基于脂类自噬探讨花生四烯酸促进草鱼肝细胞脂解的机制

One of the primary problem in the current aquaculture is excess lipid accumulation in the liver. Lipid catabolism is an important part in the reduction of lipid accumulation. Previously, the applicant has revealed that arachidonic acid (ARA) improves the hepatic lipolysis through cyclooxygenase (COX) pathway of grass carp. Interestingly, recent study in mammals strongly proved that the key lipolytic enzyme ATGL promotes lipophagy to control hepatic lipid droplet catabolism. The applicant also found that the improvement of lipid catabolism accompanied by up-regulation of autophagy-realted genes in the hepatocytes of grass carp. Thus, the applicant proposes the hypothesis that COX pathway participates in the upregulation of autophagy by ARA to improve the lipid catabolism in the liver of grass carp. Hence, in this project, hepatocytes culture in vitro would be performed as the studying method, lipid droplet staining, liquid chromatography-mass spectrometry, transmission electron microscope, fluorescent labeling, qRT-PCR and WB would be adopted to identify the regulation of COX activity, autophagy and lipolysis process in response to ARA. Afterwards, combined with the use of inhibitor/agonist, the function of autophagy in the upregulation.of lipolysis by ARA as well as intrinsic mechanism with the COX pathway would be deeply discussed. Finally, the in vivo method would be performed to further verify if dietary ARA would influence lipophagy in the liver of grass carp. The results will give a new theoretical foundation and research approach for the study of lipid metabolism and aquaculture in freshwater fish.

养殖鱼类肝脂过度蓄积是当前产业的主要问题之一，而脂解是减少脂肪蓄积的重要环节。申请人前期研究发现花生四烯酸（ARA）能够通过环氧合酶（COX）代谢途径提高草鱼肝脏的脂肪水解。近期在哺乳动物中发现脂肪水解关键酶ATGL是通过促进脂类自噬来控制肝脏脂滴分解的，申请人也发现草鱼肝细胞脂解提高的同时伴随了自噬基因的上调。为此提出ARA通过COX通路提高草鱼肝细胞的自噬，进而促进脂滴动员的假说。本项目拟通过离体草鱼肝细胞培养的方式，采用脂滴染色、液相-质谱联用、透射电镜、荧光标记、实时定量和WB等细胞分子生物学手段明确外源ARA对细胞COX活性、自噬和脂解的调控；随后结合使用抑制剂和激动剂，深入阐释自噬在ARA提高草鱼脂解过程中的作用及其与COX通路的内在关系；最后，采用在体饲喂的方式，进一步验证ARA能否提高草鱼肝脏的脂类自噬。研究结果将为养殖鱼类脂质蓄积调控提供理论依据，为生产实践提供参考资料。

养殖鱼类肝脂过度蓄积是当前产业的主要问题之一，而脂解是减少脂肪蓄积的重要环节。申请人前期研究发现花生四烯酸（ARA）能够通过环氧合酶（COX）代谢途径提高草鱼肝脏的脂肪水解。在哺乳动物中脂肪水解关键酶ATGL是通过促进脂滴自噬来控制肝脏脂滴分解的，为此提出ARA通过COX通路提高草鱼肝脏的自噬，进而促进脂滴动员的假说。本项目基于假设，采用油红O染色、透射电镜、荧光标记、实时定量、试剂盒检测等手段，深入探讨了脂滴自噬在ARA诱发草鱼肝细胞脂解过程中的作用及重要分子机制。获得如下结果：1）脂滴自噬是草鱼肝细胞脂解的有效途径，ARA在细胞营养缺乏时动员其COX代谢途径，降低细胞内脂滴的蓄积；2）抑制自噬减缓了ARA对草鱼肝细胞的降脂作用，脂滴自噬参与ARA促进细胞脂解的过程；3）COX通路是ARA诱发草鱼肝细胞脂滴自噬的必要途径，其中PGF2α发挥重要的作用，而15d-PGJ2反而促进脂肪蓄积，两种代谢物在影响肝脂蓄积过程中相互制约；4）在体饲喂草鱼ARA激活COX通路和促进PGF2α生成，诱发肝脏脂滴自噬，降低肝脏内脂肪沉积。此外，项目执行过程中还补充了研究内容：1）脂滴自噬可能是草鱼脂肪细胞脂解的途径，且ARA代谢与之呈正相关；2）斑马鱼肝细胞在营养缺乏时动员生成PGF2α，外源添加PGF2α促进细胞脂肪水解、脂滴自噬和线粒体发育；3）微管蛋白的合成是斑马鱼肝细胞脂滴自噬过程中的重要环节，但ARA及其代谢物与微管蛋白的关系尚需进一步探讨。综上，本项目明确ARA的COX通路是影响鱼类肝脂蓄积的有效途径，其中PGF2α可能是诱发脂滴自噬的关键分子。此外，COX途径中不同代谢物的产出在不同条件下表现出精巧调控的特点，对脂肪蓄积的影响存在差异。研究结果可为养殖鱼类脂质蓄积调控提供理论依据，为生产实践提供参考资料。