Hepatitis B virus (HBV) infection is currently a global public health problem. According to the World Health Organization, an estimated 296 million people worldwide were chronically infected with HBV in 2019, with about 1.5 million new infections each year (WHO, 2021). In 2019, it was estimated to have killed 820,000 people. According to the Polaris International Epidemiology Collaboration, in 2016, the prevalence of hepatitis B surface antigen (HBsAg) in the general population was 6.1%, and the number of chronic HBV infected patients was 86 million (Polaris Observatory Collaborators, 2018). Despite the widespread availability of effective vaccines and therapy, including immunomodulators (IFN-α-2B and PEG-IFN-α-2A) and reverse transcriptase inhibitors (nucleosides or nucleotide analogues) (Cui et al., 2017), the clinical treatment of chronic hepatitis B still falls short of achieving a satisfactory complete cure. HBV infection continues to be a leading cause of liver cirrhosis and hepatocellular carcinoma. The presence of HBeAg in serum is a marker of active HBV replication and is linked to more severe liver disease. Therefore, in this experiment, we choose to divide the chronic hepatitis B patients into e antigen positive and negative groups, in order to provide early indications for the clinical treatment of chronic hepatitis B.

Autophagy, a cellular homeostatic regulatory system, mediates the degradation of damaged organelles and misfolded proteins via the lysosomal pathway. Its initiation depends on the formation of the class III phosphatidylinositol 3-kinase (class III PI3K) complex mediated by Beclin1 (Liu et al., 2023). HBV bidirectionally regulates autophagy via viral proteins across stages (initiation, autophagosome formation, lysosomal degradation). Moderate autophagy suppresses viral replication via clearance, while excessive mitophagy enhances HBV replication via mitochondrial dysfunction (Lin et al., 2020). Notably, whether hepatitis B e antigen (HBeAg) participates in modulating autophagy homeostasis remains unclear.

During HBV infection, oxidative stress represents another core pathological mechanism. The virus induces reactive oxygen species (ROS) bursts, directly damaging hepatocyte genomic DNA and lipid membrane structures (Jomova et al., 2023; Popa and Popa, 2022; Klieser et al., 2019). The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway constitutes the central regulatory network for cellular antioxidant defense. Under steady-state conditions, Keap1 acts as a negative regulator of Nrf2, maintaining low Nrf2 expression via ubiquitin-proteasomal degradation. Oxidative stress disrupts the Keap1-Nrf2 complex, enabling Nrf2 nuclear translocation to activate the transcription of antioxidant genes, including glutathione (GSH), glutathione S-transferases (GSTs), and heme oxygenase-1 (HO-1). Importantly, autophagy interacts with the Nrf2 pathway through molecular crosstalk: the autophagy receptor protein p62/SQSTM1 selectively binds Keap1 via its Keap1-interacting region (KIR) domain, targeting Keap1 for co-degradation with autophagosomes (Thiruvengadam et al., 2023; Bender and Hildt, 2019; Liu et al., 2015; Miyakawa et al., 2022). This process relieves Keap1-mediated inhibition of Nrf2, establishing a positive feedback regulatory loop.

Based on these mechanisms, this study enrolled HBeAg-positive and HBeAg-negative chronic hepatitis B (CHB) patients. Autophagy levels were assessed by measuring the expression of key markers (Beclin1 and p62) via RT-qPCR. The antioxidant capacity of the Nrf2-Keap1 pathway was evaluated by quantifying Nrf2 and Keap1 expression (RT-qPCR) and downstream antioxidant enzymes (GSH, GSTs, HO-1) via ELISA. Group comparisons and correlation analyses were performed to explore the relationship between HBeAg status and autophagy-oxidative stress dynamics. The scientific significance of this study lies in elucidating whether autophagy-oxidative stress can play a role in the serological conversion of HBeAg, thereby providing theoretical foundations for personalized therapeutic strategies targeting autophagy dynamics modulation or Nrf2 pathway activation. This study utilized peripheral blood mononuclear cells (PBMCs), leveraging their non-invasive and easily accessible characteristics to serve as a surrogate tissue for liver samples in the investigation within this specific experimental context. Standardized techniques facilitated clinical translation. However, limitations include their inability to accurately mirror intrahepatic pathology, inherent cellular heterogeneity, and potential ex vivo stress artifacts. Future research should integrate hepatocyte models and mechanistic experiments to validate the underlying pathways.

Autophagy and oxidative stress can exist in the progression of inflammation and fibrosis. The above processes must exist during the development and progression of hepatitis B to cirrhosis and Hepatic carcinoma (Lin et al., 2020; Zhang, 2020; Jiang et al., 2021; Xie et al., 2018; Sartorius et al., 2020). It is well-known that the important step in the course of hepatitis B cure is the negative conversion of e antigen. Therefore, in this study, hepatitis B patients were divided into HBeAg (+) and HBeAg (−) groups, and the levels of autophagy and oxidative stress in peripheral blood mononuclear cells (PBMCs) and plasma were analyzed respectively. In this study, it can be found that HBeAg (+) patients have the ability to activate autophagy, but their antioxidant capacity is decreased, and the downstream expression enzyme is also decreased. And we also analyzed the level of autophagy in patients during the LVL/LLV stage and the normal group (Hao et al., 2024). Compared with LVL group, Beclin1 expression in peripheral blood of normal group was lower. Clinically, autophagy was found to have a greater impact on ALT, whereas oxidative stress had a more pronounced effect on bilirubin levels.

In our study, P62 expression in peripheral blood of HBeAg (+) group was lower compared with that of the HBeAg (−) group. Furthermore, we concluded that Beclin1 is negatively correlated with Nrf2 and positively correlated with Keap1. In other researches, they found Keap1 binds to the Neh2 domain of Nrf2 through its Kelch domain, inhibiting Nrf2’s transcriptional activity and promoting its degradation. During oxidative stress, Keap1 undergoes a conformational change, leading to the release of Nrf2 from the Keap1 complex. Nrf2 then translocates to the nucleus and activates downstream antioxidant genes. Also, P62 binds to Keap1 through its PB1 domain, disrupting the interaction between Keap1 and Nrf2, facilitating the accumulation of Nrf2 and activating antioxidant responses. Through our research, this mechanism may also works.

In liver diseases such as NASH, autophagy levels in the liver tissue of NASH patients are reduced, leading to the worsening of many characteristics of NAFLD, such as fat droplet accumulation, increased damage to hepatocytes, and elevated endoplasmic reticulum stress (Ren et al., 2024). During liver fibrosis, a key marker of the activation of hepatic stellate cells (HSCs) is the disappearance of fat droplets. The digestion of fat droplets by autophagy is considered an energy source driving HSC activation. Studies have found that inhibiting autophagy can downregulate the fibrotic state of HSCs. Autophagy also inhibits fibrosis, as it has been reported that autophagy-mediated miR-30a-5p/ATG5 axis can alleviate liver fibrosis through induced apoptosis suppression. A large body of evidence suggests that the mechanisms underlying the pathogenesis of alcoholic liver disease (ALD) are associated with alcohol-induced abnormal autophagy. In cancer research, autophagy has a dual role in promoting and inhibiting tumors. Autophagy can suppress tumor growth in the early stages of tumorigenesis; however, it can also provide energy to starving tumor cells. Autophagy can lead to multidrug resistance in tumor cells thereby reducing their sensitivity to radiation. In current research on autophagy and oxidative stress, during acute events, autophagy or its regulation of inflammatory signals can prevent organs from developing severe inflammatory responses, while excessive activation of autophagy can have the opposite effect (Ma et al., 2024; Shen et al., 2024). In chronic diseases, such as COPD and liver fibrosis, increased autophagy levels have been observed, contributing to disease progression (Ren et al., 2024; Lin et al., 2024). In inflammation-related autoimmune diseases, such as UC and other gastrointestinal diseases (Kim et al., 2012), numerous studies have focused on autophagy and oxidative stress. In the occurrence and development of tumors, autophagy also plays a role in promoting or inhibiting tumor progression and in drug resistance.

While our study highlights the clinical correlations between Beclin-1, the Nrf2-Keap1 pathway, and HBeAg seroconversion in chronic hepatitis B (CHB), it is critical to contextualize these findings within the intricate interplay of HBV pathogenesis, autophagy, and oxidative stress. On one hand, (HBx) has been shown to hijack the autophagic machinery by activating the AMPK/mTOR axis to promote viral envelopment and secretion, while simultaneously suppressing autophagic degradation through ubiquitination-mediated destabilization of autophagy receptors (e.g., p62/SQSTM1) (Wang et al., 2020; Kim et al., 2022). On the other hand, Beclin-1 upregulation during HBeAg seroconversion might represent a compensatory mechanism to counteract viral-induced ER (endoplasmic reticulum) stress, as HBV X protein (HBx) disrupts mitochondrial bioenergetics and increases ROS (reactive oxygen species) production, further exacerbating hepatocyte vulnerability to inflammatory damage. During HBeAg seroconversion, the upregulation of Beclin-1 may represent a compensatory mechanism to counteract virus-induced ER stress. This ER stress is exacerbated by HBx-mediated disruption of mitochondrial bioenergetic metabolism and increased ROS production, thereby heightening hepatocyte susceptibility to inflammatory injury. Current studies have demonstrated that HBV can hijack the host hypoxia-inducible factor (HIF) signaling pathway through evolutionarily conserved hypoxia-responsive elements (HREs), leveraging the hypoxic microenvironment of the liver to enhance its own transcription and replication. Concurrently, the hypoxic microenvironment may promote viral persistence and liver injury in chronic hepatitis B (CHB) via HIF-mediated pathways (Wing et al., 2021). P62, a key adaptor protein, has been implicated in mitochondrial ubiquitination. Emerging evidence highlights the dual role of the ubiquitination system in HBV-induced hepatocellular carcinoma (HCC). While ubiquitination contributes to host antiviral defense mechanisms, it is also exploited by the virus to facilitate viral replication and oncogenesis (Yamada et al., 2019). This “double-edged sword” characteristic underscores the complex interplay between the ubiquitin-proteasome system and HBV pathogenesis, balancing protective responses against viral infection with inadvertent promotion of viral survival and carcinogenesis (Kar et al., 2024). HBx, a multifunctional viral regulator, interacts directly with the mitochondrial outer membrane, compromising its structural integrity (Zhong et al., 2015, 2017; Zhou et al., 2016). This interaction is mediated through HBx binding to voltage-dependent anion channel 3 (VDAC3), a key component of the permeability transition pore (PTP) (Lee et al., 2018), which disrupts PTP function by elevating cytoplasmic and mitochondrial Ca²⁺ concentrations (Reina et al., 2016). These events culminate in mitochondrial membrane potential collapse and excessive ROS generation. Notably, both HBx and large hepatitis B surface protein (LHBs) act as transcriptional co-activators that induce c-Raf phosphorylation, a prerequisite for HBV-dependent Nrf2 activation (Kalantari et al., 2023). Activated Nrf2 translocates to the nucleus and binds antioxidant response element (ARE) sequences, upregulating cytoprotective genes such as NAD(P)H quinone oxidoreductase 1 (NQO1) and heme oxygenase-1 (HO-1). And according to the analysis, it can be roughly estimated that autophagy has a greater effect on ALT, while oxidative stress has a greater effect on bilirubin. This point, which has been overlooked in recent studies, is the highlight of this study. Thus, in the future clinical work, early detection of clinical indicators in patients with chronic hepatitis B, especially for patients with abnormal ALT and bilirubin levels. Specific measurement of patients’ peripheral blood autophagy and antioxidant indicators should be conducted to guide specific medication for patients. Our findings suggest that Beclin-1 and Nrf2-Keap1 pathway dysregulation during HBeAg seroconversion reflect HBV’s subversion of autophagy and oxidative stress networks to sustain hepatic inflammation and metabolic dysfunction. Future studies should explore spatial multi-omics approaches to map autophagy-ROS crosstalk in HBV-infected liver niches, alongside preclinical validation of pathway-specific therapies to improve CHB outcomes.

Although there are previous studies on autophagy and oxidative stress in hepatitis B virus infection, the samples were mostly collected from liver cancer patients caused by hepatitis B. However, this study is relatively novel in reflecting autophagy and oxidative stress abilities through peripheral blood related indicators in hepatitis B patients. Nevertheless, there are defects in this experiment: PBMCs cannot completely simulate the intracellular environment. Moreover, because hepatitis B patients rarely undergo liver biopsy, there is a lack of samples. However, according to the research results of related articles, autophagy is also associated with immune cells within PBMCs (Son et al., 2021; Yoon et al., 2023; Geertsema et al., 2023; Ren et al., 2024; Chandrasekaran et al., 2023; Debnath et al., 2023). Yet, this experiment did not subdivide each immune cell group in PBMCs. This study focused on the early phase of HBeAg seroconversion (defined as HBeAg loss with anti-HBe emergence), aiming to characterize autophagy and oxidative stress dynamics. HBeAg seroconversion primarily reflects immune transition, whereas HBsAg/HBV-DNA changes (e.g., HBsAg clearance) are delayed events. Thus, our design prioritized molecular mechanisms over long-term virological correlations. We plan to conduct a future study investigating the regulatory effects of HBV-DNA load on autophagy/oxidative stress pathways and exploring the association between HBsAg kinetics and immune microenvironment remodeling post-seroconversion. In addition, this study is single-center and lacks a verification set, and the number of subjects included is not large, which may lead to errors. An important limitation is the lack of systematic assessment of antiviral medication use among participants. As these agents may directly influence oxidative stress and autophagy pathways–key mechanisms under investigation–the absence of medication screening constitutes a potential confounding factor in our analysis.