Rosmarinic acid alleviates E. miricola neuroinflammation in bullfrogs with TLR5/MyD88/NF-κB suppression

The “torticollis disease” in bullfrogs poses a severe threat to aquaculture, and infection with E. miricola has been identified as a key causative factor. This study aimed to develop targeted prevention and control strategies. Through in vitro antibacterial screening, rosmarinic acid (RosA) was identified to exhibit significant inhibitory activity against E. miricola , with stable activity after heat treatment at 120 °C for 5 min. Animal experiments showed that dietary supplementation with RosA (1 and 4 g/kg) for 8 weeks significantly reduced the morbidity (15%–20%) and mortality (25%–30%) of bullfrogs after E. miricola challenge. Mechanistic studies revealed that RosA intervention significantly suppressed the overactivated inflammatory signaling pathways (e.g., TLR, NF-κB) in the brain tissues of infected bullfrogs, leading to downregulated expression of key genes ( tlr5 , myd88 , nfκb , tnfα , and il1β ). Further validation using the frog kidney epithelial cell model demonstrated that RosA (100–200 μM) effectively antagonized E. miricola infection-induced cell apoptosis and the significant upregulation (3–15 fold) of key inflammatory genes ( tlr5 , myd88 , nfκb , tnfα , il1β , and cxcl8 ). Notably, the combined use of RosA and a MyD88-specific inhibitor produced a synergistic effect, significantly enhancing the inhibitory effect on downstream genes ( nfκb , cxcl8 , tnfα , and il1β ), confirming that the TLR5/MyD88/NF-κB signaling axis is an important pathway for E. miricola -induced inflammation. This study reveals that E. miricola infection is associated with the activation of the TLR5/MyD88/NF-κB pathway in neuroinflammation, and demonstrates that RosA exerts dual antibacterial and anti-inflammatory effects, which include the suppression of this pathway's activation. These findings suggest a potential mechanism for RosA-mediated protection and provide important theoretical support for developing natural compound-based feed additives to control aquatic bacterial diseases.