维生素C对四氯苯醌诱导细胞凋亡的抑制作用及其机理研究

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol，which is one of persistent organic pollutants (POPs). Generally, researchers attribute TCBQ-induced toxicity to ROS produced by TCBQ. Recent studies suggest that TCBQ-enriched 5-hydromethylcytosine (5hmC) leads a significant difference in expression of apoptosis-related genes and we suspect that it is another mechanism of TCBQ-induced apoptosis. Vitamin C is an antioxidant. In addition, Vc mediates 5hmC level and then regulate cell apoptosis. Based on these results, we hypothesize that Vc scavenges ROS generated from TCBQ and represses TCBQ-induced 5hmC enrichment to ameliorate TCBQ-caused apoptosis. To test our hypothesis, we firstly determine the effect of Vc on TCBQ-regulated apoptosis and following that we carrying out the effect of Vc+TCBQ and TCBQ on ROS, mitochondria membrane potential and the expression of apoptosis-related protein. We also aim to assess the effect of Vc+TCBQ and TCBQ on 5hmC level, 5hmC enrichment at the promoter of apoptosis-related genes and the expression of apoptosis genes to infer the mechanism of vitamin C against of TCBQ-induced apoptosis. Our research illuminates the dual mechanisms of Vc against of TCBQ-induced apoptosis and uncovers the novel mechanism of the cytotoxicity and detoxification of TCBQ.

四氯苯醌（TCBQ）是持久性污染物五氯苯酚的活性代谢物，其严重的毒性效应驱使我们建立更有效的应对方法。本项目拟围绕维生素C（VC）抑制TCBQ诱导细胞凋亡开展研究，针对诱导ROS是TCBQ细胞毒性的主要原因，考察VC对ROS和线粒体膜电位的影响，分析由此带来的凋亡蛋白表达的变化，确定VC影响ROS抑制TCBQ诱导的凋亡；同时针对TCBQ调节DNA甲基化，分析TCBQ对整体5hmC的影响，进一步确定对凋亡相关基因启动子区5hmC的影响，筛选出一批变化显著的基因，评估其表达的变化，从而阐明TCBQ通过调节5hmC促进细胞凋亡的机理。针对VC促进5hmC的产生，考查VC对凋亡基因启动子区5hmC的调节，进而对基因表达的影响，确定VC调节5hmC抑制TCBQ凋亡的机理。本项目通过阐明TCBQ富集5hmC调节细胞凋亡机理及VC抑制TCBQ的凋亡机理，为TCBQ的毒性和解毒机理提供新方向。

四氯苯醌（TCBQ）是持久性污染物五氯苯酚的活性代谢物，其严重的毒性效应驱使我们建立更有效的应对方法。通过本项目的开展我们发现，维生素C能够有效缓解TCBQ诱导的细胞毒性，同时对其他苯醌类化合物的细胞毒性亦有抑制作用；机理研究表明维生素C通过清扫TCBQ产生的ROS，缓解其造成的线粒体膜电位下降，以及部分逆转其引发的凋亡基因mRNA和蛋白表达的变化缓解其细胞毒性。同时针对文献报道TCBQ调节DNA 5-羟甲基胞嘧啶（5hmC）水平，我们利用5hmC-免疫共沉淀（5hmC-DIP）测序技术研究发现TCBQ能够改变基因mRNA启动子区5hmC甲基化水平，揭示了TCBQ可能通过调节5hmC水平诱导细胞凋亡的机理以及可能的信号通路；维生素C通过5hmC路径缓解TCBQ细胞毒性的机理以及可能的信号通路。另外，我们还建立了一种基于DNA末端保护和金纳米粒子检测蛋白质的新方法，该方法选择性好，准确度高，生物兼容性好，方法设计简单，无需标记和酶放大，省时经济，无需昂贵仪器。本项目阐明了TCBQ通过5hmC路径调节细胞凋亡的可能机理及VC抑制TCBQ毒性的双重机理，为TCBQ的毒性和解毒机制提供新方向。