Identification of Binding Regions of Bilirubin in the Ligand-Binding Pocket of the Peroxisome Proliferator-Activated Receptor-A (PPARalpha).

Recent work has shown that bilirubin has a hormonal function by binding to the peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor that drives the transcription of genes to control adiposity. Our previous in silico work predicted three potential amino acids that bilirubin may interact with by hydrogen bonding in the PPARα ligand-binding domain (LBD), which could be responsible for the ligand-induced function. To further reveal the amino acids that bilirubin interacts with in the PPARα LBD, we harnessed bilirubin's known fluorescent properties when bound to proteins such as albumin. Our work here revealed that bilirubin interacts with threonine 283 (T283) and alanine 333 (A333) for ligand binding. Mutational analysis of T283 and A333 showed significantly reduced bilirubin binding, reductions of 11.4% and 17.0%, respectively. Fenofibrate competitive binding studies for the PPARα LBD showed that bilirubin and fenofibrate possibly interact with different amino acid residues. Furthermore, bilirubin showed no interaction with PPARγ. This is the first study to reveal the amino acids responsible for bilirubin binding in the ligand-binding pocket of PPARα. Our work offers new insight into the mechanistic actions of a well-known molecule, bilirubin, and new fronts into its mechanisms.

Bilirubin as a metabolic hormone: the physiological relevance of low levels.

Recent research on bilirubin, a historically well-known waste product of heme catabolism, suggests an entirely new function as a metabolic hormone that drives gene transcription by nuclear receptors. Studies are now revealing that low plasma bilirubin levels, defined as "hypobilirubinemia," are a possible new pathology analogous to the other end of the spectrum of extreme hyperbilirubinemia seen in patients with jaundice and liver dysfunction. Hypobilirubinemia is most commonly seen in patients with metabolic dysfunction, which may lead to cardiovascular complications and possibly stroke. We address the clinical significance of low bilirubin levels. A better understanding of bilirubin's hormonal function may explain why hypobilirubinemia might be deleterious. We present mechanisms by which bilirubin may be protective at mildly elevated levels and research directions that could generate treatment possibilities for patients with hypobilirubinemia, such as targeting of pathways that regulate its production or turnover or the newly designed bilirubin nanoparticles. Our review here calls for a shift in the perspective of an old molecule that could benefit millions of patients with hypobilirubinemia.

Bilirubin in metabolic syndrome and associated inflammatory diseases: New perspectives.

Diabetes mellitus is one of the major global health issues, which is closely related to metabolic dysfunction and the chronic inflammatory diseases. Multiple studies have demonstrated that serum bilirubin is negatively correlated with metabolic syndrome and associated inflammatory diseases, including atherosclerosis, hypertension, etc. However, the roles of bilirubin in metabolic syndrome and associated inflammatory diseases still remain unclear. Here, we explain the role of bilirubin in metabolic syndrome and chronic inflammatory diseases and its therapeutic potential. Understanding the role of bilirubin activities in diabetes may serve as a therapeutic target for the treatment of chronic inflammatory diseases in diabetic patients.

A link between bilirubin levels and atrial fibrillation recurrence after catheter ablation.

BACKGROUND: Bilirubin is associated with different cardiovascular diseases. The relationship between bilirubin and atrial fibrillation (AF) remains unclear. The aim of this study is to determine the association between bilirubin and AF recurrence after catheter ablation. METHODS: A total of 212 patients who received AF ablation were retrospectively studied. The total bilirubin level, clinical characteristics, and echocardiographic findings were analyzed to predict the outcome of AF ablations. RESULTS: During a mean follow-up period of 12.2 ± 5.8 months, 61 (28.8%) patients had AF recurrence after catheter ablation. The patients with AF recurrence had a larger left atrial (LA) diameter (39.8 ± 6.3 versus 36.7 ± 5.8 mm; p = 0.001) and higher total bilirubin levels (0.82 ± 0.37 versus 0.63 ± 0.29 mg/dL; p < 0.001) than those without recurrence. The patients with recurrence had higher direct and indirect bilirubin levels than patients without recurrence. The total bilirubin level remained an independent predictor of AF recurrence after multivariate analysis (odds ratio, 4.95; 95% CI, 1.65-14.83; p = 0.004). We identified a cut point of the total bilirubin level for predicting AF recurrence by receiver operator characteristic curve (cut point, 0.7 mg/dL; area under the curve, 0.65; p < 0.001). The total bilirubin levels were positively correlated with the neutrophil counts. However, there were no associations among the total bilirubin level, left atrial (LA) diameter, and voltage. CONCLUSION: Higher serum bilirubin levels were associated with AF recurrence in paroxysmal AF patients following catheter ablation.

Bilirubin: from an unimportant waste product to important myocardial infarction predictor.

Bilirubin is the major catabolic product of heme degradation. It has long been regarded as an unimportant waste product. However, within the last twenty-five years, it has been demonstrated to play a very important role in maintaining the bodys redox equilibrium. Disturbances of this equilibrium - increased oxidative stress - are currently considered one of the major risk factors for the development of non-communicable diseases. Although the exact mechanism is not known, a number of human studies have proved a reduced incidence of a number of (especially cardiovascular but also cancer) diseases in individuals with mildly elevated bilirubin concentrations. Key words: bilirubin - cardiovascular disease - morbidity - mortality.

Systemic regulation of bilirubin homeostasis: Potential benefits of hyperbilirubinemia.

Neurotoxic bilirubin is the end product of heme catabolism in mammals. Bilirubin is solely conjugated by uridine diphospho-glucuronosyltransferase 1A1, which is a membrane-bound enzyme that catalyzes the transfer of glucuronic acid. Due to low function of hepatic and intestinal uridine diphospho-glucuronosyltransferase 1A1 during the neonatal period, human neonates develop mild to severe physiological hyperbilirubinemia. Accumulation of bilirubin in the brain leads to the onset of irreversible brain damage, termed kernicterus. Breastfeeding is one of the most significant factors that increase the risk of developing kernicterus in infants. Why does this most natural way of feeding increase the risk of brain damage or even death? This question leads to the hypothesis that breast milk-induced hyperbilirubinemia might bring certain benefits that outweigh those risks. While bilirubin is neurotoxic and cytotoxic, this compound is also a potent antioxidant. There are studies showing improved clinical conditions in patients with hyperbilirubinemia. Accumulating evidence also shows that genetic polymorphisms linked to hyperbilirubinemia are beneficial against various diseases. In this review article, we first introduce the production, metabolism, and transport of bilirubin. We then discuss the potential benefits of neonatal and adult hyperbilirubinemia. Finally, epigenetic factors as well as metabolomic information associated with hyperbilirubinemia are described. This review article advances the understanding of the physiological importance of the paradoxical compound bilirubin. (Hepatology 2018;67:1609-1619).

Hyperbilirubinemia, Hypertension, and CKD: the Links.

PURPOSE OF REVIEW: This review aims to highlight recent advances on the role of hyperbilirubinemia in hypertension and chronic kidney disease, with a focus on the pathophysiological mechanisms explaining the protective effects of bilirubin. An overview of pharmacologic induction of hyperbilirubinemia will also be discussed. RECENT FINDINGS: The findings depict a protective role of bilirubin in the development of hypertension and cardiovascular diseases. Hyperbilirubinemia is also negatively correlated with the development and progression of chronic kidney disease. Commonly used drugs play a role in pharmacologic induction of hyperbilirubinemia. Bilirubin is therefore an exciting target for new therapeutic interventions for its antioxidant properties can be pivotal in the management of hypertension and in preventing and halting the progression of chronic kidney disease. Longitudinal studies are warranted to evaluate the prospective association between bilirubin levels and incident hypertension and chronic kidney disease in the general population. Interventions to induce hyperbilirubinemia need to be explored as a novel therapeutic approach in fighting disease burden.

Mortality outcomes after busulfan-containing conditioning treatment and haemopoietic cell transplantation in patients with Gilbert's syndrome: a retrospective cohort study.

BACKGROUND: Gilbert's syndrome is a common inherited disorder of bilirubin metabolism, characterised by mild, unconjugated hyperbilirubinaemia. However, the effect of Gilbert's syndrome on the disposition of some drugs can lead to unexpected toxicity. We tested the hypothesis that patients undergoing myeloablative conditioning and haemopoietic cell transplantation would have different mortality outcomes depending on whether or not they had laboratory evidence of Gilbert's syndrome. METHODS: In this retrospective cohort study, we used clinical and laboratory data of patients who had haemopoietic cell transplantation from Jan 1, 1991, to Dec 31, 2011. Patients were included if they had received high-dose conditioning regimens of cyclophosphamide plus total body irradiation (CY/TBI), busulfan plus cyclophosphamide (BU/CY), busulfan plus melphalan plus thioTEPA (BUMELTT), or melphalan before transplant. Patients were excluded if their original consent forms to report transplant outcomes were not signed, if consent was withdrawn, or if they were a prisoner. Patients with Gilbert's syndrome were defined as having laboratory values before the start of conditioning therapy for unconjugated serum bilirubin concentrations of at least 17·1 µmol/L (≥1 mg/dL), normal conjugated serum bilirubin, and no evidence of hepatitis, cholestasis, or haemolysis. We assessed the association of Gilbert's syndrome with overall mortality and non-relapse mortality using adjusted Cox regression models at day 200 after transplantation. FINDINGS: Our study cohort was 3379 patients-1855 (55%) allograft and 1524 (45%) autograft recipients. 211 (6%) patients had Gilbert's syndrome and 3168 (94%) did not have this condition. Most patients were adults (median age 45·8 years [IQR 33·2-55·5]) with haematological malignancies. For overall mortality 664 (20%) patients had died by day 200 after transplant (47 [22%] of 211 who had Gilbert's syndrome vs 617 [19%] of 3168 who did not have Gilbert's syndrome), and for non-relapse mortality 499 (92%) patients had died before relapse was recorded (38 [18%] who had Gilbert's syndrome vs 461 [15%] who did not have Gilbert's syndrome). The effect of Gilbert's syndrome on the risk of overall mortality and non-relapse mortality by transplant day 200 varied between the conditioning regimens and donor groups. In patients conditioned with a myeloablative regimen that contained busulfan (n=1131), those with Gilbert's syndrome (n=60) were at a significantly increased risk of death and non-relapse mortality by day 200 compared with those without Gilbert's syndrome (n=1071; hazard ratio [HR] 2·30, 95% CI 1·47-3·61, p=0·00030; and 2·77, 1·71-4·49, p<0·0001). In patients who received CY/TBI or melphalan conditioning regimens, those with Gilbert's syndrome had similar outcomes to those without Gilbert's syndrome (overall mortality at day 200 HR 0·90, 95% CI 0·60-1·34, p=0·60; non-relapse mortality at day 200: 0·90, 0·56-1·45, p=0·65). Analyses of causes of death and busulfan disposition provided no mechanistic explanation for the differences in mortality. INTERPRETATION: Overall mortality and non-relapse mortality at day 200 after transplant were significantly worse in patients with Gilbert's syndrome who received busulfan-containing myeloablative conditioning regimens, compared with non-Gilbert's syndrome patients. Patients with Gilbert's syndrome should receive busulfan-containing myeloablative conditioning regimens with caution. FUNDING: US National Institutes of Health.

Cholestatic liver (dys)function during sepsis and other critical illnesses.

OBJECTIVE: In ICU patients, abnormal liver tests are common. Markers of cholestasis are associated with adverse outcome. Research has focused on the possibility that mild hyperbilirubinemia, instead of indicating inadvertent cholestasis, may be adaptive and beneficial. These new insights are reviewed and integrated in the state-of-the-art knowledge on hepatobiliary alterations during sepsis and other critical illnesses. DATA SOURCES: Relevant publications were searched in Medline with search terms bile, bile acids, cholestasis, critical illness, intensive care, sepsis, alone or in combination. DATA SYNTHESIS: Studies have shown that bilirubin, but also bile acids, the main active constitutes of bile, are increased in plasma of patients with critical illnesses. In particular the conjugated fractions of bilirubin and bile acids are high, indicating that during critical illness the liver is capable of converting these molecules to less toxic forms. In human liver biopsies of prolonged critically ill patients, expression of bile acid excretion pumps towards the bile canaliculi was lower, while alternative transporters towards the systemic circulation were upregulated. Remarkably, in the presence of increased circulating bile acids, expression of enzymes controlling synthesis of bile acids was not suppressed. This suggested loss of feedback inhibition of bile acids synthesis, possibly explained by the observed cytoplasmic retention of the nuclear FXR/RXR heterodimer. As macronutrient restriction during acute critical illness, an intervention that improved outcome, was found to further increase plasma bilirubin while reducing other markers of cholestasis, a potentially protective role of hyperbilirubinemia was suggested. CONCLUSION: The increase in circulating levels of conjugated bile acids and bilirubin in response to acute sepsis/critical illnesses may not necessarily point to cholestasis as a pathophysiological entity. Instead it may be the result of an adaptively altered bile acid production and transport back towards the systemic circulation. How these changes could be beneficial for survival should be further investigated.

Bilirubin scavenges chloramines and inhibits myeloperoxidase-induced protein/lipid oxidation in physiologically relevant hyperbilirubinemic serum.

Hypochlorous acid (HOCl), an oxidant produced by myeloperoxidase (MPO), induces protein and lipid oxidation, which is implicated in the pathogenesis of atherosclerosis. Individuals with mildly elevated bilirubin concentrations (i.e., Gilbert syndrome; GS) are protected from atherosclerosis, cardiovascular disease, and related mortality. We aimed to investigate whether exogenous/endogenous unconjugated bilirubin (UCB), at physiological concentrations, can protect proteins/lipids from oxidation induced by reagent and enzymatically generated HOCl. Serum/plasma samples supplemented with exogenous UCB (≤250µM) were assessed for their susceptibility to HOCl and MPO/H2O2/Cl(-) oxidation, by measuring chloramine, protein carbonyl, and malondialdehyde (MDA) formation. Serum/plasma samples from hyperbilirubinemic Gunn rats and humans with GS were also exposed to MPO/H2O2/Cl(-) to: (1) validate in vitro data and (2) determine the relevance of endogenously elevated UCB in preventing protein and lipid oxidation. Exogenous UCB dose-dependently (P<0.05) inhibited HOCl and MPO/H2O2/Cl(-)-induced chloramine formation. Albumin-bound UCB efficiently and specifically (3.9-125µM; P<0.05) scavenged taurine, glycine, and N-α-acetyllysine chloramines. These results were translated into Gunn rat and GS serum/plasma, which showed significantly (P<0.01) reduced chloramine formation after MPO-induced oxidation. Protein carbonyl and MDA formation was also reduced after MPO oxidation in plasma supplemented with UCB (P<0.05; 25 and 50µM, respectively). Significant inhibition of protein and lipid oxidation was demonstrated within the physiological range of UCB, providing a hypothetical link to protection from atherosclerosis in hyperbilirubinemic individuals. These data demonstrate a novel and physiologically relevant mechanism whereby UCB could inhibit protein and lipid modification by quenching chloramines induced by MPO-induced HOCl.

Serum bilirubin and the risk of hypertension.

BACKGROUND: Experimental studies suggest oxidative stress could lead to the development of hypertension. Serum bilirubin is a major contributor to the antioxidant capacity in blood plasma and has been identified as an independent cardiovascular risk factor in cohort studies. However, data on the relationship between bilirubin and blood pressure are scarce and inconclusive. METHODS: We analysed data from the National Health and Nutrition Examination Surveys (NHANES) 1999-2012 (N=31069). Fifty multiple imputed data sets were generated and analysed to avoid selection/confounding bias due to excluding individuals/variables with missing values. A minimal sufficient adjustment set of variables (MSAS) needed to estimate the unconfounded effect of bilirubin on blood pressure and hypertension (systolic/diastolic blood pressure ≥ 140/90 mmHg or using antihypertensive medication) was identified using the back-door criterion and included in all regression models. RESULTS: After adjustment for the MSAS variables, systolic blood pressure decreased progressively up to -2.5 mmHg (p<0.001) and the prevalence of hypertension was up to 25% lower (P<0.001) in those with bilirubin ≥ 1.0 mg/dl-the highest two deciles-compared with those with 0.1-0.4 mg/dl-the lowest decile. Sensitivity analyses showed these results were unlikely to be explained by residual confounding or selection bias. CONCLUSIONS: High serum bilirubin may decrease the risk of hypertension by inactivating and inhibiting the synthesis of reactive oxygen species in vascular cells. Strategies to boost the bioavailability of circulating and tissue bilirubin or to mimic bilirubin's antioxidant properties could have a significant impact on prevention and control of hypertension as well as coronary heart disease.

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice.

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6­Ugt1(-/-) mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1(-/-) mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1(-/-) but not in C57BL/6-Ugt1(-/-) mice. Survival of FVB/NJ-Ugt1(-/-) mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1(-/-) mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0-P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1(-/-) mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1(-/-) mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions.

The multifunctional role and therapeutic potential of HO-1 in the vascular endothelium.

SIGNIFICANCE: Heme oxygenases (HO-1 and HO-2) catalyze the degradation of the pro-oxidant heme into carbon monoxide (CO), iron, and biliverdin, which is subsequently converted to bilirubin. In the vasculature, particular interest has focused on antioxidant and anti-inflammatory properties of the inducible HO-1 isoform in the vascular endothelium. This review will present evidence that illustrates the potential therapeutic significance of HO-1 and its products, with special emphasis placed on their beneficial effects on the endothelium in vascular diseases. RECENT ADVANCES: The understanding of the molecular basis for the regulation and functions of HO-1 has led to the identification of a variety of drugs that increase HO-1 activity in the vascular endothelium. Moreover, therapeutic delivery of HO-1 products CO, biliverdin, and bilirubin has been shown to have favorable effects, notably on endothelial cells and in animal models of vascular disease. CRITICAL ISSUES: To date, mechanistic data identifying the downstream target genes utilized by HO-1 and its products to exert their actions remain relatively sparse. Likewise, studies in man to investigate the efficacy of therapeutics known to induce HO-1 or the consequences of the tissue-specific delivery of CO or biliverdin/bilirubin are rarely performed. FUTURE DIRECTIONS: Based on the promising in vivo data from animal models, clinical trials to explore the safety and efficacy of the therapeutic induction of HO-1 and the delivery of its products should now be pursued further, targeting, for example, patients with severe atherosclerotic disease, ischemic limbs, restenosis injury, or at high risk of organ rejection.

Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway.

Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway.

Unconjugated bilirubin, a potent endogenous antioxidant, is decreased in patients with non-alcoholic steatohepatitis and advanced fibrosis.

BACKGROUND AND AIM: Oxidative stress is considered a key element in the progression of non-alcoholic fatty liver to non-alcoholic steatohepatitis (NASH). Unconjugated bilirubin is the main endogenous lipid antioxidant and is cytoprotective in different tissues and organs. In this study, it was evaluated if unconjugated bilirubin levels are associated with the degree of liver injury in patients with non-alcoholic fatty liver disease. METHODS: Two hundred and eighty-five patients were retrospectively evaluated with biopsy-confirmed non-alcoholic fatty liver disease. Multiple logistic regression models were used to assess the relationship of steatosis, inflammation, and fibrosis levels to the features of patients. RESULTS: Unconjugated bilirubin levels differed significantly according to inflammation and fibrosis scores. Unconjugated bilirubin was lower in patients with moderate-severe inflammation compared with those with absent-mild (P = 0.001) and in patients with moderate-severe fibrosis compared with those with absent-mild (P < 0.001), whereas no difference was observed for steatosis grades. At logistic regression analysis, low unconjugated bilirubin levels were associated with moderate-severe inflammation (odds ratio, 0.11; 95% confidence interval 0.02-0.76; P = 0.025) and moderate-severe fibrosis (odds ratio, 0.013; 95% confidence interval 0.001-0.253; P = 0.004). CONCLUSIONS: Low unconjugated bilirubin levels are independent predictors of advanced inflammation and fibrosis in patients with steatohepatitis, indicating the lack of antioxidant protection as a possible molecular determinant for the progression of liver injury.

Unconjugated bilirubin restricts oligodendrocyte differentiation and axonal myelination.

High levels of serum unconjugated bilirubin (UCB) in newborns are associated with axonal damage and glial reactivity that may contribute to subsequent neurologic injury and encephalopathy (kernicterus). Impairments in myelination and white matter damage were observed at autopsy in kernicteric infants. We have recently reported that UCB reduces oligodendrocyte progenitor cell (OPC) survival in a pure OPC in vitro proliferative culture. Here, we hypothesized that neonatal hyperbilirubinemia may also impair oligodendrocyte (OL) maturation and myelination. We used an experimental model of hyperbilirubinemia that has been shown to mimic the pathophysiological conditions leading to brain dysfunction by unbound (free) UCB. Using primary cultures of OL, we demonstrated that UCB delays cell differentiation by increasing the OPC number and reducing the number of mature OL. This finding was combined with a downregulation of Olig1 mRNA levels and upregulation of Olig2 mRNA levels. Addition of UCB, prior to or during differentiation, impaired OL morphological maturation, extension of processes and cell diameter. Both conditions reduced active guanosine triphosphate (GTP)-bound Rac1 fraction. In myelinating co-cultures of dorsal root ganglia neurons and OL, UCB treatment prior to the onset of myelination decreased oligodendroglial differentiation and the number of myelinating OL, also observed when UCB was added after the onset of myelination. In both circumstances, UCB decreased the number of myelin internodes per OL, as well as the myelin internode length. Our studies demonstrate that increased concentrations of UCB compromise myelinogenesis, thereby elucidating a potential deleterious consequence of elevated UCB.

Bilirubin participates in protecting of heme oxygenase-1 induction by quercetin against ethanol hepatotoxicity in cultured rat hepatocytes.

To attenuate alcohol liver disease (ALD) is extremely urgent since ALD has been emerged as a major liver disease. The aim of the present study is to investigate the hepatoprotective effect against ethanol-induced injury of bilirubin, a product of heme metabolism degradation via HO and biliverdin reductase catalysis. Ethanol-incubated primary rat hepatocytes (100 mmol/L) were treated by quercetin, bilirubin, inflammatory factors, and/or HO-1 inducer/inhibitor for 24 h, and the cellular damage was assayed. Quercetin lowered ethanol-induced glutathione depletion and superoxide dismutase inactivation, inhibited the overproduction of malondialdehyde and reactive oxygen species, and decreased the leakage of cellular aspartate aminotransferase and lactate dehydrogenase, accompanying the normalization of bilirubin level. The effect of quercetin was mimicked by exogenous bilirubin in a dose-dependent manner to some extent (within 25 µmol/L) and pharmacological HO-1 inducer hemin, but abolished by HO-1 inhibitor zinc protoporphyrin-IX. Inflammatory challenge of TNF-α plus IL-6 further aggravated ethanol-induced oxidative damage, which was also attenuated by bilirubin in part. These findings shed a light on the anti-oxidative and anti-inflammatory role of bilirubin released from quercetin/HO-1 and biliverdin reductase pathway against ethanol hepatotoxicity and highlight a prospective strategy of nutritional intervention for ALD by naturally occurring quercetin to induce HO-1 with the release of bioactive end-products.