronic diseases including cancer. In this study, we evaluated potential therapeutic properties of Genistein against d-Galactosamine (d-GalN) induced inflammation and hepatotoxicity in male Wistar rats. Fulminant hepatic failure (FHF) was induced in rats by intraperitoneal injection of d-GalN (700mg/kgBW). Genistein (5mg/kgBW/day) was given as pre-treatment for 30days via intra-gastric route followed by d-GalN (700mg/kgBW) injection. The hepatoprotective and curative effects of Genistein were evident from a significant decrease in the serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels as well as prevention of histological damage by pre-treatment of Genistein. Genistein pre-treatment significantly inhibited the increased protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), thereby reducing nitric oxide (NO) and prostaglandin-E2 (PGE) levels, respectively. In addition Genistein significantly suppressed the production of d-GalN-induced proinflammatory cytokines, including tumor necrosis factor-α (TNF-α) and interleukin (IL)-1β. These inhibitory effects were associated with the suppression of nuclear factor-kappa B (NF-ĸB) activation, IKKα/β and Mitogen activated protein kinase (MAPK) phosphorylation by Genistein in d-GalN-treated animals. In conclusion, our results suggest that Genistein may serve as a potential supplement in the prevention of hepatic and inflammatory diseases. Furthermore Genistein is able to maintain the redox potential and strengthens the antioxidant defense system of a cell.

D-galactosamine/lipopolysaccharide-induced hepatotoxicity downregulates sirtuin 1 in rat liver: role of sirtuin 1 modulation in hepatoprotection.

D-Galactosamine/Lipopolysaccharide (D-GalN/LPS) is a well known model of hepatotoxicity that closely resembles acute liver failure (ALF) seen clinically. The role of sirtuin 1 in this model has not yet been documented. However, there have been a number of studies about the cytoprotective effects of resveratrol, a SIRT1 activator, in the liver. This study was aimed at elucidating the roles of SIRT1 protein expression or catalytic activity in D-GalN/LPS model of hepatotoxicity. ALF was induced in male Wistar rats by intraperitoneal injection of D-GalN and LPS. Some groups of animals were pretreated with resveratrol and/or EX-527 (SIRT1 inhibitor). The effects of these treatments were evaluated by biochemical and Western blot studies. D-GalN/LPS treatment was able to induce hepatotoxicity and significantly increase all markers of liver damage and lipid peroxidation. A dramatic decrease of SIRT1 levels in response to D-GalN/LPS treatment was also documented. Resveratrol pretreatment attenuated D-GalN/LPS-induced hepatotoxicity. EX-527 blocked the cytoprotective effects of resveratrol. However, both resveratrol and EX-527 pretreatments did not exhibit any significant effect on SIRT1 protein expression. Collectively, these results suggest that downregulation of SIRT1 expression is involved in the cytotoxic effects of D-GalN/LPS model and SIRT1 activity contributes to the cytoprotective effects of resveratrol in the liver.

The effect of cholinesterase inhibition on liver dysfunction in experimental acute liver failure: a randomized controlled study

Introduction Acute liver failure (ALF), similar to sepsis, is associated with an overwhelming activation of the immune response in which hepatic and circulating inflammatory cytokines play a pivotal role. The cholinesterase inhibitor neostigmine has been shown to have anti-inflammatory properties in experimental sepsis. We investigated the role of neostigmine in attenuating d-galactosamine (d-GalN)-induced ALF. Materials and methods Thirty-six female Wistar rats were randomly allocated to three groups: the control group, the d-GalN group receiving a single intraperitoneal injection of d-GalN (400 mg/kg body weight), and the neostigmine-treated d-GalN group receiving a single intraperitoneal injection of d-GalN followed 24 h later by intraperitoneal injection of neostigmine methylsulfate 0.25% (80 μg/kg body weight) three times daily for 3 successive days. Rats were killed 24 h after the last injection. Plasma levels of alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total proteins, albumin, prothrombin, total bilirubin, and hepatic levels of superoxide dismutase and malondialdehyde were measured. Liver expression of cytokines (HMGB-1, TNF-α, and IL-10) and histopathology were evaluated. Results Neostigmine attenuated liver dysfunction and improved liver synthetic and excretory functions. It reduced proinflammatory cytokine HMGB-1 [95% confidence interval (CI) 0.33-1.09] and TNF-α (95% CI 1.26-2.06) expression compared with the d-GalN group (95% CI 2.67-4.73 and 7.33-14.53, respectively, P P Conclusion Neostigmine improved liver function in a rat ALF model through an anti-inflammatory activity.

MafK positively regulates NF-κB activity by enhancing CBP-mediated p65 acetylation

Reactive oxygen species, produced by oxidative stress, initiate and promote many metabolic diseases through activation/suppression of redox-sensitive transcription factors. NF-κB and Nrf2 are important regulators of oxidation resistance and contribute to the pathogenesis of many diseases. We identified MafK, a novel transcriptional regulator that modulates NF-κB activity. MafK knockdown reduced NF-κB activation, whereas MafK overexpression enhanced NF-κB function. MafK mediated p65 acetylation by CBP upon LPS stimulation, thereby facilitating recruitment of p65 to NF-κB promoters such as IL-8 and TNFα. Consistent with these results, MafK-depleted mice showed prolonged survival with a reduced hepatic inflammatory response after LPS and D-GalN injection. Thus, our findings reveal a novel mechanism by which MafK controls NF-κB activity via CBP-mediated p65 acetylation.

Scoparone attenuates d-galactosamine/lipopolysaccharide-induced fulminant hepatic failure through inhibition of toll-like receptor 4 signaling in mice

The purpose of this study was to investigate the protective effects and molecular mechanisms of scoparone on d-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure (FHF) in mice. FHF was induced in mice by intraperitoneal injection of D-GalN (800 mg/kg)/LPS (40 μg/kg). Mice were treated intraperitoneally with scoparone 1h before D-GalN/LPS treatment. Treatment with d-GalN/LPS markedly increased mortality, serum aminotransferase activity, and tolllike receptor 4 (TLR4) protein expression, and these increases were attenuated by scoparone. Treatment with d-GalN/LPS markedly increased myeloid differentiation primary response gene 88 protein expression, phosphorylation of p38, extracellular signal-regulated kinase and c-Jun N-terminal kinase, nuclear protein expression of nuclear factor κB and phosphorylated c-Jun, and levels of serum tumor necrosis factor-α and interleukin-6 and these increases were attenuated by scoparone. In addition, increased levels of toll-receptor-associated activator of interferon protein expression, phosphorylation of interferon (IFN) regulatory factor 3, and serum IFN-β level in D-GalN/LPS-treated mice were attenuated by scoparone. Our results suggest that scoparone attenuates d-GalN/LPSinduced liver damage by inhibition of the TLR-mediated inflammatory pathway.

Efficacy of MK615 for the treatment of patients with liver disorders

To investigate the hepatoprotective effect of MK615, a Japanese apricot extract, in an animal model, and its clinical therapeutic effect. Wistar rats were administered physiological saline (4 mL/kg) or MK615 solution (4 mL/kg) for 7 d. On the sixth d, acute hepatic injury was induced by administering a single intraperitoneal injection (ip) of D-galactosamine hydrochloride (D-GalN) (600 mg/kg). Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined, and liver tissues were used for histopathological analysis. Fifty-eight patients with liver disorders [hepatitis C (n = 40), non-alcoholic fatty liver disease (n = 15), and autoimmune liver disease (n = 3)] were orally administered commercially available Misatol ME-containing MK615 (13 g/d) daily for 12 wk. Blood and urine were sampled immediately before and 6 wk, 12 wk, and 16 wk after the start of intake to measure various biochemical parameters. The percentage change in ALT and AST levels after 12 wk from the pre-intake baseline served as a primary endpoint. D-GalN effectively induced acute hepatic injury in the rats. At 48 h after the ip injection of D-GalN, the plasma levels of ALT (475.6 ± 191.5 IU/L vs 225.3 ± 194.2 IU/L, P < 0.05) and AST (1253.9 ± 223.4 IU/L vs 621.9 ± 478.2 IU/L, P < 0.05) in the MK615 group were significantly lower than the control group. Scattered single cell necrosis, loss of hepatocytes, and extensive inflammatory cell infiltration were observed in hepatic tissue samples collected from the control group. However, these findings were less pronounced in the group receiving MK615. At the end of the clinical study, serum ALT and AST levels were significantly decreased compared with pre-intake baseline levels from 103.5 ± 58.8 IU/L to 71.8 ± 39.3 IU/L (P < 0.05) and from 93.5 ± 55.6 IU/L to 65.5 ± 34.8 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 26 (45%) of the 58 patients, while 25 (43%) patients exhibited similar AST level reductions. The chronic hepatitis C group exhibited significant ALT and AST level reductions from 93.4 ± 51.1 IU/L to 64.6 ± 35.1 IU/L (P < 0.05) and from 94.2 ± 55.5 IU/L to 67.2 ± 35.6 IU/L (P < 0.05), respectively. A reduction of ≥ 30% from the pre-study baseline ALT level was observed in 20 (50%) of the 40 patients. ALT levels in both the combined ursodeoxycholic acid (UDCA) treatment and the UDCA uncombined groups were significantly lower after Misatol ME administration. MK615 protected hepatocytes from D-GalN-induced cytotoxicity in rats. Misatol ME decreased elevated ALT and AST levels in patients with liver disorders. These results suggest that MK615 and Misatol ME are promising hepatoprotective agents for patients with liver disorders.

Granulocyte colony-stimulating factor treatment ameliorates liver injury and improves survival in rats with d-galactosamine-induced acute liver failure

Only liver transplantation is currently available therapy for the patients with acute liver failure (ALF). This study was designed to determine whether administration of granulocyte colony-stimulating factor (G-CSF) has therapeutic efficacy in animals with ALF. Female Sprague-Dawley (SD) rats were intraperitoneally injected with a single dose of d-galactosamine ( d-GalN, 1.4 g/kg) to induce ALF. After 2 h, the rats were randomized to receive G-CSF (50 μg/kg/day), or saline vehicle injection for 5 days. Rats were observed for survival and assessed for liver injury by serum alanine transaminase (ALT) measurement and histological analysis. CD34+ cells in bone marrow were assessed by flow cytometry. CD34+ cells and Ki-67+ hepatocytes in liver tissue were evaluated by immunohistochemistry. In the ALF model, 5-day survival after d-GalN injection was 33.3% (10/30), while G-CSF administration following d-GalN resulted in 53.3% (16/30) survival ( p = 0.027). G-CSF treated rats had lower ALT level and less hepatic injury compared with saline vehicle rats. The increases of CD34+ cells in bone marrow and liver tissue and Ki-67+ cells in liver tissue in G-CSF treated rats were higher than those in saline rats. No correlation was observed between CD34+ cells and Ki-67+ hepatocytes in liver tissue in both G-CSF and vehicle rats. It is suggested that G-CSF increases survival rate, decreases liver injury and enhances hepatocyte proliferation in rats with d-GalN-induced ALF possibly through actions including but not limiting to CD34+ cell mobilization, and that G-CSF may be of potential value in treating ALF.

Low susceptibility of NC/Nga mice to the lipopolysaccharide-mediated lethality withd-galactosamine sensitization and the involvement of fewer natural killer T cells

The LPS-mediated lethality of NC/Nga mice, having fewer NKT cells, was examined by using d-galactosamine (d-GalN)-sensitization. The NC/Nga mice were not killed by a simultaneous administration of d-GalN and LPS whereas all C57BL/6 (B6) control mice were killed. The injection of d-GalN and LPS failed to elevate the levels of serum alanine aminotransferase and caspase 3 in the liver tissues of NC/Nga mice. Further, the nitric oxide (NO) level of the d-GalN- and LPS-injected NC/Nga mice was much lower than those of the B6 mice. The expression of an inducible NO synthase (iNOS) was significantly reduced in the livers of NC/Nga mice. However, there was no significant difference in LPS-induced TNF-α production between B6 mice and NC/Nga mice. The NC/Nga mice had an impaired expression of IFN-γ protein and mRNA in response to d-GalN and LPS. The pretreatment with α-galactosylceramide (α-GalCer), which activates Vα14(+) NKT cells and induces the production of IFN-γ, rendered NC/Nga mice more susceptible to the LPS-mediated lethality. The livers of NC/Nga mice had fewer NKT cells compared to B6 mice. Taken together, it is suggested that the resistance of NC/Nga mice to the LPS-mediated lethality with d-GalN sensitization depended on the impaired IFN-γ production caused by fewer NKT cells and reduced NO production that followed.

Baicalein inhibits nuclear factor-κB and apoptosis via c-FLIP and MAPK in d-GalN/LPS induced acute liver failure in murine models

The hepatoprotective effects and molecular mechanisms of baicalein on acute liver failure induced by d-galactosamine ( d-GalN)/lipopolysaccharides (LPS) were investigated in vivo. Mice were administered with different doses of baicalein (50, 100 or 150 mg/kg, p.o.) 1 h before injection of d-GalN (700 mg/kg)/LPS (10 μg/kg) and then sacrificed 6 h after treatment with d-GalN/LPS. Pretreatment with baicalein prevented d-GalN/LPS-induced liver damage by preventing associated increases of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and by reducing serum tumor necrosis factor α (TNF-α), nitric oxide (NO) or inducible nitric oxide synthase (iNOS) expressions. The molecular mechanisms involved in baicalein-induced inhibition of d-GalN/LPS-caused apoptosis were associated with the protection of mitochondria, increasing the Bcl-2/Bax ratio, blocking the release of cytochrome c, and suppressing the phosphorylation of IκBα, ERK and JNK. Moreover, baicalein activated c-FLIP L, XIAP and cIAP2 proteins, potentially blocking the recruitment of NF-κB signaling molecules. The results support the investigation of baicalein as a therapeutic candidate for acute liver apoptosis or injury and indicate that baicalein might inhibit liver apoptosis by mediating one or more of these pathways.

Akt Cys-310-targeted Inhibition by Hydroxylated Benzene Derivatives Is Tightly Linked to Their Immunosuppressive Effects

The hydroxylated benzene metabolite hydroquinone (HQ) is mainly generated from benzene, an important industrial chemical, and is also a common dietary component. Although numerous reports have addressed the tumorigenesis-inducing effects of HQ, few papers have explored its molecular regulatory mechanism in immunological responses. In this study we characterized Akt (protein kinase B)-targeted regulation by HQ and its derivatives, in suppressing inflammatory responses using cellular, molecular, biochemical, and immunopharmacological approaches. HQ down-regulated inflammatory responses such as NO production, surface levels of pattern recognition receptors, and cytokine gene expression with IC(50) values that ranged from 5 to 10 microm. HQ inhibition was mediated by blocking NF-kappaB activation via suppression of its translocation pathway, which is composed of Akt, I kappaB alpha kinase beta, and I kappaB alpha. Of the targets in this pathway, HQ directly targeted and bound to the sulfhydryl group of Cys-310 of Akt and sequentially interrupted the phosphorylation of both Thr-308 and Ser-473 by mediation of beta-mercaptoethanol, according to the liquid chromatography/mass spectroscopy analysis of the interaction of HQ with an Akt-derived peptide. Therefore, our data suggest that Akt and its target site Cys-310 can be considered as a prime molecular target of HQ-mediated immunosuppression and for novel anti-Akt-targeted immunosuppressive drugs.