SCA-22 capsules resist Aeromonas hydrophila through enhanced resistance and maintained microbial homeostasis in Megalobrama amblycephala

Given the challenges of enzymatic degradation and low bioavailability in oral cytokine delivery for aquaculture, this study developed sodium alginate/carboxymethyl chitosan/astaxanthin (SA/CMCS/ASTA, SCA) capsules encapsulating recombinant Megalobrama amblycephala interleukin-22 (SCA-22). Optimal preparation parameters (1.5 % SA, 0.96 % CMCS, 0.01 % ASTA, 6.5 % CaCl 2 ) achieved an encapsulation efficiency of 87.00 ± 2.65 %. In vivo imaging revealed prolonged intestinal retention of SCA-22 compared to free rMaIL-22. Histopathological analysis confirmed the biocompatibility of SCA-22 with no adverse effects on intestinal morphology or hepatosplenic tissues after 7-day administration. Challenge experiments demonstrated SCA-22 significantly enhanced resistance against Aeromonas hydrophila infection, alleviating characteristic enteritis pathologies including submucosal edema and lamina propria swelling. Subsequently, we elucidated the possible protective pathways of SCA-22 after A. hydrophila infection. The results showed that SCA-22 significantly improved the intestinal mucosal barrier function by promoting the proliferation of goblet cells, improving the continuity of the mucus layer, and restoring the expression of zona occludens 1 (ZO-1) protein. Additionally, SCA-22 promoted nuclear factor erythroid 2-related factor 2 (NRF2) nuclear expression, enhanced the activity of CAT and T-SOD, and inhibited ROS accumulation to strengthen antioxidant defense. In inflammation regulation, SCA-22 exerted anti-inflammatory effects by curbing pro-inflammatory factors (nlrp3 , asc , caspase-1 , il-1β , tnf-α , and il-6 ) while boosting anti-inflammatory mediators ( il-10 and tgf-β ). At the microbial level, SCA-22 contributed to the maintenance of intestinal microbial homeostasis, significantly reduced the abundance of the pathogenic Aeromonas genus, and enhanced the functions of intestinal microbes in aspects such as antibiotic biosynthesis and metabolic pathways. In conclusion, these findings highlight the potential of SCA-22 in combating bacterial enteritis in aquaculture and advance the application of recombinant cytokines in aquaculture disease management.