cAMP-PKA介导的高糖抑制团头鲂呼吸爆发功能的作用机制研究

Our objective of study is to carry out the study of dietary high carbohydrate on molecular signal transduction mechanism for respiratory burst dysfunction for fish based on Wuchang bream feeding with high carbohydrate and white cells in vitro under high glucose content. By using cell culture, flow cytometry, fluorescent quantitative PCR and western method of bolting, and confocal microscopy, electron microscopy technologies, we will check the changes of key enzymes(G6PD, NOX, ROS, SOD,MPO), respiratory burst activity, signal transduction passway (cAMP, ERK, CJNK, p38MAPK, extracelluar signal-regulate kinase 5) and organism structure. In order to illustrate the relationship between G6PD, NOX, cAMP/PKA and respiratory burst dysfunction, to further explore the signal mechanism for respiratory burst dysfunction induced by dietary high carbohydrate, we use the G6PD specific inhibitor (Dehydrooepian-drosterane)in the metabolism of G6PD, PKA stimulator(Forskokin) and specific PKA inhibitor (PKI) in the pathway of cAMP-PKA in the experiment. We expect to clarify signal mechanism for respiratory burst dysfunction induced by dietary high carbohydrate of Wuchang bream mediated by cAMP-PKA, to reveal some reasons of lower carbohydrate requirement for Wuchang bream as herbivorous animal, and to offer some research ideas of non-specific immunity of high carbohydrate for the fish.The results will provide a theroetical basis for the development efficient feed and health for Wuchang bream.

本项目针对高糖导致鱼体呼吸爆发功能障碍的关键问题，以团头鲂为研究对象，采用整体高糖投喂动物实验和离体高葡萄糖胁迫鱼体白细胞的手段，运用细胞培养、流式细胞仪、荧光定量PCR和Western bolting以及激光共聚焦显微镜、电子显微镜等方法，采用G6PD抑制剂阻断G6PD代谢途径，重点阐明G6PD、NOX、ROS、SOD、MPO与高糖抑制团头鲂呼吸爆发功能的关系，解析高糖导致团头鲂呼吸爆发功能障碍除抑制G6PD活性外，是否还存在其他抑制机制。在此基础上，采用PKA特异性抑制剂PKI和激活剂Forskolin阻断cAMP-PKA信号通路，探索高糖导致团头鲂呼吸爆发功能障碍与CAMP-PKA信号转导通路的关系，进一步阐明cAMP-PKA通路及下游通路在高糖胁迫导致团头鲂呼吸爆发功能障碍的可能信号转导机制，为团头鲂作为草食鱼类却不能利用高糖提供证据，也为高糖影响团头鲂非特异免疫提供理论基础。

项目针对高糖导致鱼体呼吸爆发功能障碍的关键问题，以团头鲂为研究对象，采用在体高糖投喂动物实验和离体高葡萄糖胁迫鱼体白细胞的手段，采用G6PD抑制剂阻断G6PD代谢途径，重点解析G6PD、NOX、ROS、SOD、MPO与高糖抑制团头鲂呼吸爆发功能的关系，探讨糖代谢关键酶G6PD对高糖导致团头鲂呼吸爆发功能障碍的作用机制，并采用PKA特异性抑制剂PKI和激活剂Forskolin阻断cAMP-PKA信号通路，进一步探索了高糖导致团头鲂呼吸爆发功能障碍与CAMP-PKA信号转导通路的关系。结果表明：（1）口服高糖引起团头鲂呼吸爆发减弱并造成肝脏的氧化应激损伤，此外持续高血糖可引起鱼体脂肪代谢紊乱，肝脏细胞出现脂肪积累和早期凋亡现象，并通过抑制PI3K/AKT通路阻断抗氧化应激酶对机体的保护作用，NRF2通路被激活表现出“代偿性”表达升高，以修复氧化应激损伤。（2）高糖通过降低团头鲂外周血白细胞G6PD活性, 伴随降低NOX活性和降低NADPH的活化，影响团头鲂外周血白细胞的呼吸爆发作用，进一步发现高糖能够引起团头鲂白细胞炎症反应，引起细胞凋亡。（3）cAMP/PKA通路可通过正调控p53的表达参与高糖引起的团头鲂肝脏急性炎症和肝细胞凋亡的发生。高糖日粮影响cAMP/PKA通路的表达，进一步通过MAPK/ACC/PGC-1α通路和p38mapk参与呼吸爆发功能及其关键酶p47phox的调控。以上实验结果阐明了cAMP-PKA通路及下游通路在高糖胁迫导致团头鲂呼吸爆发功能障碍的可能作用机制，为团头鲂作为草食鱼类却不能利用高糖提供证据，也为高糖影响团头鲂非特异免疫提供理论基础。