Triflusal targeting COX-1 suppresses HBV infection in vitro and in vivo

Background : Chronic hepatitis B virus infection poses a persistent global public health burden with limited therapeutic options, and host-targeted antiviral strategies are urgently needed. Cyclooxygenase-1, encoded by the PTGS1 gene, has emerged as a potential host factor regulating viral replication, while triflusal, a specific COX-1 inhibitor, modulates intracellular cyclic adenosine monophosphate levels and its anti-HBV activity remains uncharacterized. Methods : Huh7 cells were transfected with gradient doses of HBV 1.3-fold overlength plasmid and treated with triflusal (0–20 μM). Real-time quantitative PCR was used to detect PTGS1 mRNA and HBV 3.5-kb RNA levels; enzyme-linked immunosorbent assay measured hepatitis B surface antigen, hepatitis B e antigen and intracellular cAMP levels. MTT assay assessed triflusal cytotoxicity. PTGS1 overexpression and siRNA knockdown were performed to verify its role in HBV replication. An acute HBV mouse model (AAV-HBV1.3) was used to evaluate in vivo anti-HBV efficacy of triflusal (30/50 mg/kg). Western blot and functional assays explored the involvement of the cAMP/protein kinase A (PKA)/cAMP response element-binding protein (CREB) signaling pathway. Results : PTGS1 mRNA expression and HBV replication markers (3.5-kb RNA, HBsAg, HBeAg) were upregulated in a plasmid dose-dependent manner in HBV-transfected Huh7 cells. Triflusal inhibited HBV antigen secretion and 3.5-kb RNA transcription in a concentration-dependent manner in vitro, with no obvious cytotoxicity at effective antiviral doses. PTGS1 overexpression enhanced HBV replication, while its knockdown suppressed viral markers, confirming a positive correlation between PTGS1 and HBV replication. Triflusal treatment elevated intracellular cAMP levels by inhibiting COX-1/TXA ₂ signaling, activating the PKA/CREB pathway, which suppressed HBV core promoter activity and disrupted viral core particle assembly. In vivo, triflusal significantly reduced serum HBeAg and hepatic HBV 3.5-kb RNA levels in HBV-infected mice, with good biocompatibility and no acute toxicity. Conclusions : Triflusal exerts potent anti-HBV effects in vitro and in vivo by targeting COX-1 to activate the cAMP/PKA/CREB signaling pathway, which suppresses HBV replication at both transcriptional and post-transcriptional levels. Our findings identify COX-1 as a promising host-directed therapeutic target for HBV infection and provide a theoretical basis for repurposing triflusal as a novel adjunctive treatment for chronic HBV infection.