Abstract

Aeromonas hydrophila infection is a major bacterial disease that seriously threatens the healthy development of crucian carp aquaculture, highlighting the important practical value of developing safe and effective antibiotic alternatives. This study investigated the protective effects of the natural flavonoid luteolin against A. hydrophila infection in crucian carp and explored its underlying molecular mechanisms. The results showed that dietary supplementation with luteolin (20 μg/g diet per day for 4 weeks) significantly improved the survival rate of infected fish and alleviated histopathological damage in the spleen and liver. Mechanistic analyses revealed that luteolin effectively suppressed excessive apoptosis in the spleen induced by infection, reducing TUNEL-positive signals by 8.2-fold, while downregulating the expression of the pro-apoptotic genes IL1β, bax, and caspase3. Further in vitro experiments demonstrated that luteolin inhibited A. hydrophila-induced cell death and suppressed bacterial replication. Meanwhile, luteolin significantly inhibited the activation of the NF-κB p65 promoter and reduced its nuclear translocation. At the late stage of infection, luteolin also suppressed the mRNA expression of the inflammatory cytokines IL1β and IL-6, as well as the apoptotic cascade. Additional experiments showed that both caspase-3 activity and apoptosis were attenuated by luteolin. In the SN50-treated EPC cell model, luteolin acted synergistically with SN50 to suppress the expression of IL1β and bax, while upregulating the anti-apoptotic factor bcl-2. In contrast, supplementation with the inflammatory inducer recombinant IL1β (rIL1β) restored the apoptosis that had been suppressed by luteolin. Taken together, these findings suggest that luteolin may alleviate A. hydrophila-induced tissue damage and enhance disease resistance in crucian carp by inhibiting the inflammation-apoptosis axis.