TNFR1–Caspase−2 apoptotic signaling is associated with reduced HBV replication markers during free fatty acid exposure in vitro

The coexistence of chronic hepatitis B (CHB) and metabolic dysfunction–associated steatotic liver disease (MASLD) is increasingly prevalent. Clinical and experimental evidence suggests an inverse association between hepatic steatosis and hepatitis B virus (HBV) replication; however, the underlying molecular mechanisms remain poorly defined. This study aimed to investigate the effect of free fatty acid–induced steatosis on HBV replication and the associated apoptotic signaling pathways in vitro. HepG2.2.15 cells, a stable HBV-producing hepatocyte cell line, were treated with a free fatty acid (FFA) mixture of sodium oleate and sodium palmitate at a 2:1 ratio to induce steatosis. Intracellular triglyceride content and Oil Red O staining were used to confirm lipid accumulation. Apoptosis was assessed by flow cytometry. The expression levels of tumor necrosis factor-alpha (TNF-α), tumor necrosis factor receptor 1 (TNFR1), Caspase−2, Caspase−8, and HBV DNA were analyzed before and after FFA treatment. TNF-α neutralization and pharmacological inhibition of Caspase−2 were performed to assess the contribution of endogenous TNF-α signaling and Caspase−2 enzymatic activity. FFA treatment induced lipid accumulation in HepG2.2.15 cells and was accompanied by a modest increase in apoptosis. FFA-treated cells exhibited elevated TNF-α secretion and increased TNFR1, Caspase−8, and Caspase−2 expression, along with reduced HBV DNA, pgRNA, HBsAg, and HBeAg levels. TNF-α neutralization partially restored HBV replication markers and attenuated TNFR1–Caspase−8/Caspase−2 signaling. Pharmacological inhibition of Caspase−2 activity with Z-VDVAD-FMK reduced Caspase−2 enzymatic activity and partially reversed the FFA-induced reduction of HBV replication markers. Free fatty acid–induced steatosis was accompanied by activation of TNF-α/TNFR1/Caspase−8/Caspase−2 signaling and reduced HBV replication markers in HepG2.2.15 cells. TNF-α neutralization and Caspase−2 inhibition partially reversed these effects, suggesting that TNF-α/TNFR1–Caspase−2 signaling contributes to, but may not fully account for, the reduction of HBV replication markers during lipotoxic stress.