The precise mechanisms underlying the development of NAFLD are not yet fully understood. Recent studies have identified a connection between NAFLD and redox imbalance. Prolonged oxidative stress disrupts the equilibrium between ROS and the body's antioxidant defenses in liver cells, leading to lipid peroxidation, insulin resistance, damage to cellular organelles, and activation of hepatic stellate cells (30). Elevated levels of superoxide can stimulate iron-containing compounds to release Fe2+, which plays a critical role in the catalytic subunit of lipoxygenase, promoting lipid peroxidation (31). During the progression of NAFLD, ROS significantly advances the condition to non-alcoholic steatohepatitis (NASH) (32). ROS-induced lipid peroxidation is a key factor in the development of NAFLD. Ferroptosis, a type of cell death reliant on iron and characterized by lipid peroxidation, is recognized as a pathological characteristic of NAFLD (33). Glutathione (GSH), the primary intracellular antioxidant and a cofactor for GSH peroxidase 4 (GPX4), helps shield cells from oxidative harm, thereby preventing ferroptosis.
This study identified key genes associated with ferroptosis in NAFLD and confirmed the presence of ferroptosis in MCD diet-induced steatohepatitis. In the MCD liver, a significant increase in Fe2+ production, ROS levels, and the lipid peroxidation marker MDA was observed, accompanied by a notable decrease in GPX4 and GSH levels. Additionally, under MCD conditions, there was a reduction in mitochondrial size, diminished ridges, increased density, and occasional rupture of the outer membrane, consistent with the known morphological characteristics of mitochondria affected by ferroptosis.
HMOX1 acts as a stress-induced enzyme that breaks down heme into carbon monoxide, iron, and biliverdin, playing a crucial role in counteracting oxidative processes (34). Numerous studies have shown that HMOX1 exhibits anti-inflammatory and anti-apoptotic properties in various obesity-induced metabolic syndromes (35–37). In cases of hepatocyte injury, HMOX1 activation can trigger an adaptive stress response, protecting hepatocytes from oxidative damage (38),(39). The data in this study highlight the significance of HMOX1 in regulating FRGs, demonstrating a notable reduction in NAFLD in both animal models and cell cultures. Specifically, mice fed the MCD diet exhibited decreased HMOX1 expression and elevated levels of ALT and AST. However, hemin treatment significantly decreased ALT, AST, TG, LDL, and lipid peroxidation levels compared to the MCD group. These results align with previous research indicating that higher HMOX1 expression is associated with less severe NAFLD progression in animal models (40, 41). Furthermore, hemin administration reversed the decrease in GSH and GPX4 levels induced by the MCD diet, while also enhancing mitochondrial ridges and improving mitochondrial outer membrane integrity. The findings from both animal and cell studies support the conclusion that HMOX1 alleviates NAFLD and inhibits ferroptosis.
NAFLD is regulated by various transcription factors and associated signaling pathways. The JAK/STAT signaling pathway is a widely expressed intracellular signal transduction pathway that plays a crucial role in several essential biological processes, including cell proliferation, differentiation, apoptosis, immune regulation, and adipogenesis (42). JAKs interact noncovalently with cytokine receptors, leading to receptor phosphorylation and the recruitment of one or more STAT proteins. Once phosphorylated, STATs form dimers and translocate into the nucleus to modulate specific genes. Current research on this pathway in relation to disease and drug development has primarily focused on inflammatory and neoplastic conditions (43, 44).
The research data indicated that blocking hepatic STAT3 activation can prevent liver fibrosis caused by NAFLD and act as a protective signal against lipotoxicity. This study confirmed the activation of the JAK/STAT pathway in OA-induced NAFLD cells and observed improved outcomes after treatment with AZD1480. The results support the role of STAT3 as a crucial pro-inflammatory signal in NAFLD, regulating liver inflammation. Another study highlighted the importance of the IL-22-mediated JAK1/STAT3/BAX signaling pathway in reducing NAFLD progression (45). Jung et al.'s research using a NASH-associated HCC model demonstrated that inhibiting STAT3 with a small molecule conferred resistance to NASH-related damage, thereby reducing liver fibrosis (46). Tron et al. found that HMOX1 SBE3 triggers HMOX1 gene expression via the IL-6-induced JAK/STAT pathway (47). Yu et al. also showed that IFNγ treatment increased the sensitivity of adrenocortical carcinoma cells to erastin-induced ferroptosis through the JAK/STAT pathway (48). This study revealed that targeted upregulation of HMOX1 suppressed the JAK/STAT signaling pathway, thereby inhibiting ferroptosis in NAFLD. Additionally, treatment with the ferroptosis inducer erastin reversed the effects of HMOX1, further improving NAFLD and suppressing ferroptosis. Ultimately, inhibiting the JAK/STAT pathway led to better NAFLD outcomes, suggesting that HMOX1 exerts its anti-ferroptosis effects by modulating the JAK/STAT pathway to alleviate NAFLD.
As previously discussed, diet-induced steatohepatitis via MCD and steatosis through OA can lead to the downregulation of HMOX1 and the activation of the JAK/STAT signaling pathway. However, the impact of HMOX1 regulation on the JAK/STAT signaling pathway has been infrequently addressed in NAFLD. This research introduces a novel discovery regarding the relationship between HMOX1 and the JAK/STAT signaling pathway in NAFLD, offering a new avenue for investigating the disease's pathogenesis. Targeting the JAK/STAT signaling pathway for inhibition may serve as a potential therapeutic strategy for NAFLD prevention. Nevertheless, this study has some limitations. First, because the clinical liver tissue specimens are difficult to obtain, the clinical sample size used for analysis is relatively small. Second, the anti-ferroptotic function of HMOX1 was not assessed using HMOX1+/+ or HMOX1−/− mice to conduct a more convincing validation. Future research should aim to elucidate the precise mechanisms linking HMOX1 and the JAK/STAT signaling pathway in NAFLD.
In summary, this research highlights the crucial role of HMOX1 in the development of NAFLD and provides new evidence that HMOX1 suppresses ferroptosis by modulating the JAK/STAT signaling pathway in NAFLD. These findings suggest a novel therapeutic approach for treating NAFLD.