Reactive Oxygen Species Production In Hepatocytes Research Articles

Rosmarinic acid exerts anti-inflammatory effect and relieves oxidative stress via Nrf2 activation in carbon tetrachloride-induced liver damage.

Rosmarinic acid (RA) has biological and pharmaceutical properties and shows hepatoprotective potential. However, the hepatoprotective mechanism of RA needs to be further elucidated in vivo and in vitro. This study was aimed to evaluate the protective effect of RA on carbon tetrachloride (CCl4)-induced liver injury and elucidate the hepatoprotective mechanism of RA in vivo and in vitro. In vivo, the mice were orally administrated with RA (10, 20, and 40 mg/kg bw) daily for 28 consecutive days, and 1% CCl4 (5 mL/kg bw, dissolved in peanut oil) was used to induce liver injury. In vitro, the big rat liver (BRL) hepatocytes were pretreated with RA (0.2, 0.4, and 0.8 mg/mL) for 3 h, and then the hepatocytes were treated with CC14 (final concentration, 14 mM) for 3 h to induce cell injury. The related indexes, including hepatic function, oxidative stress, protein expression of nuclear-factor erythroid 2-related factor 2 (Nrf2) pathway, inflammation, histopathological change, hepatocyte apoptosis, and mitochondrial membrane potential, were evaluated. Oral administration of RA to mice considerably decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT), alkaline phosphatase (ALP), triacylglycerols (TG), total cholesterol (TC), total bilirubin (TBIL), hepatic reactive oxygen species (ROS), malondialdehyde (MDA), nitric oxide (NO), 8-hydroxydeoxyguanosine (8-OHdG), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-8 (IL-8). RA also increased the levels of hepatic glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) and the protein expressions of Nrf2, quinine oxidoreductase (NQO1), and heme oxygenease-1 (HO-1). Histopathological examinations indicated that RA (20 and 40 mg/kg bw) alleviated the liver tissue injury induced by CCl4. Moreover, RA inhibited the hepatocyte apoptosis caused by CCl4 based on TUNEL assay. In vitro, RA pretreatment remarkably recovered the cell viability and reduced the CCl4-induced elevation of AST, ALT, lactate dehydrogenase (LDH), ROS, and 8-OHdG. Immunohistochemistry staining demonstrated that pretreatment with RA markedly inhibited the expression of IL-6, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and Caspase-3 in CCl4-treated hepatocytes. Additionally, RA pretreatment significantly decreased the elevation of mitochondrial membrane potential in CCl4-treated hepatocytes. RA exerted a protective effect against CCl4-induced liver injury in mice through activating Nrf2 signaling pathway, reducing antioxidant damage, suppressing inflammatory response, and inhibiting hepatocyte apoptosis. RA could attenuate BRL hepatocyte ROS production, DNA oxidative damage, inflammatory response, and apoptosis induced by CCl4 exposure.

Dual Regulation of Tank Binding Kinase 1 by BRG1 in Hepatocytes Contributes to Reactive Oxygen Species Production.

Excessive accumulation of reactive oxygen species (ROS) is considered a major culprit for the pathogenesis of non-alcoholic fatty liver disease (NAFLD). We have previously shown that deletion of Brahma related gene 1 (BRG1) mitigated NAFLD in mice in part by attenuating ROS production in hepatocyte. Here we report that BRG1 deletion led to simultaneous down-regulation in expression and phosphorylation of tank binding kinase 1 (TBK1) in vivo and in vitro. On the one hand, BRG1 interacted with AP-1 to bind to the TBK1 promoter and directly activated TBK1 transcription in hepatocytes. On the other hand, BRG1 interacted with Sp1 to activate the transcription of c-SRC, a tyrosine kinase essential for TBK1 phosphorylation. Over-expression of c-SRC and TBK1 corrected the deficiency in ROS production in BRG1-null hepatocytes whereas depletion of TBK1 or c-SRC attenuated ROS production. In conclusion, our data suggest that dual regulation of TBK1 activity, at the transcription level and the post-transcriptional level, by BRG1 may constitute an important mechanism underlying excessive ROS production in hepatocytes.

A Cross Talk Between BRG1 and Males Absent on the First Contributes to Reactive Oxygen Species Production in a Mouse Model of Nonalcoholic Steatohepatitis.

Aims: Accumulation of reactive oxygen species (ROS) in hepatocytes in response to excessive nutrients and the ensuing liver damages caused by ROS constitute a key pathophysiological event in nonalcoholic steatohepatitis (NASH). In the present study, we investigated the epigenetic mechanism underlying ROS production in NASH pathogenesis. Results: NASH was induced by feeding the mice with a methionine-and-choline-deficient (MCD) diet for 4 weeks. Compared with the control mice (wild type [WT]), mice with hepatocyte-specific deletion of Brg1 (HepcKO), a core component of the mammalian chromatin remodeling complex, developed a less severe form of NASH when fed on the MCD diet. Importantly, ROS levels were attenuated in HepcKO mice as opposed to WT mice. Brahma-related gene 1 (Brg1) deficiency downregulated the transcription of NADPH oxidases (NOX1, NOX2, and NOX4) both in vivo and in vitro. Mechanistically, Brg1 deletion rendered a more repressive chromatin structure surrounding the NOX promoters as characterized by reduced levels of acetylated histones. In addition, Brg1 interacted with the histone H4K16 acetyltransferase males absent on the first (MOF) to activate NOX transcription. MOF knockdown by small interfering RNA or pharmaceutical inhibition by MG149 suppressed NOX transcription and ameliorated ROS levels. Innovation: Our data highlight a novel epigenetic mechanism through which Brg1 and MOF cooperate to regulate ROS production in hepatocytes in response to pro-NASH stimuli. Conclusion: A cross talk between Brg1 and MOF epigenetically activates NOX transcription and elevates ROS synthesis contributing to NASH pathogenesis.

Targeting Liver Fibrosis with a Cell-penetrating Protease-activated Receptor-2 (PAR2) Pepducin

Chronic liver inflammation and fibrosis in nonalcoholic steatohepatitis can lead to cirrhosis and liver failure for which there are currently no approved treatments. Protease-activated receptor-2 (PAR2) is an emerging new target expressed on liver stellate cells and hepatocytes that regulates the response to liver injury and inflammation. Here, we identified a pepducin to block the deleterious actions of PAR2 in promoting liver fibrosis. Non-alcoholic fatty liver disease and early fibrosis were induced by the methionine-choline-deficient diet in mice. Fibrotic liver disease was induced by administering carbon tetrachloride for 8 weeks. Mice were treated with the pepducin PZ-235 either from onset of the experiment or after fibrosis was established. Hepatic fibrosis, collagen content, inflammatory cytokines, steatosis, triglycerides, and NAFLD activity score were assessed as primary outcome parameters depending on the model. The activity of the PAR2 pepducin on cultured stellate cell activation and hepatocyte reactive oxygen species production was evaluated. PZ-235 significantly suppressed liver fibrosis, collagen deposition, inflammatory cytokines, NAFLD activity score, steatosis, triglycerides, aspartate transaminase, alanine transaminase, and stellate cell proliferation by up to 50-100%. The PAR2 inhibitor afforded significant protective effects against hepatocellular necrosis and attenuated PAR2-mediated reactive oxygen species production in hepatocytes. PZ-235 was distributed to liver and other mouse tissues and was found to form a well structured α-helix that closely resembles the juxtamembrane helical region of the analogous TM6 and third intracellular region of the intact receptor that is critical for coupling to internal G proteins. The ability of PZ-235 to effectively suppress fibrosis, hepatocellular necrosis, reactive oxygen species production, steatosis, and inflammation indicates the potential for PAR2 pepducin inhibitors to be broadly efficacious in the treatment of liver fibrosis.

Lopimune-induced mitochondrial toxicity is attenuated by increased uncoupling protein-2 level in treated mouse hepatocytes.

Although the protease inhibitor (PI) Lopimune has proven to be effective, no studies have examined the side effects of Lopimune on mitochondrial bioenergetics in hepatocytes. The objective of the present study is to evaluate mitochondrial respiration, production of reactive oxygen species (ROS) and expression of uncoupling protein-2 (UCP2) in mouse hepatocytes following Lopimune administration. Mitochondria were extracted from mouse liver using differential centrifugation and hepatocytes were isolated by the collagenase perfusion procedure. Mitochondrial respiration was measured using a Rank Brothers oxygen electrode. ROS production in hepatocytes was monitored by flow cytometry using a 2',7'-dichlorofluorescin diacetate probe and UCP2 protein expression was detected by Western blotting. We found that Lopimune induced a significant decrease of approximately 30% in the respiratory control ratio (RCR) starting from day 4 until day 9 of treatment. This decrease was due to an increase in state 4 respiration, reflecting an increase in mitochondrial proton leak. State 2 and state 3 respirations were not affected. Moreover, ROS production significantly increased by about 2-fold after day 1 of treatment and decreased after day 3, returning to the resting level on day 5. Interestingly, UCP2 which is absent from control hepatocytes, was expressed starting from day 4 of treatment. Our findings indicate that Lopimune-induced proton leak, mediated by UCP2, may represent a response to inhibit the production of ROS as a negative feedback regulatory mechanism. These results imply a potential involvement of UCP2in the regulation of oxidative stress and add new insights into the understanding of mitochondrial toxicity induced by PIs.

Niacin inhibits fat accumulation, oxidative stress, and inflammatory cytokine IL-8 in cultured hepatocytes: Impact on non-alcoholic fatty liver disease

ObjectiveNon-alcoholic fatty liver disease (NAFLD) is a common disorder characterized by excessive hepatic fat accumulation, production of reactive oxygen species (ROS), inflammation and potentially resulting in non-alcoholic steatohepatitis (NASH), cirrhosis and end-stage liver disease. Recently, we have shown that niacin significantly prevented hepatic steatosis and regressed pre-existing steatosis in high-fat fed rat model of NAFLD. To gain further insight into the cellular mechanisms, this study investigated the effect of niacin on human hepatocyte fat accumulation, ROS production, and inflammatory mediator IL-8 secretion. Materials and methodsHuman hepatoblastoma cell line HepG2 or human primary hepatocytes were first stimulated with palmitic acid followed by treatment with niacin or control for 24h. ResultsThe data indicated that niacin (at 0.25 and 0.5mmol/L doses) significantly inhibited palmitic acid-induced fat accumulation in human hepatocytes by 45–62%. This effect was associated with inhibition of diacylglycerol acyltransferase 2 (DGAT2) mRNA expression without affecting the mRNA expression of fatty acid synthase (FAS) and carnitine palmitoyltransferase 1 (CPT1). Niacin attenuated hepatocyte ROS production and it also inhibited NADPH oxidase activity. Niacin reduced palmitic acid-induced IL-8 levels. ConclusionsThese findings suggest that niacin, through inhibiting hepatocyte DGAT2 and NADPH oxidase activity, attenuates hepatic fat accumulation and ROS production respectively. Decreased ROS production, at least in part, may have contributed to the inhibition of pro-inflammatory IL-8 levels. These mechanistic studies may be useful for the clinical development of niacin and niacin-related compounds for the treatment of NAFLD/NASH and its complications.

Growth hormone resistance exacerbates cholestasis-induced murine liver fibrosis.

Growth hormone (GH) resistance has been associated with liver cirrhosis in humans but its contribution to the disease remains controversial. In order to elucidate whether GH resistance plays a causal role in the establishment and development of liver fibrosis, or rather represents a major consequence thereof, we challenged mice lacking the GH receptor gene (Ghr(-/-), a model for GH resistance) by crossing them with Mdr2 knockout mice (Mdr2(-/-)), a mouse model of inflammatory cholestasis and liver fibrosis. Ghr(-/-);Mdr2(-/-) mice showed elevated serum markers associated with liver damage and cholestasis, extensive bile duct proliferation, and increased collagen deposition relative to Mdr2(-/-) mice, thus suggesting a more severe liver fibrosis phenotype. Additionally, Ghr(-/-);Mdr2(-/-) mice had a pronounced down-regulation of hepatoprotective genes Hnf6, Egfr, and Igf-1, and significantly increased levels of reactive oxygen species (ROS) and apoptosis in hepatocytes, compared to control mice. Moreover, single knockout mice (Ghr(-/-)) fed with a diet containing 1% cholic acid displayed an increase in hepatocyte ROS production, hepatocyte apoptosis, and bile infarcts compared to their wild-type littermates, indicating that loss of Ghr renders hepatocytes more susceptible to toxic bile acid accumulation. Surprisingly, and despite their severe fibrotic phenotype, Ghr(-/-);Mdr2(-/-) mice displayed a significant decrease in tumor incidence compared to Mdr2(-/-) mice, indicating that loss of Ghr signaling may slow the progression from fibrosis/cirrhosis to cancer in the liver. GH resistance dramatically exacerbates liver fibrosis in a mouse model of inflammatory cholestasis, therefore suggesting that GH resistance plays a causal role in the disease and provides a novel target for the development of liver fibrosis treatments.

Protective Role ofPsoralea corylifoliaL. Seed Extract against Hepatic Mitochondrial Dysfunction Induced by Oxidative Stress or Aging

The accumulation of oxidative damage and mitochondrial dysfunction is an important factor that contributes to aging. The Psoralea corylifolia seeds (PCS), commonly known as “Boh-Gol-Zhee” in Korea, have been used traditionally as a medicinal remedy. We investigated whether an extract of PCS has protective effects on oxidative stress and mitochondrial function in hepatocytes. The PCS extract showed an antisenescence effect on human diploid fibroblasts as evidenced by a decreased expression of p16INK4a mRNA and senescence-associated β-galactosidase staining. PCS extract treatment reduced H2O2-induced reactive oxygen species (ROS) production in HepG2 cells, inhibited ROS production in hepatocytes of aged mice, and increased superoxide dismutase activity. In H2O2-treated HepG2 cells, PCS extract treatment recovered ATP production. PCS extract treatment recovered the oxygen consumption rate and inhibited reduction of mitochondrial membrane potential induced by oxidative stress, suggesting improvement of mitochondrial function. In addition, PCS extract treatment recovered peroxisome proliferator-activated receptor γ coactivator 1α and carnitine palmitoyltransferase 1 mRNA and protein expression, and inhibited mitochondrial genome damage. Treatment with the major component of PCS extract, bakuchiol, also recovered mitochondrial dysfunction. On the basis of these results, we conclude that PCS extract inhibits ROS production and mitochondrial dysfunction induced by oxidative stress in hepatocytes.

Activation of CD40 with platelet derived CD154 promotes reactive oxygen species dependent death of human hepatocytes during hypoxia and reoxygenation.

BackgroundHypoxia and hypoxia-reoxygenation (H-R) are pathogenic factors in many liver diseases that lead to hepatocyte death as a result of reactive oxygen species (ROS) accumulation. The tumor necrosis factor super-family member CD154 can also induce hepatocyte apoptosis via activation of its receptor CD40 and induction of autocrine/paracrine Fas Ligand/CD178 but the relationship between CD40 activation, ROS generation and apoptosis is poorly understood. We hypothesised that CD40 activation and ROS accumulation act synergistically to drive human hepatocyte apoptosis.MethodsHuman hepatocytes were isolated from liver tissue and exposed to an in vitro model of hypoxia and H-R in the presence or absence of CD154 and/or various inhibitors. Hepatocyte ROS production, apoptosis and necrosis were determined by labelling cells with 2′,7′-dichlorofluorescin, Annexin-V and 7-AAD respectively in a three-colour reporter flow cytometry assay.ResultsExposure of human hepatocytes to recombinant CD154 or platelet-derived soluble CD154 augments ROS accumulation during H-R resulting in NADPH oxidase-dependent apoptosis and necrosis. The inhibition of c-Jun N-terminal Kinase and p38 attenuated CD154-mediated apoptosis but not necrosis.ConclusionsCD154-mediated apoptosis of hepatocytes involves ROS generation that is amplified during hypoxia-reoxygenation. This finding provides a molecular mechanism to explain the role of platelets in hepatocyte death during ischemia-reperfusion injury.

Reactive oxygen species mediate human hepatocyte injury during hypoxia/reoxygenation

Increasing evidence shows that reactive oxygen species (ROS) may be critical mediators of liver damage during the relative hypoxia of ischemia/reperfusion injury (IRI) associated with transplant surgery or of the tissue microenvironment created as a result of chronic hepatic inflammation or infection. Much work has been focused on Kupffer cells or liver resident macrophages with respect to the generation of ROS during IRI. However, little is known about the contribution of endogenous hepatocyte ROS production or its potential impact on the parenchymal cell death associated with IRI and chronic hepatic inflammation. For the first time, we show that human hepatocytes isolated from nondiseased liver tissue and human hepatocytes isolated from diseased liver tissue exhibit marked differences in ROS production in response to hypoxia/reoxygenation (H-R). Furthermore, several different antioxidants are able to abrogate hepatocyte ROS-induced cell death during hypoxia and H-R. These data provide clear evidence that endogenous ROS production by mitochondria and nicotinamide adenine dinucleotide phosphate oxidase drives human hepatocyte apoptosis and necrosis during hypoxia and H-R and may therefore play an important role in any hepatic diseases characterized by a relatively hypoxic liver microenvironment. In conclusion, these data strongly suggest that hepatocytes and hepatocyte-derived ROS are active participants driving hepatic inflammation. These novel findings highlight important functional/metabolic differences between hepatocytes isolated from normal donor livers, hepatocytes isolated from normal resected tissue obtained during surgery for malignant neoplasms, and hepatocytes isolated from livers with end-stage disease. Furthermore, the targeting of hepatocyte ROS generation with antioxidants may offer therapeutic potential for the adjunctive treatment of IRI and chronic inflammatory liver diseases.

Is There an Association Between Radon Exposure and the Incidence of Thyroid Cancer?

Is There an Association Between Radon Exposure and the Incidence of Thyroid Cancer?

Endocannabinoid Degradation and Oxidative Defense Mechanisms Determine Anandamide-induced Cell Death in Liver Cell Populations

Background: We have shown that the endogenous cannabinoid anandamide (AEA) induces reactive oxygen species (ROS)-dependent cell death in primary hepatic stellate cells (HSCs), but not in primary hepatocytes. Aim: To determine cellular factors that control AEA-induced cell death in hepatic cell populations. Methods: Fatty acid amide hydrolase (FAAH) expression was analyzed by western blot and real time PCR in primary rat hepatocytes and primary rat HSCs. FAAH activity was determined colorimetrically. Cell death was analyzed by LDH release, propidium iodide uptake and western blot for caspase 3- and PARP cleavage. Glutathione (GSH) levels were measured by the DTNB method. mRNA expression of the endocannabinoid receptors CB1, CB2 and VR1 was determined by real time PCR. ROS production was monitored by DCFDA fluorescence. Results: FAAH mRNA, FAAH protein and FAAH activity were extremely high in primary rat hepatocytes, but virtually absent in primary rat HSCs. Hepatocytes from FAAH -/- mice, or wildtype hepatocytes which had been pretreated with the FAAH inhibitor URB597 were susceptible to AEA-mediated death (45% and 40% at 50 μM after 24h, respectively). Conversely, FAAH overexpression prevented AEA-induced death in HSCs. Anandamide induced an increase in ROS production in hepatocytes which had been pretreated with the FAAH inhibitor URB597 or depleted of GSH by BSO. GSH levels were 10-fold higher in hepatocytes than in HSCs. GSH depletion rendered hepatocytes susceptible to AEA-mediated death while GSH pretreatment prevented cell death in HSCs. The combination of FAAH inhibition and GSH depletion had additive effects on AEA-mediated hepatocyte cell death resulting in almost 70% death after 24h at 50 μM AEA and lowered the threshold for AEA-induced cell death to 500 nM. AEA-induced hepatocyte death after BSO and URB597 pretreatment was purely necrotic and occurred independently of the endocannabinoid receptors CB1, CB2 and VR1 which were virtually absent (CB1, CB2) or expressed at low levels (VR1) in hepatocytes. Conclusion: FAAH and GSH determine endocannabinoid-induced necrosis in hepatocytes and HSCs, independently of expression of cannabinoid receptors. High expression of FAAH and high intracellular GSH levels efficiently prevent AEA-induced cell death in hepatocytes. FAAH-mediated endocannabinoid degradation in hepatocytes leads to a new physiological concept allowing the specific targeting of HSCs in liver fibrosis.

Acute and chronic ethanol increases reactive oxygen species generation and decreases viability in fresh, isolated rat hepatocytes.

Although reactive oxygen species (ROS) have been implicated in the etiology of alcohol-induced liver disease, neither their relative contribution to cell death nor the cellular mechanisms mediating their formation are known. The purpose of this study was to test the hypothesis that acute and chronic ethanol exposure enhances the mitochondrial generation of ROS in fresh, isolated hepatocytes. Acute ethanol exposure stimulated ROS production, increased the cellular NADH/NAD+ ratio, and decreased hepatocyte viability slightly, which was prevented by pretreatment with 4-methylpyrazole (4-MP), an inhibitor of alcohol dehydrogenase. Similarly, xylitol, an NADH-generating compound, enhanced hepatocyte ROS production and decreased viability. Incubation with pyruvate, an NADH-oxidizing compound, and cyanamide, an inhibitor of aldehyde dehydrogenase, significantly decreased ROS levels in acute ethanol-treated hepatocytes. Chronic ethanol consumption produced a sixfold increase in hepatocyte ROS production compared with levels measured in controls. Hepatocytes from ethanol-fed rats were less viable compared with controls, e.g., viability was 68% +/- 2% (ethanol) versus 83% +/- 1% (control) after 60 minutes of incubation. Antimycin A increased ROS production and decreased cell viability; however, the toxic effect of antimycin A was more pronounced in ethanol-fed hepatocytes. These results suggest that acute and chronic ethanol exposure exacerbates mitochondrial ROS production, contributing to cell death.