基于MAPK-NF-κB/AP-1信号通路探讨大豆皂甙致大菱鲆肠道炎性损伤的机制

Soybean meal induced enteritis (SBMIE) limits its application in carnivorous fish and soyasaponins in soybean meal play a key role in SBMIE. Our previous work proved that soyasaponins could induce enteritis to turbot Scophthalmus maximus, which is an important carnivorous farmed fish in China. However, the molecular mechanisms on soyasaponins-induced enteritis are still unclear. MAPK-NF-κB/AP-1 is one of the most vital signaling pathways to regulate the inflammatory responses, but there is no information on the influence of soyasaponins to this signaling pathway in fish. The present project is to work on turbot, first investigates the influence of soyasaponins on the primary cultured intestinal epithelial cells inflammatory responses, oxidative stress, MAPK-NF-κB/AP-1 signalling pathway genes transcription, protein expression and phosphorylation, NF-κB/AP-1 DNA binding activity. The role of five signaling molecules of this pathway in mediating soyasaponins-induced inflammatory responses was also investigated by applying their specific signaling inhibitors. And then, by conducting feeding experiment, the present project investigates the influence of dietary soyasaponins on the inflammatory responses, oxidative stress and MAPK-NF-κB/AP-1 signaling pathway of intestine of turbot. Finally, based on above in vitro and in vivo research, the present project will clarify the mechanisms that how soyasaponins induce enteritis by MAPK-NF-κB/AP-1 signaling pathway and lay the foundation for further control SBMIE by regulating related signaling pathways.

豆粕引起肠道炎性损伤是限制其在肉食性鱼饲料中应用的主要因素之一，而大豆皂甙是豆粕致炎的关键因素。前期研究证明大豆皂甙能够引起我国重要的肉食性养殖鱼类大菱鲆肠道炎性损伤，但其致炎机制尚不明确。MAPK-NF-κB/AP-1信号通路是调控机体炎性反应的关键信号通路之一，但大豆皂甙对鱼类该信号通路的影响尚无报道。本研究以大菱鲆作为研究对象，首先探索大豆皂甙对大菱鲆原代培养肠道上皮细胞的炎性反应、氧化应激水平和MAPK-NF-κB/AP-1信号通路相关基因的转录水平、相关蛋白表达和磷酸化水平以及转录因子NF-κB和AP-1的DNA结合活性等指标的影响，并利用抑制剂检测各信号分子在介导大豆皂甙致炎反应中的作用。其次，通过养殖实验探索大菱鲆炎性反应、氧化应激水平水平和该信号通路随大豆皂甙浓度梯度的动态变化。最后，综合离体和在体水平研究阐明大豆皂甙引起大菱鲆肠道炎性损伤的机制，为下一步通过调节相关信号分子防治这种炎性损伤奠定基础。

豆粕引起肠道炎性损伤是限制其在肉食性鱼饲料中应用的主要因素之一，而大豆皂甙是豆粕致炎的关键因素，但其致炎机制尚不明确。MAPK-NF-κB/AP-1信号通路是调控机体炎性反应的关键信号通路之一，但豆粕和大豆皂甙对鱼类该信号通路的影响尚无深入研究。基于以上背景，本研究旨在揭示MAPK-NF-κB/AP-1信号通路在介导豆粕和大豆皂甙引起大菱鲆肠道损伤中的作用。此外，通过该信号通路来解释缓解豆粕和大豆皂甙引起大菱鲆肠道炎性损伤的有益物质的作用机制。首先，本研究通过肠道结构观察、免疫组化、实时定量PCR等方法，证明大豆皂甙是豆粕中造成大菱鲆肠道炎性损伤的重要抗营养因子。其次，本研究结合细胞实验和养殖实验，通过实时定量PCR等和免疫印迹技术，证明大豆皂甙通过MAPK-NF-κB/ AP-1信号通路介导大菱鲆肠道炎性损伤。最后，本项目证明，谷氨酰胺、壳寡糖和壳聚糖均可通过影响上述信号通路而缓解豆粕和大豆皂甙造成的炎性损伤。研究结果不仅有助于揭示植物蛋白源及其包含的抗营养因子造成肉食性鱼类炎性损伤的作用机制，而且相关肠道保护剂的开发提供理论依据。

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