Abstract:The intestine is not only the primary organ responsible for digestion, absorption, and transport of nutrients in fish, but also one of the most important immune defense barriers, playing a crucial role in maintaining physiological homeostasis and overall health. Under intensive aquaculture conditions, increasing stocking density, environmental stressors, and elevated exposure to pathogens have markedly increased the incidence of intestinal diseases. Among these, enteritis has emerged as a major constraint to the sustainable development of the aquaculture industry due to its high morbidity, strong lethality, and substantial economic losses. In commercially important marine fish species such as the leopard coral trout (Plectropomus leopardus), enteritis severely impairs growth performance, feed utilization efficiency, and survival rates, thereby threatening both production efficiency and economic viability.At present, the prevention and treatment of enteritis in P. leopardus primarily rely on chemical drugs and antibiotics. However, the long-term or excessive use of these agents can disrupt intestinal mucosal immune homeostasis, alter the intestinal microbiota, induce antimicrobial resistance, and ultimately reduce the overall disease resistance of fish. Therefore, there is an urgent need to establish reliable, reproducible, and biologically relevant intestinal inflammation models that can facilitate systematic investigations into the mechanisms underlying fish enteritis and support the development of effective and environmentally friendly prevention strategies. Given the pivotal role of bile acids in maintaining intestinal barrier integrity, regulating inflammatory signaling pathways, and modulating gut microbial composition, they have been increasingly recognized as promising green additives for alleviating intestinal inflammation. Nevertheless, the evaluation of such functional compounds requires a well-established and standardized experimental enteritis model. In this context, the present study aimed to evaluate and compare the suitability of three commonly used chemical inducers—dextran sulfate sodium (DSS), oxazolone (OXZ), and 2,4,6-trinitrobenzenesulfonic acid (TNBS)—for establishing an acute intestinal inflammation model in juvenile P. leopardus. Specifically, this study sought to clarify the dose-dependent effects and pathological characteristics induced by these agents, thereby identifying an optimal modeling strategy for subsequent mechanistic and intervention studies.To achieve this objective, juvenile P. leopardus were fed a basal diet supplemented with different concentrations of chemical inducers for one week: DSS at 0.5% and 1.0%, OXZ at 0.2%, 0.4%, and 0.6%, and TNBS at 0.5% and 1.0%. During the experimental period, growth performance parameters were monitored, and the disease activity index (DAI) was evaluated based on behavioral changes, feeding activity, and external pathological signs. At the end of the feeding trial, intestinal tissues were collected for histological examination to assess morphological alterations, while myeloperoxidase (MPO) activity was measured as an indicator of inflammatory cell infiltration. In addition, the expression levels of inflammation-related cytokine genes and tight junction proteins were analyzed to elucidate the molecular responses associated with intestinal inflammation.The results demonstrated that high doses of chemical inducers, particularly 1.0% DSS and 1.0% TNBS, successfully induced pronounced pathological features of enteritis in juvenile P. leopardus. These features included marked intestinal congestion and redness, intestinal distension, and abnormal mucus secretion. Histological analysis revealed severe structural damage to the intestinal mucosa, characterized by a significant reduction in mucosal fold height, disruption of epithelial architecture, and extensive infiltration of inflammatory cells. Consistently, MPO activity was significantly elevated in these groups (P < 0.05), confirming the occurrence of acute inflammatory responses. At the molecular level, fish fed DSS-supplemented diets exhibited significant upregulation of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β), whereas TNBS treatment predominantly induced marked increases in IL-1β and interferon-γ (IFN-γ) expression (P < 0.05). Moreover, both DSS and TNBS treatments resulted in significant downregulation of the tight junction proteins zonula occludens-1 (ZO-1) and Claudin-1, indicating impaired intestinal barrier integrity. In contrast, OXZ caused relatively mild damage to the intestinal mucosal structure; however, it significantly suppressed the expression of the anti-inflammatory cytokine IL-10 and markedly inhibited growth performance (P < 0.05), suggesting a different inflammatory mechanism and higher systemic burden.Notably, a lower concentration of TNBS (0.5%) was sufficient to induce mild but recognizable pathological features of enteritis, whereas OXZ required relatively higher concentrations to elicit evident inflammatory symptoms. Furthermore, the severity of intestinal inflammation increased significantly with increasing doses of all three chemical inducers. Among the tested treatments, the 0.75% DSS group exhibited no mortality throughout the experimental period while still displaying typical enteritis characteristics. This concentration effectively balanced fish survival and inflammatory induction, and was therefore identified as the optimal modeling condition in the present study.In conclusion, DSS and TNBS are more suitable than OXZ for establishing acute intestinal inflammation models in juvenile P. leopardus, although they induce inflammation through distinct immunopathological mechanisms. The selection of chemical inducers for fish enteritis models should comprehensively consider species-specific physiological characteristics, administration routes, and target immune pathways. This study provides a systematic comparison of DSS-, TNBS-, and OXZ-induced intestinal inflammation in P. leopardus, offering valuable methodological references for the construction of enteritis models in economically important fish species. More importantly, it lays a solid scientific foundation for future investigations into the pathogenesis of fish enteritis and the development of effective, sustainable, and antibiotic-independent prevention and control strategies.
