基于“中药药性细胞色素P450酶学”假说探讨“热者寒之、寒者热之”的科学内涵

The metabolism of traditional Chinese medicine (TCM) is closely related with its efficacy and property. Looking for biomarkers the levels of which can be measured can make it easy to elucidate the objective differences between heat and cold syndrome. It can also reveal the expression mechanism of cold and heat property. The CYP450s are important functional proteins in metabolic network. Studies have shown that the expression of CYP450s had correlation with various diseases. This project, which is inspired by research productions of gene technology, metabonomics, pharmacokinetic and pharmacology in property theory of TCM, firstly proposes "Hypothesis of CYP450s for property theory of TCM". We assume that the expression of CYP450s is specific in heat (cold) syndrome and the regulatory effect of cold (heat) TCM has its regularity. This project, using methods of enzyme chemistry and molecular biology and so on, will verify the rationality of the hypothesis from multiple perspectives, such as the activity of CYP450s, gene expression and protein expression. The theoretical achievements of this project have exploratory value on disclosing objectivity of cold-heat property of TCM, and on interpreting the scientific connotation of "Treating the cold with heat, the heat with cold". We expect to build a technology platform, which uses CYP450s as the biomarker to evaluate the property of TCM, as the experimental achievements of this project, and then innovate research thoughts and methods on property theory of TCM.

中药的代谢环节与其药效-药性密切相关，寻找水平可测的生物标志物，可化繁就简的阐明代谢环节上热证、寒证的客观差异、揭示寒、热药性的表达机制。CYP450酶为机体代谢网络中重要的功能蛋白，研究表明，其表达与多种疾病具有相关性。本项目受基因技术、代谢组学、药物动力学、药理学等在寒热药性理论中的研究成果启发，首次提出"中药药性CYP450酶学"假说，设想中医热证（寒证）下CYP450酶的表征具有特异性、而寒性（热性）中药对其调控作用具有规律性。并运用酶化学、分子生物学相关方法，从CYP450同工酶的活性、基因表达、蛋白表达等多角度、系统性验证该假说的合理性。本项目的理论成果对揭示中药寒热药性差异的客观性，诠释"热者寒之、寒者热之"科学内涵具有探索性价值；实验成果预期建立以CYP450酶作为生物标志物评价寒热药性的技术平台，以此创新中药药性理论的研究思路与方法。

中药的代谢环节与其药效-药性密切相关，寻找水平可测的生物标志物，可化繁就简的阐明代谢环节上热证、寒证的客观差异、揭示寒、热药性的表达机制。CYP450酶为机体代谢网络中的重要的功能蛋白，研究表明，其表达与多种疾病具有相关性。本项目考察了寒证（热证）大鼠肝脏CYP450酶的表征特点及热性（寒性）中药的调控效果。以知母-石膏-黄柏-龙胆草制备寒性造模药，附子-干姜-肉桂制备热性造模药，对SD大鼠诱导后复制证候模型；另分别取药性明确的热性中药吴茱萸、附子、肉桂分别干预寒证大鼠，黄连、栀子、知母分别干预热证大鼠，制备治疗组；生理盐水为对照组。分光光度法测定肝微粒体蛋白浓度、CYP450酶总含量。Cocktail法评价CYP450亚酶活性变化。实时荧光定量PCR 法测定CYP450亚酶的mRNA表达；蛋白免疫印迹法检测CYP450亚酶的蛋白表达。结果寒性造模药对大鼠CYP3A1酶的活性具有显著的抑制作用，热性中药肉桂具有一定的调控作用，并且这种调控发生在mRNA水平。热性造模药对大鼠CYP3A1酶具有诱导作用，给予寒性中药黄连和知母后，酶活性在mRNA和蛋白水平被调控。寒性造模药对大鼠CYP1A2酶的活性具有抑制作用，热性造模药对CYP1A2酶具有诱导作用。分别给予肉桂和黄连后，酶活性回调，但调控机制未发生在mRNA和蛋白水平。热性造模药对CYP2E1酶具有诱导作用，黄连在mRNA和蛋白水平均具有调控作用。药物对CYP450酶活性的影响机制包括酶的基因表达、转录水平、翻译水平等多方面。因此，本项目从多角度系统评价和验证寒证和热证机体CYP450酶的表征特点及热性和寒性中药的调控效果。此外，在对机体功能的影响上，寒性药主要呈现抑制作用，而热性药主要呈现兴奋作用，本研究进一步证实了中药的寒热药性差异具有客观性。而寒性（热性）中药对其调控作用是否具有规律性以及调控机制有待进一步深入阐明。