Primary Cultures Of Rat Hepatocytes Research Articles

Effect of serotonin on phosphorylation of ribosomal p70 S6 kinase in primary cultures of adult rat hepatocytes

Effect of serotonin on phosphorylation of ribosomal p70 S6 kinase in primary cultures of adult rat hepatocytes

Cytoprotective Properties of Carnosine against Isoniazid-Induced Toxicity in Primary Cultured Rat Hepatocytes

Background: Drug-induced liver injury is a critical clinical complication. Hence, finding new and safe protective agents with potential clinical application is of value. Isoniazid (INH) is an antituberculosis agent widely used against Mycobacterium tuberculosis infection in human. On the other hand, hepatotoxicity is a clinical complication associated with isoniazid therapy. Oxidative stress and its associated events are major mechanisms identified for INH-induced liver injury. Carnosine is an endogenously found peptide widely investigated for its hepatoprotective effects. On the other hand, robust antioxidant and cytoprotective effects have been attributed to this peptide. Methods: The current study designed to evaluate the potential cytoprotective properties of carnosine against INH-induced cytotoxicity in drug-exposed primary cultured rat hepatocytes. Primary cultured rat hepatocytes were incubated with INH (1.2 mM). Results: INH treatment caused significant increase in cell death and lactate dehydrogenase (LDH) release. On the other hand, it was found that markers of oxidative stress including reactive oxygen species were significantly increased in INH-treated cells. Cellular glutathione reservoirs were also depleted in INH-treated group. Carnosine treatment (50 and 100 µM) significantly diminished INH-induced oxidative stress and cytotoxicity. Conclusion: These data mention carnosine as a potential protective agent with therapeutic capability against INH hepatotoxicity.

Glutathione inhibits expression of the proinflammatory biomarker inducible nitric oxide synthase in hepatocytes

Background: Intracellular glutathione (GSH) plays an important regulatory role in the host response to liver injury. However, there have been few scientific reports on the anti-inflammatory effects of GSH. In the inflamed liver, proinflammatory cytokines stimulate liver cells, followed by expression of inducible nitric oxide synthase (iNOS). Excessive nitric oxide (NO) levels produced by iNOS are one of the factors involved in liver injury. Therefore, inhibiting iNOS induction is important for preventing liver injury. This study aimed to investigate the protective effects of GSH on the liver by examining interleukin (IL)-1β-stimulated hepatocytes.Methods: Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of GSH. Induction of iNOS and its signaling pathway were analyzed.Results: Addition of GSH decreased IL-1β-induced iNOS protein and mRNA expression levels, which resulted in inhibition of NO production. GSH also decreased tumor necrosis factor (TNF)-α and IL-6 mRNA expression. GSH blocked “type I IL-1 receptor upregulation”, one of the essential signaling pathways for iNOS induction, through inactivation of an upstream kinase, phosphatidylinositol 3-kinase/Akt. In contrast, GSH had no effects on degradation of IκB and activation of NF-ĸB (nuclear translocation and its DNA binding). Transfection experiments revealed that GSH reduced iNOS mRNA levels at the promoter transactivation and mRNA stabilization steps. Delayed administration of GSH after IL-1β addition also inhibited iNOS induction. Conclusions: Our study suggests that GSH affects induction of inflammatory mediators, including iNOS and TNF-α, indicating its therapeutic potential for organ injuries, especially for the liver.Keywords: glutathione, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, type I interleukin-1 receptor, tumor necrosis factor-α

Assessment of the cytotoxic potential of an aqueous-ethanolic extract from Thalassia testudinum angiosperm marine grown in the Caribbean Sea.

Reported antioxidant, anti-inflammatory and neuroprotective properties for one aqueous-ethanolic extract from Thalassia testudinum which grows in the Caribbean Sea compelled us to explore about extract cytotoxic effects. Cell viability was assayed on tumour (HepG2, PC12, Caco-2 and 4T1) and non-tumour (VERO, 3T3, CHO, MCDK and BHK2) cell lines. The extract effects upon primary cultures of rat and human hepatocytes and human lymphocytes were assayed. The extract exhibited cytotoxicityagainst cancer cells compared to normal cells, and the IC50 values were 102μg/ml for HepG2, 135μg/ml for PC12, 165μg/ml for Caco-2 and 129μg/ml for 4T1 cells after 48h, whereas IC50 could not be calculated for normal cells. Additional data from a high-content screening multiparametric assay indicated that after 24-h exposure, the extract (up to 100μg/ml) induced death in HepG2 cells through oxidative stress-associated mechanism, DNA damage and hypercalcaemia. Comet assay corroborated extract-induced DNA damage. Thalassia testudinum extract is more cytotoxic and produced more DNA damage on human hepatoma cells than to other non-tumour cells. A possible mechanism is suggested for extract-induced cytotoxicity based on oxidative stress, nuclear damage and hypercalcaemia in HepG2 cells. T.testudinum may be a source for antitumour agents.

River pollution in the Kosovo: Cyto- and genotoxic effects of water samples in the primary rat hepatocyte assay.

The actual stage of the development of Kosovo is characterized by the concerning levels of environmental pollution and the serious health problems attributed to the emission of pollutants into air, soil and water. In this context, river pollution is one of the main threats due to the discharge of untreated urban and industrial waste waters that affect the quality of surface and ground water. In addition, urban and agricultural discharges are affecting the river water quality. In this article, we are presenting data on the cyto- and genotoxic potential of water samples from three rivers (Sitnica, Drenica and Lepenci) in the Kosovo as determined in the cultures of primary rat hepatocytes. Sitnica and Drenica (as the most important Sitnica tributary) drain into the Black Sea, whereas the Lepenci river drains into the Aegean Sea. These rivers are polluted mainly by industry in the Kosovo together with municipal discharges. The results of this study show that the samples have primarily a cytotoxic potential by causing necrotic cell death that was not accompanied by any increase of the rate of micronucleated cells as an indicator for a genotoxic potential. The different effects in 2 consecutive years can be attributed to variations in physico-chemical parameters such as water levels, intake of pollutants, and so on, indicating the need for continuous monitoring and risk assessment.

Erratum to "N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems" and "High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture".

Erratum to "N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems" and "High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture".

Monitoring the Effects of Pharmacological Reagents on Mitochondrial Morphology.

This protocol describes how to apply appropriate pharmacological controls to induce mitochondrial fusion or fission in studies of mitochondria morphology for four different mammalian cell types, HepG2 human liver hepatocellular carcinoma cells, MCF7 human breast adenocarcinoma cells, HEK293 human embryonic kidney cells, and collagen sandwich culture of primary rat hepatocytes. The protocol provides methods of treating cells with these pharmacological controls, staining mitochondria with commercially available MitoTracker Green and TMRE dyes, and imaging the mitochondrial morphology in live cells using a confocal fluorescent microscope. It also describes the cell culture methods needed for this protocol. © 2018 by John Wiley & Sons, Inc.

L-Carnitine has a liver-protective effect through inhibition of inducible nitric oxide synthase induction in primary cultured rat hepatocytes

Background: L-Carnitine has protective effects on various injured organs. However, it has not been reported whether L-carnitine influences the induction of inducible nitric oxide synthase (iNOS) expression during inflammation. Nitric oxide (NO) produced by iNOS is an inflammatory indicator in organs which become inflamed, including the liver.Objective: This study aimed to examine whether L-carnitine influences the induction of iNOS gene expression in inflammatory cytokine-stimulated hepatocytes and the mechanisms involved in the action. Methods: L-Carnitine was added into the primary cultures of rat hepatocytes stimulated by interleukin-1β (an in vitro liver injury model). The production of NO and induction of iNOS and its signaling pathway were analyzed.Results: Transfection experiments with iNOS promoter-luciferase constructs revealed how L-carnitine inhibited iNOS mRNA synthesis activity and reduced its stability. In support of this observation, L-carnitine reduced iNOS mRNA and iNOS protein expression levels, resulting in reduced NO production. L-Carnitine blocked two essential pathways for iNOS induction: IκB kinase (IκB degradation/NF-κB activation) and phosphatidylinositol 3-kinase/Akt (type I IL-1 receptor upregulation).Conclusions: L-Carnitine inhibited the induction of inflammatory mediator iNOS, partially through inhibition of NF-κB activation, which demonstrated L-carnitine has protective effects in an in vitro liver injury model. L-Carnitine may have therapeutic potential for organ injuries, including the liver.Keywords: L-carnitine, hepatic encephalopathy, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor

Metformin inhibits expression of the proinflammatory biomarker inducible nitric oxide synthase in hepatocytes

Background: Metformin is used to treat patients with type II diabetes. However, there are few scientific reports on its anti-inflammatory effects. In the inflamed liver, proinflammatory cytokines stimulate liver cells, followed by inducible nitric oxide synthase (iNOS) expression. Excessive NO levels produced by iNOS have been implicated as a factor in liver injury. As a result, it is essential to inhibit iNOS induction to prevent liver injury.Objective: This study aimed to investigate liver protective effects of metformin by examining interleukin (IL)-1β-stimulated hepatocytes. Methods: Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of metformin. iNOS induction and its signaling pathway were analyzed.Results: Metformin decreased iNOS protein and mRNA expression, resulting in the inhibition of hepatic NO production. Metformin also reduced tumor necrosis factor (TNF)-α and IL-6 mRNA expression. Metformin inhibited an essential signaling pathway for iNOS induction, type I IL-1 receptor upregulation. Transfection experiments revealed that metformin reduced iNOS mRNA levels through both promoter transactivation and mRNA stabilization. Delayed metformin administration after IL-1β addition also inhibited iNOS induction. Conclusions: Metformin affects the induction of inflammatory mediators including iNOS and TNF-α, demonstrating its therapeutic potential for organ injuries, including the liver.Keywords: metformin, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, type I interleukin-1 receptor, tumor necrosis factor-α

Induction of xenobiotic-metabolizing enzymes in hepatocytes by beta-naphthoflavone: Time-dependent changes in activities, protein and mRNA levels

In the present study, time-dependency of the induction effect of a selective inducer on the activity, protein and mRNA levels of cytochromes P450 1A1/2 (CYP1A1/2), NAD(P)H:quinone oxidoreductase 1 (NQO1) and glutathione S-transferases (GSTA), in primary culture of rat hepatocytes was tested and evaluated. To show the differences in responses of tested enzymes, the common aryl hydrocarbon receptor (AhR) ligand agonist, beta-naphthoflavone (BNF), was used. Induction of CYP1A1/2 by BNF was detected at all time intervals and at all levels (i.e., mRNA, protein, enzyme activity). Different responses of NQO1 and GSTA upon BNF treatment were observed. Our results demonstrate that the responses of different xenobiotic-metabolizing enzymes to the inducer vary in time and depend on the measured parameter. For these reasons, an induction study featuring only one-time interval treatment and/ or one parameter testing could produce misleading information.

N-Acetyl-2-Aminofluorene (AAF) Processing in Adult Rat Hepatocytes in Primary Culture Occurs by High-Affinity Low-Velocity and Low-Affinity High-Velocity AAF Metabolite-Forming Systems

N-acetyl-2-aminofluorene (AAF) is a procarcinogen used widely in physiological investigations of chemical hepatocarcinogenesis. Its metabolic pathways have been described extensively, yet little is known about its biochemical processing, growth cycle expression, and pharmacological properties inside living hepatocytes-the principal cellular targets of this hepatocarcinogen. In this report, primary monolayer adult rat hepatocyte cultures and high specific-activity [ring G-3 H]-N-acetyl-2-aminofluorene were used to extend previous observations of metabolic activation of AAF by highly differentiated, proliferation-competent hepatocytes in long-term cultures. AAF metabolism proceeded by zero-order kinetics. Hepatocytes processed significant amounts of procarcinogen (≈12 μg AAF/106 cells/day). Five ring-hydroxylated and one deacetylated species of AAF were secreted into the culture media. Extracellular metabolite levels varied during the growth cycle (days 0-13), but their rank quantitative order was time invariant: 5-OH-AAF > 7-OH-AAF > 3-OH-AAF > N-OH-AAF > aminofluorene (AF) > 1-OH-AAF. Lineweaver-Burk analyses revealed two principal classes of metabolism: System I (high-affinity and low-velocity), Km[APPARENT] = 1.64 × 10-7 M and VMAX[APPARENT] = 0.1 nmol/106 cells/day and System II (low-affinity and high-velocity), Km[APPARENT] = 3.25 × 10-5 M and VMAX[APPARENT] = 1000 nmol/106 cells/day. A third system of metabolism of AAF to AF, with Km[APPARENT] and VMAX[APPARENT] constants of 9.6 × 10-5 M and 4.7 nmol/106 cells/day, was also observed. Evidence provided in this report and its companion paper suggests selective roles and intracellular locations for System I- and System II-mediated AAF metabolite formation during hepatocarcinogenesis, although some of the molecules and mechanisms responsible for multi-system processing remain to be fully defined.

High-Affinity Low-Capacity and Low-Affinity High-Capacity N-Acetyl-2-Aminofluorene (AAF) Macromolecular Binding Sites Are Revealed During the Growth Cycle of Adult Rat Hepatocytes in Primary Culture.

Long-term cultures of primary adult rat hepatocytes were used to study the effects of N-acetyl-2-aminofluorene (AAF) on hepatocyte proliferation during the growth cycle; on the initiation of hepatocyte DNA synthesis in quiescent cultures; and, on hepatocyte DNA replication following the initiation of DNA synthesis. Scatchard analyses were used to identify the pharmacologic properties of radiolabeled AAF metabolite binding to hepatocyte macromolecules. Two classes of growth cycle-dependent AAF metabolite binding sites-a high-affinity low-capacity site (designated Site I) and a low-affinity high-capacity site (designated Site II)-associated with two spatially distinct classes of macromolecular targets, were revealed. Based upon radiolabeled AAF metabolite binding to purified hepatocyte genomic DNA or to DNA, RNA, proteins, and lipids from isolated nuclei, Site IDAY 4 targets (KD[APPARENT] ≈ 2-4×10-6 M and BMAX[APPARENT] ≈ 6pmol/106cells/24 h) were consistent with genomic DNA; and with AAF metabolized by a nuclear cytochrome P450. Based upon radiolabeled AAF binding to total cellular lysates, Site IIDAY 4 targets (KD[APPARENT] ≈ 1.5×10-3 M and BMAX[APPARENT] ≈ 350pmol/106cells/24 h) were consistent with cytoplasmic proteins; and with AAF metabolized by cytoplasmic cytochrome P450s. DNA synthesis was not inhibited by concentrations of AAF that saturated DNA binding in the neighborhood of the Site I KD. Instead, hepatocyte DNA synthesis inhibition required higher concentrations of AAF approaching the Site II KD. These observations raise the possibility that carcinogenic DNA adducts derived from AAF metabolites form below concentrations of AAF that inhibit replicative and repair DNA synthesis.

The Phenomenon of Albumin-Mediated Hepatic Uptake of Organic Anion Transport Polypeptide Substrates: Prediction of the In Vivo Uptake Clearance from the In Vitro Uptake by Isolated Hepatocytes Using a Facilitated-Dissociation Model.

The effects of bovine serum albumin and human serum albumin on the unbound hepatic uptake clearance (PSu,inf) of the organic anion-transporting polypeptide substrates 1-anilino-8-naphthalene sulfonate (ANS) and pitavastatin (PTV) were determined using primary cultured rat hepatocytes and isolated human hepatocytes, respectively. The PSu,inf value of hepatocytes was estimated by dividing the initial uptake rate of these anions by their unbound concentrations. The PSu,inf values for ANS and PTV were enhanced in the presence of albumin, thereby demonstrating the phenomenon of "albumin-mediated" hepatic uptake. We previously constructed a "facilitated-dissociation" model, in which the interaction of the ligand-albumin complex with the cell surface enhanced the dissociation of that complex to provide unbound ligand for uptake to the hepatocytes [J Pharmacokinet Biopharm 16:165-181 (1988)]. That model was able to describe accurately the relationship between the enhancement of the PSu,inf values and the albumin concentration. By considering the enhancement of hepatic uptake clearance by albumin using this facilitated-dissociation model, we could predict accurately the PSu,inf in vivo from that obtained in isolated hepatocytes. In the light of these findings, we suggest that the facilitated-dissociation model is applicable to describing the phenomenon of albumin-mediated hepatic uptake via organic anion transporters and to evaluating hepatic uptake clearance in vivo.

Interspecies differences in the cytochrome P450 activity of hepatocytes exposed to PLGA and silica nanoparticles: an in vitro and in vivo investigation.

Nanomedicines represent a promising approach in the treatment and diagnosis of numerous disorders. The majority of the injected dose of nanoparticles (NPs) is sequestrated in the liver. Despite this hepatic tropism, the interaction of NPs with the detoxification function of the liver remains unclear. The present study consists of evaluating the impact of biodegradable poly(lactide-co-glycolide) (PLGA) and silica NPs on cytochrome P450 (CYP) activities. The effects of NPs were evaluated in vitro on human and rat hepatocytes in primary cultures and in vivo by intravenous injections in healthy rats. More than the physicochemical properties, the composition of NPs (organic, inorganic) dramatically influenced the detoxification function of the liver. Silica NPs modulated the CYP activity both in rat and human hepatocytes, in contrast to PLGA NPs. A CYP isoform-dependent effect was reported and the modulation of the metabolic hepatic activity was species-dependent. Human hepatocytes were sensitive to an exposure to PLGA NPs, whereas no marked effect was detected in rat hepatocytes. The in vitro data obtained in rat hepatocytes were correlated with the in vivo data. This study emphasizes the interest to set up relevant in vitro models using human hepatic cells to evaluate the hepatotoxicity of nanomedicines.

Signal Transduction Mechanism for 5-HT<sub>2B</sub> Receptor-Stimulated Autocrine Secretion of Transforming Growth Factor-α in Primary Cultures of Adult Rat Hepatocytes

Signal Transduction Mechanism for 5-HT<sub>2B</sub> Receptor-Stimulated Autocrine Secretion of Transforming Growth Factor-α in Primary Cultures of Adult Rat Hepatocytes

Serotonin-induced DNA synthesis and proliferation are mediated by autocrine secretion of transforming growth factor-α in primary cultures of adult rat hepatocytes

Serotonin-induced DNA synthesis and proliferation are mediated by autocrine secretion of transforming growth factor-α in primary cultures of adult rat hepatocytes

Sulforaphane Alters β-Naphthoflavone-Induced Changes in Activity and Expression of Drug-Metabolizing Enzymes in Rat Hepatocytes.

Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, exerts many beneficial effects on human health such as antioxidant, anti-inflammatory, and anticancer effects. The effect of SFN alone on drug-metabolizing enzymes (DMEs) has been investigated in numerous in vitro and in vivo models, but little is known about the effect of SFN in combination with cytochrome P450 (CYP) inducer. The aim of our study was to evaluate the effect of SFN on the activity and gene expression of selected DMEs in primary cultures of rat hepatocytes treated or non-treated with β-naphthoflavone (BNF), the model CYP1A inducer. In our study, SFN alone did not significantly alter the activity and expression of the studied DMEs, except for the glutathione S-transferase (GSTA1) mRNA level, which was significantly enhanced. Co-treatment of hepatocytes with SFN and BNF led to a substantial increase in sulfotransferase, aldoketoreductase 1C, carbonylreductase 1 and NAD(P)H:quinone oxidoreductase 1 activity and a marked decrease in cytochrome P450 (CYP) Cyp1a1, Cyp2b and Cyp3a4 expression in comparison to the treatment with BNF alone. Sulforaphane is able to modulate the activity and/or expression of DMEs, thus shifting the balance of carcinogen metabolism toward deactivation, which could represent an important mechanism of its chemopreventive activity.

Differential effects of epidermal growth factor (EGF) receptor ligands on receptor binding, downstream signalling pathways and DNA synthesis in hepatocytes

Hepatocytes are responsive to mitogenic effects of several ligands acting via EGFR. Studying primary cultures of rat hepatocytes, we found that, as compared to EGF, HB-EGF had a markedly higher affinity of the EGFR, while AR and TGFα had lower affinity. HB-EGF was also more potent compared to the other growth factors regarding phosphorylation of EGFR, Shc, ERK1/2 and Akt. All ligands induced phosphorylation of ErbB2, indicating receptor heterodimerization. TGFα, despite having much lower receptor affinity, was about equally potent and efficacious as HB-EGF as a stimulator of DNA synthesis. In contrast, EGF had relatively high affinity but markedly lower efficacy in stimulation of DNA synthesis. The results suggest that amplifying and/or inhibitory mechanisms may modulate the mitogenic responses downstream of the initial signalling steps, and that this may affect the effects of the EGFR ligands differentially.

Curcumin protects liver inflammation by suppressing expression of inducible nitric oxide synthase in primary cultured rat hepatocytes

Background: Curcumin has beneficial effects on organ metabolism. However, there is little evidence that curcumin affects inflammatory mediators, such as tumor necrosis factor (TNF)-α and nitric oxide (NO). In an inflamed liver, proinflammatory cytokines stimulate liver cells, followed by the induction of inducible NO synthase (iNOS). Excessive NO produced by iNOS is one of the factors in liver injury. Therefore, inhibiting iNOS induction for preventing liver injury is important.Objective: This study aimed to investigate liver protective effects of curcumin by examining interleukin (IL)-1β-stimulated hepatocytes.Methods: Primary cultured rat hepatocytes were treated with IL-1β in the presence or absence of curcumin. Induction of NO production and iNOS, and the signaling pathway of iNOS were analyzed.Results: Simultaneous addition of IL-1β and curcumin decreased expression levels of iNOS protein and mRNA, resulting in inhibition of NO production. Curcumin also reduced mRNA expression of TNF-α and IL-6. Curcumin inhibited two essential signaling pathways for iNOS induction, NF-κB activation and type I IL-1 receptor upregulation. Transfection experiments revealed that curcumin reduced iNOS mRNA levels at the promoter activation and mRNA stabilization steps. Delayed administration of curcumin after IL-1β addition also inhibited iNOS induction.Conclusions: Curcumin affects induction of inflammatory mediators, such as iNOS and TNF-α, in part through the inhibition of NF-κB activation in hepatocytes. Curcumin may have therapeutic potential for organ injuries, including the liver.Key words: curcumin, inducible nitric oxide synthase, liver injury, primary cultured hepatocytes, nuclear factor-κB, type I interleukin-1 receptor, tumor necrosis factor-α.

Elental® amino acid component has protective effects on primary cultured hepatocytes and a rat model of acute liver injury

Amino acids can exert protective effects on the liver either when administered as a medication or following an operation. In this study, we examined the protective effects of amino acids on the liver using in vitro and in vivo models by studying their influence on the induction of inducible nitric oxide synthase (iNOS) and nitric oxide production as a liver injury marker in cultured hepatocytes and liver-protective effects in d-galactosamine and lipopolysaccharide (GalN/LPS)-treated rats, respectively. Primary cultured rat hepatocytes were treated with interleukin (IL)-1β in the presence or absence of Elental® amino acid component (EleAA; 17 amino acids). Rats were pretreated with either EleAA or a diet containing selected amino acids followed by GalN/LPS injection. Survival rate and mRNA expression were analyzed. EleAA inhibited iNOS induction through reduction of mRNA synthesis and stability in cultured hepatocytes, indicating prevention of liver injury, but did not show a liver-protective effect in GalN/LPS rats. Among EleAA, Lys, Trp, His, and Arg (4AA) markedly decreased nitric oxide production and inhibited nuclear factor-κB (NF-κB) activation. In GalN/LPS rats, 4AA (3% of each amino acid in diet) increased survival rate by 50% and decreased mRNA expression of iNOS, tumor necrosis factor-α, and cytokine-induced neutrophil chemoattractant-1 in the liver. 4AA reduced NF-κB activation induced by GalN/LPS. 4AA inhibited the expression of inflammatory mediators, in part through inhibition of NF-κB activation in cultured hepatocytes and GalN/LPS-treated rats. The results suggest that EleAA has therapeutic potential for organ injuries including liver.