基于短路电流研究藿香正气水影响肠道离子通道的机制及其活性成分

Huoxiang Zhengqi Shui (HZS) was from “Taiping Huimin Hejiju Fang”, which is a classic clearing damp prescription of TCM. It has been mainly used to treat gastrointestinal diseases such as acute gastroenteritis and has many changs of drug form. But its intestinal mechanism and the active components are still not fully clear, which has seriously restricted its improvement of quality standardand and its safety and effective. Abdominal pain and diarrhea due to the abnormal intestinal ion transport. We found HZS acted on intestinal calcium channel and chloride channel and it could inhibit intestinal secretion only after intestinal metabolism. But the mechanism and active components of HZS affecting on ion channels has not been reported. Short circuit current (Isc) technology, ion substitution, and adding the blockers of ion channels and cotransporters or the regulatory factors such as neurotransmitters, gastrointestinal hormone and inflammatory mediators will be used in this project to explore its mechanism. We will set up a intestinal mucosa screening model by Ussing room and use intestinal and serum kinetics, △Isc, LC-MS/MS or GC-MS, spectrum effect relationship, and biotransformation in vitro in this project to explore its active components. The dynamic changes of metabolism, absorption and effect will be kepted consistently, but not separated. The pain relieving and antidiarrheal mechanism and active components of HZS will be elucidated from the new perspective and new idea, which will be a demonstration effect to gastrointestinal medicine, especially compound chinese herbal medicine. This is advantageous to the TCM which have advantages to the world and it has an important practical significance to the global public health.

藿香正气水（HZS）出自《太平惠民和剂局方》，是中医祛湿经典方，治疗急性胃肠炎等病疗效显著，剂型更改多，但其肠道作用机制和活性成分研究不足，严重制约了制剂质量标准提高及安全有效。腹痛腹泻本质是肠道离子转运异常。课题组发现该方作用于肠道钙通道和氯通道、只有肠道代谢后才能抑制肠道分泌的现象。但其影响离子通道的机制和活性成分尚无报道。本项目拟采用短路电流（Isc）技术，离子替代、加入离子通道及共转运体阻断剂、神经递质、胃肠激素和炎症介质等方法探索其机制；建立Ussing室肠黏膜筛选模型，基于肠道和血清动力学、△Isc效应、LC-MS/MS或GC-MS全息表征体内成分，谱效相关，体外生物转化探索其活性成分。保持成分体内代谢、吸收和效应的动态变化而不分离研究。从新角度和思路阐明该方止痛止泻机制及其活性成分。对胃肠道中药特别是复方中药研究有示范意义，对优势病种中药走向世界和全球公共卫生有重要现实意义。

本研究通过电流-时间法考察了藿香正气水（HZS）对肠道氯通道和钙通道离子转运的影响，从离子通道水平阐明HZS肠道止泻止痛的机制及药效成分，为该药全面质量控制奠定基础。利用HPLC-MS/MS分析不同时间点HZS小鼠肠道内容物，在体外生物转化提取分离多种成分；考察氯通道激动剂（肾上腺素、ATP、毛猴素、乙酰唑胺）和NKCC和CFTR氯通道抑制剂（布美他尼、DPC）的影响，发现HZS作用于NKCC通道（有效成分为和厚朴酚、厚朴酚、甘草苷与光甘草定）和CFTR通道（有效成分为橙皮素、橙皮苷、桔皮素与甘草次酸），抑制小肠细胞Cl-外流，达到止泻效果；考察钠、钾通道试剂（苯妥英钠、硝苯地平和KCl）和激动剂Ach对钙通道的影响，发现Ach直接影响，钠、钾通道也可以间接影响，达到止痛效果。当KCl为激动剂，与苯妥英钠相似，通过影响钠、钾通道间接影响钙通道，或通过抑制细胞内外钾浓度变化对细胞通道信号的改变使小肠收缩抑制（有效成分为甘草次酸、厚朴酚与甘草素）；当Ach为激动剂，与苯妥英钠相似，通过影响钠、钾通道来影响钙通道，或者通过影响M受体来影响Ach的作用来抑制小肠张力（有效成分为甘草苷、伞形花内酯、异欧前胡素与桔皮素）。此外，还开发高灵敏度微电极并应用，为进一步研究肠道和中药作用机制奠定技术基础。