IRE1α介导高脂诱发黑鲷炎症反应发生机制的研究

High lipids could induce inflammation of organisms, however, inflammation could reduce the immunity and easy to cause diseases. This phenomenon has been taken seriously. However, the mechanism of high lipids induce inflammatory response in fish is still unknown. When the body intake high lipids, it can cause the endoplasmic reticulum stress, thus activating the IRE1α signal pathway in the unfold-protein reaction. Moreover, the activated IRE1α will form a polymer with TRAF2 to activate the downstream NF- κB signal pathway. NF-κB signal pathway plays a vital role in the inflammatory response, because NF-κB could be transferred into the nucleus and then initiate transcription of associated inflammatory factors. In the present project, in vitro experiment, the technology of gene silencing and immunofluorescence were used to illustrate the important role of IREα in the inflammatory response, which induced by high lipids. Furthermore, in vivo experiment, the application of western blot and immunohistochemistry were used to perform verification experiments. In conclusion, the present project aimed to demonstrate the occurrence mechanism of high lipids induce inflammation mediated by IRE1α in black seabream (Acanthopagrus schlegelii). Then obtain the regulatory target, and provide a theoretical basis for the nutrition strategy of responding to this inflammatory reaction. Besides, it is could also promote the development of effective anti-inflammatory reaction feed additives.

高脂能诱发炎症反应进而造成机体免疫力降低，极易感染疾病，该现象已经逐渐得到重视，然而关于高脂在鱼体中引发炎症反应的发生机制还尚不明确。当机体摄入高脂后，会造成内质网应激，从而激活未折叠蛋白反应中的IRE1α信号通路，而激活的IRE1α会与TRAF2形成聚合物进而激活下游的NF-κB信号通路。此外，NF-κB信号通路在机体炎症反应中具有重要的作用，游离的NF-κB可以转入细胞核中，最终启动相关炎症因子的转录。本研究拟在离体实验中利用基因沉默和免疫荧光等技术探明IRE1α在高脂所介导的炎症反应中的重要作用；再通过在体实验，应用蛋白质印迹和免疫组化等技术进一步加以验证。本项目力图清晰的阐明IRE1α通过激活下游NF-κB途径介导高脂诱发黑鲷炎症反应的发生机制，从而确定调控靶点，为提出应对该炎症反应发生的营养学策略提供理论依据，更为推动开发研制有效的抗炎饲料添加剂提供重要的学术价值。

鱼类营养性脂肪肝问题严重制约了水产养殖业快速和健康发展，并已引起学界和产业界的高度重视。本项目采用分子营养学的解决策略以病因形成机制的角度力图从中间阻断高脂引发鱼类营养性脂肪肝，因此必须要清晰而全面地掌握高脂饲料产生营养性脂肪肝的重要途径，进而确定调控靶点。在本项目的资助下，申请人围绕着研究内容开展实验并获得了以下重要结果：（1）黑鲷肝细胞系的构建：采用组织块迁移法进行细胞分离，获得状态稳定的黑鲷肝细胞系，目前已稳定传至37代。（2）完成了内质网关键基因的克隆、序列分析和组织特异性表达：本研究克隆并鉴定了黑鲷grp78完整的cDNA序列（GeneBank，登入号：MT451934）以及ire1α部分cDNA核心序列（GeneBank，登入号：OL361769）。（3）通过在体水平研究高脂对黑鲷肝脏内质网应激、炎症反应和细胞凋亡的影响：结果表明摄入高脂饲料后使脂肪代谢紊乱，导致脂肪沉积和肝脏脂肪变性，引发氧化应激和内质网应激，造成炎症反应和细胞凋亡，证实了高脂可以正向调控Ire1ɑ，Nf-κb 和Jnk。（4）在细胞水平探明Ire1α介导高脂诱发黑鲷炎症反应和细胞凋亡的作用机制：进一步的研究表明Ire1α可通过对Jnk和Nf-κb 的调控作用从而介导高引发黑鲷肝脏炎症反应和细胞凋亡，且Ire1α是一个重要的调节靶蛋白，通过抑制或干扰其表达可减轻高脂引发的黑鲷营养性脂肪肝问题。综上所述，本项目已按要求完成了研究计划，研究结果有助于解决高脂饲料在海水鱼中产生的营养性脂肪肝问题，从而促进产业发展，但能否通过调控ERS途径从而缓解高脂饲料引发的黑鲷炎症反应和细胞凋亡还需进一步开展深入的研究。