Liu Binghua, Li Guangling, Yang Jun, Li Xiaohui, Wang Hao, Yang Jing, Wen Haishen, He Feng
Key Laboratory of Mariculture, Ocean University of China, Ministry of Education, Qingdao, 266003, PR China.
Key Laboratory of Mariculture, Ocean University of China, Ministry of Education, Qingdao, 266003, PR China.
Fish Shellfish Immunol. 2022 Apr;123:152-163. doi: 10.1016/j.fsi.2022.02.036. Epub 2022 Feb 24.
Apoptosis genes Egr2, Fas and FasL are related to immune responses. However, the mechanism of these genes inducing apoptosis in fish are still not very clear. An acute hypoxia treatment (1.73 ± 0.06 mg/L) for 24 h was carried out on Japanese flounder (Paralichthys olivaceus). The increasingly dense apoptotic signals at 3 h, 6 h, 12 h by TUNEL in skeletal muscle indicated that hypoxia could quickly affect muscle growth and development. Furthermore, we concluded that the Egr2-FasL-Fas signal pathway, which was located at the upstream of apoptotic executor protein caspases, was related to the apoptosis by quantitative real-time PCR, protein concentration detection in ELISA and double gene in situ hybridization methods. The mechanism of the pathway was researched in transcription regulation and epigenetic modification by dual-luciferase reporter assay and bisulfite modified method, respectively. Egr2, as a transcription factor, could up-regulate the expression of FasL gene. And its binding site was mainly between -479 to -1 of FasL gene promoter. The 5th CpG dinucleotides (-514) methylation levels in FasL gene were significantly affected by hypoxia, and they were negatively correlated with its expressions. These suggested that the -514 site may be a very important site to regulate the FasL gene expression. Above results, we concluded that hypoxia activated the immune related signal pathway Egr2-FasL-Fas to induced skeletal muscle apoptosis to affect growth and development of Japanese flounder. The study revealed the mechanism of hypoxia induced apoptosis, which could provide a reference for fish immunity and aquaculture management.
凋亡基因Egr2、Fas和FasL与免疫反应相关。然而，这些基因在鱼类中诱导凋亡的机制仍不是很清楚。对牙鲆（Paralichthys olivaceus）进行了24小时的急性缺氧处理（1.73±0.06mg/L）。TUNEL法检测发现，在3小时、6小时、12小时时，骨骼肌中凋亡信号越来越密集，这表明缺氧可迅速影响肌肉的生长和发育。此外，通过定量实时PCR、ELISA法检测蛋白浓度以及双基因原位杂交方法，我们得出位于凋亡执行蛋白半胱天冬酶上游的Egr2-FasL-Fas信号通路与凋亡相关。分别通过双荧光素酶报告基因检测和亚硫酸氢盐修饰法从转录调控和表观遗传修饰方面研究了该信号通路的机制。Egr2作为一种转录因子，可以上调FasL基因的表达。其结合位点主要在FasL基因启动子的-479至-1之间。FasL基因第5个CpG二核苷酸（-514）的甲基化水平受缺氧影响显著，且与FasL基因的表达呈负相关。这些表明-514位点可能是调节FasL基因表达的一个非常重要的位点。基于以上结果，我们得出缺氧激活免疫相关信号通路Egr2-FasL-Fas从而诱导骨骼肌凋亡，进而影响牙鲆的生长和发育。该研究揭示了缺氧诱导凋亡的机制，可为鱼类免疫和水产养殖管理提供参考。
