Zhang Anying, Guo Yafei, Zhang Shengnan, Fan Xinchi, Wang Xinyan, Zhou Xiaofei, Yang Kun, Zhou Hong
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, PR China.
School of Life Science and Technology, University of Electronic Science and Technology of China, Chengdu, PR China.
Fish Shellfish Immunol. 2015 Oct;46(2):550-6. doi: 10.1016/j.fsi.2015.07.016. Epub 2015 Jul 22.
In mammals, the cytokine effects of heat shock protein 70 (HSP70) have been well documented. However, the precise role and molecular mechanism of fish HSP70 in cytokine production are poorly understood. In the present study, recombinant grass carp HSP70 (rgcHSP70) was prepared by using the E. coli expression system and its effect on interleukin (IL)-1β (gcil1β) and tumor necrosis factor (TNF)-α (gctnfα) mRNA expression was examined in grass carp head kidney leukocytes (HKLs). Compared with its heated-inactivated protein, rgcHSP70 enhanced the mRNA levels of these two cytokines in a dose- and time-dependent manner. More importantly, the results of enzyme-linked immunoabsorbent assay (ELISA) showed that rgcHSP70 markedly stimulated gcIL-1β and gcTNF-α secretion from the HKLs, for the first time indicating the ability of HSP70 to regulate cytokine release in fish. Moreover, rgcHSP70 displayed more potential to increase the cytokines' release instead of their transcription in the same cell model. Subsequently, the signaling mechanisms governing the cytokine effect of rgcHSP70 were investigated, showing that rgcHSP70-induced gcil1β mRNA expression was suppressed by inhibiting p38 MAPK and NF-κB pathways, while the stimulation of rgcHSP70 on gctnfα mRNA expression were blocked by inhibiting JNK MAPK and NF-κB pathways. In parallel, rgcHSP70 was also shown to activate NF-κB pathway through regulating grass carp IκBα protein levels in a time-dependent oscillation. These findings provide further understanding of cytokine effect and regulatory mechanism of HSP70 in fish and new insights into the roles of HSP70 in teleost immunity.
在哺乳动物中，热休克蛋白70（HSP70）的细胞因子效应已有充分记载。然而，鱼类HSP70在细胞因子产生中的精确作用和分子机制却知之甚少。在本研究中，利用大肠杆菌表达系统制备了重组草鱼HSP70（rgcHSP70），并检测了其对草鱼头肾白细胞（HKLs）中白细胞介素（IL）-1β（gcil1β）和肿瘤坏死因子（TNF）-α（gctnfα）mRNA表达的影响。与热灭活蛋白相比，rgcHSP70以剂量和时间依赖性方式提高了这两种细胞因子的mRNA水平。更重要的是，酶联免疫吸附测定（ELISA）结果表明，rgcHSP70显著刺激了HKLs中gcIL-1β和gcTNF-α的分泌，首次表明HSP70具有调节鱼类细胞因子释放的能力。此外，在同一细胞模型中，rgcHSP70在增加细胞因子释放方面比转录方面更具潜力。随后，研究了调控rgcHSP70细胞因子效应的信号机制，结果表明，抑制p38 MAPK和NF-κB途径可抑制rgcHSP70诱导的gcil1β mRNA表达，而抑制JNK MAPK和NF-κB途径可阻断rgcHSP70对gctnfα mRNA表达的刺激。同时，rgcHSP70还被证明可通过调节草鱼IκBα蛋白水平以时间依赖性振荡激活NF-κB途径。这些发现进一步加深了对鱼类中HSP70细胞因子效应和调控机制的理解，并为HSP70在硬骨鱼免疫中的作用提供了新的见解。
