Sodium Butyrate Mitigates Pseudomonas aeruginosa Infection in bMECs Associated with the Modulation of TLR4/MAPK Pathway and Improvement of Autophagic Markers.

Pseudomonas aeruginosa (PA) is a formidable environmental pathogen. It causes severe and refractory bovine mastitis. The escalating threat of antimicrobial resistance requires new non-antibiotic therapies. These alternative therapies should focus on targeting host-directed responses. Sodium butyrate (SB) is a prominent short-chain fatty acid. It possesses potent immunomodulatory properties. However, its protective mechanisms against PA-induced mammary injury remain elusive. This study investigated the efficacy and underlying molecular mechanisms of SB. bMECs were pretreated with 0.5 mmol/L SB for 18 h prior to challenge with P. aeruginosa (1 × 107 CFU/mL, 6 h). We evaluated its ability to alleviate PA-induced cytotoxicity in bovine mammary epithelial cells (bMECs). Flow cytometry and ELISA demonstrated the strong protective effects of SB. SB pretreatment significantly reduced PA-induced cellular apoptosis. It also suppressed the hypersecretion of pro-inflammatory cytokines, including IL-6 and TNF-α. Next, transcriptomic sequencing (RNA-seq) was performed. We identified 589 differentially expressed genes (DEGs) between the PA-challenged and SB-treated groups. These DEGs were significantly enriched in the Toll-like receptor (Tlr), Mapk, and autophagy signaling pathways. We subsequently conducted molecular validations via RT-qPCR, Western blotting, and immunofluorescence. The results revealed that SB significantly suppressed the overactivation of the TLR4/MAPK cascade. Specifically, SB significantly downregulated the expression of TLR4. It also decreased the downstream phosphorylation levels of p38, ERK, and JNK. Furthermore, PA infection induced a severe blockade of autophagic flux. This dysfunction was evidenced by the concurrent cellular accumulation of LC3-II and the autophagic substrate p62. Remarkably, SB intervention was associated with the reduction in autophagic marker accumulation, evidenced by facilitated lysosomal clearance of p62. Collectively, sodium butyrate protects bMECs against PA-induced inflammation and apoptosis. These protective effects are closely associated with the suppression of the TLR4/MAPK signaling cascade and the alleviation of autophagic marker accumulation. This highlights the potential of SB as a promising preventive strategy for the clinical management of bovine mastitis.