基于砷甲基化相关通路研究雄黄的遗传毒性及其配伍减毒机制

Genotoxicity is an important content of drug non-clinical safety studies, and is also a major research field that is related to carcinogenesis and teratogenicity. Realgar and its formula are widely used in clinical, but some studies have shown that realgar has potential genetic toxicity. On the basis of the literature search and the previous studies, we propose hypotheses: classical prescription "Niuhuangjiedu tablet" compatibility can reduce the genetic toxicity caused by realgar through affecting the realgar methylation and Nrf2-ARE pathway. To confirm this hypothesis, we first use the genotoxic technology to completely evaluate the effect of Niuhuangjiedu compatibility on the genotoxicity induced by realgar from different ends of genetics in vitro and vivo. Then using modern biomedical technology to compare and analyze the differences between before and after compatibility of realgar from the arsenic methylation pathway, oxidative stress induced by arsenic methylation and oxidative stress-related Nrf2-ARE pathway, then to reveal the mechanisms of realgar’s compatibility attenuated mechanisms. The project will be the first study on genotoxicity of realgar and it’s compatibility attenuated mechanisms.The results may provide a necessary basis for reasonable and safe application of realgar in clinical, a solid basis for the study on the development of realgar’s toxic antagonists or inhibitors, the realgar’s reducing toxicity and increasing effectiveness as well as new ideas and methods for studying the compatibility attenuated mechanisms of Chinese Medicine (formula).

遗传毒性是药物非临床安全性研究的重要内容，亦是与致癌、致畸密切相关的重要研究领域。雄黄及其复方制剂在中医临床应用甚广，但雄黄具有潜在的遗传毒性。本课题在文献检索和前期研究的基础上，提出研究假说:经典名方牛黄解毒片配伍可以通过影响雄黄甲基化及相关Nrf2-ARE通路发挥减低雄黄所致的遗传毒性。为证实这一假说，本课题先采用遗传毒理学技术，从体内外不同遗传学终点全面评价牛黄解毒片配伍对雄黄所致遗传毒性的减低作用。然后从配伍对砷甲基化代谢通路的影响、配伍对砷甲基化所致氧化应激的影响、配伍对甲基化相关Nrf2-ARE通路的诱导三个方面揭示配伍减低雄黄所致遗传毒性的机制。本项目首次基于砷甲基化相关通路研究雄黄的遗传毒性及其配伍减毒机制，能为临床安全合理应用雄黄及其复方提供必要的实验依据，为开发靶点明确的雄黄毒性拮抗剂或抑制剂、减毒增效提供研究基础，同时为中药（复方）配伍减毒机制研究提供新思路、新方法。

雄黄具有潜在的遗传毒性，本课题在前期研究的基础上，提出研究假说:经典名方牛黄解毒片配伍可以通过影响雄黄甲基化及相关Nrf2-ARE通路发挥减低雄黄所致的遗传毒性。为证实这一假说，本课题先从不同遗传学终点评价牛黄解毒片配伍对雄黄所致遗传毒性的减低作用，然后从配伍对甲基化代谢通路的影响、配伍对甲基化所致氧化应激的影响、配伍对Nrf2-ARE通路的诱导三个方面揭示配伍减低雄黄所致遗传毒性的机制。.主要研究结果：①牛黄解毒片方药配伍对雄黄引起的染色体畸变、DNA损伤、微核形成等有减低作用；②雄黄甲基化代谢产物DMA、MMA较雄黄本身具有更高的遗传毒性；③牛黄解毒片配伍可促进雄黄的甲基化代谢，表现为配伍后As3+、As5+的明显下降和一甲基化率（PMI）的明显升高。④牛黄解毒片配伍通过提高甲基化代谢通路中关键酶（GSTO1-1、PNP、AS3MT）合成以及砷甲基供体（SAM）含量来发挥其促进雄黄甲基化代谢作用。⑤降低氧化损伤作用是配伍减低雄黄遗传毒性的重要机制，可溶性砷化物（雄黄毒性成分）可引起L-02细胞ROS、8-OHdG、MDA等生成增加，而甘草（牛黄解毒片配伍减毒有效药味）提取物联用后ROS、8-OHdG、MDA等生成降低。⑥配伍减低雄黄遗传毒性作用与Nrf2-ARE通路分子（Nrf2、HO-1、NQO1）mRNA和蛋白表达水平改变有关。综上所述，牛黄解毒片配伍可通过促进雄黄的甲基化代谢，减少雄黄甲基化引起的氧化损伤，改变Nrf2通路的分子表达，从而降低雄黄的遗传毒性。本项目围绕项目任务书确立的内容展开研究，如期完成研究内容，课题结果可为临床安全合理应用雄黄及其复方提供必要的实验依据。