The Nrf2 Pathway in Liver Diseases

Abstract

Oxidative stress is the leading cause of most liver diseases, such as drug-induced liver injury, viral hepatitis, and alcoholic hepatitis caused by drugs, viruses, and ethanol. The Kelch-like ECH-associated protein 1-NFE2-related factor 2 (Keap1-Nrf2) system is a critical defense mechanism of cells and organisms in response to oxidative stress. Accelerating studies have clarified that the Keap1-Nrf2 axis are involved in the prevention and attenuation of liver injury. Nrf2 up-regulation could alleviate drug-induced liver injury in mice. Moreover, many natural Nrf2 activators can regulate lipid metabolism and oxidative stress of liver cells to alleviate fatty liver disease in mice. In virus hepatitis, the increased Nrf2 can inhibit hepatitis C viral replication by up-regulating hemeoxygenase-1. In autoimmune liver diseases, the increased Nrf2 is essential for mice to resist liver injury. In liver cirrhosis, the enhanced Nrf2 reduces the activation of hepatic stellate cells by reducing reactive oxygen species levels to prevent liver fibrosis. Nrf2 plays a dual function in liver cancer progression. At present, a Nrf2 agonist has received clinical approval. Therefore, activating the Nrf2 pathway to induce the expression of cytoprotective genes is a potential option for treating liver diseases. In this review, we comprehensively summarized the relationships between oxidative stress and liver injury, and the critical role of the Nrf2 pathway in multiple liver diseases.

Keywords: kelch-like ECH-associated protein 1; liver diseases; nuclear factor-erythroid 2-related factor 2; oxidative stress; reactive oxygen species.

FIGURE 1

Nrf2 associated pathways. Proteases degrade nrf2 ubiquitinated by the Keap1-CUL3 complex under normal conditions in the cytoplasm. However, under oxidative stress conditions, Nrf2 dissociates from Keap1, accumulates in the cytoplasm, and is transported into the nucleus to bind to target genes. The selective autophagy substrate p62 could compete with Nrf2 for Keap1 binding at the bottom of the DC domain, dislocating Nrf2 from Keap1 and leading to the accumulation of Nrf2, initiating the transcription of antioxidant protective genes and phase II detoxification enzyme genes. PKC phosphorylates Ser40 in Neh2, dissociating the Keap1 homodimer, and transporting Nrf2 into the nucleus to recognize and bind the ARE. Neh6 in Nrf2 can be phosphorylated by GSK-3β, leading to degradation through being recognized by β-TrCP. PI3K-AKT signaling could inhibit GSK-3β through phosphorylation. ARE, antioxidant responsive element; β-TrCP, β-transducin repeats-containing protein; GSK-3, phosphorylated by glycogen synthase kinase 3; Keap1, Kelch-like ECH-associated protein 1; Nrf2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; sMAF, small musculoaponeurotic fibrosarcoma oncogene homologue.

FIGURE 2

Structure and interaction of Keap1 and Nrf2 (A) Keap1 contains BTB domain, IVR domain, DGR domain and CTR domain. Its dimerization is mediated by BTB domain. IVR domain and CUL3 interact to form Keap1-CUL3 complex, which ubiquitinates Nrf2 under normal condition. The DGR and CTR domain are collectively called DC domain, which directly interact with the DLG and ETGE of Neh2 of Nrf2. (B) The DLG and ETGE domain on Neh2 could bind with DC domain of Keap1 homodimer. sMAF can bind with Neh1 of Nrf2 to form heterodimers that bind to DNA. Neh6 could be phosphorylated by GSK-3, leading to the degradation of Nrf2. There are several types of Nrf2 activators, such as Keap1 cysteine-targeting drugs and drugs that disrupt the Nrf2-Keap1 protein-protein interactions. BTB, bric-a-brac domain; CBP, cAMP responsive element binding protein; CTR, carboxyl terminal region; CUL3, CULLIN3; DGR, double glycine repeat; GSK-3, glycogen synthase kinase 3; IVR, intermediate region; Neh1, Nrf2-ECH homology domain-1; sMAF, small musculoaponeurotic fibrosarcoma oncogene homologue.

FIGURE 3

Drug-induced liver injury and Nrf2 drugs. Drugs metabolized by CYP are more likely to cause DILI. Reactive metabolites give rise to mitochondrial dysfunction, endoplasmic reticulum (ER) stress, or DNA damage. ROS and electrophiles activate Nrf2 to compound antioxidant proteins, alleviating DILI. For example, APAP can be metabolized by CYP to produce NAPQI, which detoxified by GSH at first. As APAP dosage increases, GSH exhausts and NAPQI accumulates, resulting in cytotoxicity. Reactive metabolites like ROS could stimulate Nrf2, initiating the transcription of target genes. CDDO and limonin could activate Nrf2 to compound NQO1, HO-1 and GCLC, which alleviate AILI. Omega-3 and AITC could also stimulate Nrf2 to compound HO-1 for alleviating AILI. But JNK phosphorylates Nrf2 to downregulate the transcriptional expression of cytoprotective genes in AILI. SFN, catalpol, Vitamin C and arctiin alleviate the liver damage induced by triptolide by activating Nrf2 to compound corresponding antioxidant proteins. AITC, allyl isothiocyanate; APAP, acetaminophen; CYP, cytochrome P450 proteins; DILI, drug-induced liver injury; GCLC, catalytic subunit of glutamate-cysteine ligase; GSH, reduced glutathione; HO-1, hemeoxygenase-1; JNK, c-Jun NH2 -terminal kinase; NAPQI, N-acetyl-1,4-benzoquinone imine; NQO1, quinone oxidoreductase 1; Nrf2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; SOD, superoxide dismutase; UGT1A6, UDP-glucuronosyltransferase 1A6.

FIGURE 4

Roles of Nrf2 in FLD. Mitochondria in NAFLD is impaired, leading to overproduction of ROS which triggers lipid peroxidation. The generated ROS and lipid peroxidation products will further damage the function of the respiratory chain, resulting in a vicious circle. ROS also activates Nrf2 to compound antioxidant proteins, alleviating NAFLD. Expression of Nrf2 is much higher in the livers of mice which fed with high-fat diet than those fed with normal chow. However, the accumulation of Nrf2 is inhibited when the intake of Met and Tyr is restricted in high-fat feeding mice. CDDO-Im, DMF, curcumin and ginkgolide B can alleviate NAFLD through activating Nrf2. Aucubin stimulates Nrf2 to compound HO-1 and SOD, alleviating NAFLD. Alcohol is metabolized to acetaldehyde in liver cells by alcohol dehydrogenase and CYP2E1. Acetaldehyde can destroy mitochondria, resulting in the production of ROS. ROS activates Nrf2 to alleviate ALD. SFN stimulates Nrf2 to compound HMOX1, NQO1 and GSTM3 for alleviating NAFLD. The up-regulation of Nrf2 was observed in knockout SNX10 mice. And SOD was compounded to alleviate ALD. ALD, alcoholic liver disease; CYP2E1, cytochrome P450 2E1; DMF, dimethyl fumarate; GSH, reduced glutathione; GSTM3, glutathione S-transferase 3; HMOX1, heme oxygenase 1; HO-1, hemeoxygenase-1; Keap1, Kelch-like ECH-associated protein 1; Met, methionine; NAFLD, non-alcoholic fatty liver disease; NQO1, quinone oxidoreductase 1; Nrf2, nuclear factor-erythroid 2-related factor 2; ROS, reactive oxygen species; SFN, sulforaphane; SNX10, sorting nexin 10; SOD, superoxide dismutase; SQSTM1, sequestosome 1; Tyr, tyrosine.

FIGURE 5

Roles of Nrf2 in viral hepatitis, autoimmune liver disease and liver fibrosis. HCV core protein and NS5A protein induce mitochondrial dysfunction, CYP2E1 and NOX expression in hepatocytes producing large amounts of ROS. HCV core protein and NS5A protein also induce Nrf2 phosphorylation, resulting in up-regulation of HO-1 and NQO1 which alleviate HCV. However, HCV could cause sMAF to delocalize and connect with extranuclear NS3, and then bind to Nrf2 in the cytoplasm, preventing Nrf2 from entering the nucleus. SFN, celastrol and caffeic acid can up-regulate the expression of HO-1 through the Nrf2-associated pathway to inhibit HCV viral replication. The HBx protein of HBV can induce intense stimulation of Nrf2. At the same time, HBV enhances the reciprocity between p62 and Keap1, forming a HBx-p62-Keap1 complex in the cytoplasm, prompting the dissociation of Keap1-Nrf2, which contributes to activation of Nrf2. Oxidative damage products, dihydroquercetin (DHQ) and pristimerin increase the expression of Nrf2 in the cytoplasm, significantly enhancing the transcriptional expression of the HO-1 alleviating AIH. Ursodeoxycholic acid (UDCA) enhances the activation of Nrf2 in liver cells of PBC patients, increasing TRX and TrxR1 protein which alleviate PBC. Huangqi Decoction (HQD) up-regulates the expression of Nrf2 for alleviating PSC. ROS is one of the activating factors of hepatic stellate cells (HSC). It also promotes the activation of Nrf2 which suppresses HSC. Andrographolide and dimethyl fumarate (DMF) can significantly ameliorate the stimulation of HSC by enhancing Nrf2 and increasing the expression of antioxidant proteins. The damage of sinusoidal endothelial cells (LSEC) during acute liver injury can aggravate the oxidative stress response and activate HSC to promote liver fibrosis. The increase of p62 level caused by impaired autophagy will trigger the stimulation of Nrf2 and the up-regulation of its target gene, alleviating liver fibrosis. AIH, autoimmune hepatitis; APAP, acetaminophen; CCl₄, carbon tetrachloride; CYP2E1, cytochrome P450 2E1; DHQ, dihydroquercetin; DMF, dimethyl fumarate; HO-1, hemeoxygenase-1; HQD, Huangqi Decoction; HSC, hepatic stellate cell; Keap1, Kelch-like ECH-associated protein 1; LSEC, liver sinusoidal endothelial cell; Nrf2, nuclear factor-erythroid 2-related factor 2; NOX, NADPH oxidase; PBC, primary biliary cholangitis; PSC, primary sclerosing cholangitis; ROS, reactive oxygen species; SFN, sulforaphane; sMAF, small musculoaponeurotic fibrosarcoma oncogene homologue; TRX, thioredoxin; TrxR1, thioredoxin reductase 1; UDCA, ursodeoxycholic acid.

FIGURE 6

The relationship between Nrf2 and tumor. Reasonable Nrf2 levels may inhibit tumor, but abnormal stimulation of Nrf2 can promote tumor progression. Therefore, dose of Nrf2-drugs should be carefully determined and pharmacological activation of Nr2 for therapy should not exceed the therapeutic window. Nrf2, nuclear factor-erythroid 2-related factor 2.