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

## TL;DR

Luteolin, a natural compound, inhibits bovine viral diarrhea virus replication and reduces inflammation by targeting viral processes and immune pathways.

## Contribution

This is the first study to show luteolin's dual antiviral and anti-inflammatory effects against BVDV via the NF-κB/STAT3-NLRP3 pathway.

## Key findings

- Luteolin inhibits BVDV replication by targeting internalization and replication stages.
- Luteolin suppresses NF-κB/STAT3-NLRP3 signaling, reducing inflammatory mediators like TNF-α and IL-8.
- Luteolin shows no cytotoxicity at effective concentrations and inhibits NS5B polymerase activity.

## Abstract

This study focuses on the inhibitory effect and underlying mechanisms of luteolin, a natural flavonoid compound, against bovine viral diarrhea virus (BVDV). Experiments on MDBK cells revealed that luteolin exhibits no cytotoxicity at concentrations below 20 μM and inhibits the replication of BVDV-1m strain in a dose-dependent manner. Its core mechanisms involve two aspects: first, it directly targets the internalization and replication stages of the viral life cycle, inhibits the activity of NS5B polymerase, and exerts no direct effect on viral attachment, release, or the viral particles themselves; second, it blocks the activation of the NF-κB/STAT3-NLRP3 signaling axis by inhibiting the phosphorylation of p65 (Ser536) and STAT3 (Ser727), downregulating the transcriptional expression of NLRP3 and pro-caspase-1, preventing the cleavage of caspase-1 into the p20 subunit, and inhibiting the maturation of IL-1β/IL-18, thereby reducing the excessive production of inflammatory mediators such as TNF-α and IL-8. This study is the first to confirm that luteolin possesses dual antiviral and anti-inflammatory activities, providing crucial experimental evidence and a theoretical basis for the development of flavonoid-based drugs against Flaviviridae viruses. Meanwhile, it points out that the clinical application of luteolin needs to address issues such as low bioavailability, potential immunosuppression, and viral drug resistance.

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.