TNBS诱导的草鱼肠炎模型

A TNBS-induced enteritis model in grass carp

通过建立草鱼肠道炎症模型，为经济鱼类炎症性肠病发病机理研究及药物筛选提供实验平台。于水温28℃条件下，从草鱼肛门插管注入0．25mL浓度为2．5％的三硝基苯磺酸（TNBS）50％乙醇溶液，诱发草鱼肠炎，注入等体积0．7％生理盐水作为对照，观察草鱼机体损伤程度、肠道组织病理，分析不同肠段组织内髓过氧化物酶（MPO）活性及IL-1β、IL-8和TNF-a等炎性相关基因表达量。病理观察结果显示，实验期间实验鱼未出现死亡现象，经TNBS诱导后出现较严重的肠道炎症，肠灌TNBS后1d，草鱼肠粘膜严重充血溃烂，肠道组织中出现大量炎性细胞浸润；3d后出现腹水，损伤部位招募大量炎性细胞，溃疡开始愈合；7d后炎性细胞逐渐减少，出现杯状细胞，呈慢性炎症；14d时腹水减少，肠粘膜中杯状细胞丰富，21d仅有少量炎性细胞，肠道组织基本恢复正常。TNBS诱导后1d，MPO活性极显著升高，3d后明显下降，至第7天趋于正常，与组织病理变化趋势一致。TNBS致炎后第1天，IL-1β、IL-8和TNF-a等炎性相关基因表达量极显著升高，3d时开始下降，3～14d表达量略高于对照组，急性炎症转为轻微的慢性炎症，21d时基本恢复到正常水平。结果还显示，不同肠段间的炎症反应存在差异，第三、四肠段明显较第一、二肠段严重。本研究构建的草鱼TNBS肠炎模型稳定，可作为鱼类肠炎病理机制研究和新型药物筛选的良好工具。

Fish models of intestinal inflammation are still fairly scarce though dozens of such models have been established in other animals. In this study, we aimed to develop a TNBS-induced enteritis model in grass carp. To induce the intestinal inflammation ,0.25 mL TNBS/ethanol solution （at a concentration of 2.5% TNBS in 50% ethanol ） was instilled into the intestine of each fish （70 ±5 g ） via the anus at a water temperature of 28℃. An equal volume of 0.7% physiological saline was instilled as control. After TNBS enema, the intestinal inflammation was assessed by examining the degree of body injury, and the intestinal histopathological characteristics, and by analyzing myeloperoxidase （MPO）activities and gene expression patterns of inflammatory cytokines, IL-1β, IL-8 and TNF-a, in different intestinal segments. The pathological results indicated that all fish survived during the whole observation period following TNBS treatment, and one day after TNBS enema,serious congestion and fester in intestinal mucosa were observed, and abundant inflammatory cells infiltrated into intestinal tissues. On the third day, severe abdominal dropsy occurred, large amounts of inflammatory cells were recruited at the damaged site, and healing of intestinal ulcers appeared. On the seventh day, the number of inflammatory cells decreased gradually, while that of goblet cells increased. Afterwards the abdominal dropsy reduced, the goblet cells densely arranged in the intestinal mucus layer. On the twenty-first day after TNBS enema, the fish recovered nearly into normal conditions, and only a few inflammatory cells could be found. In addition, our results showed that TNBS administration induced similar changes in MPO activities between different intestinal segments. MPO activities in the intestine remarkably increased after TNBS treatment, peaked on the first day. Subsequently, the activities decreased rapidly. On the seventh day, there was no significant difference between the TNBS-induced and the control groups. This pattern in MPO activity appeared to be consistent with those results obtained from the histopathological observations. Moreover, we also found that, after TNBS enema, the mRNA expression patterns of inflammatory cytokine genes, IL-1β, IL-8 and TNF-a, showed roughly the same trend as those of MPO activities. Our results also showed that different intestinal segments exhibited significant differences in inflammatory response,and TNBS induced more severe inflammatory signs in the 3rd and 4th segments than the 1st and 2na ones. Overall,these results suggest that we have developed a reliable and reproducible TNBS- induced enteritis model in grass carp. This grass carp model will not only extend our understanding in pathological mechanisms involved in intestinal inflammation of teleost fish,but also provide a tool to screen novel drugs for the treatment of inflammatory diseases in grass carp and other aquacultural animals.