The Protective Role of the Heme Catabolic Pathway in Hepatic Disorders

Abstract

Significance:

As the central metabolic organ, the liver is exposed to a variety of potentially cytotoxic, proinflammatory, profibrotic, and carcinogenic stimuli. To protect the organism from these deleterious effects, the liver has evolved a number of defense systems, which include antioxidant substrates and enzymes, anti-inflammatory tools, enzymatic biotransformation systems, and metabolic pathways.

Recent Advances:

One of the pivotal systems that evolved during phylogenesis was the heme catabolic pathway. Comprising the important enzymes heme oxygenase and biliverdin reductase, this complex pathway has a number of key functions including enzymatic activities, but also cell signaling, and DNA transcription. It further generates two important bile pigments, biliverdin and bilirubin, as well as the gaseous molecule carbon monoxide. These heme degradation products have potent antioxidant, immunosuppressive, and cytoprotective effects. Recent data suggest that the pathway participates in the regulation of metabolic and hormonal processes implicated in the pathogenesis of hepatic and other diseases.

Critical Issues:

This review discusses the impact of the heme catabolic pathway on major liver diseases, with particular focus on the involvement of cellular targeting and signaling in the pathogenesis of these conditions.

Future Directions:

To utilize the biological consequences of the heme catabolic pathway, several unique therapeutic strategies have been developed. Research indicates that pharmaceutical, nutraceutical, and lifestyle modifications positively affect the pathway, delivering potentially long-term clinical benefits. However, further well-designed studies are needed to confirm the clinical benefits of these approaches. Antioxid. Redox Signal. 35, 734–752.

The Heme Catabolic Pathway

The heme catabolic pathway belongs to a phylogenetically old and conserved system (23, 42, 200) long believed to represent the only means of eliminating potentially toxic and pro-oxidant heme molecules.

The entire heme degradation pathway (Figs. 1–2) is activated by the heme oxygenase (HMOX) enzyme. Containing two isoforms (HMOX1, OMIM *141250 and HMOX2, OMIM *141251) (141, 200), this ubiquitous microsomal enzyme is found throughout the human body. HMOX produces biologically significant amounts of bilirubin (∼300 mg [500 μmoles] in a 70 kg human per day), mostly from hemoglobin originating in senescent erythrocytes (16, 187). HMOX1, believed to be the most inducible enzyme in the human body (104, 110), plays a major role in protecting against exaggerated oxidative stress. HMOX1 gene expression falls under the control of nuclear factor erythroid 2-related factor 2 (Nrf2). This evolutionarily conserved, multifunctional transcription factor is responsible for cellular redox balance regulation, antioxidant protection, and phase II detoxification responses in mammals (100).

FIG. 1.

The major steps of the heme catabolic pathway. The cyclic tetrapyrrole heme is cleaved by the action of HMOX into linear tetrapyrrole biliverdin, which releases ferrous iron and CO. Biliverdin is subsequently reduced to bilirubin. BLVR, biliverdin reductase; CO, carbon monoxide; HMOX, heme oxygenase.

FIG. 2.

The heme catabolic pathway and bilirubin metabolism in the human body. The steps of the heme catabolic pathway described in Figure 1 occur primarily in the spleen. Upon its release into circulation, bilirubin binds to albumin and is then transported to the liver, the bilirubin biotransformation organ. In the liver sinusoids, bilirubin is transferred to hepatocytes using specific transporters belonging to the OATP family of transporting proteins. In the liver microsomes, bilirubin is conjugated with two molecules of glucuronic acid by the action of UGT1A1. The bilirubin diglucuronosid formed is then secreted into the bile duct system and actively transported by MRP2 to the canalicular membrane of the liver cell. Bilirubin entering the gut is deconjugated (mostly by the intestinal bacteria) and then reduced into a series of products known as urobilinoids. Under specific conditions, unconjugated bilirubin may be reabsorbed into the portal vein, or even escape into systemic circulation (known as eneterohepatic and enterosystemic circulation of bilirubin, respectively). BLVRA, biliverdin reductase A; MRP2, multidrug resistance-associated protein; OATP, organic anion-transporting polypeptide; UCB, unconjugated bilirubin; UGT1A1, UDP-glucuronosyltransferase 1A1. Color images are available online.

Although not widely distributed in the body, HMOX2 is highly expressed in hepatocytes (141). HMOX1 is overexpressed in Kupffer cells, liver-specific fixed macrophages (15), and, surprisingly, also in hepatic stellate cells (HSC) (207). These topological characteristics have pathophysiological consequences, particularly for iron metabolism in the liver and for protection against liver inflammation (15) and hepatic fibrogenesis (207). The basic function of HMOX is to split the cyclic tetrapyrrole heme into the linear tetrapyrrole structure of biliverdin, while simultaneously releasing ferrous iron and carbon monoxide (CO). Heme also induces ferritin, which sequesters released iron and prevents iron-mediated oxidative damage (9). The CO generated significantly contributes to hepatic sinusoidal tone and proper vascular functioning in liver tissue (167).

Like its immediate precursor bilirubin, biliverdin boasts potent antioxidant properties. Whether these effects are produced under in vivo conditions in the human body is debatable, since biliverdin is reduced to bilirubin almost immediately by ubiquitous biliverdin reductase (BLVR). Biliverdin has direct antioxidant effects, preventing both lipoperoxidation (162, 165) and hypochlorite-mediated pro-oxidation (164) and, similar to bilirubin, inhibiting NADPH oxidase-induced superoxide generation (43).

BLVR in the human body consists of two isoforms: BLVR A and B (BLVRA/B, OMIM Nos. *109750 and *600941, respectively). Apart from its enzymatic activity, BLVR also has an important signaling function. This key enzyme acts as a rare multispecific kinase (serine/threonine/tyrosine) that is particularly active in the insulin signaling pathway (91), posing considerable metabolic consequences (119). Among the most potent of the endogenous antioxidants (44), bilirubin is conjugated in liver cells with glucuronic acid by microsomal bilirubin UDP-glucuronosyltransferase 1A1 (UGT1A1), another member of the heme catabolic pathway. Its partial deficiency results in mild elevation of serum unconjugated bilirubin concentrations typically observed in Gilbert's syndrome (187).

Surprisingly, bilirubin was recently found to act as a ligand of several specific cell receptors, including the aryl hydrocarbon receptor (AhR) and peroxisome proliferator-activated receptors (PPAR) α and γ, which are implicated in the development of a variety of metabolic diseases such as obesity, diabetes, and metabolic syndrome (183). Based on this evidence, bilirubin seems to function like an endocrine molecule, as previously suggested (182) (Fig. 3).

FIG. 3.

The beneficial roles of bilirubin in the human body. When mildly elevated, bilirubin has general cytoprotective effects on various human body organs and tissues, including the liver, cardiovascular system, brain, and immune system. It also protects against hepatic, metabolic, cardiovascular, autoimmune, and cancer diseases. Dotted lines indicate mutual interactions. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis. Color images are available online.

The Heme Catabolic Pathway and Regulation of Oxidative Stress in Liver Pathology

The liver is a major organ attacked by reactive oxygen and nitrogen species (ROS and RNS, respectively). Hepatic cell subpopulations, which include hepatocytes, Kupffer cells, HSC, endothelial cells, cholangiocytes, and hepatic progenitors, are all functionally affected by increased oxidative stress (93, 112). ROS and RNS induce HMOX1 expression in the liver, at least partially, by Nrf2 (7), a key modulator of HMOX1 expression (100). Most liver diseases are associated with oxidative stress, which plays a pivotal role in the following complications: pathogenesis of alcoholic liver disease, inflammatory hepatic conditions, drug-induced liver injury, autoimmune liver disorders, metabolic dysfunction-associated fatty liver disease (MAFLD), liver fibrosis, liver cirrhosis, and liver cancer (53, 93, 144, 194). In mitigating the progression of a variety of liver diseases, the enzymes and intermediates of the heme catabolic pathway, best exemplified by HMOX1, are understood to have significant protective effects (60, 122, 148).

Heme oxygenase 1

HMOX1 is expressed mainly in Kupffer cells of the liver, and also in hepatocytes in response to various noxious stimuli (122). HMOX1 plays important roles in protecting hepatocytes from oxidative damage, as documented in previous studies of hepatic ischemia/reperfusion injury (IRI) (29, 92, 97; see also section “Products of the Heme Catabolic Pathway As Protective Factors in Hepatic IRI and Liver Transplantation” below) and viral infections of the liver (216).

The central protective role of HMOX1 against pathogenic pathways in liver diseases has been documented also in clinical studies of HMOX1 promoter gene polymorphisms. Those gene variations resulting in higher HMOX1 activity are associated with improved outcomes in liver transplant rejection (20; see also section “Products of the Heme Catabolic Pathway As Protective Factors in Hepatic IRI and Liver Transplantation” below), pediatric MAFLD (24), and liver cirrhosis progression (38).

BLVRA-biliverdin-bilirubin

BLVRA plays a crucial role in the regulation of oxidative stress in the liver. An in vitro study of various liver cell types revealed a negative association between BLVRA expression and ROS production (49). This finding is consistent with animal data by Chen et al., who demonstrated exaggerated systemic oxidative stress in Blvra knockout mice (26). Similarly, a recent in vitro study of a murine Blvra-deficient hepatocyte cell line reported increased ROS production and oxidative stress (50). Complementing the negative association between Blvra expression and oxidative stress, it has been shown that biliverdin has a similar protective effect on bile acid-induced oxidative stress (49). These findings correspond with experimental models of neurodegenerative diseases, which confirm the accumulation of oxidatively modified proteins in the brain cortices of BLVRA-deficient mice (88).

Previous comprehensive studies have documented the antioxidant activities of bilirubin (188) and the effects of biliverdin on bile acid-induced ROS generation (49). With potent antioxidant effects on liver tissue, bilirubin probably functions as the major endogenous buffer against oxidative stress induced by bile acids accumulated during cholestasis (111). In addition to unconjugated bilirubin, its conjugates such as bilirubin ditaurate (used as a surrogate for bisglucuronosyl bilirubin accumulated during cholestasis) (163 –165) and delta bilirubin (a bilirubin covalently bound to albumin accumulated in circulation during cholestasis) (203) have similar antioxidant effects.

The protective effect of unconjugated bilirubin is most pronounced in the lipid membranes of liver cells and cell organelles. While the lipid-binding properties (219) of bilirubin help to counteract membrane lipid peroxidation, water-soluble antioxidants such as glutathione are better at protecting water-soluble proteins (153). Studies have shown that the antioxidant effects of bilirubin are strongly potentiated by the bilirubin/biliverdin redox cycle, protecting cells against 10,000-fold higher concentrations of hydrogen peroxide (10, 11). It should be noted, however, that not all authors support the biological relevance of this cycle, a subject that has provoked much debate (71, 103, 107).

In correlation with systemic bilirubin levels, intracellular concentrations of bilirubin reflect the various conditions of oxidative stress in the liver and other organs, as demonstrated in HepG2 hepatoblastoma cells and a rat animal model (212). In one in vitro study performed using various cell types (including those of hepatic origin), different pro- and antioxidant thresholds were observed for intracellular bilirubin concentrations, indicating that the antioxidant functions of bilirubin are highly varied (18). The antioxidant and potentially cytotoxic effects of bilirubin and lumirubin (a photo-oxidation product generated during phototherapy for hyperbilirubinemia) were recently investigated. Using HepG2 hepatoblastoma cells, the study demonstrated a dependent relationship between the potential toxicity of high bilirubin concentration and the glycolytic reserve in these cells (36).

In addition to this mechanism, several additional pathogenic pathways have been implicated in the toxicity associated with severe hyperbilirubinemia. These effects include neuron dysfunction due to the binding of bilirubin to myelin-rich membranes (196), bilirubin-induced DNA fragmentation (52), and decreased mitochondrial respiration, which alters membrane potential and permeability, leading to apoptosis (117, 138).

Conversely, it has been reported that under reduced and nontoxic concentrations, both pigments have beneficial biological impacts on mitochondria, substantially affecting lipid and glucose metabolism (36). Our animal study of aged hyperbilirubinemic Gunn rats found that mildly elevated serum bilirubin concentrations had the following positive impacts on various biological functions: attenuation of oxidative stress, improved anthropometric parameters, decreased inflammatory status, increased glucose tolerance, fewer signs of cellular senescence, and enhanced mitochondrial function (211). These findings also apply to the anti-inflammatory activity of bilirubin, as confirmed in primary hepatocytes exposed to the compound. Hyperbilirubinemic Gunn rats treated with bacterial lipopolysaccharide (LPS) were found to exhibit lower levels of liver injury and inflammatory markers compared with their normobilirubinemic littermates (178).

Carbon monoxide

Endogenously produced CO is an important regulator of the liver vasculature. CO also interacts with pathways responsible for protecting against oxidative stress, including the Nrf2 system, antioxidant-responsive (ARE) genes, and glutathione metabolism (81). These observations are supported by a number of experimental studies demonstrating the protective effects of CO gas and CO donors on the following: hepatic IRI (89; see also section “Products of the Heme Catabolic Pathway As Protective Factors in Hepatic IRI and Liver Transplantation” below), hemorrhagic shock (120), systemic inflammation-associated liver injury (19), and steatohepatitis (177). All of the above studies found that CO actively suppressed the generation of ROS.

Products of the Heme Catabolic Pathway As Protective Factors Against MAFLD/Metabolic Dysfunction-Associated Steatohepatitis Development

Key pathogenic aspects

MAFLD/metabolic dysfunction-associated steatohepatitis (MASH) is a complex metabolic disease that covers a spectrum of conditions ranging from simple steatosis to steatohepatitis, the latter potentially progressing to liver fibrosis, cirrhosis, and ultimately, liver cancer (Fig. 4). Increasing in global prevalence, MAFLD/MASH substantially affects life quality and expectancy, with severe social and economic impacts (94). The complex pathogenesis of this metabolic disease is rooted in deteriorating lipid metabolism, hepatic overflow of free fatty acids, increased de novo hepatic lipogenesis, and impaired free fatty acid elimination (94). These processes initiate the generation of ROS, development of low-grade inflammation, induction of proinflammatory pathways (such as nuclear factor kappa B [NF-κB]), activation of specific toll-like receptors (such as TLR2 and 4), and production of inflammatory cytokines (34). These phenomena further impair coexistent insulin resistance (94).

FIG. 4.

The role of the heme catabolic pathway in MAFLD/MASH. All parts of the heme catabolic pathway, including HMOX1, BLVRA, bilirubin, and CO, protect against the development of MAFLD/MASH via mutual interactions leading to the modulation of various signaling pathways. AMPK, AMP-activated protein kinase; BLVRA, biliverdin reductase A; FGF21, fibroblast growth factor 21; GSK3β, glycogen synthase kinase-3β; HMOX1, heme oxygenase 1; MAFLD, metabolic dysfunction-associated fatty liver disease; MASH, metabolic dysfunction-associated steatohepatitis; mTOR, mammalian target of rapamycin; PPAR, peroxisome proliferator-activated receptor; PGC1α, peroxisome proliferator-activated receptor gamma coactivator 1-α; SREBP-1, sterol regulatory element-binding protein-1. Color images are available online.

On the cellular level, the master regulatory effectors of energy metabolism—which include PPAR-γ coactivator-1α (PGC1α) (activated by free fatty acid-induced AMP-activated protein kinase [AMPK]) and the PPARs—are all dysregulated in MAFLD/MASH (34). Combined with the impact of impaired adipokine signaling (129), the vicious circle of this disease is perpetuated.

Heme oxygenase 1

Due to its effects on the metabolism of glucose and lipids, HMOX1 is understood to significantly affect the pathogenesis of liver steatosis and its subsequent transition to steatohepatitis and fibrosis (149, 159). Although most of the available reports are derived from experimental data, they clearly show the impacts of HMOX1 and its products on the pathogenesis of MAFLD/MASH. HMOX1 upregulation has been shown to ameliorate disease progression in animal models of MAFLD. A study by Wang et al. found that HMOX1 induction not only reduced hepatic steatosis and inflammation, but also prevented fibrotic changes in mice placed on a methionine-choline-deficient (MCD) diet (192).

Similarly, Salley et al. documented the MASH-ameliorating effects of HMOX1 induction, observing improvements in both inflammatory and fibrotic lesions in a rat model of diabetes (142). Another study reported decreased fat accumulation following HMOX1 induction in a murine model of obesity. Importantly, the authors also detailed the modulation of cellular metabolic targets, including PPARα, fibroblast growth factor 21 (FGF21), and PGC1α (59). The key role of PGC1α was also confirmed in an animal study by Raffaele et al. (136). These data tally with the results of a clinical study on pediatric MAFLD patients by Chang et al. They found that children with reduced activity of HMOX1 promoter gene polymorphisms were more susceptible to the development of MAFLD (24).

Biliverdin reductase A

As proven in a liver-specific Blvra knockout mouse model (55), BLVRA regulates hepatic lipid metabolism by directly affecting the key enzymes implicated in lipid metabolism. These include AMPK, an important cellular energy sensor (181), acetyl-CoA carboxylase, an essential and rate-limiting enzyme in fatty acid metabolism (25), glycogen synthase kinase-3β (GSK3β), one of the most active cellular kinases and boasting more than 100 known targets, involved in the regulation of multiple cellular functions, including lipid and glucose metabolism, among others (1, 17).

In addition, BLVRA has been shown to regulate nuclear receptor PPARα and its downstream effectors (55), key elements in the energy homeostasis pathways of various metabolic diseases (35). The metabolic role of BLVRA has been recently confirmed. In vitro study by Miralem et al. (108) proved the effect of BLVRA on regulation of GSK3β. Lanzillota and colleagues confirmed the dysregulation of both AMPK and mammalian target of rapamycin (mTOR), an evolutionarily conserved nutrient-sensing protein kinase that regulates metabolism (157) hyperactivation in Blvra-deficient mice (88). The essential role of Blvra in the lipid metabolism and pathogenesis of obesity was verified in mice with adipocyte-specific deletion of Blvra demonstrating multiple cellular and systemic metabolic effects (158). These results were confirmed by an in vitro study (by the same group) of a murine BLVRA-deficient hepatocyte cell line. The authors reported increased ROS generation, reduced numbers of mitochondria, and deterioration of mitochondrial metabolism (50).

BLVRA, which acts as a potent multispecific kinase, significantly modulates the insulin signaling pathway (47, 91) implicated in the pathogenesis of MAFLD/MASH (33). In a recent human clinical study, low BLVRA expression was associated with MASH, adipose tissue inflammation, and insulin-signaling dysregulation (30). As demonstrated in recent animal studies (13, 156), insulin resistance, also present in Alzheimer's disease (172), falls under the direct control of BLVRA, which is impaired in this brain disorder (14).

Bilirubin

Serum bilirubin concentrations are negatively associated with metabolic syndrome, type 2 diabetes mellitus (115, 118), and MAFLD/MASH in both pediatric and adult patients (86, 96, 134). A large study by Hjelkrem et al. found that MASH occurrence was 16 times less likely in subjects with Gilbert's syndrome (benign hyperbilirubinemia) (61). In another study, the presence of unconjugated hyperbilirubinemia was associated with a much milder course of MAFLD, with lower rates of progression to MASH and fibrosis (84). This finding was replicated in a study by Salomone et al. They reported serum unconjugated bilirubin concentration to be an independent predictor of advanced inflammation and fibrosis in MASH patients (143).

These data were further confirmed by Anwar et al. in their systemic review of seven clinical studies examining the association between serum bilirubin concentrations and MAFLD risk (6). However, not all results support the protective effect of bilirubin on MAFLD, as recently demonstrated by two Mendelian randomization studies (85, 102). Therefore, to elucidate all of the potential factors affecting this putative association, further well-designed controlled studies are required.

It should also be noted that hyperbilirubinemic subjects with Gilbert's syndrome have substantially lower BMI, glucose, insulin, C-peptide, and triacylglycerol serum concentrations, most likely due to the activation of AMPK, PPARα/γ, and PgC1α (109). Improved metabolic health was reported in a study by Seyed Khoei et al. (155) and in other studies reviewed by Bulmer and colleagues (21).

The largely positive clinical data on the protective effects of bilirubin are supported by several important experimental studies on hyperbilirubinemic and bilirubin-treated animals. In a diet-induced obesity mouse model, bilirubin treatment led to improved cholesterol and glucose metabolism and increased adiponectin production. Enhancements were also observed for mRNA expression of PPARγ, sterol regulatory element-binding protein-1 (SREBP-1), and the insulin receptor, which likely contributed to increased insulin sensitivity and glucose tolerance in this animal model (98). Amelioration of liver steatosis in mice fed a high-fat diet was clearly demonstrated in genetically engineered hyperbilirubinemic mice phenotypically resembling Gilbert's syndrome (58). This improvement was mediated via bilirubin-induced activation of PPARα (58), a selective intracellular target of bilirubin previously identified by the same authors (160).

Bilirubin has been shown to remodel white adipose tissue in mice via modulation of mitochondrial activity (51); a similar effect on mitochondrial metabolism was observed in our recent study (36). One research group recently identified lower levels of hepatic steatosis, MASH, and hepatic fibrosis in humanized transgenic hyperbilirubinemic mice fed a high-fat diet (87). In another animal study of mice with diet-induced MAFLD treated with pegylated bilirubin nanoparticles, a marked improvement in liver disease was observed (57).

As discussed above, in MASH patients, mitochondria are typically overloaded with free fatty acids, principally due to increased fat intake and de novo lipogenesis. This situation is further worsened by the presence of insulin resistance, which in turn leads to mitochondrial dysfunction and overgeneration of ROS (106). Vitamin E, which prevents lipid oxidation, has been proposed as a potentially powerful therapeutic agent (37). The synergistic interaction between bilirubin and vitamin E has also been demonstrated (165). Although yet to receive specific attention, the antioxidant effects of bilirubin can be assumed to mitigate the pathophysiological mechanism behind MASH development.

The association between physical inactivity and the risk of MAFLD/MASH is widely accepted (139). Analogously, increased energy expenditure due to both aerobic and resistance exercises is associated with the prevention and amelioration of fatty liver disease (179). The negative correlation between body mass index and serum bilirubin concentrations is also acknowledged (4, 155). In obese subjects, bilirubin is most likely overconsumed due to obesity-induced oxidative stress (131). In this context, notable data from a recent animal study revealed exercise-induced elevation of plasma bilirubin concentration, which was accompanied by substantial improvements in glucose and lipid metabolism (56). These findings tally with observations by Swift et al., who documented significant increases in serum bilirubin concentration in previously sedentary postmenopausal women placed on an exercise regimen (171). Similar bilirubin elevations have been found in Polish (201) and elite Czech athletes (202).

Carbon monoxide

According to recent experimental research, CO (whether endogenously produced or administered as a therapeutic agent) has considerable therapeutic potential for inhibiting MAFLD/MASH progression. In dietary-induced obese mice treated with a CO donor (carbon monoxide releasing molecule A1 [CORM-A1]), chronic treatment with this therapeutic substance reversed adiposity, led to remodeling of white adipose tissue, and normalized insulin resistance independently of changes in food intake or activity (63, 64). The most likely mechanism is modulation of mitochondrial metabolism, since CO is a potent regulator of both mitochondrial biogenesis and mitochondrial respiration (99, 145, 169).

Similarly, the therapeutic activities of CO inhalation on hepatic steatosis have been reported in mice placed on an MCD diet; activation of AMPK and inhibition of mTOR complex I were identified as the cellular mechanisms responsible for these effects (78). In another study of obese mice administered a high-fat diet and exposed to CO inhalation, mitochondrial metabolism substantially improved. This treatment resulted in reduced hepatic lipid accumulation as well as enhanced mitochondria biogenesis and metabolism (77). More recently, another animal model of MASH documented similar benefits of CO (in the form of CORM-A1) on mitochondrial functions in mice fed a high-fat/high-fructose diet (177).

Products of the Heme Catabolic Pathway As Protective Factors Against Liver Inflammation

Key pathogenic aspects

The anti-inflammatory role of the heme catabolic pathway is well-documented. Its complex impact on the inflammatory response enlists a number of mechanisms (Fig. 5). These include regulation of cytokine gene expression via TLRs and the control of inflammasome-dependent cytokine maturation and macrophage polarization [for a review, see Ryter (140)]. Specific inflammatory pathways affected by BVRA and bilirubin, a potent immunosuppressant, are discussed in detail below. It should be noted that the anti-inflammatory and metabolic activities of the heme catabolic pathway are interlinked. These mechanisms, which comprise joint signaling pathways such as TLR4, the regulation of mTOR target genes including protein kinase B (Akt), protein kinase C (PKC), and AMPKα, and the modulation of mitogen-activated protein kinases (MAPKs) and NF-κB (discussed in detail below), are important in counteracting the development of metabolic syndrome and MAFLD (34; see also section “Products of the Heme Catabolic Pathway As Protective Factors Against MAFLD/MASH Development” above).

FIG. 5.

The role of the heme catabolic pathway in liver inflammation. All parts of the heme catabolic pathway, including HMOX1, BLVRA, bilirubin, and CO, are involved in general immunosuppressive activities that protect against hepatic inflammation. Akt, protein kinase B; IL-10, interleukin 10; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; TLR4, toll-like receptor 4. Color images are available online.

Heme oxygenase 1

HMOX1 is the most important inflammation regulator in the heme catabolic pathway (125). In addition to its anti-inflammatory potential (127), the gene has a virostatic function, as confirmed in a study of viral hepatitis C (216). It is involved in the degradation of proinflammatory and pro-oxidant free heme, iron recycling, and the production of the anti-inflammatory molecules bilirubin and CO (69).

Biliverdin reductase A

BLVRA, the second enzyme in the heme catabolic pathway, significantly contributes to the control of inflammation, as documented in our own study. We demonstrated the protective role of BLVRA in hepatitis C viral infection. Compared with healthy control subjects, patients with chronic HCV infection displayed increased mRNA levels of BLVRA in peripheral white blood cells. Interestingly, individuals who responded to standard antiviral therapy had significantly upregulated BLVRA expression in peripheral blood leukocytes, whereas those without BLVRA overexpression were nonresponsive. In addition, mRNA levels of BLVRA in peripheral white blood cells closely correlated with levels in liver tissue, indicating that peripheral blood cells could be used as a surrogate tissue to access the heme catabolic pathway in the liver (166).

Significantly, it is not only enzymatic activity per se, but also the specific signaling of BLVRA expressed on the cytoplasmic membranes of macrophages—mediated via phosphatidylinositol 3-kinase and Akt signaling pathways—that has potent anti-inflammatory effects on the inhibition of proinflammatory cytokine production (198). Upon exposure to biliverdin, BLVRA translocates to the nucleus of mouse macrophages, from where it blocks specific gene expression.

It also acts as a transcription factor in a variety of signaling pathways, including TLR4 (124). In a study by Zhang et al., biliverdin inhibited TLR4 signaling in leukocytes and triggered the phosphorylation of mTOR-specific targets, including Akt, PKC, and AMPKα (213). These findings support the modulatory function of biliverdin and BLVRA in the TLR4 signaling pathway. The critical role of BLVRA in macrophage polarization and anti-inflammatory cytokine production has been demonstrated both in vitro and in an animal model of kidney disease (65). The effects of BLVRA on various signaling pathways, including MAPK, Akt, and NF-κB activation, have been documented in LPS-induced inflammation in macrophage cells (79).

As previously mentioned, low BLVRA expression in peripheral blood leukocytes is associated with inflammation of adipose tissue in patients with MASH (30), representing yet another function of BLVRA.

Bilirubin

Bilirubin has immunosuppressive effects on almost all effectors of both innate and adaptive immune responses. The compound also protects against inflammation-induced liver injury, the extensive clinical consequences of which have been reviewed in detail (22, 70). In agreement with these findings, our recent study of hyperbilirubinemic Gunn rats exposed to bacterial LPS confirmed the ameliorative effect of bilirubin on liver injury (178). Like biliverdin and BLVRA, bilirubin inhibits TLR4 signaling (67), an important factor in the pathogenesis of liver disease. In a mouse model of colitis, bilirubin was shown to inhibit TLR4, suppress the production of proinflammatory cytokines, and regulate changes in the intestinal microbiome and gut barrier (214).

Based on a recent review by Corral-Jara et al., all components of the heme catabolic pathway, including heme, HMOX, biliverdin, bilirubin, and CO, are likely to be involved in the pathogenesis of various types of viral hepatitis (31). In addition to regulating specific immune responses against hepatitis viral infections, both biliverdin and bilirubin have direct antiviral effects, as demonstrated in herpetic and hepatitis C viruses (113, 146, 215).

According to one study, bilirubin considerably alleviated sterile inflammation in mice fed a high-fat diet (87), a finding consistent with the profound effects of the pigment on immune system responses being in play also in MASH development (22, 70).

Carbon monoxide

CO is not only a powerful modulator of immune responses, but it also affects many inflammatory signal transduction pathways [for a review, see Hoetzel et al. (62)]. As well as having direct effects on immune-competent cells, CO has been shown to inhibit the activation of monocytes, macrophages, leukocytes, and T cells (126, 147). Previous studies have highlighted CO-induced suppression of proinflammatory cytokine production and NF-κB activation through the modulation of several important cellular proinflammatory signaling pathways (123, 147). Yan et al. confirmed that these anti-inflammatory effects also apply to liver pathology. In a murine model of acute liver failure induced by LPS/D-galactosamine, treatment with CORM-3 was found to suppress proinflammatory cytokine production, increase anti-inflammatory IL10 cytokine secretion, and inhibit nuclear expression of NF-κB (205).

Products of the Heme Catabolic Pathway As Protective Factors in Hepatic IRI and Liver Transplantation

Hepatic IRI is a multifactorial process triggered by transient tissue deprivation of oxygen and subsequent reoxygenation. This complication can occur as a result of various clinical conditions such as shock of any etiology, hepatic resection, and liver transplantation. The mechanisms of hepatic IRI particularly involve dysregulation of redox status and activation of the innate immune system (176). During the initial ischemic phase, oxygen in liver cells is depleted, with ROS generation increasing during the second reperfusion phase. The consequent massive flux and activation of inflammatory cells—including macrophages, Kupffer cells, neutrophils, and T cells—perpetuate a number of apoptotic processes [for a review, see Hirao et al. (60)].

Pointing to its important therapeutic potential, the heme catabolic pathway is understood to be implicated in protection against IRI as well as transplant rejection in various organs and tissues (29, 121). The pathway comprises a complex protective system that counteracts inflammation, apoptosis, cell proliferation, and pro-oxidation, and also modifies other immune responses, as discussed above (121).

Heme oxygenase

HMOX expression and activity seem to be of key importance in preventing IRI (97), driven primarily by HMOX expressed on both infiltrating macrophages and resident Kupffer cells (114). A number of experimental studies have documented the essential role of HMOX1 in alleviating IRI in both cold-preserved (2, 74) and aged livers (193). A large study by Buis et al. involving more than 300 liver transplant patients highlighted the beneficial effects of HMOX. Subjects with high expression of the HMOX1 genotype were protected from unfavorable outcomes and had better graft survival rates than individuals with low expression (20). As reported in a clinical study by Geuken et al., the ability to upregulate HMOX1 activity during liver transplantation seems to be more important than steady expression in protecting liver grafts (46). In another recent study, conducted by Kadono et al., the human biopsies of liver transplant recipients were evaluated. The authors found negative associations between innate (represented by CD68, cathepsin G, TLR4, CXCL10), adaptive (such as CD4, CD8, IL17), and costimulatory (CD28, CD80, CD86) immune system molecules. Most importantly, liver transplant recipients with high HMOX1 expression exhibited a trend toward improved overall survival, indicating the direct effect of the HMOX system on immune responses in liver transplantation (72).

Biliverdin/bilirubin

The protective function of biliverdin was documented in an experimental rat model of IRI by Fondevila et al. Following cold ischemia of liver grafts and syngeneic orthotopic liver transplantation, biliverdin therapy extended animal survival, increasing from 50% in untreated controls to 90%–100%. This effect correlated with improved liver functions and preserved hepatic histology (41). The beneficial effects of biliverdin therapy observed in both donors and recipients were observed in a swine model of liver transplantation (5). Interestingly, a significant improvement in liver function was reported in one model of a rat liver exposed to cold ischemia following bilirubin rinse. This rinsing of liver grafts with bilirubin before reperfusion substantially improved the survival rate (75). Significantly, the cytoprotective effects of biliverdin and bilirubin seem to apply to other organ transplantations, as reviewed by Ollinger and colleagues (121).

Carbon monoxide

Boasting impressive vasoactive properties, CO exerts an important protective influence on IRI, as evidenced by a number of experimental animal studies (97). These include models of rats with CO-enriched blood and rat livers perfused either with CO or different CORM compounds (3, 68, 132, 161, 170, 199).

The collective experimental and clinical data strongly support the beneficial role played by the entire heme catabolic pathway in hepatic IRI and liver transplantation.

Products of the Heme Catabolic Pathway As Protective Factors Against Liver Fibrogenesis

The heme catabolic pathway significantly contributes to the process of fibrogenesis (Fig. 6). Inhibition of the TLR4 signaling pathway seems to be important not only in the immune response but also in liver fibrogenesis (133, 197). Accordingly, biliverdin, BLVRA, and bilirubin may protect against liver fibrogenesis by blocking HSC activation, a finding reported in a study by Tang et al. Their in vitro study documented the inhibition of HSC activation and a decreased mRNA ratio for tissue inhibitor of metalloproteinase-1/matrix metalloproteinase-2 (TIMP-1/MMP-2) upon exposure to only moderately increased bilirubin concentrations (173). Apart from hepatic fibrogenesis, MMPs are also involved in hepatocellular cancer (HCC) development and the pathogenesis of other liver diseases (45). Bilirubin was shown to inhibit MMPs in other pathological conditions such as nasopharyngeal cancer (32) and wound healing in diabetic rats (137). The same effects of bilirubin were observed in a murine model of UVB-induced skin photodamage (8) and in an experimental model of breast cancer involving HMOX1 induction (95). The HMOX gene itself, which is expressed on HSC, inhibits hepatic fibrogenesis via proapoptotic effects on cells mediated by NF-κB (207). As mentioned above, the protective effects of HMOX1 induction on hepatic fibrogenesis were reported in MASH animal models (142, 192).

FIG. 6.

The role of the heme catabolic pathway in liver fibrogenesis and HCC. All parts of the heme catabolic pathway, including HMOX1, BLVRA, bilirubin, and CO, are involved in the protection against the development of HCC via mutual interactions leading to the modulation of various proliferative and inflammatory signaling pathways. AhR, aryl hydrocarbon receptor; HCC, hepatocellular cancer; HSC, hepatic stellate cells; MMP, matrix metalloproteinase; VCAM-1, vascular cell adhesion molecule-1. Color images are available online.

CO also boasts a number of antifibrotic effects, as demonstrated in an experimental model of kidney disease (116). Heme is a potent biomolecule that acts as a molecular switch, since different concentrations of free heme generate different cellular responses during both inflammation and fibrosis [for a review, see Lundvig et al. (101)]. Although exposure to high levels of heme has been shown to induce hepatic fibrosis (175), an experimental Mdr2 knockout mouse model with reduced HSC activation in liver tissue demonstrated that pharmacological induction of HMOX1 prevents and even reverts liver fibrosis (12). Interestingly, similar effects of HMOX1 induction have been observed in the pancreas. Nutraceutical upregulation of HMOX1 was found to prevent pancreatic stellate cell activation and development of pancreatic fibrosis (151), with these effects principally driven by the HMOX1/CO-modulated p38 MAPK pathway (152). Based on a recent review, CO also significantly contributes to fibrogenesis in pancreatic tissue (150).

AhR-mediated fibrogenetic pathways are among the other potential cell targets affected by bilirubin, a potent ligand of this nuclear receptor (130). AhR is significantly involved in the process of hepatic fibrogenesis, as confirmed by fibrotic changes in the liver tissue of Ahr-deficient mice (39). This may be at least partially due to the beneficial role of AhR in the activation of HSC, as recently described in an in vitro study on primary mouse and human HSC (206).

As previously mentioned, bilirubin was shown to improve both the gut microbiome and barrier functions in an animal model of colitis (214), with similar results reported for treatment of murine acute colitis with hyaluronic acid bilirubin nanoparticles (90, 189). In a murine study by Vogel and Zucker (190) and in another work by the same authors (221), bilirubin ameliorated dextran sodium sulfate-induced colitis, helping to preserve colonic functions and prevent trafficking of leukocytes by inhibition of vascular cell adhesion molecule-1 (VCAM-1)-mediated signaling. Bilirubin has also been shown to inhibit VCAM-1-dependent airway inflammation (76).

It is widely accepted that dysfunctional intestinal permeability increases hepatic exposure to gut bacteria products, which act as TLR4 ligands that trigger hepatic fibrogenesis (154) and ultimately the development of liver cirrhosis (73).

In summary, the above collective data clearly demonstrate the profound impact of the entire heme catabolic pathway on hepatic fibrogenesis.

Products of the Heme Catabolic Pathway As Protective Factors Against Liver Carcinogenesis

Heme oxygenase

The inconclusive role of HMOX in carcinogenesis is the subject of conflicting reports in the literature (195) (Fig. 6). A study by Park et al. found that HMOX1 expression did not seem to influence the prognosis of HCC patients (128). However, another clinical study revealed low expression of Nrf2 and HMOX1 in HCC biopsies, pointing to the beneficial role of HMOX1 in this cancer (28). Supporting these clinical data, an experimental in vitro study by Zou et al. found that HMOX1 inhibited HepG2 hepatoblastoma cell growth mediated via microRNA pathways (218). In another experimental in vitro animal study by the same authors, the inhibitory effects of HMOX1 on HepG2 cell growth and migration were observed, with CO identified as an important contributing factor (217).

Biliverdin reductase A

BLVRA mRNA expression was found to be a predictor in our small study of patients with HCC. Compared with controls, mRNA levels of BLVRA in the livers of HCC patients increased by up to threefold. This finding correlated with BLVRA expression in peripheral leukocytes, a result perhaps attributable to a feedback mechanism for controlling increased oxidative stress associated with HCC progression (83). Our results were corroborated by an in vitro study on human HCC cell lines by Huan et al., who found that RNA interference of BLVRB suppressed HCC cell growth, despite BLVRB overexpression having an opposite effect (66).

Bilirubin

To date, there have been no epidemiological studies on the association of bilirubin with HCC. However, data in relation to overall cancer prevalence indicate a clear negative association between serum/plasma bilirubin concentration and cancer risk. Yamamoto et al. found a negative correlation between high serum bilirubin and cancer development in a large cohort of Japanese adults (n = 78,000) with no hepatobiliary disease (204). In our own study of the Polish arm of the HAPIEE cohort (n = 1700), bilirubin was associated with lower mortality, driven particularly by cancer mortality (186). The large NHANES study by Zucker et al. (covering 176,748,000 individuals) reported a negative association between serum bilirubin and the risk of nondermatological cancers, particularly gastrointestinal cancer (220). Interestingly, a study by Han et al. found a negative association between liver donor preoperative total bilirubin concentration and risk of HCC recurrence in transplanted patients (54).

Carbon monoxide

Although no specific research has confirmed the involvement of CO in HCC development, recent data from research on different cancers, recently summarized in a review article by Kourti et al. and corroborated by an experimental study by Zou et al. (217), suggest that CO may interfere with HCC pathogenesis (82), This hypothesis is consistent with our own in vitro and animal data on the therapeutic effect of CO on pancreatic cancer (185). Although promising results regarding CO treatment in MASH (HCC precarcinosis) have recently been published, further detailed research into the effects of CO on liver carcinogenesis is warranted.

Therapeutic Potential of Bilirubin Metabolism Modulation

To improve metabolic and liver health outcomes, there have been concerted efforts to explore therapeutic options aimed at modulating the heme catabolic pathway as well as its specific intermediates and products. Recently, several comprehensive reviews on the topic have highlighted the potential of various treatments (44, 159, 184, 191).

Several promising methods have been developed, with the possibilities of HMOX1 induction receiving the most attention of researchers (159). Many natural products commonly used as nutraceuticals have been shown to upregulate HMOX1 and improve MAFLD according to a number of experimental and clinical reports [comprehensively reviewed in Stec and Hinds (159)]. For example, silymarin flavonolignans are widely acknowledged to protect against a wide range of liver diseases (174). These agents also act as partial inhibitors of UGT1A1, mildly elevating the hepatic (168) and systemic (40) concentrations of bilirubin. Therefore, partial inhibition of UGT1A1 using either medical xenobiotics or better nutraceutical substances (aimed at potentiating bilirubin signaling activities) represents a promising therapeutic approach for patients with liver diseases (184).

A similar chemopreventive effect is offered by naturally occurring tetrapyrroles, chemical compounds that resemble the structures of biliverdin and bilirubin. The most promising of these agents include pulverized bovine gallstones, which have been used for centuries in Traditional Chinese Medicine (135, 210), green-plant chlorophylls (180), and phycocyanobilin from blue-green algae (80, 105). Modulation of the signaling and immunomodulatory functions of BLVRA has also been investigated, especially in the treatment of metabolic diseases such as obesity, diabetes, and MAFLD (47, 48).

Finally, with the advent of modern nanotechnologies, incorporation of bilirubin into various forms of nanoparticles has demonstrated remarkable biological benefits in a number of clinical pathologies, as recently reviewed in several comprehensive studies (27, 189, 208, 209). The same nanomedical approach has been notably applied to BLVRA-based peptides to successful effect, indicating the broad potential of this therapeutic modality in metabolic disease prevention (48).

Conclusion

The accumulating body of evidence indicates that the heme catabolic pathway, including its key proteins and pigments, has significant impacts not only on heme degradation but also on the metabolic functions of the human body. Primarily localized in the liver, a central metabolic organ, the pathway unsurprisingly plays a significant role in modulating the pathogenesis of a variety of liver diseases. However, further clinical studies are needed to elucidate the full range of pathogenetic mechanisms that drive this therapeutic pathway.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by grants NV18-07-00342 and RVO-VFN64165/2021 from the Czech Ministry of Health, and Progress Q25/LF1 from Charles University.

Abbreviations Used

References

Ahmad

and Woodgett

. Emerging roles of GSK-3alpha in pathophysiology: emphasis on cardio-metabolic disorders. Biochim Biophys Acta Mol Cell Res, 1867: 118616, 2020.

Amersi

, Buelow

, Kato

, Ke

, Coito

, Shen

, Zhao

, Zaky

, Melinek

, Lassman

, Kolls

, Alam

, Ritter

, Volk

, Farmer

, Ghobrial

, Busuttil

, and Kupiec

. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest, 104: 1631–1639, 1999.

Amersi

, Shen

, Anselmo

, Melinek

, Iyer

, Southard

, Katori

, Volk

, Busuttil

, Buelow

, and Kupiec

. Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway. Hepatology, 35: 815–823, 2002.

Andersson

, Weeke

, Fosbol

, Brendorp

, Kober

, Coutinho

, Sharma

, Van Gaal

, Finer

, James

, Caterson

, Rode

, and Torp-Pedersen

. Acute effect of weight loss on levels of total bilirubin in obese, cardiovascular high-risk patients: an analysis from the lead-in period of the Sibutramine Cardiovascular Outcome trial. Metabolism, 58: 1109–1115, 2009.

Andria

, Bracco

, Attanasio

, Castaldo

, Cerrito

, Cozzolino

, Di Napoli

, Giovannoni

, Mancini

, Musumeci

, Mezza

, Nasti

, Scuderi

, Staibano

, Lavitrano

, Otterbein

, and Calise

. Biliverdin protects against liver ischemia reperfusion injury in swine. PLoS One, 8: e69972, 2013.

Anwar

MS.

Systematic review: association of serum bilirubin and non-alcoholic fatty liver disease: a feasible therapeutic avenue?. World J Pharmacol, 3: 209–216, 2014.

Ayer

, Zarjou

, Agarwal

, and Stocker

. Heme oxygenases in cardiovascular health and disease. Physiol Rev, 96: 1449–1508, 2016.

Bai

, Liu

, You

, Li

, Ma

. Intraperitoneally administered biliverdin protects against UVB-induced skin photo-damage in hairless mice. J Photochem Photobiol B, 144: 35–41, 2015.

Balla

, Vercellotti

, Jeney

, Yachie

, Varga

, Jacob

, Eaton

, and Balla

. Heme, heme oxygenase, and ferritin: how the vascular endothelium survives (and dies) in an iron-rich environment. Antioxid Redox Signal, 9: 2119–2137, 2007.

10.

Baranano

, Rao

, Ferris

, and Snyder

. Biliverdin reductase: a major physiologic cytoprotectant. Proc Natl Acad Sci U S A, 99: 16093–16098, 2002.

11.

Baranano

and Snyder

. Neural roles for heme oxygenase: contrasts to nitric oxide synthase. Proc Natl Acad Sci U S A, 98: 10996–11002, 2001.

12.

Barikbin

, Neureiter

, Wirth

, Erhardt

, Schwinge

, Kluwe

, Schramm

, Tiegs

, and Sass

. Induction of heme oxygenase 1 prevents progression of liver fibrosis in Mdr2 knockout mice. Hepatology, 55: 553–562, 2012.

13.

Barone

, Di Domenico

, Cassano

, Arena

, Tramutola

, Lavecchia

, Coccia

, Butterfield

, and Perluigi

. Impairment of biliverdin reductase-A promotes brain insulin resistance in Alzheimer disease: a new paradigm. Free Radic Biol Med, 91: 127–142, 2016.

14.

Barone

, Di Domenico

, Cenini

, Sultana

, Cini

, Preziosi

, Perluigi

, Mancuso

, and Butterfield

. Biliverdin reductase-a protein levels and activity in the brains of subjects with Alzheimer disease and mild cognitive impairment. Biochim Biophys Acta, 1812: 480–487, 2011.

15.

Bauer

and Bauer

. Heme oxygenase-1: redox regulation and role in the hepatic response to oxidative stress. Antioxid Redox Signal, 4: 749–758, 2002.

16.

Berk

, Rodkey

, Blaschke

, Collison

, and Waggoner

. Comparison of plasma bilirubin turnover and carbon monoxide production in man. J Lab Clin Med, 83: 29–37, 1974.

17.

Beurel

, Grieco

, and Jope

. Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases. Pharmacol Ther, 148: 114–131, 2015.

18.

Bianco

, Dvorak

, Capková

, Gironde

, Tiribelli

, Furger

, Vitek

, Bellarosa

. The extent of intracellular accumulation of bilirubin determines its anti- or pro-oxidant effect. Int J Mol Sci, 21: 1–17, 2020.

19.

Brugger

, Schick

, Brock

, Baumann

, Muellenbach

, Roewer

, and Wunder

. Carbon monoxide has antioxidative properties in the liver involving p38 MAP kinase pathway in a murine model of systemic inflammation. Microcirculation, 17: 504–513, 2010.

20.

Buis

, van der

, Visser

, Nolte

, Hepkema

, Nijsten

, Slooff

, and Porte

. Heme oxygenase-1 genotype of the donor is associated with graft survival after liver transplantation. Am J Transplant, 8: 377–385, 2008.

21.

Bulmer

, Verkade

, and Wagner

. Bilirubin and beyond: a review of lipid status in Gilbert's syndrome and its relevance to cardiovascular disease protection. Prog Lipid Res, 52: 193–205, 2013.

22.

Canesin

, Hejazi

, Swanson

, and Wegiel

. Heme-derived metabolic signals dictate immune responses. Front Immunol, 11: 66, 2020.

23.

Celis

and DuBois

. Making and breaking heme. Curr Opin Struct Biol, 59: 19–28, 2019.

24.

Chang

, Lin

, Liu

, Yeh

, and Ni

. Heme oxygenase-1 gene promoter polymorphism and the risk of pediatric nonalcoholic fatty liver disease. Int J Obes (Lond), 39: 1236–1240, 2015.

25.

Chen

, Duan

, Wei

, Ning

, Bi

, Zhao

, Qin

, and Li

. Acetyl-CoA carboxylase (ACC) as a therapeutic target for metabolic syndrome and recent developments in ACC1/2 inhibitors. Expert Opin Investig Drugs, 28: 917–930, 2019.

26.

Chen

, Maghzal

, Ayer

, Suarna

, Dunn

, and Stocker

. Absence of the biliverdin reductase-a gene is associated with increased endogenous oxidative stress. Free Radic Biol Med, 115: 156–165, 2018.

27.

Chen

, Vong

, Gao

, Chen

, Wu

, Wang

, and Wang

. Bilirubin nanomedicines for the treatment of reactive oxygen species (ROS)-mediated diseases. Mol Pharm, 17: 2260–2274, 2020.

28.

Cheng

, Lu

, Chen

, Shen

, and Liu

. Liver expression of Nrf2-related genes in different liver diseases. Hepatobiliary Pancreat Dis Int, 14: 485–491, 2015.

29.

Cheng

and Rong

. Therapeutic potential of heme oxygenase-1/carbon monoxide system against ischemia-reperfusion injury. Curr Pharm Des, 23: 3884–3898, 2017.

30.

Cimini

, Arena

, Barchetta

, Tramutola

, Ceccarelli

, Lanzillotta

, Fontana

, Bertoccini

, Leonetti

, Capoccia

, Silecchia

, Di Cristofano

, Chiappetta

, Di Domenico

, Baroni

, Perluigi

, and Cavallo

, Barone

. Reduced biliverdin reductase-A levels are associated with early alterations of insulin signaling in obesity. Biochim Biophys Acta Mol Basis Dis, 1865: 1490–1501, 2019.

31.

Corral-Jara

, Trujillo-Ochoa

, Realpe

, Panduro

, Roman

, and Fierro

. Rethinking the immune properties of bilirubin in viral hepatitis: from bench to bedside. Clin Transl Immunology, 4: e54, 2015.

32.

Deng

, Xu

, Li

, Luo

, Zhu

, Jiang

, Zhang

, Lei

, Lian

, Zou

, You

, Chen

, Feng

, Bei

, Chen

, and Zeng

. Unconjugated bilirubin is a novel prognostic biomarker for nasopharyngeal carcinoma and inhibits its metastasis via antioxidation activity. Cancer Prev Res (Phila), 9: 180–188, 2016.

33.

Dongiovanni

, Rametta

, Meroni

, and Valenti

. The role of insulin resistance in nonalcoholic steatohepatitis and liver disease development-a potential therapeutic target?. Expert Rev Gastroenterol Hepatol, 10: 229–242, 2016.

34.

Dornas

and Schuppan

. Mitochondrial oxidative injury: a key player in nonalcoholic fatty liver disease. Am J Physiol Gastrointest Liver Physiol, 319: G400–G411, 2020.

35.

Duszka

, Gregor

, Guillou

, Konig

, and Wahli

. Peroxisome proliferator-activated receptors and caloric restriction-common pathways affecting metabolism, health, and longevity. Cells, 9: 1708, 2020.

36.

Dvorak

, Pospisilova

, Zizalova

, Capkova

, Muchova

, Vecka

, Vrzackova

, Krizova

, Zelenka

, and Vitek

. The effects of bilirubin and lumirubin on metabolic and oxidative stress markers. Front Pharmacol, 12: 567001, 2021.

37.

El Hadi

, Vettor

, and Rossato

. Vitamin E as a treatment for nonalcoholic fatty liver disease: reality or myth?. Antioxidants (Basel), 7: 12, 2018.

38.

Ellakany

, Mahmoud MoheyEldin

, Invernizzi

, Mahmoud ElKady

, Eldin Fathy Abou Elkheir

, Abdel Haleem Abo Elwafa

, and Ellakany

. Study of the influence of heme oxygenase 1 gene single nucleotide polymorphism (rs2071746) on esophageal varices among patients with cirrhosis. Eur J Gastroenterol Hepatol, 30: 888–892, 2018.

39.

Fernandez-Salguero

, Pineau

, Hilbert

, McPhail

, Lee

, Kimura

, Nebert

, Rudikoff

, Ward

, and Gonzalez

. Immune system impairment and hepatic fibrosis in mice lacking the dioxin-binding Ah receptor. Science, 268: 722–726, 1995.

40.

Flaig

, Gustafson

, Su

, Zirrolli

, Crighton

, Harrison

, Pierson

, Agarwal

, and Glode

. A phase I and pharmacokinetic study of silybin-phytosome in prostate cancer patients. Invest New Drugs, 25: 139–146, 2007.

41.

Fondevila

, Shen

, Tsuchiyashi

, Yamashita

, Csizmadia

, Lassman

, Busuttil

, Kupiec-Weglinski

, and Bach

. Biliverdin therapy protects rat livers from ischemia and reperfusion injury. Hepatology, 40: 1333–1341, 2004.

42.

Frankenberg-Dinkel

Bacterial heme oxygenases. Antioxid Redox Signal, 6: 825–834, 2004.

43.

Fujii

, Inoguchi

, Sasaki

, Maeda

, Zheng

, Kobayashi

, and Takayanagi

. Bilirubin and biliverdin protect rodents against diabetic nephropathy by downregulating NAD(P)H oxidase. Kidney Int, 78: 905–919, 2010.

44.

Gazzin

, Vitek

, Watchko

, Shapiro

, and Tiribelli

. A novel perspective on the biology of bilirubin in health and disease. Trends Mol Med, 22: 758–768, 2016.

45.

Geervliet

, and Bansal

. Matrix metalloproteinases as potential biomarkers and therapeutic targets in liver diseases. Cells, 9: 1212, 2020.

46.

Geuken

, Buis

, Visser

, Blokzijl

, Moshage

, Nemes

, Leuvenink

, de Jong

, Peeters

, Slooff

, and Porte

. Expression of heme oxygenase-1 in human livers before transplantation correlates with graft injury and function after transplantation. Am J Transplant, 5: 1875–1885, 2005.

47.

Gibbs

, Lerner-Marmarosh

, Poulin

, Farah

, and Maines

. Human biliverdin reductase-based peptides activate and inhibit glucose uptake through direct interaction with the kinase domain of insulin receptor. FASEB J, 28: 2478–2491, 2014.

48.

Gibbs

, Miralem

, Lerner-Marmarosh

, and Maines

. Nanoparticle delivered human biliverdin reductase-based peptide increases glucose uptake by activating IRK/Akt/GSK3 axis: the peptide is effective in the cell and wild-type and diabetic ob/ob mice. J Diabetes Res, 2016: 4712053, 2016.

49.

Gonzalez-Sanchez

, Perez

, Nytofte

, Briz

, Monte

, Lozano

, Serrano

, and Marin

. Protective role of biliverdin against bile acid-induced oxidative stress in liver cells. Free Radic Biol Med, 97: 466–477, 2016.

50.

Gordon

, Adeosun

, Ngwudike

, Anderson

, Hall

, Hinds

Jr ., and Stec

. CRISPR Cas9-mediated deletion of biliverdin reductase A (BVRA) in mouse liver cells induces oxidative stress and lipid accumulation. Arch Biochem Biophys, 672: 108072, 2019.

51.

Gordon

, Neifer

, Hamoud

, Hawk

, Nestor-Kalinoski

, Miruzzi

, Morran

, Adeosun

, Sarver

, Erhardt

, McCullumsmith

, Stec

, and Hinds

Jr . Bilirubin remodels murine white adipose tissue by reshaping mitochondrial activity and the coregulator profile of peroxisome proliferator-activated receptor alpha. J Biol Chem, 295: 9804–9822, 2020.

52.

Grojean

, Koziel

, Vert

, and Daval

. Bilirubin induces apoptosis via activation of NMDA receptors in developing rat brain neurons. Exp Neurol, 166: 334–341, 2000.

53.

, Shin

, Feitelson

, and Yu

. Oxidative stress and antioxidants in hepatic pathogenesis. World J Gastroenterol, 16: 6035–6043, 2010.

54.

Han

, Yang

, Sinn

, Ko

, Kim

, Shin

, Son

, Gwak

, Joh

, and Kim

. Higher bilirubin levels of healthy living liver donors are associated with lower posttransplant hepatocellular carcinoma recurrence. Transplantation, 100: 1933–1938, 2016.

55.

Hinds

Jr. , Burns

, Hosick

, McBeth

, Nestor-Kalinoski

, Drummond

, AlAmodi

, Hankins

, Vanden Heuvel

, and Stec

DE.

Biliverdin reductase A attenuates hepatic steatosis by inhibition of glycogen synthase kinase (GSK) 3beta phosphorylation of serine 73 of peroxisome proliferator-activated receptor (PPAR) alpha. J Biol Chem, 291: 25179–25191, 2016.

56.

Hinds

Jr. , Creeden

, Gordon

, Spegele

, Britton

, Koch

, and Stec

DE.

Rats genetically selected for high aerobic exercise capacity have elevated plasma bilirubin by upregulation of hepatic biliverdin reductase-A (BVRA) and Suppression of UGT1A1. Antioxidants (Basel), 9: 889, 2020.

57.

Hinds

Jr. , Creeden

, Gordon

, Stec

, Donald

, and Stec

DE.

Bilirubin nanoparticles reduce diet-induced hepatic steatosis, improve fat utilization, and increase plasma beta-hydroxybutyrate. Front Pharmacol, 11: 594574, 2020.

58.

Hinds

Jr. , Hosick

, Chen

, Tukey

, Hankins

, Nestor-Kalinoski

, and Stec

DE.

Mice with hyperbilirubinemia due to Gilbert's syndrome polymorphism are resistant to hepatic steatosis by decreased serine 73 phosphorylation of PPARalpha. Am J Physiol Endocrinol Metab, 312: E244–E252, 2017.

59.

Hinds

Jr. , Sodhi

, Meadows

, Fedorova

, Puri

, Kim

, Peterson

, Shapiro

, Abraham

, and Kappas

Increased HO-1 levels ameliorate fatty liver development through a reduction of heme and recruitment of FGF21. Obesity (Silver Spring), 22: 705–712, 2014.

60.

Hirao

, Dery

, Kageyama

, Nakamura

, and Kupiec-Weglinski

. Heme oxygenase-1 in liver transplant ischemia-reperfusion injury: from bench-to-bedside. Free Radic Biol Med, 157: 75–82, 2020.

61.

Hjelkrem

, Morales

, Williams

, and Harrison

. Unconjugated hyperbilirubinemia is inversely associated with non-alcoholic steatohepatitis (NASH). Aliment Pharmacol Ther, 35: 1416–1423, 2012.

62.

Hoetzel

, Dolinay

, Schmidt

, Choi

, and Ryter

. Carbon monoxide in sepsis. Antioxid Redox Signal, 9: 2013–2026, 2007.

63.

Hosick

, AlAmodi

, Hankins

, and Stec

. Chronic treatment with a carbon monoxide releasing molecule reverses dietary induced obesity in mice. Adipocyte, 5: 1–10, 2016.

64.

Hosick

, AlAmodi

, Storm

, Gousset

, Pruett

, Gray

, 3rd, Stout

, and Stec

. Chronic carbon monoxide treatment attenuates development of obesity and remodels adipocytes in mice fed a high-fat diet. Int J Obes (Lond), 38: 132–139, 2014.

65.

, Pei

, Wang

, Yang

, Zhu

, Guo

, Wang

, Yao

, Zeng

, Liao

, and Xu

. Biliverdin reductase A (BVRA) mediates macrophage expression of interleukin-10 in injured kidney. Int J Mol Sci, 16: 22621–22635, 2015.

66.

Huan

, Bao

, Chen

, Li

, Lian

, Ding

, Huang

, Liang

, and He

. MicroRNA-127-5p targets the biliverdin reductase B/nuclear factor-kappaB pathway to suppress cell growth in hepatocellular carcinoma cells. Cancer Sci, 107: 258–266, 2016.

67.

Idelman

, Smith

, and Zucker

. Bilirubin inhibits the up-regulation of inducible nitric oxide synthase by scavenging reactive oxygen species generated by the toll-like receptor 4-dependent activation of NADPH oxidase. Redox Biol, 5: 398–408, 2015.

68.

Ikeda

, Ueki

, Nakao

, Tomiyama

, Ross

, Stolz

, Geller

, and Murase

. Liver graft exposure to carbon monoxide during cold storage protects sinusoidal endothelial cells and ameliorates reperfusion injury in rats. Liver Transpl, 15: 1458–1468, 2009.

69.

Immenschuh

, Baumgart-Vogt

, and Mueller

. Heme oxygenase-1 and iron in liver inflammation: a complex alliance. Curr Drug Targets, 11: 1541–1550, 2010.

70.

Jangi

, Otterbein

, and Robson

. The molecular basis for the immunomodulatory activities of unconjugated bilirubin. Int J Biochem Cell Biol, 45: 2843–2851, 2013.

71.

Jansen

and Daiber

. Direct antioxidant properties of bilirubin and biliverdin. Is there a role for biliverdin reductase? Front Pharmacol , 3: 30, 2012.

72.

Kadono

, Dery

, Hirao

, Ito

, Kageyama

, Nakamura

, Oncel

, Aziz

, Kaldas

, Busuttil

, and Kupiec-Weglinski

. Heme oxygenase-1 dictates innate—adaptive immune phenotype in human liver transplantation. Arch Biochem Biophys, 671: 162–166, 2019.

73.

Karthikeyan

, Mohan

, Chinnakali

, and Vairappan

. Elevated systemic zonula occludens 1 is positively correlated with inflammation in cirrhosis. Clin Chim Acta, 480: 193–198, 2018.

74.

Kato

, Amersi

, Buelow

, Melinek

, Coito

, Ke

, Busuttil

, and Kupiec-Weglinski

. Heme oxygenase-1 overexpression protects rat livers from ischemia/reperfusion injury with extended cold preservation. Am J Transpl, 1: 121–128, 2001.

75.

Kato

, Shimazu

, Kondo

, Uchida

, Kumamoto

, Wakabayashi

, Kitajima

, and Suematsu

. Bilirubin rinse: a simple protectant against the rat liver graft injury mimicking heme oxygenase-1 preconditioning. Hepatology, 38: 364–373, 2003.

76.

Keshavan

, Deem

, Schwemberger

, Babcock

, Cook-Mills

, and Zucker

. Unconjugated bilirubin inhibits VCAM-1-mediated transendothelial leukocyte migration. J Immunol, 174: 3709–3718, 2005.

77.

Kim

, Kwon

, Choe

, Hong

, Yoo

, Goto

, Kawada

, Choi

, Joe

, Chung

, and Yu

. Quercetin reduces obesity-induced hepatosteatosis by enhancing mitochondrial oxidative metabolism via heme oxygenase-1. Nutr Metab (Lond), 12: 33, 2015.

78.

Kim

, Joe

, Kim

, Park

, Chen

, Kim

, Ryu

, Cho

, Surh

, Ryter

, Kim

, and Chung

. Carbon monoxide protects against hepatic steatosis in mice by inducing sestrin-2 via the PERK-eIF2alpha-ATF4 pathway. Free Radic Biol Med, 110: 81–91, 2017.

79.

Kim

, Kim

, Jo

, Cho

, Park

, Lee

, Choi

, Yeo

, Park

, Kim

, Yu

, Kim

, Cho

, Han

, Park

, Eum

, and Choi

. Tat-biliverdin reductase A inhibits inflammatory response by regulation of MAPK and NF-kappaB pathways in Raw 264.7 cells and edema mouse model. Mol Immunol, 63: 355–366, 2015.

80.

Konickova

, Vankova

, Vanikova

, Vanova

, Muchova

, Subhanova

, Zadinova

, Zelenka

, Dvorak

, Kolar

, Strnad

, Rimpelova

, Ruml

, Wong

, and Vitek

. Anti-cancer effects of blue-green alga Spirulina platensis, a natural source of bilirubin-like tetrapyrrolic compounds. Ann Hepatol, 13: 273–283, 2014.

81.

Korbut

, Brzozowski

, and Magierowski

. Carbon monoxide being hydrogen sulfide and nitric oxide molecular sibling, as endogenous and exogenous modulator of oxidative stress and antioxidative mechanisms in the digestive system. Oxid Med Cell Longev, 2020: 5083876, 2020.

82.

Kourti

, Jiang

, and Cai

. Aspects of carbon monoxide in form of CO-releasing molecules used in cancer treatment: more light on the way. Oxid Med Cell Longev, 2017: 9326454, 2017.

83.

Kubickova

, Subhanova

, Konickova

, Matousova

, Urbanek

, Parobkova

, Kupec

, Pudil

, and Vitek

. Predictive role BLVRA mRNA expression in hepatocellular cancer. Ann Hepatol, 15: 881–887, 2016.

84.

Kumar

, Rastogi

, Maras

, and Sarin

. Unconjugated hyperbilirubinemia in patients with non-alcoholic fatty liver disease: a favorable endogenous response. Clin Biochem, 45: 272–274, 2012.

85.

Kunutsor

, Frysz

, Verweij

, Kieneker

, Bakker

SJL

, and Dullaart

RPF

. Circulating total bilirubin and risk of non-alcoholic fatty liver disease in the PREVEND study: observational findings and a Mendelian randomization study. Eur J Epidemiol, 35: 123–137, 2020.

86.

Kwak

, Kim

, Chung

, Kang

, Park

, Kim

, Yoon

, and Lee

. Serum bilirubin levels are inversely associated with nonalcoholic fatty liver disease. Clin Mol Hepatol, 18: 383–390, 2012.

87.

Landerer

, Kalthoff

, Paulusch

, and Strassburg

. A Gilbert syndrome-associated haplotype protects against fatty liver disease in humanized transgenic mice. Sci Rep, 10: 8689, 2020.

88.

Lanzillotta

, Zuliani

, Vasavda

, Snyder

, Paul

, Perluigi

, Di Domenico

, and Barone

. BVR-A deficiency leads to autophagy impairment through the dysregulation of AMPK/mTOR axis in the brain-implications for neurodegeneration. Antioxidants (Basel), 9: 671, 2020.

89.

Lee

, Kaizu

, Toyokawa

, Zhang

, Ross

, Stolz

, Huang

, Gandhi

, Geller

, and Murase

. Carbon monoxide induces hypothermia tolerance in Kupffer cells and attenuates liver ischemia/reperfusion injury in rats. Liver Transpl, 17: 1457–1466, 2011.

90.

Lee

, Sugihara

, Gillilland

, 3rd, Jon

, Kamada

, and Moon

. Hyaluronic acid-bilirubin nanomedicine for targeted modulation of dysregulated intestinal barrier, microbiome and immune responses in colitis. Nat Mater, 19: 118–126, 2020.

91.

Lerner-Marmarosh

, Shen

, Torno

, Kravets

, Hu

, and Maines

. Human biliverdin reductase: a member of the insulin receptor substrate family with serine/threonine/tyrosine kinase activity. Proc Natl Acad Sci U S A, 102: 7109–7114, 2005.

92.

, Fujino

, Takahara

, and Li

. Protective role of heme oxygenase-1 in fatty liver ischemia-reperfusion injury. Med Mol Morphol, 52: 61–72, 2019.

93.

, Tan

, Wang

, Zhang

, Lao

, Wong

, and Feng

. The role of oxidative stress and antioxidants in liver diseases. Int J Mol Sci, 16: 26087–26124, 2015.

94.

Lim

, Taskinen

, and Boren

. Crosstalk between nonalcoholic fatty liver disease and cardiometabolic syndrome. Obes Rev, 20: 599–611, 2019.

95.

Lin

, Shen

, Hou

, Yang

, and Chen

. Heme oxygenase-1 inhibits breast cancer invasion via suppressing the expression of matrix metalloproteinase-9. Mol Cancer Ther, 7: 1195–1206, 2008.

96.

Lin

Y-C

, Chang

P-F

, Hu

F-C

, Chang

M-H

, and Ni

Y-H

. Variants in the UGT1A1 gene and the risk of pediatric nonalcoholic fatty liver disease. Pediatrics, 124: E1221–E1227, 2009.

97.

Liu

and Qian

. Cytoprotective role of heme oxygenase-1 in liver ischemia reperfusion injury. Int J Clin Exp Med, 8: 19867–19873, 2015.

98.

Liu

, Dong

, Zhang

, Cao

, Song

, Pan

, Bulmer

, Adams

, Dong

, and Wang

. Bilirubin increases insulin sensitivity by regulating cholesterol metabolism, adipokines and PPARgamma levels. Sci Rep, 5: 9886, 2015.

99.

Lo Iacono

, Boczkowski

, Zini

, Salouage

, Berdeaux

, Motterlini

, and Morin

. A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species. Free Rad Biol Med, 50: 1556–1564, 2011.

100.

Loboda

, Damulewicz

, Pyza

, Jozkowicz

, and Dulak

. Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism. Cell Mol Life Sci, 73: 3221–3247, 2016.

101.

Lundvig

, Immenschuh

, and Wagener

. Heme oxygenase, inflammation, and fibrosis: the good, the bad, and the ugly?. Front Pharmacol, 3: 81, 2012.

102.

Luo

, An

, Jia

, Yue

, Zheng

, Liu

, Chen

, An

, Winkler

, and Duan

. Genetically regulated bilirubin and risk of non-alcoholic fatty liver disease: a Mendelian randomization study. Front Genet, 9: 662, 2018.

103.

Maghzal

, Leck

, Collinson

, Li

, and Stocker

. Limited role for the bilirubin-biliverdin redox amplification cycle in the cellular antioxidant protection by biliverdin reductase. J Biol Chem, 284: 29251–29259, 2009.

104.

Maines

MD.

The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol, 37: 517–554, 1997.

105.

Markova

, Konickova

, Vankova

, Lenicek

, Kolar

, Strnad

, Hradilova

, Sachova

, Rasl

, Klimova

, Vomastek

, Nemeckova

, Nachtigal

, and Vitek

. Anti-angiogenic effects of the blue-green alga Arthrospira platensis on pancreatic cancer. J Cell Mol Med, 24: 2402–2415, 2020.

106.

Masarone

, Rosato

, Dallio

, Gravina

, Aglitti

, Loguercio

, Federico

, and Persico

. Role of oxidative stress in pathophysiology of nonalcoholic fatty liver disease. Oxid Med Cell Longev, 2018: 9547613, 2018.

107.

McDonagh

AF.

The biliverdin-bilirubin antioxidant cycle of cellular protection: missing a wheel?. Free Radic Biol Med, 49: 814–820, 2010.

108.

Miralem

, Lerner-Marmarosh

, Gibbs

, Jenkins

, Heimiller

, and Maines

. Interaction of human biliverdin reductase with Akt/protein kinase B and phosphatidylinositol-dependent kinase 1 regulates glycogen synthase kinase 3 activity: a novel mechanism of Akt activation. FASEB J, 30: 2926–2944, 2016.

109.

Molzer

, Wallner

, Kern

, Tosevska

, Schwarz

, Zadnikar

, Doberer

, Marculescu

, and Wagner

. Features of an altered AMPK metabolic pathway in Gilbert's Syndrome, and its role in metabolic health. Sci Rep, 6: 30051, 2016.

110.

Morse

and Choi

. Heme oxygenase-1: the “emerging molecule” has arrived. Am J Respir Cell Mol Biol, 27: 8–16, 2002.

111.

Muchova

, Vanova

, Zelenka

, Lenicek

, Petr

, Vejrazka

, Sticova

, Vreman

, Wong

, and Vitek

. Bile acids decrease intracellular bilirubin levels in the cholestatic liver: implications for bile acid-mediated oxidative stress. J Cell Mol Med, 15: 1156–1165, 2011.

112.

Muriel

Role of free radicals in liver diseases. Hepatol Int, 3: 526–536, 2009.

113.

Nakagami

, Taji

, Takahashi

, and Yamanishi

. Antiviral activity of a bile pigment, biliverdin, against human herpesvirus 6 (HHV-6) in vitro. Microbiol Immunol, 36: 381–390, 1992.

114.

Nakamura

, Zhang

, Kageyama

, Ke

, Fujii

, Sosa

, Reed

, Datta

, Zarrinpar

, Busuttil

, Araujo

, and Kupiec-Weglinski

. Macrophage heme oxygenase-1-SIRT1–p53 axis regulates sterile inflammation in liver ischemia-reperfusion injury. J Hepatol, 67: 1232–1242, 2017.

115.

Nano

, Muka

, Cepeda

, Voortman

, Dhana

, Brahimaj

, Dehghan

, and Franco

. Association of circulating total bilirubin with the metabolic syndrome and type 2 diabetes: a systematic review and meta-analysis of observational evidence. Diabetes Metab, 42: 389–397, 2016.

116.

Neto

, Nakao

, Toyokawa

, Nalesnik

, Romanosky

, Kimizuka

, Kaizu

, Hashimoto

, Azhipa

, Stolz

, Choi

, and Murase

. Low-dose carbon monoxide inhalation prevents development of chronic allograft nephropathy. Am J Physiol Renal Physiol, 290: F324–F334, 2006.

117.

Noir

, Boveris

, Garaza Pereira

, and Stoppani

. Bilirubin: a multi-site inhibitor of mitochondrial respiration. FEBS Lett, 27: 270–274, 1972.

118.

Novak

, Jackson

, Zhao

, and Yin

. Bilirubin in metabolic syndrome and associated inflammatory diseases: new perspectives. Life Sci, 257: 118032, 2020.

119.

O'Brien

, Hosick

, John

, Stec

, and Hinds

Jr . Biliverdin reductase isozymes in metabolism. Trends Endocrinol Metab, 26: 212–220, 2015.

120.

Ogaki

, Taguchi

, Maeda

, Watanabe

, Ishima

, Otagiri

, and Maruyama

. Kupffer cell inactivation by carbon monoxide bound to red blood cells preserves hepatic cytochrome P450 via anti-oxidant and anti-inflammatory effects exerted through the HMGB1/TLR-4 pathway during resuscitation from hemorrhagic shock. Biochem Pharmacol, 97: 310–319, 2015.

121.

Ollinger

, Wang

, Yamashita

, Wegiel

, Thomas

, Margreiter

, and Bach

. Therapeutic applications of bilirubin and biliverdin in transplantation. Antioxid Redox Signal, 9: 2175–2185, 2007.

122.

Origassa

and Camara

. Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury. World J Hepatol, 5: 541–549, 2013.

123.

Otterbein

, Bach

, Alam

, Soares

, Tao

, Wysk

, Davis

, Flavell

, and Choi

. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med, 6: 422–428, 2000.

124.

Otterbein

, Hedblom

, Harris

, Csizmadia

, Gallo

, and Wegiel

. Heme oxygenase-1 and carbon monoxide modulate DNA repair through ataxia-telangiectasia mutated (ATM) protein. Proc Natl Acad Sci U S A, 108: 14491–14496, 2011.

125.

Pae

and Chung

. Heme oxygenase-1: its therapeutic roles in inflammatory diseases. Immune Netw, 9: 12–19, 2009.

126.

Pae

, Oh

, Choi

, Chae

, Kim

, Chung

, and Chung

. Carbon monoxide produced by heme oxygenase-1 suppresses T cell proliferation via inhibition of IL-2 production. J Immunol, 172: 4744–4751, 2004.

127.

Paine

, Eiz-Vesper

, Blasczyk

, and Immenschuh

. Signaling to heme oxygenase-1 and its anti-inflammatory therapeutic potential. Biochem Pharmacol, 80: 1895–1903, 2010.

128.

Park

, Eom

, Ahn

, Jang

, Hwang

, and Lee

. Can heme oxygenase-1 be a prognostic factor in patients with hepatocellular carcinoma?. Medicine (Baltimore), 98: e16084, 2019.

129.

Parola

and Marra

. Adipokines and redox signaling: impact on fatty liver disease. Antioxid Redox Signal, 15: 461–483, 2011.

130.

Phelan

, Winter

, Rogers

, Lam

, and Denison

. Activation of the Ah receptor signal transduction pathway by bilirubin and biliverdin. Arch Biochem Biophys, 357: 155–163, 1998.

131.

Pietrocola

and Bravo-San Pedro

. Targeting autophagy to counteract obesity-associated oxidative stress. Antioxidants (Basel), 10: 102, 2021.

132.

Pizarro

, Rodriguez

, Mamprin

, Fuller

, Mann

, Motterlini

, and Guibert

. Protective effects of a carbon monoxide-releasing molecule (CORM-3) during hepatic cold preservation. Cryobiology, 58: 248–255, 2009.

133.

Pradere

, Troeger

, Dapito

, Mencin

, and Schwabe

. Toll-like receptor 4 and hepatic fibrogenesis. Semin Liver Dis, 30: 232–244, 2010.

134.

Puri

, Nobili

, Melville

, Corte

, Sartorelli

, Lopez

, Feldstein

, and Alkhouri

. Serum bilirubin level is inversely associated with nonalcoholic steatohepatitis in children. J Pediatr Gastroenterol Nutr, 57: 114–118, 2013.

135.

Qin

XF.

Bilirubin would be the indispensable component for some of the most important therapeutic effects of Calculus Bovis (Niuhuang). Chin Med J (Engl), 121: 480, 2008.

136.

Raffaele

, Bellner

, Singh

, Favero

, Rezzani

, Rodella

, Falck

, Abraham

, and Vanella

. Epoxyeicosatrienoic intervention improves NAFLD in leptin receptor deficient mice by an increase in PGC1alpha-HO-1-PGC1alpha-mitochondrial signaling. Exp Cell Res, 380: 180–187, 2019.

137.

Ram

, Singh

, Kumawat

, Kant

, Tandan

, and Kumar

. Bilirubin modulated cytokines, growth factors and angiogenesis to improve cutaneous wound healing process in diabetic rats. Int Immunopharmacol, 30: 137–149, 2016.

138.

Rodrigues

, Sola

, Brites

. Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology, 35: 1186–1195, 2002.

139.

Romero-Gomez

, Zelber-Sagi

, and Trenell

. Treatment of NAFLD with diet, physical activity and exercise. J Hepatol, 67: 829–846, 2017.

140.

Ryter

SW.

Heme oxygenase-1, a cardinal modulator of regulated cell death and inflammation. Cells, 10: 515, 2021.

141.

Ryter

, Alam

, and Choi

. Heme oxygenase-1/carbon monoxide: from basic science to therapeutic applications. Physiol Rev, 86: 583–650, 2006.

142.

Salley

, Mishra

, Tiwari

, Jadhav

, and Ndisang

. The heme oxygenase system rescues hepatic deterioration in the condition of obesity co-morbid with type-2 diabetes. PLoS One, 8: e79270, 2013.

143.

Salomone

, Li Volti

, Rosso

, Grosso

, and Bugianesi

. Unconjugated bilirubin, a potent endogenous antioxidant, is decreased in patients with non-alcoholic steatohepatitis and advanced fibrosis. J Gastroenterol Hepatol, 28: 1202–1208, 2013.

144.

Sanchez-Valle

, Chavez-Tapia

, Uribe

, and Mendez-Sanchez

. Role of oxidative stress and molecular changes in liver fibrosis: a review. Curr Med Chem, 19: 4850–4860, 2012.

145.

Sandouka

, Balogun

, Foresti

, Mann

, Johnson

, Tayem

, Green

, Fuller

, and Motterlini

. Carbon monoxide-releasing molecules (CO-RMs) modulate respiration in isolated mitochondria. Cell Mol Biol, 51: 425–432, 2005.

146.

Santangelo

, Mancuso

, Marchetti

, Di

, Pani

, and Fadda

. Bilirubin: an endogenous molecule with antiviral activity in vitro. Front Pharmacol, 3: 36, 2012.

147.

Sarady

, Otterbein

, Liu

, Otterbein

, and Choi

. Carbon monoxide modulates endotoxin-induced production of granulocyte macrophage colony-stimulating factor in macrophages. Am J Respir Cell Mol Biol, 27: 739–745, 2002.

148.

Sass

, Barikbin

, and Tiegs

. The multiple functions of heme oxygenase-1 in the liver. Z Gastroenterol, 50: 34–40, 2012.

149.

Sasson

, Kristoferson

, Batista

, McClung

, Abraham

, and Peterson

. The pivotal role of heme oxygenase-1 in reversing the pathophysiology and systemic complications of NAFLD. Arch Biochem Biophys, 697: 108679, 2021.

150.

Schwer

CI.

Carbon monoxide and the pancreas. Curr Pharm Biotechnol, 13: 813–818, 2012.

151.

Schwer

, Guerrero

, Humar

, Roesslein

, Goebel

, Stoll

, Geiger

, Pannen

, Hoetzel

, and Schmidt

. Heme oxygenase-1 inhibits the proliferation of pancreatic stellate cells by repression of the extracellular signal-regulated kinase1/2 pathway. J Pharmacol Exp Ther, 327: 863–871, 2008.

152.

Schwer

, Mutschler

, Stoll

, Goebel

, Humar

, Hoetzel

, and Schmidt

. Carbon monoxide releasing molecule-2 inhibits pancreatic stellate cell proliferation by activating p38 mitogen-activated protein kinase/heme oxygenase-1 signaling. Mol Pharmacol, 77: 660–669, 2010.

153.

Sedlak

, Saleh

, Higginson

, Paul

, Juluri

, and Snyder

. Bilirubin and glutathione have complementary antioxidant and cytoprotective roles. Proc Natl Acad Sci U S A, 106: 5171–5176, 2009.

154.

Seki

, De Minicis

, Osterreicher

, Kluwe

, Osawa

, Brenner

, and Schwabe

. TLR4 enhances TGF-beta signaling and hepatic fibrosis. Nat Med, 13: 1324–1332, 2007.

155.

Seyed Khoei

, Grindel

, Wallner

, Molzer

, Doberer

, Marculescu

, Bulmer

, and Wagner

. Mild hyperbilirubinaemia as an endogenous mitigator of overweight and obesity: implications for improved metabolic health. Atherosclerosis, 269: 306–311, 2018.

156.

Sharma

, Tramutola

, Lanzillotta

, Arena

, Blarzino

, Cassano

, Butterfield

, Di Domenico

, Perluigi

, and Barone

. Loss of biliverdin reductase-A favors Tau hyper-phosphorylation in Alzheimer's disease. Neurobiol Dis, 125: 176–189, 2019.

157.

Stallone

, Infante

, Prisciandaro

, and Grandaliano

. mTOR and aging: an old fashioned dress. Int J Mol Sci, 20: 2774, 2019.

158.

Stec

, Gordon

, Nestor-Kalinoski

, Donald

, Mitchell

, Creeden

, and Hinds

Jr . Biliverdin reductase A (BVRA) knockout in adipocytes induces hypertrophy and reduces mitochondria in white fat of obese mice. Biomolecules, 10: 387, 2020.

159.

Stec

and Hinds

Jr . Natural product heme oxygenase inducers as treatment for nonalcoholic fatty liver disease. Int J Mol Sci, 21: 9493, 2020.

160.

Stec

, John

, Trabbic

, Luniwal

, Hankins

, Baum

, and Hinds

Jr . Bilirubin binding to PPARalpha inhibits lipid accumulation. PLoS One, 11: e0153427, 2016.

161.

Steiger

, Wollborn

, Gutmann

, Zehe

, Wunder

, and Meinel

. Controlled therapeutic gas delivery systems for quality-improved transplants. Eur J Pharm Biopharm, 97: 96–106, 2015.

162.

Stocker

Antioxidant activities of bile pigments. Antioxid Redox Signal, 6: 841–849, 2004.

163.

Stocker

and Ames

. Potential role of conjugated bilirubin and copper in the metabolism of lipid peroxides in bile. Proc Natl Acad Sci U S A, 84: 8130–8134, 1987.

164.

Stocker

and Peterhans

. Antioxidant properties of conjugated bilirubin and biliverdin: biologically relevant scavenging of hypochlorous acid. Free Radic Res Commun, 6: 57–66, 1989.

165.

Stocker

and Peterhans

. Synergistic interaction between vitamin-E and the bile-pigments bilirubin and biliverdin. Biochim Biophys Acta, 1002: 238–244, 1989.

166.

Subhanova

, Muchova

, Lenicek

, Vreman

, Luksan

, Kubickova

, Kreidlova

, Zima

, Vitek

, and Urbanek

. Expression of biliverdin reductase A in peripheral blood leukocytes is associated with treatment response in HCV-infected patients. PLoS One, 8: e57555, 2013.

167.

Suematsu

, Goda

, Sano

, Kashiwagi

, Egawa

, Shinoda

, and Ishimura

. Carbon monoxide: an endogenous modulator of sinusoidal tone in the perfused rat liver [see comments]. J Clin Invest, 96: 2431–2437, 1995.

168.

Suk

, Jasprova

, Biedermann

, Petraskova

, Valentova

, Kren

, Muchova

, and Vitek

. Isolated silymarin flavonoids increase systemic and hepatic bilirubin concentrations and lower lipoperoxidation in mice. Oxid Med Cell Longev, 2019: 6026902, 2019.

169.

Suliman

, Carraway

, Tatro

, and Piantadosi

. A new activating role for CO in cardiac mitochondrial biogenesis. J Cell Sci, 120: 299–308, 2007.

170.

Sun

, Guo

, Yang

, Liu

, Hu

, Jiang

, Dirsch

, Dahmen

, Dong

, and Liu

. Carbon monoxide ameliorates hepatic ischemia/reperfusion injury via sirtuin 1-mediated deacetylation of high-mobility group box 1 in rats. Liver Transpl, 23: 510–526, 2017.

171.

Swift

, Johannsen

, Earnest

, Blair

, and Church

. Effect of different doses of aerobic exercise training on total bilirubin levels. Med Sci Sports Exerc, 44: 569–574, 2012.

172.

Talbot

, Wang

, Kazi

, Han

, Bakshi

, Stucky

, Fuino

, Kawaguchi

, Samoyedny

, Wilson

, Arvanitakis

, Schneider

, Wolf

, Bennett

, Trojanowski

, and Arnold

. Demonstrated brain insulin resistance in Alzheimer's disease patients is associated with IGF-1 resistance, IRS-1 dysregulation, and cognitive decline. J Clin Invest, 122: 1316–1338, 2012.

173.

Tang

, Zhang

, Zhu

, Chen

, and Yu

. Low concentrations of bilirubin inhibit activation of hepatic stellate cells in vitro. Mol Med Rep, 15: 1647–1653, 2017.

174.

Tighe

, Akhtar

, Iqbal

, and Ahmed

. Chronic liver disease and silymarin: a biochemical and clinical review. J Clin Transl Hepatol, 8: 454–458, 2020.

175.

Tolosano

, Fagoonee

, Hirsch

, Berger

, Baumann

, Silengo

, and Altruda

. Enhanced splenomegaly and severe liver inflammation in haptoglobin/hemopexin double-null mice after acute hemolysis. Blood, 100: 4201–4208, 2002.

176.

Tsuchihashi

, Fondevila

, and Kupiec-Weglinski

. Heme oxygenase system in ischemia and reperfusion injury. Ann Transplant, 9: 84–87, 2004.

177.

Upadhyay

, Jadeja

, Vyas

, Pandya

, Joshi

, Vohra

, Thounaojam

, Martin

, Bartoli

, and Devkar

. Carbon monoxide releasing molecule-A1 improves nonalcoholic steatohepatitis via Nrf2 activation mediated improvement in oxidative stress and mitochondrial function. Redox Biol, 28: 101314, 2020.

178.

Valaskova

, Dvorak

, Lenicek

, Zizalova

, Kutinova-Canova

, Zelenka

, Cahova

, Vitek

, and Muchova

. Hyperbilirubinemia in Gunn rats is associated with decreased inflammatory response in LPS-mediated systemic inflammation. Int J Mol Sci, 20: 2306, 2019.

179.

van der Windt

, Sud

, Zhang

, Tsung

, and Huang

. The effects of physical exercise on fatty liver disease. Gene Expr, 18: 89–101, 2018.

180.

Vankova

, Markova

, Jasprova

, Dvorak

, Subhanova

, Zelenka

, Novosadova

, Rasl

, Vomastek

, Sobotka

, Muchova

, and Vitek

. Chlorophyll-mediated changes in the redox status of pancreatic cancer cells are associated with its anticancer effects. Oxid Med Cell Longev, 2018: 4069167, 2018.

181.

Viollet

, Guigas

, Leclerc

, Hebrard

, Lantier

, Mounier

, Andreelli

, and Foretz

. AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therapeutic perspectives. Acta Physiol (Oxf), 196: 81–98, 2009.

182.

Vitek

Bilirubin and atherosclerotic diseases. Physiol Res, 66: S11–S20, 2017.

183.

Vitek

Bilirubin as a signaling molecule. Med Res Rev, 40: 1335–1351, 2020.

184.

Vitek

, Bellarosa

, and Tiribelli

. Induction of mild hyperbilirubinemia: hype or real therapeutic opportunity?. Clin Pharmacol Ther, 106: 568–575, 2019.

185.

Vitek

, Gbelcova

, Muchova

, Vanova

, Zelenka

, Konickova

, Suk

, Zadinova

, Knejzlik

, Ahmad

, Fujisawa

, Ahmed

, and Ruml

. Antiproliferative effects of carbon monoxide on pancreatic cancer. Dig Liver Dis, 46: 369–375, 2014.

186.

Vitek

, Hubacek

, Pajak

, Dorynska

, Kozela

, Eremiasova

, Danzig

, Stefler

, and Bobak

. Association between plasma bilirubin and mortality. Ann Hepatol, 18: 379–385, 2019.

187.

Vitek

and Ostrow

. Bilirubin chemistry and metabolism; harmful and protective aspects. Curr Pharm Des, 15: 2869–2883, 2009.

188.

Vitek

and Schwertner

. The heme catabolic pathway and its protective effects on oxidative stress-mediated diseases. Adv Clin Chem, 43: 1–57, 2007.

189.

Vitek

and Tiribelli

. Bilirubin, intestinal integrity, the microbiome, and inflammation. New Eng J Med, 383: 684–686, 2020.

190.

Vogel

and Zucker

. Bilirubin acts as an endogenous regulator of inflammation by disrupting adhesion molecule-mediated leukocyte migration. Inflamm Cell Signal, 3: e1178, 2016.

191.

Wagner

, Wallner

, Molzer

, Gazzin

, Bulmer

, Tiribelli

, and Vitek

. Looking to the horizon: the role of bilirubin in the development and prevention of age-related chronic diseases. Clin Sci (Lond), 129: 1–25, 2015.

192.

Wang

, Nan

, Wu

, Kong

, Han

, Zhao

, Kong

, and Yu

. Induction of heme oxygenase-1 protects against nutritional fibrosing steatohepatitis in mice. Lipids Health Dis, 10: 31, 2011.

193.

Wang

, Wang

, Zhang

, Li

, De

, Guo

, Qian

, and Fan

. Heme oxygenase-1 alleviates ischemia/reperfusion injury in aged liver. World J Gastroenterol, 11: 690–694, 2005.

194.

Wang

, Li

, Ye

, Xie

, and Li

. Oxidative stress and liver cancer: etiology and therapeutic targets. Oxid Med Cell Longev, 2016: 7891574, 2016.

195.

Was

, Dulak

, and Jozkowicz

. Heme oxygenase-1 in tumor biology and therapy. Curr Drug Targets, 11: 1551–1570, 2010.

196.

Watchko

and Tiribelli

. Bilirubin-induced neurologic damage-mechanisms and management approaches. N Engl J Med, 369: 2021–2030, 2013.

197.

Weaver

, Hamoud

, Stec

, and Hinds

Jr . Biliverdin reductase and bilirubin in hepatic disease. Am J Physiol Gastrointest Liver Physiol, 314: G668–G676, 2018.

198.

Wegiel

, Baty

, Gallo

, Csizmadia

, Scott

, Akhavan

, Chin

, Kaczmarek

, Alam

, Bach

, Zuckerbraun

, and Otterbein

. Cell surface biliverdin reductase mediates biliverdin-induced anti-inflammatory effects via phosphatidylinositol 3-kinase and Akt. J Biol Chem, 284: 21369–21378, 2009.

199.

Wei

, Chen

, de Bruyn

, Zhang

, Bremer

, and Helfrich

. Carbon monoxide-releasing molecule-2 (CORM-2) attenuates acute hepatic ischemia reperfusion injury in rats. BMC Gastroenterol, 10: 42, 2010.

200.

Wilks

Heme oxygenase: evolution, structure, and mechanism. Antioxid Redox Signal, 4: 603–614, 2002.

201.

Witek

, Scislowska

, Turowski

, Lerczak

, Lewandowska-Pachecka

, and Pokrywka

. Total bilirubin in athletes, determination of reference range. Biol Sport, 34: 45–48, 2017.

202.

Woronyczova

, Novakova

, Lenicek

, Bolek

, Cifkova

, and Vitek

. Elite athletes have mildly elevated serum bilirubin concentrations. Med Sci Sports Exerc, 52: 35, 2020.

203.

, Wu

, Li

, Mickle

, and Carey

. Albumin-bound bilirubins protect human ventricular myocytes against oxyradical damage. Biochem Cell Biol, 69: 683–688, 1991.

204.

Yamamoto

, Ikeya

, Okuyama

, Okamoto

, Fukuda

, and Kobayashi

. Negative correlation of high serum bilirubin with cancer development in adults without hepatobiliary disease. Eur J Cancer Prev, 30: 69–75, 2021.

205.

Yan

, Yang

, Li

, Zhang

, Pei

, Zhu

, Wang

, and Liu

. The therapeutic effect of CORM-3 on acute liver failure induced by lipopolysaccharide/D-galactosamine in mice. Hepatobiliary Pancreat Dis Int, 15: 73–80, 2016.

206.

Yan

, Tung

, Li

, Niu

, Garbacz

, Lu

, Bi

, Li

, He

, Xu

, Ren

, Monga

, Schwabe

, Yang

, and Xie

. Aryl hydrocarbon receptor signaling prevents activation of hepatic stellate cells and liver fibrogenesis in mice. Gastroenterology, 157: 793–806.e14, 2019.

207.

Yang

, Chen

, Zhao

, Zhang

, Chen

, Zhang

, and Zhao

. Heme oxygenase-1 exerts pro-apoptotic effects on hepatic stellate cells in vitro through regulation of nuclear factor-kappaB. Exp Ther Med, 16: 291–299, 2018.

208.

Yao

, Chen

, Ganapathy

, and Kou

. Therapeutic application and construction of bilirubin incorporated nanoparticles. J Control Release, 328: 407–424, 2020.

209.

Yao

, Jiang

, Kou

, Samuriwo

, Xu

, and Zhao

. Pharmacological actions and therapeutic potentials of bilirubin in islet transplantation for the treatment of diabetes. Pharmacol Res, 145: 104256, 2019.

210.

, Xu

, Peng

, Liu

, Zhang

, Li

, Sun

, and Wang

. Calculus bovis: a review of the traditional usages, origin, chemistry, pharmacological activities and toxicology. J Ethnopharmacol, 254: 112649, 2020.

211.

Zelenka

, Dvorak

, Alan

, Zadinova

, Haluzik

, and Vitek

. Hyperbilirubinemia protects against aging-associated inflammation and metabolic deterioration. Oxid Med Cell Longev, 2016: 6190609, 2016.

212.

Zelenka

, Muchova

, Zelenkova

, Vanova

, Vreman

, Wong

, and Vitek

. Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress. Biochimie, 94: 1821–1827, 2012.

213.

Zhang

, Amorosa

, Petrova

, Coyle

, Macor

, Nair

, Lee

, and Haimovich

. TLR4 counteracts BVRA signaling in human leukocytes via differential regulation of AMPK, mTORC1 and mTORC2. Sci Rep, 9: 7020, 2019.

214.

Zheng

, He

, Yu

, Liu

, Ge

, Li

, Wang

, Guo

, Qu

, Qin

, Jiang

, and Wang

. Unconjugated bilirubin alleviates experimental ulcerative colitis by regulating intestinal barrier function and immune inflammation. World J Gastroenterol, 25: 1865–1878, 2019.

215.

Zhu

, Wilson

, Luxon

, Brown

, Mathahs

, Bandyopadhyay

, McCaffrey

, and Schmidt

. Biliverdin inhibits hepatitis C virus nonstructural 3/4A protease activity: mechanism for the antiviral effects of heme oxygenase?. Hepatology, 52: 1897–1905, 2010.

216.

Zhu

, Wilson

, Mathahs

, Wen

, Brown

, Luxon

, and Schmidt

. Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury. Hepatology, 48: 1430–1439, 2008.

217.

Zou

, Zhang

, Li

, Xiao

, Yu

, Ke

, Zhou

, Liu

, Wang

, Huang

, Ma

, Liu

, Wang

, Zhao

, Zhou

, and Gao

. Heme oxygenase-1: a molecular brake on hepatocellular carcinoma cell migration. Carcinogenesis, 32: 1840–1848, 2011.

218.

Zou

, Zou

, Cheng

, Li

, Han

, Wang

, Jin

, Zou

, Liu

, Zhou

, Zhao

, and Du

. Heme oxygenase-1 retards hepatocellular carcinoma progression through the microRNA pathway. Oncol Rep, 36: 2715–2722, 2016.

219.

Zucker

, Goessling

, Bootle

, and Sterritt

. Localization of bilirubin in phospholipid bilayers by parallax analysis of fluorescence quenching. J Lipid Res, 42: 1377–1388, 2001.

220.

Zucker

, Horn

, and Sherman

. Serum bilirubin levels in the US population: gender effect and inverse correlation with colorectal cancer. Hepatology, 40: 827–835, 2004.

221.

Zucker

, Vogel

, Kindel

, Smith

, Idelman

, Avissar

, Kakarlapudi

, and Masnovi

. Bilirubin prevents acute DSS-induced colitis by inhibiting leukocyte infiltration and suppressing upregulation of inducible nitric oxide synthase. Am J Physiol Gastrointest Liver Physiol, 309: G841–G854, 2015.