Reactive oxygen species (ROS) are inevitable byproducts of aerobic metabolism in living organisms, including superoxide (O₂⁻), hydrogen peroxide (H₂O₂), hydroxyl radicals (HO·), and so on. Among them, hydroxyl radicals are the most reactive, capable of attacking almost all biological molecules, inducing lipid peroxidation, and causing DNA double-strand breaks, making them the most destructive type of ROS [7,8], Superoxide (O₂⁻) serves as the precursor of other ROS. When O₂ leaks from the mitochondrial respiratory chain, an O₂ molecule accepting an electron generates O₂⁻, which can be dismutated by superoxide dismutase (SOD) into H₂O₂ and O₂. H₂O₂ is not a free radical molecule but can be catalyzed to generate highly active HO· through the Fenton reaction when accepting an electron. The majority of ROS in cells originate from this process [9,10]. The excess ROS causes damage to mitochondrial DNA (mtDNA), respiratory chain complexes, lipids, and other components [11], which, in turn, further induces ROS production, creating a vicious cycle [12,13].

The other source sites of ROS include the endoplasmic reticulum (ER), a crucial site for protein synthesis, post-translational modification, processing, and folding in cells. In the ER lumen, the correct folding of most proteins requires the formation of disulfide bonds between cysteine residues to stabilize their structures. The formation of disulfide bonds is a reversible process that can be achieved through thiol–disulfide interchange. In eukaryotic cells, the folding of oxidative proteins is mainly catalyzed by a series of redox enzymes, such as protein disulfide isomerase (PDI), a member of the thioredoxin superfamily proteins [14]. Additionally, the folding of ER proteins is also dynamically regulated by the balance of the redox buffer, such as GSH/GSSG, which maintains the cellular redox state in a reduced state, GSH accounts for approximately 99% of the overall content, whereas it is found in only 50–60% in the endoplasmic reticulum [15]. PDI transfers two electrons to its substrate ERO1 (ER oxidoreductase 1), producing hydrogen peroxide. This electron transfer mode indicates the induction of ROS production [16].

Moreover, peroxisome, the organelles present in all eukaryotic cells, is believed to be the site to produce ROS. Peroxisome is closely related to the regulation of liver lipid homeostasis [17]. Medium- and long-chain fatty acids (FAs) are oxidized predominantly in mitochondria, whereas very long-chain FAs (VLCFAs) are metabolized almost exclusively by β-oxidation in the peroxisome and are involved in α-oxidation of fatty acids [18]. Peroxisomes produce acetyl coenzyme A through fatty acid β-oxidation, which plays a role in lipid signal transduction to promote lipid autophagy [19]. It is estimated that peroxisomes contribute about 35% of ROS in cells.

Moreover, NADPH oxidase, also known as NOX, is the only enzyme whose main function is to produce ROS. Researchers have identified seven isoforms, NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, and DUOX2, of which NOX2 is highly expressed in the liver [20]. NOX2 consists of 6 subunits: gp91phox, p22phox, p47phox, p67phox, p40phox, and Rac. Activation of NOX2 requires the translocation of p47phox, p67phox, p40phox, and Rac from the cytoplasm to the cell membrane, where they bind to gp91phox and p22phox to form the complex to produce the oxidants [21]. NOX2 affects signal transduction and immune function by transferring electrons from NADPH to molecular oxygen and generating large amounts of oxidants, a process involved in a variety of physiological activities such as cell proliferation and differentiation [22]. Studies have reported that in liver-resident Kupffer cells and infiltrating macrophages, NOX2-derived ROS stimulate them to produce pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β in response to a variety of factors, including oxidized low-density lipoproteins (LDL) and lipopolysaccharides (LPS) [23,24,25]. Therefore, over-activation of NOX2 leads to a surge of oxidants, which directly activates downstream inflammatory pathways and indirectly damages DNA and regulates protein phosphorylation, causing oxidative damage and exacerbating inflammation, which leads to cellular dysfunction and the onset and deterioration of diseases and affects the function and metabolism of tissues and organs [26,27]. Moreover, increased NOX2 activity has been shown in patients with NAFLD [28,29], suggesting a correlation between NOX2 and NAFLD. As mentioned above, oxidative stress serves as the “second hit” in the pathogenesis of NAFLD and is typically accompanied by a large amount of ROS. ROS can lead to the inactivation of mitochondrial respiratory chain enzymes and GAPDH, inhibit Ca²⁺ channels on the membrane, and induce liver cell damage [30]. Furthermore, ROS can induce the production of cytokines and Fas, along with their ligands, exacerbating lipid peroxidation, thus aggravating liver inflammation and hepatocyte fibrosis [31]. Cellular FFA loading can lead to changes in mitochondrial adaptability, enhancing fatty acid oxidation and upregulating electron transport, further resulting in excessive ROS production [32]. Thus, the excessive production of ROS can damage mitochondrial proteins, lipids, and DNA, a process that may play a role in the initiation of NASH. In the late stage of NASH, mitochondrial ultrastructure damage affects its function, reduces ATP synthesis, and intensifies ROS overproduction [33].

When the liver is exposed to high levels of ROS or electrophilic reagents, oxidative damage is prone to occur. At this point, the body initiates a series of antioxidant defense mechanisms [34]. The mammalian thiol-dependent antioxidant system comprises the thioredoxin (Trx) system and the GSH-glutaredoxin (Grx) system, both of which undergo interconversion between thiol (-SH) and disulfide bond (-S-S-) forms to eliminate ROS and maintain the redox homeostasis in the body [35].

The Trx system mainly consists of Trx, thioredoxin reductase (TrxR), and nicotinamide adenine dinucleotide phosphate (NADPH). It is a major thiol-dependent antioxidant system in the body, responsible for maintaining intracellular redox homeostasis and protecting cells from oxidative stress (Figure 1) [9,36]. Mammals have two isoforms of Trxs. Trx1 is present in the cytoplasm with five cysteines, two active site cysteines, and three structural cysteines, while Trx2 is located in the mitochondria with only two cysteine residues in its active center [36]. The Trx system transfers electrons to various key cellular proteins, including peroxiredoxin (Prx), which directly scavenges H₂O₂, and methionine-S-sulfoxide reductase (MsrA), which participates in oxidative stress

defense [35,37,38]. The proper functioning of the Trx system relies on TrxR accepting electrons from NADPH and transferring them to Trx. The activity and expression levels of TrxR directly determine the normal biological function of Trx and its downstream proteins. Mammalian cells have two forms of TrxR, cytoplasmic TrxR1 and mitochondrial TrxR2, both of which have FAD and NADPH binding domains as well as interface domains. At their N-terminus, they have an active site CVNVGC, and at their C-terminus, they possess a special sequence Gly-Cys-Sec-Gly (GCUG), with Sec being the catalytic active site of TrxR, which is necessary for its reduction activity [39].

Studies have shown significant changes in the Trx system in metabolic syndrome [50,51]. For example, in metabolic syndrome patients, consuming a diet rich in high-saturated fatty acids (HSFA) significantly increased the mRNA levels of Trx in adipose tissue. Simultaneously, postprandial adipose tissue TrxR1 mRNA significantly decreased.This suggests that the ability of Trx to revert from its oxidized form to its reduced form decreased, leading to a compensatory increase in Trx gene levels, indicating increased oxidative stress due to saturated fat intake [52]. The Trx system also plays a crucial role in adipocyte dysfunction and obesity [53]. In obese individuals with metabolic disorders, TrxR activity and Trx content in subcutaneous tissue are significantly increased because subcutaneous fat seems to provide good protection against increased oxidation in obese subjects compared to visceral fat. Protein levels of Trx, GPx, and CuZnSOD, as well as activities of GPx, GR, GST, TrxR, and SOD, were significantly higher in “at-risk” obese women than in metabolically healthy women [54]. While the expression of Trx-dependent peroxiredoxin (Prx3) in adipose tissue is significantly reduced [55], Prx3 is a key molecule regulating adipocyte oxidative stress and adipokine expression [56]. This may indicate that expression of various antioxidant enzymes is predominantly and specifically regulated by different transcription factors, but the detailed mechanism behind it needs further clarification.

Trx system in NASH has been shown to be changed in several studies [57,58]. Serum Trx levels are significantly increased in NASH patients compared to simple steatosis patients, making it a potential biomarker to distinguish NASH from early-stage fatty liver [59]. In animal models of choline-deficient liver steatosis, the activities of hepatic Trx1 and TrxR are upregulated at day 14 but significantly decreased at day 30 compared to day 14 [60]. Furthermore, the level of thioredoxin-interacting protein (TXNIP) in the liver of NAFLD patients is significantly elevated [61], and in mice fed a methionine and choline-deficient diet to induce NASH, the TXNIP gene was overexpressed, and expression of hepatic TrxR1 and TrxR2 decreased [62].

In addition to the Trx system, the glutathione-glutaredoxin (GSH-Grx) system is another crucial thiol-dependent redox system involved in cellular redox balance regulation [63]. It comprises NADPH, glutathione reductase (GR), and GSH coupled with Grxs, including cytosolic Grx1 and mitochondrial Grx2. The GSH-Grx systems mainly mediate redox signal transduction by reversibly catalyzing protein S-glutathionylation. GSH is the most abundant thiol small molecule, playing a vital role in maintaining intracellular redox homeostasis [64]. GSH can reduce various proteins, including glutathione peroxidases and glutathione transferases [65], and the resulting GSSG can be recycled by GR using NADPH as an electron donor [66].

Studies have shown that when Glrx1 KO mice are given a standard diet for some time, they develop obesity, hyperlipidemia, and fatty liver, resembling NAFLD. When fed a high-fat diet, Glrx1 KO mice develop fatty liver inflammation and progress to NASH [67]. In NAFLD animal models fed with a high-fat diet, S-glutathionylated protein levels increase, and an important target protein, Sirtuin-1 (SirT1), is identified. SirT1 is a NAD+-dependent class III histone deacetylase that regulates key transcription factors coordinating hepatic lipid metabolism [68,69,70]. Activation of SirT1 ameliorates Non-Alcoholic Fatty Liver (NAFL); conversely, hepatic SirT1 deficiency leads to steatosis [71], and thiol modification of SirT1 regulates lipid metabolism through acetylation of key transcription factors. S-glutathionylation inactivates SirT1 and promotes hyperacetylation and activation of downstream target proteins such as p53 and sterol regulatory element binding protein (SREBP) [68]. Activation of p53 is a cellular response to stress, resulting in the alteration of metabolism and the arrest of the cell cycle, and severe oxidative damage to hepatocytes may trigger p53 to induce cell death [72]. Studies also suggest that SirT1 overexpression can improve NAFLD [73], while Sirt1 knockout in mice leads to NAFLD (Figure 2) [74].

Grx2 has two known isoforms encoded by the same gene (GLRX2): Grx2a (mitochondrial) and Grx2c (cytoplasmic) [75,76]. The role and effects of mitochondrial Grx2-mediated glutathionylation in cellular metabolism are still poorly understood. A mouse model with a depletion of mitochondrial Grx2 fed with a standard diet had spontaneous weight gain and accumulation of lipid droplets in the liver, suggesting that Grx2 is involved in mitochondrial redox environment and lipid metabolism regulation [77]. Genes such as cytochrome P-450 7a1 (Cyp7a1), which is specifically expressed in the liver and participates in the reduction of cholesterol, were found to be significantly downregulated in the mice model [77].

The Keap1-Nrf2-ARE pathway is one of the major pathways that maintain cellular homeostasis during oxidative stress in the liver and is also regulated by Trx. Its function is to protect cells from endogenous and exogenous damage caused by oxidative stress. When Nrf2 accumulates significantly in the cell nucleus, a large number of antioxidant and phase II detoxification enzyme genes are upregulated [78]. Previous studies have identified over 200 downstream target genes regulated by Nrf2 in human cells [79], including the genes encoding NAD(P)H quinone oxidoreductase 1 (NQO1), HO-1, GST, GPx, CAT, SOD, and GR (Figure 1) [80]; It controls the expression of the glutamate–cysteine ligase catalytic subunit (GCLC) and glutamate–cysteine ligase modifier subunit (GCLM) to maintain the proper ratio of GSH to GSSG in cells [81]; Nrf2 also positively regulates some enzymes involved in liver detoxification, including Trx1, TrxR1, Gpx2, and GST [82,83]. These detoxification enzymes can eliminate H₂O₂, free radicals, and oxidized thiols in the cytoplasm, endoplasmic reticulum, and mitochondria [84]. Thus, Nrf2 represents a potential therapeutic target for NAFLD.

In addition to Nrf2, which has been shown to bind to the ARE of the Trx, TrxR, and Prx1 promoters [85,86,87], hypoxia-inducible factor (HIF)-1α synergistically up-regulates specific genes during hypoxia with another class of transcription factors, the E26 translationally specific (Ets) family of related proteins [88]. In human PC3 prostate cancer cells, the Prx1 gene promoter was significantly induced by either H₂O₂ or reoxygenation after hypoxic growth [89]. Co-transfection of constructs overexpressing Ets-1 or Ets-2 with the Prx1 promoter construct resulted in increased promoter activity, and ChIP assays also confirmed that both Ets-1 and Ets-2 bound to the Prx1 gene promoter in PC3 cells [89]. Thus, it can be hypothesized that the Ets pathway may play a role in regulating redox control during hypoxia and reoxygenation to complement the activation of the Nrf2 pathway. In addition, specificity protein 1 (Sp1), Fas/Jun, TATA box binding protein (TBP), cAMP response element binding protein (CREB), retinoic acid receptor/retinoid X receptor (RAR/RXR), and other transcription factors bind to their DNA sites to regulate Trx [85,87,90,91]. Retinoid X receptor (RAR/RXR) and other transcription factors that bind to DNA sites to regulate Trx; Octamer binding protein (Oct-1), Sp1, Sp3, and other transcription factors bind to the DNA site to regulate TrxR [92]. However, whether these transcriptional regulations are involved in NAFLD/NASH is unclear.