内质网应激在微囊藻毒素致雌性生殖细胞自噬与凋亡中的作用及机制

Microcystin-LR (MC-LR), a cellular endotoxin, exists ubiquitously in water, and the gonads are its main target organs. Recently, our research group found that MC-LR could increase the expression of endoplasmic reticulum stress and autophagy protein markers with resultant occurrence of apoptosis in female reproductive cells. These results are suggestive of a role for microcystins in initiating endoplasmic reticulum stress, autophagy and apoptosis during female reproductive toxicity. The underlying molecular mechanisms, however, remain unclear. This project seeks to understand, using in vivo and in vitro experimentation (mice ovarian granulosa cells lines and female C57BL/6mice, respectively), MC-LR-induced endoplasmic reticulum stress, autophagy, apoptosis, and the main underlying pathways involved. Key genes and proteins will be evaluated using RNA-seq and isobaric tags for relative and absolute quantitation to gain insights into the mechanisms regulating MC-LR-induced endoplasmic reticulum stress, while their connections to apoptosis and autophagy will be explored in a bid to clarify its molecular toxicity mechanisms. The goal of this project is to ultimately clarify the molecular mechanisms of endoplasmic reticulum stress regulating autophagy and apoptosis in MC-LR-induced female reproductive toxicity. The findings from the project could provide the theoretical basis and therapeutic target for preventing and treating diseases associated with microcystin toxicity, and enrich the biological effects of microcystins and reproductive toxicity theory.

微囊藻毒素-LR（Microcystin-LR）是广泛存在于水体的一类细胞内毒素，性腺是其主要靶器官。课题组近期发现MC-LR能够引起雌性生殖细胞中内质网应激和自噬标志性蛋白表达升高及凋亡发生，表明内质网应激、自噬和凋亡在MC-LR的雌性毒性作用中起着重要调控作用，但其机制目前还不清楚。因此，本项目采用小鼠卵巢颗粒细胞和C57BL/6小鼠，应用细胞siRNA干扰技术、动物体内RNA转染技术、RNA-seq和iTRAQ技术，从动物、细胞、转录组及蛋白组水平分析内质网应激状态下的细胞自噬和凋亡，筛选内质网应激状态下自噬与凋亡的关键基因和蛋白，探讨MC-LR致卵巢生殖细胞内质网应激下细胞凋亡与自噬的调控通路及其交互作用机制。本项目将阐明MC-LR致雌性生殖毒性作用过程中，内质网应激调控细胞自噬与凋亡的分子机制，为相关疾病的防治提供理论依据和治疗靶点，丰富微囊藻毒素的生物学作用及生殖毒性理论。

随着富营养化、二氧化碳水平上升和全球变暖，有害蓝藻水华的频率、强度和持续时间都在增加，并且已成为主要的公共卫生和环境问题。微囊藻毒素-LR（Microcystin-leucine arginine exposure, MC-LR）是由水中蓝藻产生的环状七肽细胞内毒素，分布广泛，具有生殖毒性。该项目以C57BL/6小鼠和小鼠颗粒细胞（KK-1）为模型，应用体内转染、RNA-seq及iTRAQ等技术深入挖掘、探讨MC-LR对雌性生殖系统的致毒效应。研究发现MC-LR可破坏卵巢微结构和超微结构，导致颗粒细胞内质网肿胀，通过增加内质网应激相关的PERK、IRE1及ATF6等水平导致内质网应激。一方面，MC-LR介导的内质网应激可通过激活IRE1通路（IRE1、XBP1和JNK）和CaMKKβ通路（CaMKKβ、AMPK、mTOR）促进自噬相关LC3Ⅱ和BECLIN1表达，抑制P62表达，导致颗粒细胞自噬体的形成进而诱导细胞自噬发生；另一方面，RNA-seq测序挖掘发现MC-LR介导的内质网应激促进CHOP（DDIT3）表达，导致DR5水平上升，BCL-2水平下降，进而通过Caspase通路引起颗粒细胞凋亡。除此之外，RNA-seq测序挖掘还发现MC-LR介导的内质网应激促进ASK1的表达，并通过激活P38/JNK通路诱导小鼠卵巢损伤和凋亡。我们进一步深入研究证实氧化应激调控MC-LR诱导的小鼠卵巢内质网应激。发现MC-LR可通过增加ROS、MDA水平及降低抗氧化相关的GSH、SOD等水平引起卵巢氧化应激，而氧化应激抑制剂NAC可通过降低内质网应激相关的XBP1、GRP78等表达缓解MC-LR引起的内质网应激，进而缓解MC-LR引起的细胞自噬和凋亡。另外，通过iTRAQ蛋白组测序发现MC-LR可以引起小鼠卵巢紧密链接的损伤。这可能与MC-LR通过内质网应激下IRE1导致的JNK/β-catenin通路激活有关。总而言之，我们的研究阐明了内质网应激在MC-LR诱导的卵巢颗粒细胞自噬与凋亡中的作用与机制。我们的研究同时证明暴露于MC-LR可对生殖健康构成严重威胁，为MC-LR暴露引起的生殖细胞自噬与凋亡的机制和治疗提供了新的见解与理论依据。