Abstract：
Exposure to cigarette smoke (CS) adversely affects ovarian health and it is currently unknown how CS exposure causes ovarian injury. This study compared the differences in proteomics between CS exposure and healthy control groups using liquid chromatography-tandem mass spectrometry quantitative proteomics to further understand the molecular mechanism of ovarian cell injury in mice exposed to CS. Furthermore, western blotting and qPCR were carried out to validate the proteomic analysis outcomes. CREB1 was selected from the differentially expressed proteins, and then the down-regulation of CREB1 and phosphorylated CREB1(Ser133) expressions were confirmed in mice ovarian tissue and human ovarian granulosa cells (KGN cells) after CS exposure. In addition, the expressions of apoptosis-related proteins BCL-2 and BCL-XL were downregulated, and BAX expression was up-regulated. Moreover, the results of cellular immunofluorescence, flow cytometry, and transmission electron microscopy (TEM) showed that cigarette smoke extract (CSE) efficiently stimulated the production of reactive oxygen species, apoptosis, G1 phase arrest, mitochondrial membrane potential decreases, and ultrastructural changes in KGN cells. KG-501 (CREB inhibitor) aggravated CSE-induced mitochondrial dysfunction and apoptosis-proliferation imbalance in KGN cells mediated by down-regulated CREB1/BCL-2 axis. In addition, CREB1 over-expression partially restores mitochondrial dysfunction and apoptosis-proliferation imbalance of KGN cells induced by CSE. The results suggested that CSE diminished ovarian reserve in mice by disrupting the CREB1-mediated ovarian granulosa cell (GCs) proliferation-apoptosis balance and provided possible therapeutic targets for the clinical intervention of premature ovarian failure (POI) caused by CS exposure.
摘要：
暴露于香烟烟雾（CS）对卵巢健康有不利影响，目前尚不清楚CS暴露如何导致卵巢损伤。本研究采用液相色谱-串联质谱定量蛋白质组学方法比较CS暴露组和健康对照组在蛋白质组学方面的差异,以进一步了解CS暴露小鼠卵巢细胞损伤的分子机制。此外，进行蛋白质印迹和qPCR以验证蛋白质组学分析结果。CREB1是从差异表达的蛋白质中选择的，然后在CS暴露后的小鼠卵巢组织和人卵巢颗粒细胞（KGN细胞）中证实了CREB1和磷酸化CREB1（Ser133）表达的下调。此外,凋亡相关蛋白BCL-2和BCL-XL表达下调,BAX表达上调。此外，细胞免疫荧光的结果，流式细胞术,和透射电子显微镜（TEM）显示，香烟烟雾提取物（CSE）有效地刺激了活性氧的产生，凋亡，G1期阻滞,线粒体膜电位降低,和KGN细胞的超微结构变化。KG-501(CREB抑制剂)通过下调CREB1/BCL-2轴,加重CSE诱导的KGN细胞线粒体功能障碍和凋亡-增殖失衡。此外,CREB1过表达可部分恢复CSE诱导的KGN细胞线粒体功能障碍和凋亡-增殖失衡.结果表明，CSE通过破坏CREB1介导的卵巢颗粒细胞（GCs）增殖-凋亡平衡而降低小鼠卵巢储备，为CS暴露引起的卵巢早衰（POI）的临床干预提供了可能的治疗靶点。
