Bilirubin ameliorates osteoarthritis via activating Nrf2/HO-1 pathway and suppressing NF-κB signalling.

Osteoarthritis (OA) is a chronic degenerative joint disease that affects worldwide. Oxidative stress plays a critical role in the chronic inflammation and OA progression. Scavenging overproduced reactive oxygen species (ROS) could be rational strategy for OA treatment. Bilirubin (BR) is a potent endogenous antioxidant that can scavenge various ROS and also exhibit anti-inflammatory effects. However, whether BR could exert protection on chondrocytes for OA treatment has not yet been elucidated. Here, chondrocytes were exposed to hydrogen peroxide with or without BR treatment. The cell viability was assessed, and the intracellular ROS, inflammation cytokines were monitored to indicate the state of chondrocytes. In addition, BR was also tested on LPS-treated Raw264.7 cells to test the anti-inflammation property. An in vitro bimimic OA microenvironment was constructed by LPS-treated Raw264.7 and chondrocytes, and BR also exert certain protection for chondrocytes by activating Nrf2/HO-1 pathway and suppressing NF-κB signalling. An ACLT-induced OA model was constructed to test the in vivo therapeutic efficacy of BR. Compared to the clinical used HA, BR significantly reduced cartilage degeneration and delayed OA progression. Overall, our data shows that BR has a protective effect on chondrocytes and can delay OA progression caused by oxidative stress.

Bilirubin Down-Regulates Oxidative Stress and Fibroblast Growth Factor 23 Expression in UMR106 Osteoblast-Like Cells.

INTRODUCTION: Fibroblast growth factor 23 (FGF23) is a major regulator of phosphate and vitamin D metabolism in the kidney, and its higher levels in plasma are associated with poorer outcomes in kidney and cardiovascular diseases. It is produced by bone cells upon enhanced oxidative stress and inhibits renal phosphate reabsorption and calcitriol (active form of vitamin D) production. Bilirubin, the final product of the heme catabolic pathway in the vascular bed, has versatile biological functions, including antioxidant and anti-inflammatory effects. This study explored whether bilirubin alters FGF23 production. METHODS: Experiments were performed using UMR106 osteoblast-like cells. Fgf23 transcript levels were determined by quantitative real-time polymerase chain reaction, C-terminal and intact FGF23 protein levels were determined by enzyme-linked immunosorbent assay, and cellular oxidative stress was assessed by CellROX assay. RESULTS: Unconjugated bilirubin down-regulated Fgf23 gene transcription and FGF23 protein abundance; these effects were paralleled by lower cellular oxidative stress levels. Also, conjugated bilirubin reduced Fgf23 mRNA abundance. CONCLUSION: Bilirubin down-regulates FGF23 production in UMR106 cells, an effect likely to be dependent on the reduction of cellular oxidative stress.

Antioxidative Hyaluronic Acid-Bilirubin Nanomedicine Targeting Activated Hepatic Stellate Cells for Anti-Hepatic-Fibrosis Therapy.

Liver fibrosis is a life-threatening and irreversible disease. The fibrosis process is largely driven by hepatic stellate cells (HSCs), which undergo transdifferentiation from an inactivated state to an activated one during persistent liver damage. This activated state is responsible for collagen deposition in liver tissue and is accompanied by increased CD44 expression on the surfaces of HSCs and amplified intracellular oxidative stress, which contributes to the fibrosis process. To address this problem, we have developed a strategy that combines CD44-targeting of activated HSCs with an antioxidative approach. We developed hyaluronic acid-bilirubin nanoparticles (HABNs), composed of endogenous bilirubin, an antioxidant and anti-inflammatory bile acid, and hyaluronic acid, an endogenous CD44-targeting glycosaminoglycan biopolymer. Our findings demonstrate that intravenously administered HABNs effectively targeted the liver, particularly activated HSCs, in fibrotic mice with choline-deficient l-amino acid-defined high-fat diet (CD-HFD)-induced nonalcoholic steatohepatitis (NASH). HABNs were able to inhibit HSC activation and proliferation and collagen production. Furthermore, in a murine CD-HFD-induced NASH fibrosis model, intravenously administered HABNs showed potent fibrotic modulation activity. Our study suggests that HABNs have the potential to serve as a targeted anti-hepatic-fibrosis therapy by modulating activated HSCs via CD44-targeting and antioxidant strategies. This strategy could also be applied to various ROS-related diseases in which CD44-overexpressing cells play a pivotal role.

Bilirubin impairs neuritogenesis and synaptogenesis in NSPCs by downregulating NMDAR-CREB-BDNF signaling.

Neonatal jaundice is one of the most common disorders in the first 2 wk after birth. Unconjugated bilirubin (UCB) is neurotoxic and can cause neurological dysfunction; however, the underlying mechanisms remain unclear. Neurogenesis, neuronal growth, and synaptogenesis are exuberant in the early postnatal stage. In this study, the impact of UCB on neuritogenesis and synaptogenesis in the early postnatal stage was evaluated both in vitro and in vivo. Primary culture neuronal stem and progenitor cells (NSPCs) were treated with UCB during differentiation, and then the neurite length and synapse puncta were measured. In the bilirubin encephalopathy (BE) animal model, DCX+-marked developing neurons were used to detect apical length and dendritic arborization. According to the data, UCB significantly reduced neurite length and synapse density, as well as decreased the apical dendrite length and dendritic arborization. Furthermore, the NMDAR subunit NR2B was downregulated in NSPCs, while pCREB expression in the hippocampus progressively decreased during disease progression in the BE model. Next, we tested the expression of NR2B, pCREB, mBDNF, and p-mTOR in NSPCs in vitro, and found that UCB treatment reduced the expression of these proteins. In summary, this suggests that UCB causes chronic neurological impairment and is related to the inhibition of NMDAR-CREB-BDNF signaling in NSPCs, which is associated with reduced neuritogenesis and synaptogenesis. This finding may inspire the development of novel pharmaceuticals and treatments.

Bilirubin impacts microglial autophagy via the Akt-mTOR signaling pathway.

Bilirubin encephalopathy is a severe complication of neonatal hyperbilirubinemia. With elevation of serum unconjugated bilirubin (UCB) levels, UCB crosses the blood-brain barrier and possibly leads to neurological dysfunction. Neuroinflammation is recognized as a prominent pathological feature in bilirubin encephalopathy. Recent studies have suggested that autophagy plays a crucial role in the inflammatory response. However, the potential effect of microglial autophagy in the pathogenesis of bilirubin encephalopathy remains uncertain. The in vitro findings verified that in primary cultured microglia, UCB significantly reduced the ratio of LC3B-II to LC3B-I and downregulated the expression of ATG5, Beclin-1, and ATG7, while increasing the expression of p62/SQSTM1. The results showed that UCB could decrease the number of mCherry-EGFP-LC3 positive puncta, even when chloroquine (CQ) was applied to block the microglial autophagy flux. Mechanistically, UCB was found to upregulate the expression of TLR4 and increase the phosphorylation levels of Akt and mammalian target of rapamycin (mTOR). Promoting microglial autophagy by treatment with Rapamycin (RAPA), an mTOR inhibitor, decreased the levels of NOD-like receptor protein 3 (NLRP3) inflammasome components and IL-1ß, rescued microglial overactivation, and improved neurological functions. These data indicated that UCB could impact microglial autophagy via the Akt-mTOR signaling pathway and synergistically promote neuroinflammatory responses. Enhancing autophagy might disrupt the assembly of NLRP3 inflammasome, attenuate UCB-induced neuroinflammation, and improve the prognosis of model rats with bilirubin encephalopathy. In conclusion, this study implies that regulating microglial autophagy might be a promising therapeutic strategy for bilirubin encephalopathy.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids.

UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.

Evaluating variations in bilirubin glucuronidation activity by protease inhibitors in canine and human primary hepatocytes cultured in a 3D culture system.

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development.

[Effects of Li-Dan-He-Ji on regulating oxidative stress and antagonising infantile cholestatic hepatic fibrosis].

OBJECTIVE: To explore the clinical effect of Li-Dan-He-Ji in the treatment of infantile cholestatic hepatic fibrosis. METHODS: Patients who met the diagnostic criteria of infantile cholestatic hepatic fibrosis in the department of integrated traditional Chinese and Western medicine and the department of gastroenterology of Wuhan Children's Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from January to December 2021 were included in the study by prospective randomized controlled trial. They were divided into the conventional treatment group and Li-Dan-He-Ji group according to the random number table. The patients in the conventional treatment group were given conventional treatment according to the guidelines. In the Li-Dan-He-Ji group, the self-made Chinese medicinal compound Li-Dan-He-Ji (prescription: Herba Artemisiae Scopariae, Fructus Forsythiae, Radix et Rhizoma Rhei preparata, Radix Polygoni Multiflori Preparata, Radix Paeoniae Rubra, Ramulus Cinnamomi, Fructus Aurantii, Rhizoma Atractylodis Macrocephalae, Fructus Schisandrae Chinensis, Carapax Trionycis, and Radix Glycyrrhizae) was given on the basis of the routine treatment, by oral, enema or nasal feeding, 60 mL each day, divided into 2 or 3 times, for 28 days. Outpatient follow-up was maintained for 4 weeks. Before and after treatment, serum liver fibrosis 4 items [type IV collagen (IV-C), hyaluronidase (HA), type III procollagen (PC III), laminin (LN)], liver function and cholestasis-related markers [total bilirubin (TBil), direct bilirubin (DBil), total bile acid (TBA), alkaline phosphatase (ALP), γ-glutamyl transpeptidase (γ-GGT), alanine aminotransferase (ALT), aspartate aminotransferase (AST)], oxidative stress markers [superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione (GSH)], liver stiffness measurement (LSM) detected by transient elastography (TE), aspartate aminotransferase-to-platelet ratio index (APRI), and liver and spleen retraction time were recorded in the two groups. RESULTS: During the observation period, a total of 40 cases of cholestatic hepatic fibrosis were treated, including 21 cases in the conventional treatment group and 19 cases in the Li-Dan-He-Ji group. Before treatment, the differences in serum liver fibrosis 4 items, serum liver function and cholestasis-related markers, oxidative stress indexes, LSM and APRI of the two groups were not statistically significant. After treatment, the liver fibrosis 4 items, liver function and cholestasis-related markers, LSM, and APRI were all significantly decreased in both groups, and the indexes in the Li-Dan-He-Ji group were significantly lower than those in the conventional treatment group [HA (ng/L): 165.81±21.57 vs. 203.87±25.88, PC III (µg/L): 69.86±9.32 vs. 81.82±7.39, IV-C (µg/L): 204.14±38.97 vs. 239.08±24.93, LN (µg/L): 162.40±17.39 vs. 190.86±15.97, TBil (µmol/L): 37.58±27.63 vs. 53.06±45.09, DBil (µmol/L): 20.55±19.34 vs. 30.08±27.39, ALP (U/L): 436.50±217.58 vs. 469.60±291.69, γ-GGT (U/L): 66.78±35.84 vs. 87.00±32.82, ALT (U/L): 64.75±50.53 vs. 75.20±50.19, AST (U/L): 77.25±54.23 vs. 96.80±59.77, TBA (µmol/L): 74.35±44.44 vs. 85.45±39.50, LSM (kPa): 5.24±0.39 vs. 7.53±3.16, APRI: 0.52±0.39 vs. 0.98±0.29, all P < 0.05]. After treatment, MDA in the two groups were significantly lower than those before treatment, and SOD and GSH were significantly higher than those before treatment. The level of SOD in the Li-Dan-He-Ji group was significantly higher than that in the conventional treatment group (kU/L: 64.56±6.69 vs. 51.58±5.98, P < 0.05). In addition, the liver retraction time (day: 20.13±10.97 vs. 24.33±13.46) and spleen retraction time (day: 25.93±13.01 vs. 29.14±14.52) in the Li-Dan-He-Ji group were significantly shorter than those in the conventional treatment group (both P < 0.05). CONCLUSIONS: The use of Li-Dan-He-Ji in the treatment of cholestatic hepatic fibrosis can effectively improve the indicators of cholestasis, hepatic fibrosis, oxidative stress and clinical symptoms in children.

HYPERBILIRUBINEMIA AGGRAVATES RENAL ISCHEMIA REPERFUSION INJURY BY EXACERBATING PINK1-PARKIN-MEDIATED MITOPHAGY.

ABSTRACT: Background: Hyperbilirubinemia is a common perioperative complication, which is associated with acute kidney injury. Bilirubin permeabilizes mitochondrial membranes leading to mitochondrial swelling and dysfunction. In this study, we aimed to determine the association between PINK1-PARKIN-mediated mitophagy and renal ischemia-reperfusion (IR) injury aggravated by hyperbilirubinemia. Methods: A C57BL/6 mouse hyperbilirubinemia model was induced via intraperitoneal injection of bilirubin solution. In addition, a hypoxia/reoxygenation (H/R) injury model of TCMK-1 cells was established. In these models, we determined the effects of hyperbilirubinemia on oxidative stress, apoptosis, mitochondrial damage, and fibrosis. Results:In vitro , colocalization of GFP-LC3 puncta and Mito-Tracker Red showed that the number of mitophagosomes increased in TCMK-1 cells under H/R and bilirubin condition. Silencing of PINK1 or inhibition of autophagy alleviated mitochondrial damage, oxidative stress, and apoptosis in H/R injury aggravated by bilirubin and decreased cell death detected by methyl-thiazolyl-tetrazolium. In vivo , hyperbilirubinemia increased serum creatinine level in the renal IR injury mice model. Hyperbilirubinemia enhanced apoptosis induced by renal IR. In addition, hyperbilirubinemia increased mitophagosomes and autophagosomes and disrupted mitochondrial cristae in the IR kidney. Inhibition of PINK1 or autophagy reduced histological damages by alleviating apoptosis in renal IR injury, aggravated by hyperbilirubinemia. 3-MA or PINK1-shRNA-AAV9 treatment decreased the area of collagen and proteins related to fibrosis in renal IR injury, aggravated by hyperbilirubinemia. Conclusions: We have demonstrated that hyperbilirubinemia aggravated oxidative stress, apoptosis, mitochondrial damage, and fibrosis in renal IR injury by exacerbating PINK1-PARKIN-mediated mitophagy.

Can 'toxic' bilirubin be put to medical use?

New view of molecule's good side inspires plans to test it as treatment for a variety of illnesses.

Vitamin C as a potential ameliorating agent against hepatotoxicity among alcoholic abusers.

OBJECTIVE: Drug and substance abuse remains a major medical problem globally. Alcohol consumption, particularly heavy drinking, is an important risk factor for many health problems and is a major contributor to the global burden of disease. Vitamin C has proven to be defensive against toxic substances and provides antioxidant and cytoprotective activity to hepatocytes. The aim of this study was to investigate vitamin C as a potential ameliorating agent against hepatotoxicity among alcohol abusers. PATIENTS AND METHODS: This study was a cross-sectional study that included eighty male hospitalized alcohol abusers and twenty healthy people as a control group. Alcohol abusers received standard treatment plus vitamin C. Total protein, albumin, total Bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), thiobarbituric acid reactive substances (TBARS), reduced glutathione (GSH), superoxide dismutase (SOD), catalase (CAT) and 8-hydroxhguanosine (8-OHdG) were investigated. RESULTS: This study reported that, in the alcohol abuser group, there was a significant increase in the total protein, bilirubin, AST, ALT, ALP, TBARS, SOD and 8-OHdG; on the other hand, there was a significant decrease in albumin, GSH and CAT compared with the control group. The alcohol abuser group treated with vitamin C showed a significant decrease in total protein, bilirubin, AST, ALT, ALP, TBARS, SOD and 8-OHdG; on the other hand, there was a significant increase in albumin, GSH and CAT compared with the control group. CONCLUSIONS: This study's findings suggest that alcohol abuse induces significant alterations in various hepatic biochemical parameters and oxidative stress and that vitamin C has a partial protective role in countering alcohol abuse-induced hepatotoxicity. Using vitamin C as an adjunctive supplement to standard treatment may be helpful in minimizing the toxic side effects of alcohol abuse.

The Combination of Sinusoidal Perfusion Enhancement and Apoptosis Inhibition by Riociguat Plus a Galactose-PEGylated Bilirubin Multiplexing Nanomedicine Ameliorates Liver Fibrosis Progression.

Chronic liver injury and continuous wound healing lead to extracellular matrix (ECM) deposition and liver fibrosis. The elevated production of reactive oxygen species (ROS) in the liver leads to the apoptosis of hepatocytes and the activation of hepatic stellate cells (HSCs). In the current study, we describe a combination strategy of sinusoidal perfusion enhancement and apoptosis inhibition enabled by riociguat together with a tailor-designed galactose-PEGylated bilirubin nanomedicine (Sel@GBRNPs). Riociguat enhanced sinusoidal perfusion and decreased the associated ROS accumulation and inflammatory state of the fibrotic liver. Concurrently, hepatocyte-targeting galactose-PEGylated bilirubin scavenged excessive ROS and released encapsulated selonsertib. The released selonsertib inhibited apoptosis signal-regulating kinase 1 (ASK1) phosphorylation to alleviate apoptosis in hepatocytes. The combined effects on ROS and hepatocyte apoptosis attenuated the stimulation of HSC activation and ECM deposition in a mouse model of liver fibrosis. This work provides a novel strategy for liver fibrosis treatment based on sinusoidal perfusion enhancement and apoptosis inhibition.

Bilirubin Nanomedicine Rescues Intestinal Barrier Destruction and Restores Mucosal Immunity in Colitis.

Inflammatory bowel disease (IBD) manifests as intestinal barrier destruction, mucosal immunity dysregulation, and disrupted gut microbiome homeostasis. Conventional anti-inflammatory medications for IBD therapy partially alleviate symptoms but are unable to restore normal barrier and immune function. Here, we report a nanomedicine comprising bilirubin (BR)-attached low-molecular-weight, water-soluble chitosan nanoparticles (LMWC-BRNPs), that promotes restoration of the intestinal barrier, mucosal immunity, and the gut microbiome, thereby exerting robust therapeutic efficacy. In a mouse model of dextran sulfate sodium salt (DSS)-induced colitis, orally administered LMWC-BRNPs were retained in the GI tract much longer than other nonmucoadhesive BRNPs owing to the mucoadhesiveness of LMWC via electrostatic interaction. Treatment with LMWC-BRNPs led to considerable recovery of the damaged intestinal barrier compared with the current IBD medication, 5-aminosalicylic acid (5-ASA). Orally administered LMWC-BRNPs were taken up by pro-inflammatory macrophages and inhibited their activity. They also concurrently increased the population of regulatory T cells, thereby leading to the recovery of dysregulated mucosal immunity. An analysis of the gut microbiome revealed that LMWC-BRNPs treatment significantly attenuated the increase Turicibacter, an inflammation-related microorganism, resulting in protection of gut microbiome homeostasis. Taken together, our findings indicate that LMWC-BRNPs restored normal functions of the intestine and have high potential for use as a nanomedicine for IBD therapy.

A Biomimetic Nanoparticle Exerting Protection against Acute Liver Failure by Suppressing CYP2E1 Activity and Scavenging Excessive ROS.

Acute liver failure (ALF) is a severe liver disease caused by many reasons. One of them is the overdosed acetaminophen (APAP), which is metabolized into N-acetyl-p-benzoquinone imine (NAPQI), an excessive toxic metabolite, by CYP2E1, resulting in excessive reactive oxygen species (ROS), exhausted glutathione (GSH), and thereafter hepatocyte necrosis. N-acetylcysteine is the Food and Drug Administration-approved drug for detoxification of APAP, but it has limited clinical application due to the short therapeutic time window and concentration-related adverse effects. In this study, a carrier-free and bilirubin dotted nanoparticle (B/BG@N) is developed, which is formed using bilirubin and 18ß-Glycyrrhetinic acid, and bovine serum albumin (BSA) is then adsorbed to mimic the in vivo behavior of the conjugated bilirubin for hitchhiking. The results demonstrate that B/BG@N can effectively reduce the production of NAPQI as well as exhibit antioxidant effects against intracellular oxidative stress via regulating the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 signal axis and reducing the production of inflammatory factors. In vivo study shows that B/BG@N can effectively improve the clinical symptom of the mice model. This study suggests that B/BG@N own increases circulation half-life, improves accumulation in the liver, and dual detoxification, providing a promising strategy for clinical ALF treatment.

Kaempferitrin, a major compound from ethanol extract of Chenopodium ambrosioides, exerts antitumour and hepatoprotective effects in the mice model of human liver cancer xenografts.

OBJECTIVES：: This study aimed to investigate antitumour effect and possible toxicity of kaempferitrin, the major compound from ethanol extract of Chenopodium ambrosioides, in the mice model of human liver cancer xenografts. METHODS：: Forty mice bearing SMMC-7721 cells xenografts were divided into control group (not treated) and three groups orally administered with ethanol extract of C. ambrosioides, kaempferol (positive control) and kaempferitrin for 30 days. Antitumour effect was evaluated by measurement of tumour growth, histological examinations of tumours, flow cytometry detection of splenic CD19+ B lymphocytes and CD161+ Natural Killer cells, biochemical measurements of serum levels of tumour necrosis factor-α, interleukin-6, interferon-γ, malonaldehyde, 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azinobis-(3-ethylbenz thiazoline-6-sulphonate) radicals. Toxicity was evaluated by histological examinations of livers and measurements of serum levels of aspartate transaminase, alanine transaminase, total bilirubin, direct bilirubin, malonaldehyde and hepatic malonaldehyde level. KEY FINDINGS: Kaempferitrin significantly (P < 0.05) decreased tumour volume, mass and cell number. Antitumour effect was due to induction of tumour cells necrosis and apoptosis, stimulation of splenic B lymphocytes, decreases of radicals and malonaldehyde. Kaempferitrin did not change liver structure, and decreased serum levels of transaminases, bilirubin, malonaldehyde and hepatic malonaldehyde level. CONCLUSIONS: Kaempferitrin exerts antitumour and hepatoprotective effects.

Bilirubin induces microglial NLRP3 inflammasome activation in vitro and in vivo.

Despite current advancements in neonatal care, hyperbilirubinemia resulting in bilirubin-induced neurological dysfunction (BIND) continues to be one of the major reasons of mortality or lifelong disability. Although the exact mechanisms underlying brain injury upon bilirubin exposure remains unelucidated, inflammation is considered to be one of the major contributors to BIND. This study investigates the role of the NLRP3 inflammasome in bilirubin-induced injury using in vitro and in vivo models. We successfully demonstrated that the upregulation of NLRP3 expression is significantly associated with the release of active caspase-1 and IL-1ß in N9 microglial cells exposed to bilirubin. Functional in vitro experiments with NLRP3 siRNA confirms that bilirubin-induced inflammasome activation and cell death are mediated by the NLRP3 inflammasome. Following injection of bilirubin into the cisterna magna of a neonatal mouse, activation of the NLRP3 inflammasome and microglia were determined by double staining with Iba1-NLRP3 and Iba1-Caspase-1. Upon injection of bilirubin into the cisterna magna, neuronal loss was significantly higher in the wild-type mouse compared to Nlrp3-/- and Caspase-1-/- strains. Collectively, these data indicate that NLRP3 inflammasome has a crucial role in microglial activation and bilirubin-induced neuronal damage.

The physiology of bilirubin: health and disease equilibrium.

Bilirubin has several physiological functions, both beneficial and harmful. In addition to reactive oxygen species-scavenging activities, bilirubin has potent immunosuppressive effects associated with long-term pathophysiological sequelae. It has been recently recognized as a hormone with endocrine actions and interconnected effects on various cellular signaling pathways. Current studies show that bilirubin also decreases adiposity and prevents metabolic and cardiovascular diseases. All in all, the physiological importance of bilirubin is only now coming to light, and strategies for increasing plasma bilirubin levels to combat chronic diseases are starting to be considered. This review discusses the beneficial effects of increasing plasma bilirubin, incorporates emerging areas of bilirubin biology, and provides key concepts to advance the field.

Bile duct ligation increased dopamine levels in the cerebral cortex of rats partly due to induction of tyrosine hydroxylase.

BACKGROUND AND PURPOSE: Liver failure is associated with psychiatric alterations, partly resulting from the increased brain dopamine levels. We investigated the relationship between increased dopamine levels and mental abnormalities using bile duct ligation (BDL) rats and the mechanism by which liver failure increased dopamine levels in SH-SY5Y cells. Behavioural tests were carried out on day 13 and 27 following BDL, along with measurements of dopamine and metabolites, expressions of enzymes and transporters related to dopamine metabolism, and its transport into the cortex and the hippocampus. SH-SY5Y cells were used to investigate whether NH4 Cl, bile acids and bilirubin affected expression of tyrosine hydroxylase or not. Tyrosine hydroxylase (TH) expression in SH-SY5Y cells co-incubated with bilirubin and signal pathway inhibitors was measured. KEY RESULTS: Open-field test results demonstrated BDL rats showed anxiety-like behaviour, accompanied by increased dopamine levels and expression of TH protein in the cortex. Membrane bound long form (MB)-COMT, slightly but significantly decreased. SH-SY5Y cells indicated that increased bilirubin levels was a factor in inducing TH expression. Both inhibitor of NF-κB pathway BAY 11-7082 and silencing NF-κB p65 reversed bilirubin-induced upregulation of TH protein. NF-κB activator TNF-α increased expression of TH protein. Roles of bilirubin in increases of TH protein expressions and dopamine levels were measured using hyperbilirubinemia rats. Anxiety-like behaviour, was associated with increased dopamine levels and TH protein expressions in hyperbilirubinemia rats. CONCLUSION AND IMPLICATIONS: BDL significantly increased dopamine levels in rat cortex partly due to bilirubin-mediated TH induction. Increased bilirubin induced TH expression via activating NF-κB signalling pathway.

Hepatoprotective potential of diosmin against hepatotoxic effect of isoniazid and rifampin in wistar rats.

OBJECTIVE: The treatment of tuberculosis with isoniazid and rifampin is associated with hepatocellular damage. Therefore, the study was designed to evaluate the hepatoprotective potential of diosmin against hepatotoxic effect of isoniazid and rifampin in Wistar rats. METHODS: Hepatotoxicity was induced by administering isoniazid and rifampin (100 mg/kg), whereas diosmin was given as treatment control. Markers of liver function (ALT, AST, ALP and bilirubin), inflammatory cytokines (TNFα, IL-6 and IL-1ß), apoptosis (caspase-3), oxidative stress parameters (LPO, GSH, CAT and SOD) and histological changes in liver were assessed in normal, hepatotoxic control and treatment groups. RESULTS: The administration of isoniazid and rifampin significantly increased markers of liver dysfunction (ALT, AST, ALP and bilirubin), cytokines (TNFα, IL-6 and IL-1ß) and apoptosis (caspase-3). However, daily dosing of diosmin significantly reduced these markers of liver dysfunction, inflammatory cytokines and apoptosis to near normal levels. Additionally, markers of hepatocellular oxidative stress parameters were significantly altered as evident from increased LPO level and decreased endogenous antioxidants such as GSH, SOD and CAT in isoniazid-and rifampin-treated hepatotoxic group. It was observed that diosmin treatment reduced high levels of LPO and demonstrated significant improvement in antioxidant levels. Histological studies of liver also supported our biochemical findings, which are also manifested as diosmin treatment exhibited protection against hepatocellular degeneration and inflammation. CONCLUSION: Results of the present study demonstrate hepatoprotective potential of diosmin against isoniazid-and rifampin-treated hepatotoxicity. Thus, we conclude that diosmin may be used along with anti-tubercular drugs (isoniazid and rifampin) in tuberculosis patients to overcome their hepatotoxic adverse effect.

Biochemical effects of copper nanomaterials in human hepatocellular carcinoma (HepG2) cells.

In dose-response and structure-activity studies, human hepatic HepG2 cells were exposed for 3 days to nano Cu, nano CuO or CuCl2 (ions) at doses between 0.1 and 30 ug/ml (approximately the no observable adverse effect level to a high degree of cytotoxicity). Various biochemical parameters were then evaluated to study cytotoxicity, cell growth, hepatic function, and oxidative stress. With nano Cu and nano CuO, few indications of cytotoxicity were observed between 0.1 and 3 ug/ml. In respect to dose, lactate dehydrogenase and aspartate transaminase were the most sensitive cytotoxicity parameters. The next most responsive parameters were alanine aminotransferase, glutathione reductase, glucose 6-phosphate dehydrogenase, and protein concentration. The medium responsive parameters were superoxide dismutase, gamma glutamyltranspeptidase, total bilirubin, and microalbumin. The parameters glutathione peroxidase, glutathione reductase, and protein were all altered by nano Cu and nano CuO but not by CuCl2 exposures. Our chief observations were (1) significant decreases in glucose 6-phosphate dehydrogenase and glutathione reductase was observed at doses below the doses that show high cytotoxicity, (2) even high cytotoxicity did not induce large changes in some study parameters (e.g., alkaline phosphatase, catalase, microalbumin, total bilirubin, thioredoxin reductase, and triglycerides), (3) even though many significant biochemical effects happen only at doses showing varying degrees of cytotoxicity, it was not clear that cytotoxicity alone caused all of the observed significant biochemical effects, and (4) the decreased glucose 6-phosphate dehydrogenase and glutathione reductase support the view that oxidative stress is a main toxicity pathway of CuCl2 and Cu-containing nanomaterials.