基于药证相关理论探讨醋甘遂-炙甘草减毒增效的配伍关系及机制

“Eighteen antagonisms”— the controversial prohibited combinations in traditional Chinese medicine, has experienced a history of thousands of years so far. Whether and how they could be used in clinic, has been a problem that people have argued without reaching a decision. The combination of gansui and gancao is Anti-drug combinations in clinic. Studies have shown that the compatibility of gancao and gansui could decreasing toxicity ,but some contrary conclusion still exits . We hold the opinion that the reason for discordance is associated with disease model. Therefore, in order to be accord with the clinical application, in this study, we will build a pathological model of malignant ascites to explore the Water-electrolyte imbalance ,which is closely related to the clinical effect of gansui.Meanwhile, the hepatic and renal function will be detected with toxicity.This viable researching method is in accordance with its own developing rules of TCM theory.To reveal the material base of compatibility,we will have a quantitative analysis of active components in rat plasma. And then,Chemicals of herbal products may cause unexpected toxicity or adverse effect by the potential for alteration of the activity of CYP450 when co-administered with other drugs.In order to explain compatibility mechanism,using in vitro and in vivo model systems ,we will investigate the activity of CYP450 isozyme CYP2C9, CYP3A4, CYP2E1 by use of high performance liquid chromatographic(HPLC) assay.By means of RT-PCR and western-blot,we will detecte the protein expression and mRNA levels in the rats suffered from malignant ascites.The results could provide a basis for guiding rational use of this prohibited combination in clinic and enriching content of theory of compatibility of traditional chinese hebs.

甘遂与甘草属于中药“十八反”范畴，是传统的配伍禁忌。但无论古今均有其在临床应用的案例。已有化学研究和药理毒理研究为甘遂与甘草配伍减毒提供了一定的数据支撑，但也有相反配伍的结论。根据中医药证相关理论，认为结论不一致的原因与机体状态不同有关。因此，本课题拟选择前期结果中减毒增效的配比，从与甘遂功效密切相关的水盐代谢方面入手，结合肝肾功能指标的变化，通过观察甘遂与甘草对癌性腹水模型大鼠的影响，探求二者在辨证论治理论指导下减毒增效的配伍关系。通过检测血浆中甘遂毒性成分和甘草水钠潴留主要成分甘草次酸的含量，揭示二者配伍关系的物质基础。采用体内外模型，观察肝微粒体CYP450同工酶的活性、基因、蛋白表达，揭示甘遂与甘草配伍是否与CYP450同工酶诱导或抑制活性成分的代谢、转化有关，结果对推进临床合理安全使用反药组合具有重要意义，同时更有助于完善甘遂-甘草反药配伍的科学内涵。

“十八反”为中药传统的配伍禁忌，认为配伍使用能够致毒增毒。甘遂与甘草药组属于中药“十八反”范畴，其中甘遂是峻下逐水药，甘草是补气药，甘草中主要成分甘草酸水解后产生甘草次酸，有类似肾上腺皮质激素样作用，能使水钠潴留。但自古以来二者同用治疗各种水饮之证的验案屡见不鲜，现代还用于肿瘤、肝硬化腹水、胸水、心包积液、胰腺炎等疾病的治疗。因此，本课题基于临床应用从水盐代谢的角度,通过检测甘遂与甘草配伍前后大鼠肝肾功能指标来研究配伍后是否有减毒作用，通过检测肝药酶的活性来揭示减毒的机制， 通过检测尿量、血尿电解质情况以及肾素、血管紧张素、醛固酮、抗利尿激素的含量以及AQP2、AVPR2、ADRB1、PIK3CG含量来探讨配伍对水盐代谢的影响及机制，通过检测配伍前后血浆及尿液中的甘草次酸含量的变化揭示物质基础，最终揭示甘遂与甘草配伍禁忌的实质。.研究结果显示，甘遂与甘草在特定的病理条件下与其他药物组成复方不但不会出现毒性反应，还可有减毒增效的作用，能增加癌性腹水大鼠的尿量，对肝脏和肾脏有一定的保护作用。研究显示甘遂与甘草配伍后，尤其甘遂半夏汤能调节预测肝损伤的血清标志物PON1的含量，以及预测肾损伤的β2-MG、KIM-1的含量，从而具有保护作用，其作用机制通过调节肝药酶的活性促进甘遂中毒性物质KC的代谢来实现，其中CYP3A4和CYP2E1在代谢过程中发挥了主要作用。根据网络药理研究显示，水液代谢与肾素-血管紧张素-醛固酮系统以及抗利尿激素通路相关因子AQP2、AVPR2、ADRB1、PIK3CG有关。研究表明，甘遂与甘草配伍通过降低肾素、醛固酮以及抗利尿激素的含量，下调AQP2、ADRB1、PIK3CG、AVPR2的表达发挥利水作用。通过检测血浆及尿液中的甘草次酸含量发现，配伍后甘草次酸的含量显著升高，甘遂半夏汤组甘草次酸含量显著降低，从物质基础角度推测甘草能够降低甘遂的毒性，增强利水疗效，与甘草次酸的免疫调节作用也有一定的关系。而甘遂半夏汤中的半夏与白芍增强了甘草次酸的减毒增效作用。综上，研究成果完善了配伍禁忌理论，为临床安全用药提供了参考，也为探索中药配伍的作用机制提供了有益的借鉴，扩展了研究思路。