基于蛋白质组学方法研究半夏对胚胎发育的影响及配伍减毒机制

Involved in prenatal and postnatal care, medication safety during pregnancy is a major research area concerned in clinical and researches on the embryotoxicity of medicines have become hot spot at home and abroad. With the advantages of good efficacy and low toxicity, traditional Chinese medicine have played an important role in the treatment of pregnancy-related illness. Zingiberis-Ginseng-Pinelliae (ZGP) Bolus is a classic formula in the "Golden Chamber" to refrain vomiting when in pregnancy and widely used in clinical application from ancient times. Combining literature retrieval and preliminary research results, we speculate that raw Pinellia has embryotoxicity, but the formula ZGP Bolus (Decoction) does not. Therefore, we intend to study the effects of raw Pinellia and ZGP Bolus (Decoction) on embryonic development by using general reproductive toxicity test methods and embryonic stem cell (ES) model. And then, with proteomics technologies, we will analysis comparatively of the protein expression profiles of uterine decidua, gastrula and ES (D3) which were treated with raw Pinellia or ZGP Bolus (Decoction), look for the key molecules and investigate the toxic mechanisms of raw Pinellia and its compatibility attenuated mechanisms. The project will be the first study on developmental security of ZGP Bolus (Decoction) and compatibility attenuated mechanisms of raw Pinellia. The results may can provide a necessary basis for reasonable and safe application of Pinellia in clinical, a solid basis for the the study in Pinellia of reducing toxicity and increasing effectiveness as well as new ideas and methods for studying the embryotoxicity mechanisms of Chinese Medicine (formula).

妊娠期安全用药涉及优生优育，是临床关注的重大研究领域，药物的胚胎毒性成为国内外研究热点。中医药以疗效好、毒性低的优势，在治疗妊娠期相关疾病方面发挥了重要作用。干姜人参半夏丸是《金匮要略》之妊娠止呕经典名方，古今临床应用广泛。通过文献检索及前期实验研究，课题组认为生半夏有胚胎毒性，而配伍后的干姜人参半夏丸（汤）无胚胎毒性。本课题采用一般生殖毒性法及胚胎干细胞（ES）模型，从动物、细胞水平上全面评价生半夏配伍前后对胚胎发育的影响；进而采用蛋白质组学技术比较分析生半夏和干姜人参半夏丸（汤）干预后子宫蜕膜组织、原肠胚以及ES（D3）的蛋白质差异表达谱，寻找关键分子，探讨生半夏毒性作用机制及其配伍减毒机制。本课题首次研究干姜人参半夏丸（汤）的发育安全性及生半夏配伍减毒机制，能够为临床安全合理应用半夏提供必要的实验依据，为半夏减毒增效提供研究基础，同时为中药（复方）胚胎毒性机制研究提供新思路、新方法。

妊娠期安全用药涉及优生优育，而中医药以疗效好、毒性低的优势，在治疗妊娠期相关疾病方面发挥了重要作用。干姜人参半夏丸是《金匮要略》之妊娠止呕经典名方，其组成中生半夏是否有胚胎发育毒性颇具争议，该复方配伍后发育安全性如何值得研究，能够为临床合理应用半夏提供必要的实验依据。本课题致畸敏感期毒性试验表明生半夏汤与干姜人参半夏汤在仲景4倍剂量下无胚胎毒性，但同等剂量下生半夏粉剂显著抑制孕鼠体重增长及胎仔身长，且有一定的免疫抑制作用；干姜人参半夏粉对小鼠的胚胎发育没有影响，具配伍减毒效应。胚胎干细胞（ES）模型也证实生半夏粉具有促进细胞凋亡、降低细胞活性、抑制干细胞分化的毒性效应，而干姜人参半夏粉对ES细胞的生长及分化无明显影响。采用液相-质谱联用（LC-MS/MS）的蛋白质组学技术研究生半夏粉和干姜人参半夏粉干预后小鼠原肠胚的差异蛋白质组以及相应的含药血清干预后ES细胞的差异蛋白质组，生物信息学分析结果表明生半夏粉的毒性作用机制可能主要在于引起小鼠心室，中枢神经，大脑皮质，骨骼，眼，免疫系统等发育相关蛋白的表达变化。采用代谢组学技术检测了生半夏粉干预后大鼠血浆样本中内源性代谢物的变化，结果表明主要为氨基酸和脂质代谢紊乱，可能是其胚胎毒性作用机制之一。本研究为中药（复方）胚胎毒性机制的研究提供了可行的思路与方法。