Naringenin alleviates LPS-induced mastitis by inhibiting inflammation and regulating gut microbiota

Bacterial bovine mastitis causes severe mammary inflammation, posing a major challenge to the global dairy industry. Combining antibiotics with anti-inflammatory agents may reduce tissue damage and improve therapeutic efficacy by reducing the inflammation induced by endotoxin release from antibiotics-lysed bacteria. Natural products offer promising anti-inflammatory alternatives. This study investigates naringenin (NG), a citrus flavonoid, for its potential in the treatment of bovine mastitis. We found that NG significantly reduced the production of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8) in lipopolysaccharide (LPS)-induced bovine and mouse mammary epithelial cells by downregulating their encoding genes. Mechanistically, the activation of the NF-κB pathway was suppressed by NG through its direct binding to IκBα (binding energy: −24.05 kcal/mol) and the subsequent inhibition of IκBα phosphorylation, thereby inhibiting proinflammatory cytokines secretion. Moreover, our LPS-induced mouse mastitis model successfully simulated the pathological properties of bovine mastitis as evidenced by H&E staining. NG pretreatment effectively alleviated LPS-induced mammary tissue damage by the regression of mammary gland swelling, the reduction of neutrophil infiltration, and the alleviation of acinar structure damage. Furthermore, the 16S rRNA sequencing showed that NG reversed LPS-induced gut dysbiosis, restored beneficial bacterial populations (such as Firmicutes and Bacteroidetes), and enhanced blood-milk barrier integrity via tight junction proteins reorganization. Collectively, our results suggest that NG ameliorates mastitis by inhibiting inflammation via NF-κB signal inhibition and regulating the gut microbiota through the gut-mammary axis, providing an experimental basis for NG as a natural therapeutic agent for the treatment of bovine mastitis.