Abstract
Backgroud and aim
With the rising prevalence of chronic hepatitis B (CHB) co-occurring with metabolic dysfunction–associated fatty liver disease (MAFLD), an in-depth investigation into the impact and mechanisms of MAFLD on CHB is urgently needed. Prior studies have reported that patients with concomitant CHB and MAFLD exhibit lower levels of hepatitis B virus (HBV) replication; however, the underlying mechanistic basis remains unclear. Accumulating evidence indicates that induction of apoptosis is a critical mechanism for HBV clearance. Therefore, it remains to be determined whether lipotoxicity-induced hepatocyte apoptosis contributes significantly to the accelerated elimination of HBV. To investigate the effects and underlying mechanisms of fatty acids against HBV in HepG2.2.15 cells.
Methods
Human liver transcriptome data were used to analyze biological changes in CHB patients with MAFLD compared to those with CHB alone. In vitro, palmitic acid (PA) was used to simulate lipotoxicity in HepG2.2.15 cells, and oleic acid (OA) was used to reverse the effects of PA. The levels of HBsAg, HBeAg, and HBV-DNA in cell supernatants and lysates were detected by ELISA and PCR. Mitochondrial membrane permeability, oxidative stress, and apoptosis were evaluated to investigate the potential mechanisms of PA’s effects on HepG2.2.15 cells.
Results
Human hepatic transcriptome enrichment analysis showed upregulated apoptosis-related pathways and downregulated mitochondrial/redox genes in patients with comorbid CHB and MASH. Cell experiments showed that PA reduced the levels of HBsAg, HBeAg, and HBV-DNA in HepG2.2.15 cell supernatants and lysates. The underlying mechanism is that PA increased mitochondrial membrane permeability and ROS levels to activate Caspase-9, PARP, and Caspase-3, thereby inducing apoptosis. However, the unsaturated fatty acid OA restored mitochondrial function, reduced oxidative stress and apoptosis, and reversed HBV antigen levels.
Conclusion
PA activates the mitochondrial apoptotic pathway to promote HBV clearance, while OA can reverse this effect. This study provides new evidence for the interaction mechanism between CHB and MAFLD and suggests new treatment strategies for patients with this comorbidity.
Supplementary Information
The online version contains supplementary material available at https://doi.org/10.1186/s12985-026-03218-x.
Keywords: Chronic hepatitis B, Metabolic associated fatty liver disease, Apoptosis, Palmitic acid, Mitochondrial dysfunction, Oxidative Stress
Background
China bears the heaviest global burden of hepatitis B worldwide. Despite the effectiveness of vaccines and antiviral treatments in reducing prevalence, there were still 79.74 million HBV-infected individuals in China in 2022 [1]. Hepatitis B is the leading cause of liver-related deaths in China [2]. Meanwhile, the incidence of metabolic-associated fatty liver disease (MAFLD) has steadily increased with improved nutritional status. Study indicated that MAFLD is the fastest-growing cause of liver disease mortality and morbidity [2]. Consequently, the co-occurrence of hepatitis B virus infection and MAFLD is becoming increasingly common. Due to differences in detection methods and diagnostic criteria, the global incidence of CHB combined with liver steatosis ranges from 14% to 60% [3]. A meta-analysis based on multiple clinical studies showed that the prevalence of MAFLD in Chinese patients with chronic hepatitis B infection is as high as 25.9% [4].
Emerging evidence suggests that liver steatosis may inhibit HBV replication and promote the clearance of HBeAg and HBsAg. In animal experiments, reduced hepatic expression of HBeAg and HBcAg has also been observed. A retrospective cohort study of 6,786 adult CHB patients demonstrated a higher HBsAg clearance rate over a 10-year follow-up period in patients with liver steatosis compared to those without [5]. Another study showed that patients with liver steatosis achieved spontaneous HBsAg clearance approximately five years earlier than those without [6]. The same research team further found no significant difference in HBsAg clearance rate between patients with mild steatosis and those without, whereas moderate to severe steatosis significantly enhanced antigen clearance [7]. These findings suggest that simple steatosis alone may not be sufficient to reduce HBsAg and HBV-DNA levels; instead, hepatocellular inflammation and death induced by moderate to severe steatosis may serve as a potential mechanism for HBV clearance.
Apoptosis is the primary driver of hepatocellular death in metabolic-associated steatohepatitis (MASH). Studies have shown significantly increased TUNEL and Cleaved-caspase 3 positivity in liver samples from MASH patients, indicating enhanced apoptosis [8]. Excessive lipid accumulation in hepatocytes impairs mitochondrial β-oxidation capacity, leading to excessive reactive oxygen species (ROS) production and depletion of cellular antioxidant reserves, which triggers oxidative stress. High ROS levels and lipotoxicity directly disrupt mitochondrial membrane permeability, activate pro-apoptotic proteins (e.g., Bax), and subsequently initiate caspase-9 and caspase-3 cascades, thereby inducing the mitochondrial apoptotic pathway and promoting hepatocellular death [9]. This process ultimately results in liver injury, inflammation, transaminase elevation, and progression to liver fibrosis or hepatocellular carcinoma.
CHB patients with liver steatosis exhibit significantly elevated transaminase levels and increased fibrosis risk, suggesting that steatosis exacerbates liver damage and mortality in CHB [6]. Animal studies have also confirmed more severe liver inflammation and fibrosis in CHB mice fed a high-fat diet compared to those on a normal diet [10]. Based on these findings, MAFLD may promote HBsAg and HBV-DNA clearance in CHB patients by inducing hepatocellular apoptosis.
In this study, we used the saturated fatty acid palmitic acid (PA) to simulate MASH-related lipotoxicity and reversed PA-induced lipotoxicity with the unsaturated fatty acid oleic acid (OA). We aimed to preliminarily clarify the effects of lipotoxicity on HBsAg and HBeAg levels in the HBV-stable replication cell line HepG2.2.15, as well as the underlying molecular mechanisms of cellular damage. Our findings may provide new evidence for the potential mechanism by which MAFLD enhances HBV clearance in CHB patients.
Methods
Culture of the HepG2.2.15 cells and preparation of PA/OA
The HepG2.2.15 cell line with HBV replication was obtained from Wuhan Walner Biotechnology Co Ltd. Cells were maintained in MEM medium with 10% (v/v) FBS, 1% (v/v) penicillin–streptomycin solution, and 0.38 mg/mL Geneticin (G418) at 37 °C in 5% CO₂ and subcultured every 5–7 days.
Palmitic acid (PA; Sigma-Aldrich, Cat. No. P0500) and oleic acid (OA; Sigma-Aldrich, Cat. No. O1008) were separately dissolved in ethanol to prepare 100 mM stock solutions (0.128 g PA in 5 mL ethanol; 0.141 g OA in 5 mL ethanol). Then, the PA stock solutions were diluted 25-fold into pre-warmed 10% fatty acid–free BSA ( Solarbio, Cat. No. A8010), followed by vortexing and incubation at 50 °C for 30 min with intermittent mixing to 4 mM PA–BSA working stock. Similarly, OA was diluted 10-fold into the same BSA solution to yield a 10 mM OA–BSA working stock. All fatty acid–BSA complexes were filter-sterilized (0.22 μm), aliquoted, and stored at − 20 °C.
Cell viability assay
HepG2.2.15 cells were plated in 96-well plates (10,000 cells/well, n = 3). After 24 h treatment with PA and/or OA, the medium was replaced with FBS-free medium containing 10 µL CCK8 reagent per well. Plates were incubated at 37 °C for 2 h, and absorbance was read at 450 nm.
HBsAg, HBeAg, and HBV-DNA quantification
HepG2.2.15 cells were cultured in 12-well plates (10,000 cells/well). After treatment with PA and/or OA for 24 h, supernatants were collected. And cells were lysed by three freeze-thaw cycles in 100 µL PBS to obtain cell lysates. HBsAg and HBeAg levels in supernatants and lysates were measured by ELISA (Kehua Biotech). HBV DNA copies were determined by quantitative PCR using fluorescence-labeled probes (Sansure Biotech).
Cell apoptosis, total ROS, and Mitochondrial membrane potential
HepG2.2.15 cells were cultured in 12-well plates at a density of 1 × 10⁴ cells per well to a 80% density. After 24 h of treatment with PA and/or OA, cells were harvested and detected by flow cytometry with Annexin V-FITC/PI apoptosis detection kit (Beyotime Biotech). HepG2.2.15 cells were plated in 96-well plates (10,000 cells/well, n = 3). After 24 h treatment with PA and/or OA, total ROS generation and mitochondrial permeability transition pore opening were detected with DCFDA probe ( Beyotime Biotech) and membrane-permeant fluorescent probe Calcein AM (Beyotime Biotechnology).
Western blotting
Immunoblotting on HepG2.2.15 protein extracts were performed with Caspase9, PARP, Bcl2, and Bax (Abmart Inc.), Caspase3(Proteintech Group, Inc), phospho-p38MAPK/p38MAPK and phospho-ERK/ERK (CST).
Transcriptome analysis
For transcriptomic analysis, raw files of GSE274114 were downloaded from the GEO public database for downstream analyses, including relative abundance, estimated counts, and effective length. The DESeq2 software package was used to perform differential gene expression analysis. After normalization and differential expression analysis, genes with adjusted p-value < 0.05 and absolute Fold Change ≥ 2 were considered differentially expressed genes (DEGs). The Metascape Database was employed for Gene Ontology (GO) pathway enrichment analysis.
Statistical analysis
All experiments were independently repeated at least three times. Analysis of variance (ANOVA) was used for comparisons among multiple groups, and Student’s t-test was employed for comparisons between two groups. Quantitative data were expressed as mean ± standard error of the mean (mean ± SEM), and a P-value < 0.05 was considered statistically significant.
Results
Upregulated apoptosis-related genes and downregulated mitochondrial/oxidative phosphorylation pathways in livers of CHB patients complicated with MASH
Differential gene expression analysis and gene enrichment analysis were performed on bulk RNA sequencing data (GSE274114) from liver samples of 11 patients with HBeAg-negative CHB alone and 9 patients with HBeAg-negative CHB complicated with MASH [11]. Compared with patients with CHB alone, 619 genes were significantly upregulated in liver biopsy tissues from patients with CHB+MASH (Fig. 1A), including several liver apoptosis-related genes such as PDX-1, ATP5IF1, CIDEB, and DEDD2 (Fig. 1B). In contrast, 568 genes were significantly downregulated, involving genes related to mitochondrial structure and function as well as oxidative stress, such as GGCT, FIS1, and SOD1 (Fig. 1A and C).
Fig. 1.
A: Volcano plot depictes differentially expressed genes in liver biopsies from CHB patients with versus without MASH. Blue and red dots represent significantly downregulated and upregulated genes, respectively, in CHB patients with MASH (absolute fold change [FC] ≥ 2 and adjusted p-value [padj] < 0.05). B-C: Box plots shows the normalized expression levels of genes associated with apoptosis and redox regulation in liver biopsies. D-E: Gene Ontology enrichment analysis illustrating the primary upregulated and downregulated biological pathways in CHB patients with MASH. F: Heatmap displaying the expression profiles of apoptosis related genes (absolute FC > 1.5 and p-value < 0.1). CHB: chronic hepatitis B; MASH: metabolic dysfunction-associated steatohepatitis
Gene Ontology (GO) enrichment analysis showed that, compared with CHB alone, apoptosis-related pathways and lipase activity were upregulated in the livers of patients with CHB+MASH (Fig. 1D), while pathways related to oxidative phosphorylation, oxidoreductase activity, and mitochondrial inner membrane structure and function were significantly downregulated (Fig. 1E). Therefore, apoptosis induced by mitochondrial dysfunction and oxidative stress in the liver may be the main mechanism of hepatic injury in patients with CHB complicated with MASH.
Focusing on apoptosis-related genes (absolute fold change [FC] > 1.5, p value < 0.1), heatmap analysis showed that compared with CHB patients, the expression of genes related to mitochondrial structure and function (such as FIS1, VDAC2, SLC25A4, TMEM14A, and ATP2A1) was significantly decreased in the livers of CHB+MASH patients. Genes related to the redox system in response to oxidative stress (such as SOD2-OT1, GPX1, GGCT, SOD1, and ALDH2) were also significantly downregulated. Similarly, genes involved in DNA repair and correct protein folding were downregulated, while the expression of genes encoding pro-apoptotic proteins (such as M) was significantly increased. However, the expression of some other pro-apoptotic genes, such as BAX and MOP1, was decreased (Fig. 1F).
Based on the above evidence, we further verified whether steatohepatitis-induced hepatic injury in CHB patients is mediated by apoptosis induced by mitochondrial damage, redox homeostasis imbalance, and stress response, thereby reducing HBsAg and HBeAg levels.
PA induces apoptosis and reduces HBsAg, HBeAg, and HBV-DNA in HepG2.2.15 Cells
Palmitic acid (PA) is the most common saturated fatty acid in fatty liver and has been reported to induce mitochondrial dysfunction, oxidative stress, and apoptosis through lipid accumulation and lipotoxicity [12]. Treatment of HepG2.2.15 cells with PA for 24 h exhibited a significant inhibitory effect on cell viability in a dose-dependent manner, with a half-maximal inhibitory concentration (IC50) of 400 µM. Specifically, cell viability was inhibited to 19.7% at 200 µM, while no significant inhibition was observed at 100 µM (Fig. 2A). Therefore, 400 µM PA was set as the maximum concentration for subsequent experiments, with a treatment duration of 24 h.
Fig. 2.
A: Cell viability of HepG2.2.15 cells treated with various concentrations of PA (0–800 µM) for 24 h was assessed by CCK-8 assay. B: Oil red O staining shows the effects of PA on lipid accumulation, cell density, and morphology of HepG2.2.15 cells (magnification, ×200). C: TUNEL fluorescence staining shows the effects of PA on apoptosis (magnification, ×200). D-G: ELISA quantification of HBsAg and HBeAg levels in the supernatant and cell lysate of HepG2.2.15 cells treated with PA.H: qPCR analysis of HBV-DNA levels in cells. All data are presented as mean ± SEM. BSA: Bovine Serum Albumin; PA: palmitic acid
Oil Red O staining showed that at 200 µM PA, cell morphology was altered, and lipid accumulation was significantly increased; in contrast, although significant lipid accumulation was also observed at 400 µM PA, the cell morphology was obviously changed with sparse density (Fig. 2B). These also confirmed that the lipotoxic effect of 400 µM PA on HepG2.2.15 cells was enhanced.To further confirm the apoptotic effect of PA on HepG2.2.15 cells, the TUNEL fluorescence assay found that PA-induced cell apoptosis increased in a dose-dependent manner, with the most significant apoptosis observed at 400 µM compared to 100 and 200 µM (Fig. 2C).
The effects of PA on HBsAg, HBeAg, and HBV-DNA levels in HepG2.2.15 cells showed that compared with the BSA group, there were no significant changes in HBsAg and HBeAg levels in the cell supernatant and cell lysate treated with 100 µM and 200 µM PA, whereas HBsAg and HBeAg levels were significantly decreased in both the cell supernatant and cell lysate at 400 µM PA (Figs. 2D-G). PA inhibited intracellular HBV-DNA levels in HepG2.2.15 cells in a dose-dependent manner. No significant difference was observed between 100 µM PA treatment and the BSA control, while intracellular HBV-DNA was reduced to 48% and 25% after treatment with 200 µM PA and 400 µM PA, respectively (Fig. 2H).
Collectively, the above results suggested that the ability of PA to inhibit HBsAg, HBeAg, and HBV-DNA levels in HepG2.2.15 cells was consistent with its ability to induce cell apoptosis, indicating that PA may clear HBV by inducing cell apoptosis.
OA reverses apoptosis and HBV clearance induced by PA
Oleic acid (OA) is the most common unsaturated fatty acid in the human diet and has been shown to exert a protective effect against PA-induced cellular lipotoxicity by inducing autophagy, alleviating PA-mediated cell apoptosis, oxidative stress, mitochondrial dysfunction, and hepatocellular inflammation [12, 13]. CCK-8 assay results showed that co-treatment of 400 µM PA with different concentrations of OA for 24 h significantly increased the viability of HepG2.2.15 cells, among which 200 µM OA restored cell viability to 74% (Fig. 3A). Therefore, 200 µM OA was used for co-incubation with HepG2.2.15 cells for 24 h in subsequent experiments to reverse 400 µM PA-induced apoptosis. Oil Red O staining results showed that after treatment with PA/OA, lipid accumulation was significantly increased and cell morphology was significantly improved compared with PA treatment alone (Fig. 3B).
Fig. 3.
A: Cell viability of HepG2.2.15 cells treated with 400 µM PA combined with increasing concentrations of OA for 24 h was assessed by CCK-8 assay. B: Oil Red O staining shows lipid accumulation, cell density, and morphological changes in HepG2.2.15 cells with different treatment (magnification, ×200). C-E: Annexin V-PI flow cytometry shows apoptosis under the indicated treatment, with quantitative analysis of early and late apoptotic rates. F-I: ELISA quantification of HBsAg and HBeAg levels in the supernatant and cell lysate of HepG2.2.15 cells following different treatments. All data are presented as mean ± SEM. OA: oil acid
The effect of OA on PA-induced apoptosis in HepG2.2.15 cells was detected by Annexin V-FITC flow cytometry. The results showed that OA had little effect on cell apoptosis. The early and late apoptosis rates induced by PA were as high as 5.4% and 21.1%, respectively; however, OA reduced the PA-induced cell apoptosis rates to 2.7% and 8.5%, indicating that OA significantly reversed PA-induced apoptosis (Figs. 3C-E).
Consistently, while reversing apoptosis, OA also reversed the levels of HBsAg and HBeAg in the supernatant and cell lysate of HepG2.2.15 cells treated with PA, restoring them to the levels of the control group and the OA-alone group (Figs. 3F-I). The reversal of cell death, HBsAg, and HBeAg levels by OA further confirmed that PA may clear HBV by inducing cell apoptosis.
PA induces apoptosis via mitochondrial dysfunction and oxidative stress
To further confirm the potential mechanisms of PA-induced apoptosis in HepG2.2.15 cells, we detected expression of apoptosis-related proteins. The results showed that compared with the BSA control group, 400 µM PA significantly upregulated the expression of apoptosis pathway-related proteins, including Cleaved-Caspase-3 (cleaved active form), Cleaved-Caspase-9, and Cleaved-PARP; moreover, the addition of 200 µM OA rescued the PA-induced increase in these apoptosis-related proteins. However, the Bax/Bcl2 ratio tended to increase but without statistical significance (Figs. 4A-B). Bax and Bcl2 are key apoptotic proteins that regulate mitochondrial membrane permeability to determine whether the apoptotic process is initiated.
Fig. 4.
A-B: Western blotting shows the expression levels of apoptosis related proteins in HepG2.2.15 cells with different treatments, and analyzed with BSA group as the standard. C-D: Fluorescence staining shows the intracellular ROS concentration. E: Fluorescence staining shows the mitochondrial permeability transition pore permeability. F-G: Western blotting detects the expression levels of p38MAPK, ERK, and phosphorylation. Data are presented as mean ± SEM. PARP: poly-ADP-ribose polymerase; ROS: reactive oxygen species; MAPK: mitogen activated protein kinases; ERK: extracellular signal regulated kinase
Therefore, ROS concentration and mitochondrial membrane permeability were detected. Consistent with the aforementioned hypothesis that mitochondrial function is impaired and oxidative stress is increased in CHB patients complicated with MASH. The results demonstrated that 400 µM PA significantly increased ROS concentration, with a marked elevation in fluorescence absorbance; in contrast, no significant difference was observed between 200 µM OA treatment and the BSA group, and co-treatment with PA and OA significantly reduced ROS concentration (Figs. 4C-D). Similarly, fluorescence detection of mitochondrial permeability transition pores showed that PA treatment significantly increased mitochondrial permeability, while combined OA treatment alleviated such mitochondrial damage (Fig. 4E). Collectively, these results indicated that PA may induce apoptosis through mitochondrial damage and oxidative stress, which is consistent with the hypothesis proposed above based on the hepatic transcriptome of human subjects.
In addition, the MAPK signaling pathway is an important molecular pathway regulated by intracellular oxidative stress and mitochondria to modulate apoptosis; thus, the main molecules of the MAPK family were detected. Western blot (WB) results showed that PA treatment significantly increased the phosphorylation levels of ERK and p38MAPK, while OA could reduce their activation (Figs. 4F-G). This suggested that PA activates the ERK and p38MAPK pathways to regulate apoptosis in HepG2.2.15 cells.
Discussion
Some clinical studies show lower serum HBsAg, HBeAg, and HBV-DNA in in patients with CHB complicated with MAFLD than CHB alone, but mechanisms remain unclear. Using human hepatic transcriptome data, we found upregulated apoptosis-related pathways and downregulated mitochondrial/redox genes in patients with CHB and MASH comorbidity. At the cellular level, saturated fatty acid PA-induced lipotoxicity increased HepG2.2.15 mitochondrial membrane permeability and ROS, activated Caspase 9, PARP, and Caspase 3, induced apoptosis, and reduced HBsAg, HBeAg, and HBV-DNA. The unsaturated fatty acid OA restored mitochondrial function, reduced oxidative stress and apoptosis, and reversed HBV antigen levels. Thus, fatty acids may promote hepatocellular apoptosis via mitochondrial damage and oxidative stress, reducing HBV markers.
Current studies suggest that inducing hepatocellular apoptosis promotes HBV-DNA clearance. A study found that antagonizing inhibitors of apoptosis proteins (IAPs) rapidly reduces serum HBV-DNA and HBsAg levels in mice with chronic HBV infection, and promotes hepatic HBcAg clearance and cccDNA reduction [14, 15]. Drugs antagonizing IAPs eliminate HBV replication and the episomal HBV genome in primary human liver organoid models. The TNFα-mediated signaling pathway induces the death of HBV-infected hepatocytes [14, 15]. Treatment of the HepG2.2.15 cell line with IFNγ and/or TNFα results in a significant increase in cell apoptosis, accompanied by decreased HBsAg and HBeAg levels [16]. The traditional Chinese medicine Liuwei Wuling Tablet has been found to clear HBV-pgRNA and HBV-DNA by increasing cell apoptosis in both HBV-infected mice and hepatocytes stably expressing HBV; moreover, compared with the non-HBV-infected mouse model, it exhibited a more significant and selective promotion of hepatic apoptosis in the HBV-replicating mouse model [17]. Humic acid has been reported to inhibit the HBx protein in HepG2.2.15 cells, suppress HBV-DNA replication, and induce cell apoptosis by inhibiting autophagy and mediating endoplasmic reticulum stress [18]. All, OA protected cells from apoptosis and reversed the reduction of HBsAg and HBeAg levels by PA in HepG2.2.15 cells, which indicated that promoting hepatocellular apoptosis can reduce HBV replication.
PA induces hepatocyte death through mitochondrial damage and oxidative stress, while OA reverses that effect. In MAFLD, dysregulated lipid metabolism and lipotoxicity initiate signaling pathways such as MAPK that disrupt intracellular homeostasis. As the main organelle responsible for lipid processing, mitochondria undergo dysfunction under the pressure of oxidizing excessive lipids, produce by-products such as ROS, lead to oxidative stress, and ultimately induce hepatocellular apoptosis [19]. Quantitative proteomic have found that PA-treated HepG2 cells exhibit abnormal mitochondrial function and excessive ROS production, while the unsaturated fatty acids eicosapentaenoic acid and OA can alleviate PA-induced abnormal ATP production and ROS generation by improving mitochondrial function and reducing inflammation [12]. Another study found that exogenous addition of PA induced apoptosis, oxidative stress, endoplasmic reticulum stress, and mitochondrial dysfunction in HepG2 cells and primary rat hepatocytes, while OA addition protected cells from PA-induced damage [13]. In the study, we also found that PA increased mitochondrial membrane permeability and ROS concentration in HepG2.2.15 cells, accompanied by activation of MAPK family molecules, suggesting that PA may regulate the MAPK signaling pathway through mitochondrial damage and oxidative stress, thereby inducing cell apoptosis; moreover, the addition of OA reversed this result.
In addition, some studies have suggested a potential role for fatty acids in reducing HBsAg and HBV-DNA levels in hepatocytes through other mechanisms. Treatment of HepG2.2.15 cells with PA has been shown to significantly inhibit HBV replication markers, including HBV DNA, pgRNA, HBsAg, and HBcrAg. The underlying mechanism involves activation of the miR-122-5p/ sex-determining region Y box 4 axis by fatty acids, which directly suppresses HBV transcriptional regulation [20]. One study demonstrated that hepatocellular steatosis induced by OA, acting through the induction of endoplasmic reticulum (ER) stress, inhibited the secretion of HBsAg and HBV-DNA in HepG2.2.15 cells [21]. Another study indicated that OA treatment led to a sustained increase in autophagy in HepG2.2.15 cells, which may compete with the autophagic machinery utilized by HBV for replication, thereby reducing HBV-DNA levels in the supernatant [22]. In the study, however, no significant effect of OA on HepG2.2.15 cells was observed. Some studies have reported that OA does not induce ER stress or autophagy and may even alleviate these responses triggered by lipotoxicity [23]; however, those findings were based on non-HBV-expressing hepatocytes. HBV-infected cells exhibit heightened sensitivity to stimuli such as oxidative stress and ER stress induced by fatty acids and other agents, leading to significantly enhanced apoptosis compared with non-infected cells [17]. In this study, HepG2.2.15 cells and non-infected hepatocytes were not assessed in parallel; therefore, it remains unclear whether PA and OA differentially promote ER stress, autophagy, and apoptosis in HBV-expressing versus non-expressing hepatocytes. Future investigations at the animal level are warranted to further explore the effects of steatohepatitis on autophagy and apoptosis in the livers of healthy mice versus mice with HBV replication.
However, promoting apoptosis to clear HBV may not be entirely beneficial. Hepatocyte apoptosis induced by lipotoxicity can accelerate hepatic injury, which may promote the progression of MASH, liver fibrosis/cirrhosis, and even hepatocellular carcinoma. Although the coexistence of CHB and hepatic steatosis has been associated with a threefold increase in the HBsAg clearance, it also confers a twofold increased risk of liver fibrosis progression [24]. A prospective study demonstrated that transcriptomic analysis of peripheral blood mononuclear cells (PBMCs) revealed enrichment of inflammatory and apoptotic pathways in patients with HBV-related acute-on-chronic liver failure compared with CHB and healthy controls. Furthermore, inhibition of hepatocellular apoptosis and inflammation ameliorated liver failure in murine models [25]. Accordingly, while fatty acids may promote HBsAg clearance and suppress HBV-DNA replication, they may also potentiate the risk of cirrhosis, liver failure, and hepatocellular carcinoma.
Conclusion
In conclusion, our study demonstrated that PA induces mitochondrial damage and oxidative stress in HepG2.2.15 cells, activates the intrinsic apoptotic pathway, thereby promoting HBV clearance, but OA can reverse that. As common chronic liver diseases, MAFLD and CHB have an increasingly high incidence of comorbidity, and their interaction on disease treatment and prognosis presents new challenges for clinical diagnosis and treatment. We provide new evidence for the potential mechanisms by which fatty acids affect HBV infection and clearance, and offer new ideas and theoretical basis for the treatment strategies of comorbid patients.
Supplementary Information
Acknowledgements
Not applicable.
Abbreviations
- BSA
Bovine Serum Albumin
- CHB
Chronic hepatitis B
- DEGs
Differentially Expressed Genes
- ER
Endoplasmic reticulum
- ERK
Extracellular signal regulated kinase
- FC
Fold Change
- HBcAg
Hepatitis B core antigen
- HBeAg
Hepatitis B e antigen
- HBsAg
Hepatitis B surface antigen
- HBV
Hepatitis B virus
- HCC
Hepatocellular carcinoma
- MAFLD
Metabolic dysfunction–associated fatty liver disease
- MAPK
Mitogen activated protein kinases
- MASH
Metabolic dysfunction-associated steatohepatitis
- PARP
Poly-ADP-ribose polymerase
- PA
Palmitic acid
- ROS
Reactive oxygen species
- OA
Oleic acid
Author contributions
Xin Zheng made substantial contributions to the conception and design of the study, and revised the manuscript; Jiayu Yu performed experiments and analyzed data, and wrote the manuscript.
Funding
Prevention and Control of Emerging and Major Infectious Diseases-National Science and Technology Major Project (2026ZD01912200). Hubei Provincial Natural Science Foundation of China, No. 2025AFA036.
Data availability
The data are publicly available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
References
- 1.Global prevalence. cascade of care, and prophylaxis coverage of hepatitis B in 2022: a modelling study. lancet Gastroenterol Hepatol. 2023;8(10):879–907. [DOI] [PubMed] [Google Scholar]
- 2.Paik JM, Golabi P, Younossi Y, Mishra A, Younossi ZM. Changes in the Global Burden of Chronic Liver Diseases From 2012 to 2017: The Growing Impact of NAFLD. Hepatology (Baltimore MD). 2020;72(5):1605–16. [DOI] [PubMed] [Google Scholar]
- 3.Diao Y, Tang J, Wang X, Deng W, Tang J, You C. Metabolic Syndrome, Nonalcoholic Fatty Liver Disease, and Chronic Hepatitis B: A Narrative Review. Infect Dis therapy. 2023;12(1):53–66. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Yang M, Wei L. Impact of NAFLD on the outcome of patients with chronic hepatitis B in Asia. Liver international: official J Int Association Study Liver. 2022;42(9):1981–90. [DOI] [PubMed] [Google Scholar]
- 5.Li J, Yang HI, Yeh ML, Le MH, Le AK, Yeo YH, Dai CY, Barnett S, Zhang JQ, Huang JF, et al. Association Between Fatty Liver and Cirrhosis, Hepatocellular Carcinoma, and Hepatitis B Surface Antigen Seroclearance in Chronic Hepatitis B. J Infect Dis. 2021;224(2):294–302. [DOI] [PubMed] [Google Scholar]
- 6.Chu CM, Lin DY, Liaw YF. Clinical and virological characteristics post HBsAg seroclearance in hepatitis B virus carriers with hepatic steatosis versus those without. Dig Dis Sci. 2013;58(1):275–81. [DOI] [PubMed] [Google Scholar]
- 7.Chu CM, Lin DY, Liaw YF. Does increased body mass index with hepatic steatosis contribute to seroclearance of hepatitis B virus (HBV) surface antigen in chronic HBV infection? Int J Obes. 2007;31(5):871–5. [DOI] [PubMed] [Google Scholar]
- 8.Tourkochristou E, Assimakopoulos SF, Thomopoulos K, Marangos M, Triantos C. NAFLD and HBV interplay - related mechanisms underlying liver disease progression. Front Immunol. 2022;13:965548. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Schwabe RF, Luedde T. Apoptosis and necroptosis in the liver: a matter of life and death. Nat reviews Gastroenterol Hepatol. 2018;15(12):738–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Zheng RD, Xu CR, Jiang L, Dou AX, Zhou K, Lu LG. Predictors of hepatic steatosis in HBeAg-negative chronic hepatitis B patients and their diagnostic values in hepatic fibrosis. Int J Med Sci. 2010;7(5):272–7. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Osmani Z, Brouwer WP, Grashof DGB, Lim Y, Doukas M, Janssen HLA, van de Werken HJG, Boonstra A. Metabolic dysfunction-associated steatohepatitis reduces interferon and macrophage liver gene signatures in patients with chronic hepatitis B. J Hepatol. 2025;82(4):594–603. [DOI] [PubMed] [Google Scholar]
- 12.Sun Y, Wang J, Guo X, Zhu N, Niu L, Ding X, Xie Z, Chen X, Yang F. Oleic Acid and Eicosapentaenoic Acid Reverse Palmitic Acid-induced Insulin Resistance in Human HepG2 Cells via the Reactive Oxygen Species/JUN Pathway. Genom Proteom Bioinform. 2021;19(5):754–71. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Chen X, Li L, Liu X, Luo R, Liao G, Li L, Liu J, Cheng J, Lu Y, Chen Y. Oleic acid protects saturated fatty acid mediated lipotoxicity in hepatocytes and rat of non-alcoholic steatohepatitis. Life Sci. 2018;203:291–304. [DOI] [PubMed] [Google Scholar]
- 14.Ebert G, Allison C, Preston S, Cooney J, Toe JG, Stutz MD, Ojaimi S, Baschuk N, Nachbur U, Torresi J, et al. Eliminating hepatitis B by antagonizing cellular inhibitors of apoptosis. Proc Natl Acad Sci USA. 2015;112(18):5803–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Clark MP, Huynh T, Rao S, Mackiewicz L, Mason H, Romal S, Stutz MD, Ahn SH, Earnest L, Sozzi V, et al. Clinical stage drugs targeting inhibitor of apoptosis proteins purge episomal Hepatitis B viral genome in preclinical models. Cell Death Dis. 2021;12(7):641. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Shi H, Guan SH. Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon-gamma and tumour necrosis factor-alpha. Liver international: official J Int Association Study Liver. 2009;29(3):349–55. [DOI] [PubMed] [Google Scholar]
- 17.Ge FL, Yang Y, Si LL, Li YH, Cao MZ, Wang J, Bai ZF, Ren ZG, Xiao XH, Liu Y. Inhibition of hepatitis B virus via selective apoptosis modulation by Chinese patent medicine Liuweiwuling Tablet. World J Gastroenterol. 2024;30(13):1911–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Pant K, Yadav AK, Gupta P, Rathore AS, Nayak B, Venugopal SK. Humic acid inhibits HBV-induced autophagosome formation and induces apoptosis in HBV-transfected Hep G2 cells. Sci Rep. 2016;6:34496. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Fromenty B, Roden M. Mitochondrial alterations in fatty liver diseases. J Hepatol. 2023;78(2):415–29. [DOI] [PubMed] [Google Scholar]
- 20.Ren G, Jia K, Yi S, Yin S, Huang G, Zhu Y. Hepatic Steatosis Inhibits Hepatitis B Virus Replication by Promoting miR-122-5p-SOX4. J Viral Hepatitis. 2026;33(3):e70151. [DOI] [PubMed] [Google Scholar]
- 21.Liu Q, Mu M, Chen H, Zhang G, Yang Y, Chu J, Li Y, Yang F, Lin S. Hepatocyte steatosis inhibits hepatitis B virus secretion via induction of endoplasmic reticulum stress. Mol Cell Biochem. 2022;477(11):2481–91. [DOI] [PubMed] [Google Scholar]
- 22.Wang Y, Li J, Wang S, Pang Y, Liu P, Xie B, Dou S, Yang T, Liu X, Shi Y, et al. The hepatitis B virus promotes the progression of non-alcoholic fatty liver disease through incomplete autophagy. Free Radic Biol Med. 2023;204:326–36. [DOI] [PubMed] [Google Scholar]
- 23.Liu X, Li X, Su S, Yuan Y, Liu W, Zhu M, Zheng Q, Zeng X, Fu F, Lu Y, et al. Oleic acid improves hepatic lipotoxicity injury by alleviating autophagy dysfunction. Exp Cell Res. 2023;429(2):113655. [DOI] [PubMed] [Google Scholar]
- 24.Mao X, Cheung KS, Peng C, Mak LY, Cheng HM, Fung J, Peleg N, Leung HH, Kumar R, Lee JH, et al. Steatosis, HBV-related HCC, cirrhosis, and HBsAg seroclearance: A systematic review and meta-analysis. Hepatology (Baltimore MD). 2023;77(5):1735–45. [DOI] [PubMed] [Google Scholar]
- 25.Yang H, Cai Q, Xin J, Liang X, Hassan HM, Chen J, He L, Sun S, Guo B, Ma S, et al. SEMA6B induces macrophage-mediated inflammation and hepatocyte apoptosis in hepatitis B virus-related acute-on-chronic liver failure. Theranostics. 2024;14(13):5200–18. [DOI] [PMC free article] [PubMed] [Google Scholar]
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Data Availability Statement
The data are publicly available from the corresponding author on reasonable request.