自噬在MC-LR诱导鱼类肝细胞毒性中的作用及调控机制

Microcystin-LR (MC-LR) is produced by cyanobacterial, which is highly hepatotoxic. Although the study of MC-LR has been extensively carried out, the toxic mechanism of MC-LR is still unclear. Recent studies have shown that autophagy is a new programmed cell death which is not different from apoptosis; however, the roles and regulation mechanisms of autophagy induced by MC-LR is still unknown. During our previous studies, we accidentally found that MC-LR could induce the autophagy in hepatocytes of grass carp, at the same time, endoplasmic reticulum stress were observed, then, we certified the result by using molecular technology. Another, we found many autophagy related genes, such as ATG12, Beclin1 were changed based on the transcriptome of grass carp induced by MC-LR, which suggest that PERK/eIF2α/ATG12/ Beclin1 and IRE1/JNK/Beclin1 pathway might be induced by MC-LR. On the base of the above reasons, grass carp and hepatocytes cultured in vitro will be exposed to MC-LR to build experimental model. The roles and mechanisms of autophagy will be studied by using confocal laser, and western blot, etc. Furthermore, PERK and IRE1 plasmid transfection and siRNA interference will used to interfere with their expression in order to investigate their regulation to autophagy. This work would provide some new insights of the hepatotoxic mechanisms of MC-LR.

微囊藻毒素-LR(MC-LR)是蓝藻水华产生的一种藻毒素，具有高度的肝毒性。尽管已对MC-LR开展了广泛的研究，但仍难以诠释其详细的致毒机制。自噬是近年来发现的一种不同于细胞凋亡的死亡形式，在哺乳动物中已有三篇文献报道MC-LR的肝毒性与自噬密切相关，然而，在鱼类中尚未开展MC-LR与自噬的相关研究。我们前期实验偶然发现MC-LR可致草鱼肝细胞自噬并伴随着内质网应激（ERS），随后又从分子水平进一步证明了ERS和自噬的产生。此外，对草鱼肝脏转录组分析发现ERS活化的两条途径PERK/eIF2α/ATG12/Beclin1和IRE1/JNK/Beclin1可能参与了MC-LR诱导细胞自噬。基于以上研究背景，本项目拟采用激光共聚焦、免疫印迹、RNA干扰等研究自噬在MC-LR诱导草鱼肝细胞毒性中的作用，并从推测的两条途径探讨对自噬的调控。本项目的完成将从新的视角解析MC-LR的致毒机制。

微囊藻毒素-LR(MC-LR)是蓝藻水华产生的一种藻毒素，具有高度的肝毒性。本项目以草鱼作为研究对象，首先从活体水平通过电镜等方法研究了MC-LR对草鱼肝细胞自噬的影响，发现MC-LR可诱导草鱼肝脏细胞自噬的产生并随着内质网应激。此外，本项目从活体水平还开展了MC-LR对草鱼肾脏凋亡的影响，结果表明MC-LR在诱导肝脏细胞自噬和凋亡的同时可诱导肾脏细胞凋亡。在开展MC-LR在活体水平上对自噬产生影响的同时，本项目还克隆了自噬相关基因（Beclin1、ATG4B、ATG12、ATG3、和ATG7），制备了相关抗体，从基因和蛋白水平上检测了不同剂量MC-LR诱导其表达变化。其次，本项目重点从细胞水平研究了MC-LR对GCO细胞自噬和凋亡的影响、自噬在MC-LR诱导细胞损伤中的作用以及α-LA对MC-LR致GCO细胞毒性的保护效应。通过采用透射电镜、MDC和TUNEL染色方法、转录组学等方法研究发现MC-LR可以降低GCO细胞活力，导致细胞空泡化、线粒体肿胀和内质网扩张等造成GCO细胞结构的损伤，诱导细胞自噬体增多，并通过激活TNFα-TNFR1-TRAF2-JNK途径上调Bax、Caspase7和Caspase9基因的表达促进GCO细胞凋亡的发生，最终导致细胞死亡。在开展自噬在MC-LR诱导细胞损伤中的作用的研究中发现当3-MA抑制GCO细胞自噬后，MC-LR会加剧GCO细胞受损，导致细胞活力下降及细胞培养上清液LDH活性升高，造成细胞形态的改变以及损伤其超微结构，进一步引起细胞周期紊乱，造成氧化应激和抑制细胞凋亡，并通过MyD88-TNF-MAPK途径诱导细胞炎症反应，最终导致细胞坏死。而α-LA可缓解MC-LR引起的氧化应激，提高细胞活力，抑制GCO细胞自噬、凋亡和炎症的发生，从而减弱MC-LR对GCO细胞的损伤。本项目从自噬角度解析了MC-LR的毒性作用机制，并重点阐释了自噬在MC-LR诱导细胞毒性中的作用，该项目为今后MC-LR污染的防治奠定了理论基础。