The liver plays a crucial role in regulating metabolism, protein synthesis, and detoxification. Following injury, the liver exhibits compensatory adaptation to maintain normal physiological function, with its capacity for repair and regeneration serving as the foundation of its homeostasis. Liver regeneration (LR) is an intricate and vital process that facilitates recovery from liver injury or partial hepatectomy (Michalopoulos and Bhushan, 2021). Hepatocyte proliferation is a key cellular mechanism governing the generation of new hepatocytes during LR, both under stable conditions and following injury (Heinke et al., 2022). Previous studies (Kim et al., 2024) have demonstrated that, after surgical resection of approximately 70% of the liver, the liver can restore total lost liver mass. This restoration occurs through the replication of hepatic epithelial cells, specifically hepatocytes, and biliary epithelial cells: proliferationmediated regeneration. Chronic liver disease (CLD) represents a significant global health challenge. CLD is characterized by progressive hepatic deterioration involving persistent inflammation and parenchymal regeneration, ultimately leading to fibrosis and cirrhosis. The primary etiological factors of CLD include viral hepatitis, chronic alcohol consumption, autoimmune disorders, and genetic conditions. Hepatitis B virus (HBV) infection induces extensive oxidative stress, multiple studies (Jabeen et al., 2021; Mansouri et al., 2018) indicate that HBV alters mitochondrial function, thereby generating oxidative stress and modulating host gene expression. During hepatic injury, necrotic hepatocytes release mitochondrial DNA (mtDNA), which activates the TLR9 and cGAS-STING pathways, promoting neutrophil infiltration and exacerbating liver inflammation (He et al., 2017). Research demonstrates that alterations in mitochondrial metabolism and increased oxidative/nitrosative stress significantly contribute to the pathogenesis and progression of chronic HBV infection (Begriche et al., 2006). Safeguarding hepatocytes from damage and enhancing liver repair and regeneration are pivotal strategies for mitigating liver disease progression (Chalasani et al., 2018). Cytochrome P450 2E1 (CYP2E1) is a major source of reactive oxygen species (ROS) production in the liver (Lu et al., 2017), with its upregulated expression being a critical factor in the development of hepatic oxidative stress (Harjumäki et al., 2021). ROS-induced oxidative stress, mediated by CYP2E1, damages hepatocytes via peroxidation of cellular macromolecules, including lipid peroxidation, protein carbonylation, and DNA oxidation, leading to increased hepatocyte apoptosis and liver fibrosis, thereby impairing the liver’s capacity for repair and regeneration (Zhang et al., 2019). While the role of CYP gene variants in the development and progression of hepatocellular carcinoma (HCC) is well established, limited data exist regarding their correlation with susceptibility to chronic hepatitis B (CHB). Several studies (Bose et al., 2013) have reported that CYP2E1 can significantly influence both the susceptibility and severity of HBV-related liver diseases. Transcriptional coregulators play a crucial role in regulating various physiological processes, including cell survival and death. Among these, nuclear receptor corepressor 1 (NCOR1) serves as an epigenetic regulator of gene transcription (Stallcup and Poulard, 2020). NCOR1 expressed by cardiomyocytes acts as a negative regulator in acute myocardial infarction/reperfusion injury, inhibiting mitochondria-mediated apoptotic pathways and inflammation via the signal transducer and activator of transcription 1 pathway (Qin et al., 2022). Lima et al. (2019) demonstrated that NCOR1 knockdown reduces mitochondrial ROS levels and prevents cell death due to lipid overload in skeletal muscle cells. Another study (Ou-Yang et al., 2018) revealed that hepatocyte-specific NCOR1 deficiency promotes adipogenesis and enhances LR following partial hepatectomy. Inflammation is considered a key factor in coordinating liver injury repair and reconstruction, with leukemia inhibitory factor (LIF), a member of the IL-6 family of inflammatory cytokines (Xie et al., 2025), playing a significant role. LIF signals through its receptor LIR (LIFR) and co-receptor gp130 to activate the JAK/STAT inflammatory pathway (Guo et al., 2021). Yao et al. (2021) demonstrated that LIFR expression was significantly downregulated in hepatocellular carcinoma tissues, and the absence of LIFR facilitated the progression of liver cancer. Deng et al. (2024) reported that LIFR promoted liver injury repair and regeneration by enhancing neutrophil recruitment and secreting hepatocyte growth factor, thereby accelerating hepatocyte proliferation and regeneration. Studies (Li et al., 2023; Frick et al., 2024) have indicated that platelet (PLT) not only play a crucial role in physiological hemostasis but also contribute significantly to liver ischemia-reperfusion injury, liver injury, tissue repair, and LR. A reduced PLT count can lead to spontaneous bleeding, infections, and other complications, which can severely impact patient prognosis. Moreover, both platelet count and serotonin levels in platelets are associated with post-hepatectomy liver dysfunction and morbidity (Amygdalos et al., 2020). Collectively, these findings underscore the critical role of PLT in promoting new cell production and LR.

Fibroblast growth factor21 (FGF21) is an atypical member of the fibroblast growth factor family, exhibiting distinct activities in cell proliferation, angiogenesis, oxidative stress response, and tissue repair and regeneration (Itoh et al., 2016). FGF21 expression is upregulated in response to both physiological and pathophysiological stress. In particular, under pathological conditions such as metabolic syndrome, FGF21 levels are compensatorily increased in response to oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction (Li et al., 2024). Recent studies have demonstrated that FGF21 exerts a protective effect on the liver following acute insults that induce damage (Ma et al., 2023; Eguchi et al., 2020). For instance, Huai et al. (2024) reported that overexpression of FGF21 in mesenchymal stem cells/stromal cells significantly enhanced therapeutic efficacy in mice with alcohol-related liver disease, likely by mitigating liver damage, steatosis, inflammatory infiltration, oxidative stress, hepatocyte apoptosis, and promoting liver regeneration. Mechanistically, FGF21 enhances the immunomodulatory function of mesenchymal stem cells on macrophages. Currently, FGF21 and hepatocyte growth factor receptor have emerged as promising therapeutic targets in LR (Falamarzi et al., 2022). Therefore, FGF21 may serve as a sensitive, specific, and clinically significant biomarker with predictive value for liver function assessment and the progression and prognosis of liver disease. In this study, we primarily utilized MethyLight technology to evaluate the methylation levels of the FGF21 promoter in patients with CHB and healthy controls (HC). Additionally, we investigated the expression levels of FGF21, LIFR, and CYP2E1 to elucidate the potential relationship between FGF21 promoter methylation and the expression of molecules involved in hepatic repair and regeneration in CHB patients.

The liver plays a crucial role in various physiological processes, including metabolism, immune defense, and detoxification. HBV infection represents a significant global public health challenge, affecting approximately 2 billion individuals worldwide, its associated liver cirrhosis and HCC (GBD, 2015 LRI Collaborators, 2017). FGF21 is a stress-responsive factor produced by the liver, whose expression and induction are believed to confer protective effects when hepatic homeostasis is disrupted by diverse stimuli (Desai et al., 2017; Ye et al., 2014). Yang et al. (2013) demonstrated that overexpression of FGF21 in mice promotes LR following partial hepatectomy. Qiang et al. (2021) revealed that FGF21 plays a critical role in hepatocyte survival, and exogenous administration of FGF21 enhances liver resilience by activating autophagy, reducing oxidative stress, and inhibiting apoptosis. These findings confirm that FGF21 activates hepatocyte autophagy via the AMPK-mTOR signaling pathway, thereby accelerating the regeneration of damaged liver cells. In a mouse model of nonalcoholic steatohepatitis using leptin-deficient mice fed a methionine and choline-deficient diet, an FGF21 analogue was shown to reverse liver inflammation and fibrosis (Stefan et al., 2023; Keinicke et al., 2020). However, Liu et al. (2022) reported that elevated FGF21 levels are associated with poorer survival rates in HCC patients, suggesting that increased serum FGF21 may serve as a prognostic indicator for HCC. While FGF21 enhances liver regeneration by inhibiting apoptosis and reducing oxidative stress, thereby improving the survival of damaged hepatocytes (Qiang et al., 2021), it has been proposed (Gómez-Sámano et al., 2017) that the initial increase in FGF21 production during the early stages of disease represents a compensatory response by the organism. Conversely, other researchers suggest that sustained elevation of serum FGF21 as the disease progresses may lead to the development of resistance to FGF21. In peripheral nerve injury (PNI), studies (Lu et al., 2019) have demonstrated that FGF21 exerts protective effects on bone marrow and nerve regeneration following PNI, potentially through inhibition of the overactivation of the ERK/Nrf-2 signaling pathway. The liver demonstrates a remarkable capacity for regeneration in response to injury or viral infection. Multiple growth factors and cytokines are intricately involved in regulating this regenerative process. The liver’s regenerative ability is essential for maintaining homeostasis; however, this capacity is markedly diminished in cases of severe or CLD (Huang et al., 2022). When viral activation results in extensive hepatocyte death and the residual functional liver mass becomes insufficient, the liver’s regenerative potential may be compromised, potentially leading to liver failure (Clavien et al., 2007). Walesky et al. (2020) demonstrated that cytokines, growth factors, compensatory mechanisms, and epigenetic factors play pivotal roles in LR. Epigenetic regulators not only influence cell proliferation and stem cell differentiation but also significantly contribute to the severity of liver injury and the maintenance of tissue microenvironment homeostasis. DNA methylation, which involves the addition of methyl groups to DNA, represents one of the most significant epigenetic modifications. Previous studies have demonstrated that DNA methylation is intricately associated with the development of cancer, oxidative stress, and validation. Given the pressing concern regarding the impact of HBV infection on liver regeneration, our study investigates the effect of FGF21 promoter methylation levels on liver function assessment and repair-regeneration capacity in the context of HBV infection.

Currently, biomarkers based on PBMCs have great potential for clinical application. Numerous studies have utilized them for early diagnosis or efficacy monitoring of diseases. Previous studies (Feng et al., 2015) have demonstrated a positive correlation between peripheral blood and liver tissue characteristics in patients with chronic HBV infection and both ALT levels and the extent of liver damage. Chen et al. (2019) utilized animal models to show that the mRNA and protein expression levels of BATF in liver tissues of HBV transgenic mice, as well as the serum concentrations of Th17 and the cytokines IL-17 and IL-22, were significantly elevated compared to those in the control group. In the author’s prior research, it was also observed that the proportion of Th17 in PBMCs was markedly higher in patients with chronic HBV infection. Fan et al. (2016) reported that the relative expression level of A20 mRNA in PBMCs from patients with HCC was significantly higher than that in CHB, and elevated A20 protein expression was similarly detected in HCC liver tissues. In the study of HBV-ACLF disease, Li et al. (2022) conducted functional synergy analysis of seven biological processes related to PBMCs responses and the top 500 differentially expressed genes (DEGs), showing that the viral process was associated with all disease stages. These findings collectively support the significant clinical translational potential of PBMCs-based biomarkers, which can be applied for disease early warning, staging, and therapeutic monitoring. In this study, we mainly used PBMCs for the research, and subsequently we utilized quantitative RT-PCR, MethLight, and ELISA to evaluate FGF21 expression and elucidate the relationship between FGF21 promoter methylation levels and liver repair-regeneration in CHB patients. Our findings revealed that PBMCs and serum FGF21 expression levels were elevated in CHB patients compared to HCs, while methylation levels were reduced. Specifically, HBeAg(+) CHB patients exhibited higher PBMCs and serum FGF21 expression levels and lower methylation levels than HBeAg(−) patients. These results suggest that the increased FGF21 expression in PBMCs of CHB patients is a compensatory response to HBV viral activation and oxidative stress-induced damage. Additionally, LIFR mRNA expression and PLT levels in PBMCs of HBeAg(+) patients were significantly lower than those in HBeAg(−) patients and healthy controls. Similarly, NCOR1 and CYP2E1 mRNA levels, as well as ALT and AST levels, were also lower in HBeAg(+) patients compared to HBeAg(−) patients. These observations indicate that LIFR and PLT play crucial roles in the repair and regeneration processes during the progression of CHB. In the correlation analysis between viral load and repair and regeneration factors, LIFR mRNA exhibited a significant negative correlation with HBeAg, while NCOR1 and CYP2E1 mRNA showed significant positive correlations with HBeAg. These findings suggest that liver repair and regeneration capacity is associated with HBeAg status. Our previous studies have demonstrated that HBeAg status reflects the body’s response to oxidative stress, which may be linked to virus activation and liver damage in the progression of CHB. Further analysis revealed that HBsAg levels were negatively correlated with LIFR mRNA and positively correlated with NCOR1 mRNA, indicating that higher viral loads impair liver repair and regeneration. We hypothesize that the expression of repair and regeneration factors in CHB patients may be influenced by the methylation status of the FGF21 promoter.

Therefore, the correlation analysis between the methylation level of the FGF21 promoter and the mRNA expression levels of LIFR, NCOR1, and CYP2E1, as well as PLT, ALT, and AST, revealed that the methylation level of FGF21 was significantly positively correlated with LIFR mRNA expression and PBMC PLT levels. Conversely, it exhibited significant negative correlations with NCOR1 and CYP2E1 mRNA expression, and ALT and AST levels, supporting our hypothesis. Additionally, we performed a correlation analysis between the methylation level of the FGF21 promoter and viral load. The results indicated a significant negative correlation between the methylation level of the FGF21 promoter and HBV-DNA, HBsAg, and HBeAg levels, further validating our conclusion that the methylation status of the FGF21 promoter is closely associated with the progression and development of CHB. This association is linked to liver injury induced by viral activation and oxidative stress, and plays a crucial role in liver repair and regeneration following injury, potentially serving as an important biomarker for these processes. In summary, this study underscores the regenerative potential of FGF21 in the context of HBV infection, offering promising prospects for the diagnosis and treatment of CHB. Biomarkers indicative of liver regenerative capacity may enhance patient outcomes in CHB management. However, our research has certain limitations. Firstly, we evaluated the methylation level of the FGF21 promoter in PBMCs, but the methylation situation in the liver is still unknown. In the future, we will examine the methylation status and gene expression in liver tissues. Secondly, future research should adopt a large-scale, multi-center approach to verify the accuracy of these findings, and conduct mechanism studies by integrating public databases and in vitro/in vivo experiments to elucidate how methylation of the FGF21 promoter affects its expression and signal transduction, and subsequently influences liver repair and regeneration functions.

The methylation status of FGF21 serves as a critical indicator for evaluating the liver’s repair and regeneration capacity in patients with CHB. This marker is closely associated with both the extent of hepatic injury and viral load.