基于微透析及激光共聚焦显微镜技术研究中药穴位贴敷透皮给药的作用机理

As the most distinctive branch of transdermal drug delivery system, acupoint-application of TCM could illustrate the essence of TCM theory best in treating asthma. The metabolomics research of local exudate(blister liquid) and blood by acupoint-application of " Winter Diseases Treated in Summer" simultaneous using microdialysis and UPLC-Q-TOF methods will be studied in this project. The topical pharmacokinetics of the points (blister liquid) penetrated by the " Winter Diseases Treated in Summer" complex prescription, the overall pharmacokinetics of the blood and pharmacology will also be analyzed by a combination of in vivo microdialysis and LC/MS/MS, followed by fitting the model with "Local PK" and "overall PK"-"PD". The changes of physical screens, immune barrier and drug permeation behavior in local skin treated by acupoint sticking will be studied with confocal laser scanning microscope. And then combination "acupoint sticking area information" and "whole body information" to completely discuss the significance of skin irritation in points to the method of " Winter Diseases Treated in Summer". This will explain not only the relationship between local and whole body but also between the micro-adjustment of blister points and macro-adjustment of the whole body. Furthermore the mechanism of "observed outside, effect inside"in this treatment and the interpretation of "attach to the skin, permeat the muscle , absorb by qi, melt at exudate " will also be studied in this project.

中药穴位贴敷治疗哮喘是中药经皮给药系统中最有特色、最能体现传统中医药理论精华的分支之一。本项目拟同步采用微透析联用UPLC-Q-TOF进行"冬病夏治"方药贴穴局部皮内渗出液(发泡液)与血液的代谢组学研究；采用微透析联用LC/MS/MS研究"冬病夏治"方药渗透穴位皮肤(发泡液)的局部药动学、血液整体药动学及药效学，进行"局部PK"/"整体PK"-"PD"的模型拟合。采用皮肤激光共聚焦显微镜等方法观察贴穴对局部皮肤的物理屏障、免疫屏障及药物渗透行为。最后融合"贴穴局部"与"机体整体"研究的多方信息，多角度阐释贴穴部位皮肤刺激（发泡）对冬病夏治疗法的意义，说明该疗法中局部与整体、发泡部位微观调节与机体整体宏观调控的关系，阐明《理瀹骈文》关于该疗法"外惹内效、""切于皮肤，彻于肉理，摄于吸气，融于渗液"的科学内涵。

中药穴位贴敷治疗哮喘是中药经皮给药系统中最有特色、最能体现传统中医药理论精华的分支之一。本项目进行"冬病夏治"方药贴穴给药后血液的代谢组学研究，结果分析显示处方对磷脂、鞘脂、嘌呤、氨基酸和肾上腺素等与哮喘相关的内源性物质代谢均有影响，对药效学指标检测结果揭示了处方通过抑制NF-κB信号通路的抗炎抗哮喘机制。对处方渗透穴位皮肤皮下组织液的局部药动学、血液整体药动学进行研究，结果表明刺激肺俞穴和大椎可促进芥子碱硫氰酸盐的经皮吸收，控制药物释放，减少血药浓度的波动。采用分子生物学等方法评价皮肤物理屏障情况，结果提示芥子挥发油降低细胞 Ca 2+ -A TP 酶活性、改变细胞内外Ca2+平衡而增加皮肤活性表皮流动性，影响HaCaT细胞中物理屏障相关蛋白，提示芥子挥发油可通过降低表皮活性细胞物理屏障来促进药物渗透皮肤。处方通过调节皮肤免疫屏障及整体免疫系统，免疫因子水平而间接起到调节Th1/Th2平衡的作用。采用皮肤激光共聚焦显微镜等方法观察药物渗透行为，结果显示作为白芥子的有效成分之一白芥子挥发油能促进方中多有效成分经皮渗透吸收，起药效与促进药物渗透皮肤双重作用。.对有效成分芥子碱硫氰酸盐、黄芩苷等进行标记，结果显示芥子碱硫氰酸盐及黄芩苷均能有效渗透HaCaT细胞，随着时间增加，荧光信号均逐渐增强,即芥子碱硫氰酸盐渗透HaCaT细胞的浓度越来越大。对哮喘豚鼠气道炎症及肠道菌群的影响研究表明，处方能改善哮喘产生的肺部炎症及气道上皮及周围胶原纤维沉积，调控肠道菌群多样性及构成，普氏菌属、拟杆菌门、变形菌、厚壁菌门等在哮喘发病及治疗中可能作为标志物发挥重要的作用。本研究是对前期项目研究结果的深入与完善，研究结果进一步科学阐释传统中药药剂学理论，为传统中药外治法提供科学依据，结果将对中医经络系统、穴位理论等方面的现代化研究，有助于推进传统中药理论的现代化、国际化进程。