从黄芪抑制附子吸收机理的角度探讨“脾主为卫”的生物学实质

At present, the concept of "the spleen being as the guard of the human body" is mainly concentrated in the field of immunology. Based on reported papers and our previous work, we assume that the concept is related to the inhibition of small intestine on the absorption of harmful substances, whose mechanism is related to the absorption barrier. Radix Astragali could reduce toxicity of Radix Aconiti Lateralis by strengthening the absorption barrier, and its effect on the barrier is related to body function. After we use in vitro and in vivo methods to prove that Radix Astragali can inhibit the absorption of harmful substances of Radix Aconiti Lateralis, we use normal and spleen-deficiency rats and cell model to research on the regulating effect of Radix Astragali, Radix Aconiti Lateralis and the drug-pair on the drug transporters, drug metabolizing enzymes and tight junction on small intestinal mucosa. All the work is to clarify the mechanism of absorption of Astragalus aconite toxicity reduction. We use Radix Astragali as model drug and aconite to test toxicity. In different functional states, the regulating effect of Radix Astragali on absorption barriers is studied to verify the relationship among the concept of "the spleen being as the guard of the human body", the concept of "replenishing qi to invigorate the spleen" and absorption barriers. We then clarify the biological essence of "the spleen being as the guard of the human body" and expand the understanding of "replenishing qi to invigorate the spleen". Subjected with "the spleen being as the guard of the human body" theory as a guide, our work is to study the effect of Radix Astragali reduce on the reduced toxicity of Radix Aconiti Lateralis, provide new ideas for theory of compatibility of traditional Chinese Medicine for reducing toxicity. The research results of this project will also guide Radix Astragali compound prescription, completing the theoretical basis for the safety of the original prescription.

目前，“脾主为卫”的现代认识主要集中在免疫学等领域，课题根据文献论述和前期实验结果，提出“脾主为卫与小肠抑制有害物质的吸收相关，其机制与强化吸收屏障相关；黄芪可通过强化吸收屏障减附子毒性，且其对吸收屏障的作用与机体机能状态有关”假说，在运用体内外2种方法证明黄芪可抑制附子有害物质吸收后，运用正常和脾虚大鼠及细胞模型，研究黄芪、附子、黄芪—附子配伍对小肠黏膜上的外排药物转运器、药物代谢酶和紧密连接等吸收屏障的调节作用，从而从吸收角度阐明黄芪减附子毒的机理。课题以黄芪为模型，以附子为毒性工具，研究不同机能状态下，黄芪对吸收屏障的调节作用，以探明脾主为卫、益气健脾与吸收屏障的关系，从而阐明“脾主为卫”的生物学实质、拓展“益气健脾”功效的认识；课题以“脾主为卫”理论为指导，研究黄芪减附子毒机理，为中药配伍减毒理论研究提供新思路。项目研究结果也可指导黄芪配伍组方，为提高原方安全性提供理论依据。

课题实施前，“脾主为卫”的认识主要集中在免疫学等领域。本课题运用灌服小承气汤、结合暴饮暴食、劳累过度的方法构建了脾虚模型动物构建方法。运用正常大鼠外翻肠囊模型、单向灌流模型和Caco-2细胞模型及脾虚大鼠外翻肠囊模型、单向灌流模型研究证实了黄芪提取物可以抑制附子提取物6种生物碱的吸收影响。运用Caco-2细胞模型和转运蛋白抑制剂为探针研究确证了外排药物转运器P-gp、MRP2、BCRP参与了附子生物碱的转运过程。研究证实了黄芪无论是作用于脾虚大鼠、正常大鼠还是Caco-2细胞，均可上调转运蛋白的表达；附子对药物转运器表达的影响随转运器类型及机能状态不同而不同；黄芪和附子配伍对转运蛋白表达的影响主要表现为上调。研究证实黄芪和黄芪—附子配伍作用于正常大鼠和Caco-2细胞后对3种外排转运蛋白转录影响的趋势与其对蛋白表达的影响趋势一致；而附子对3种外排转运蛋白转录影响的趋势与其对蛋白表达的影响趋势不完全一致。研究证实黄芪无论是作用于脾虚大鼠、正常大鼠还是Caco-2细胞，均可上调紧密连接蛋白的表达；附子以及黄芪—附子配伍对紧密连接蛋白表达的影响整体表现为上调。研究证实黄芪、附子和黄芪—附子配伍作用于正常大鼠和Caco-2细胞后对紧密连接蛋白转录影响的趋势与其对蛋白表达的影响趋势基本一致。研究证实黄芪活性成分对附子6种生物碱的代谢具有抑制作用；提取物可以降低附子生物碱整体动物血药浓度，且证实这种作用主要是由于黄芪减少附子生物碱的吸收。这些研究结果证明了黄芪可以通过强化吸收屏障而抑制附子生物碱吸收，验证了“脾主为卫与小肠抑制有害物质的吸收相关，其机制与强化吸收屏障相关；黄芪可通过强化吸收屏障减附子毒性，且其对吸收屏障的作用与机体机能状态有关”假说，从而初步从吸收角度阐明了“脾主为卫”的生物学实质、拓展了“益气健脾”功效的认识。