Luteolin Inhibits Bovine Viral Diarrhea Virus Replication by Disrupting Viral Internalization and Replication and Interfering with the NF-κB/STAT3-NLRP3 Inflammasome Pathway

Bovine viral diarrhea virus (BVDV) causes severe mucosal inflammation in cattle, and effective treatment options remain limited. Dysregulated activation of the NLRP3 inflammasome, driven by NF-κB and STAT3 signaling, may exacerbate disease pathogenesis, highlighting this axis as a potential therapeutic target. Although traditional Chinese medicine has shown promise in antiviral and anti-inflammatory applications, it remains unclear whether it can inhibit BVDV replication via the NF-κB/STAT3-NLRP3 pathway. The present study aimed to clarify the inhibitory effect of luteolin on bovine viral diarrhea virus (BVDV) replication, and to elucidate its underlying mechanisms from two perspectives: interference with viral internalization and replication processes, as well as regulation of the NF-κB/STAT3-NLRP3 inflammasome pathway. Collectively, this work intended to provide experimental evidence and theoretical support for the development of luteolin as a natural anti-BVDV agent. To this end, BVDV-infected MDBK cells were treated with gradient concentrations of luteolin, followed by quantification of viral load using qRT-PCR and Western blot assays. Meanwhile, the activation status of the NF-κB/STAT3-NLRP3 signaling pathway was evaluated via immunofluorescence staining and luciferase reporter gene assays. Our results demonstrate that luteolin exhibits potent dual antiviral activity against cytopathic BVDV-1m in MDBK (Madin-Darby Bovine Kidney) cells, effectively suppressing both viral replication and inflammatory responses. At non-cytotoxic concentrations, luteolin specifically inhibited the internalization and replication stages of the viral lifecycle, accompanied by reduced NS5B polymerase activity. Importantly, luteolin disrupted the NF-κB/STAT3-NLRP3 axis by suppressing phosphorylation of p65 (Ser536) and STAT3 (Ser727), downregulating NLRP3 and pro-caspase-1 expression, and inhibiting caspase-1 cleavage (p20) as well as maturation of IL-1β and IL-18. Consequently, it attenuated the overexpression of TNF-α and IL-8. To our knowledge, this is the first report of a single compound simultaneously targeting multiple stages of the BVDV lifecycle and counteracting NLRP3-mediated immunopathology, offering a strategic basis for developing flavonoid-based therapies against Flavivirus infections.