基于DNA甲基化的寒、热药性辨析研究

It is important to study on cold or hot property of the Traditional Chinese Medicine (TCM) for elucidating the theory of TCM properties. The breakthrough point is multi-disciplinary integration on study properties of TCM under the guidance of Chinese medicine theory. DNA methylation is one of the research contents of epigenetics which is a new subject developing in recent years. DNA methyltransferases(DNMTs) catalyze methylation reactions followed by gene silencing and demethylases catalyze demethylation reactions followed by gene reactivating. The cold medicine or hot medicine correlates closely to energy metabolism. The physical activities of body can be reduced by cold medicine and increased by hot medicine. On the basis of the above discussion, we put forward a hypothesis that cold/hot property of TCM is corresponded with DNA methylation/DNA demthylation. Our previous research showed that mRNA levels of genes encoding DNMT1 and DNMT3a were down-regulated by three cold medicines including Radix Gentianae which could not availably alter mRNA level of one demethylase(MBD4). However, Alpinia officinarum Hance was contrary to Radix gentianae. Four kinds of cold medicines (Rhizoma Coptidis, Radix gentianae, Radix Rehmanniae, Fructus Ligustri Lucidi) and four kinds of hot medicines (Fructus evodiae, Pricklyash, Alpinia officinarum Hance, Rhizoma zingiberis) will be selected as objects of study. We intend to clarify the hypothesis through detecting genes expression of DNMTs and DNA demethylases as well as methylation status of target gene. If the hypothesis is confirmed, the project will offer an objective method to distinguish between cold property and hot property of TCM, and it will provide a new vision for study on the theory of TCM properties.

中药寒、热药性的现代辨析是药性理论研究的重点和难点。在中医药理论指导下，采取多学科交叉研究是重要的突破点。甲基化是新兴学科- - 表观遗传学的研究内容之一。DNA甲基转移酶催化DNA甲基化伴随基因表达关闭，DNA去甲基酶催化去甲基化伴随基因表达开启。而寒、热药与能量代谢关系密切，寒药能降低机体机能活动，热药增加机能活动。基于此，提出"寒/热药性与DNA甲基化/去甲基化存在对应关系"假说。前期研究显示龙胆草等三味寒药显著增加大鼠DNA甲基转移酶1和3a的表达，龙胆草对DNA去甲基酶MBD4的表达影响不明显；热药高良姜的作用结果则相反。本项目选择四味寒凉药（黄连、龙胆草、生地黄、女贞子）和四味温热药（吴茱萸、花椒、高良姜、干姜），系统研究其对正常动物及对应热/寒证动物DNA甲基转移酶和去甲基酶的表达、靶基因的甲基化修饰和表达的影响，为寒、热药性提供客观的辨析手段，为中药药性提供新的研究视角。

本项目以细胞色素氧化酶（COX）的亚基基因Cox4i1为靶基因，探讨寒热药性与基因甲基化/去甲基化的对应关系。研究了4味热药（吴茱萸、花椒、干姜、高良姜）对正常大鼠和利血平所致寒证大鼠肝脏和骨骼肌Cox4i1的甲基化频率、mRNA水平、蛋白水平、DNA甲基转移酶和去甲基化相关蛋白的mRNA水平与蛋白水平的影响；并研究了4味寒药（黄连、龙胆草、生地黄、女贞子）对正常大鼠肝脏这些指标的影响，及对正常大鼠骨骼肌Cox4i1的甲基化频率、蛋白水平的影响；研究了生地和女贞子对酵母所致热证大鼠肝脏和骨骼肌Cox4i1的甲基化频率、mRNA水平、蛋白水平、DNA甲基转移酶和去甲基化相关蛋白的mRNA水平和蛋白水平的影响；研究了黄连和龙胆草对酵母所致热证大鼠骨骼肌Cox4i1的甲基化频率的影响，以及其对大鼠肝脏总DNA甲基化频率、基因表达谱、DNMT3A和TET1蛋白的影响。研究结果显示：（1）4味热药对正常大鼠肝脏和骨骼肌Cox4i1甲基化频率影响不明显；4味热药对利血平所致虚寒证大鼠肝脏Cox4i1甲基化频率影响也不明显，除低剂量花椒对寒证大鼠骨骼肌的Cox4i1甲基化频率有一定的升高作用（P<0.05），其它3味寒药均无此显著的效应。（2）除高剂量生地黄对正常大鼠肝脏Cox4i1甲基化频率有明显的降低效应（P<0.05）外，其它寒药对Cox4i1甲基化频率无显著影响；黄连、龙胆草对酵母所致热证大鼠的肝脏总DNA的甲基化率也无显著影响。（3）4味热药对于正常大鼠肝脏DNMT3A蛋白有显著降低作用（P<0.01）；高良姜还能显著降低正常大鼠肝脏和骨骼肌Dnmt1、Dnmt3a、Tet2、Tet3基因的mRNA水平；吴茱萸和花椒能显著增加寒证大鼠肝脏COX4I1蛋白水平和Dnmt3a mRNA水平；吴茱萸和花椒也能降低寒证大鼠骨骼肌Cox4i1、Dnmts、Tets基因的mRNA水平。（4）4味寒药能显著降低正常大鼠肝脏COX4I1蛋白水平和COX酶活性；黄连和龙胆草都能显著影响热证大鼠肝脏核糖体蛋白基因表达谱。本项目研究结果提示：（1）寒热药性与Cox4i1基因的甲基化/去甲基化可能不存在对应关系；（2）热药对大鼠的甲基化酶和去甲基化相关蛋白有一定作用；（3）寒药可能对蛋白的翻译过程有一定的作用。