【摘要】 【目的】利用系统生物学分析和鸡胚十二指肠上皮细胞模型探究热应激诱导肠道损伤的作用机制，筛选出家禽热应激诱导肠道损伤的作用靶标，为缓解热应激诱导的肠细胞氧化应激提供理论支撑。【方法】基于NCBI数据库筛选热应激诱导肠道损伤的作用靶标，通过GO功能注释和KEGG信号通路富集分析获得相关代谢通路；分离鸡胚十二指肠上皮细胞构建42℃热应激模型，通过检测细胞损伤病理、细胞活性、乳酸脱氢酶（LDH）活性、抗氧化酶活性、丙二醛（MDA）含量及关键基因（Nrf2和HSP70）表达情况等，进一步确认热应激对肠细胞的损伤作用。【结果】将热应激作用靶标与肠道损伤作用靶标取交集，共获得23个热应激诱导肠道损伤的作用靶标，分别是BCL2、CD4、CFTR、ERN1、FOXM1、FOXP3、HMOX1、HSF1、HSP90AA1、HSP70、HSPA4、HSPA8、JUN、MAPK1、MTOR、NFKB1、NLRP3、PLK1、RHOB、SIRT1、SUMO2、TP53和YAP1。KEGG信号通路富集分析发现热应激诱导肠道损伤与NOD-样受体信号通路、沙门氏菌感染、凋亡、细胞衰老及内质网内蛋白加工等有关；GO功能注释分析发现热应激诱导肠损伤与细胞缺氧反应、细胞对镉离子反应、细胞对热反应及热休克蛋白结合等有关。鸡胚十二指肠上皮细胞体外热应激模型验证发现，热应激能导致鸡胚十二指肠上皮细胞空泡变性，严重时则导致细胞核浓缩深染的变性坏死；热应激还会引起鸡胚十二指肠上皮细胞的活性降低，而细胞上清液中的LDH含量升高；此外，随着热应激时间的延长，鸡胚十二指肠上皮细胞内的MDA含量不断上升，而超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的活性均呈下降趋势；热应激导致鸡胚十二指肠上皮细胞内Nrf2基因表达下调、HSP70基因表达上调。【结论】细胞内氧化—抗氧化失衡是热应激诱导肠道损伤的重要因素，而热休克蛋白是机体激活细胞抵御热应激损伤的重要保护机制。更多还原

【Abstract】 【Objective】Based on systems biology analysis and chicken embryo duodenal epithelial cell model,this paper explored the mechanism of heat stress-induced intestinal injury,to screen the action targets of heat stress-induced intestinal damage in poultry,and to provide theoretical support for alleviating heat stress-induced oxidative stress in enterocytes.【Method】The NCBI database was used to screen the action targets of heat stress-induced intestinal injury,and the relevant metabolism pathways were obtained by GO functional annotation and KEGG signaling pathway enrichment analysis. Chicken embryo duodenal epithelial cells were separated to construct the heat stress model at 42 ℃. And by using pathological detection,cell activity,lactate dehydrogenase(LDH)activity,antioxidant enzyme activity,malondialdehyde(MDA)content and expression of key genes(Nrf2 and HSP70),the damages of heat stress on intestinal cells were confirmed.【Result】By taking the intersection of heat stress action targets and intestinal injury action targets,a total of 23 action targets of heat stress-induced intestinal injury were obtained,including BCL2,CD4,CFTR,ERN1,FOXM1,FOXP3,HMOX1,HSF1,HSP90AA1,HSP70,HSPA4,HSPA8,JUN,MAPK1,MTOR,NFKB1,NLRP3,PLK1,RHOB,SIRT1,SUMO2,TP53 and YAP1. KEGG signaling pathway enrichment analysis found that heat stressinduced intestinal injury were associated with NOD-like receptor signaling pathways,salmonella infection,apoptosis,cellular senescence,and protein processing in the endoplasmic reticulum. GO functional annotation analysis found that heat stress-induced intestinal injury were related to cell hypoxia response,cellular response to cadmium ion,cell response to heat,and heat shock protein binding. The heat stress model of chicken embryo duodenal epithelial cells in vitro showed that heat stress could cause cells vacuolar degeneration and in severe cases cause nucleus concentrated deep staining that leading degenerative necrosis;heat stress could reduce cell activity of chicken embryo duodenal epithelial cells and increase LDH content in cell supernatant;in addition,with the prolongation of heat stress,the MDA content in the chicken embryo duodenal epithelial cells increased,while the activities of superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)and catalase(CAT)activities showed a decreasing trend;heat stress resulted in down-regulation of Nrf2 gene and up-regulation of HSP70 gene in chicken embryo duodenal epithelial cells.【Conclusion】Intracellular oxidative-antioxidative imbalance is an important factor in heat stress-induced intestinal injury,and heat shock proteins are important protective mechanism against heat stress-induced injury.更多还原