基于Caco-2/HT29-MTX细胞模型阐述桂枝“宜导诸药”的科学内涵

Chinese herbal formulas composed of monarch herb, minister herb, assistant herb and guiding herb were widely applied for thousands of years. As a traditional Chinese medicine (TCM), Ramulus Cinnamomi (GuiZhi) is often described as “suitable guidance for various drugs”, “guidance for all drugs first” and “need to use cinnamon as a guide” in classic ancient books. GuiZhi is suspected to promote the intestinal absorption of drugs due to the commonly oral routs of its decoction. Our previous study found that when GuiZhi was combined with Caulis Sinomenii, it greatly promoted intestinal absorption of sinomenine with higher AUC and Cmax. Therefore, we speculate that one or several components of GuiZhi have the function of breaking through one or several kinds of intestinal barriers. This project intends to study the pharmacokinetics of simomenine which is combined with different extractions from GuiZhi (decoction, 70% alcohol extract, 30% alcohol extract and volatile oils) to select the extraction with highest enhancement of absorption for simomenine. To clarify the absorption enhancing mechamisms of GuiZhi, effects of GuiZhi on intestinal epithelial cell barrier, tight junction and intestinal mucus barrier are to be investigated by the Caco-2/HT29-MTX co-cultures. Furthermore, the absorption enhancing components of GuiZhi can be explored by selecting best enhancing extraction, cinnamaldehyde and cinnamic as model drugs. The implement of this project will be helpful to explore the mechanism of promoting drug intestinal absorption by GuiZhi. It will reveal the scientific connotation of the "suitable guidance for various drugs" and enrich the scientific significance of guide herbs in the TCM theoretical system including "monarch herb, minister herb, assistant herb and guide herb". It also provides a new idea for screening the absorption accelerator from the treasure house of TCM.

由君药、臣药、佐药、使药构成的中药方剂，自古以来应用广泛。桂枝在经典古籍中常有“宜导诸药”、“为诸药先聘通使”、“需用桂枝以为向导”的记载，由于古时以口服汤剂居多，我们猜想桂枝促进了药物的肠道吸收。前期研究发现桂枝与青风藤配伍后，大大促进了青藤碱的口服吸收。据此我们推测桂枝中的某些成分可以改善肠道的吸收环境。本课题拟将桂枝水煎液、70%醇提液、30%醇提液以及挥发油分别与青藤碱配伍后进行药动学研究，筛选出促吸收的最佳组分;以最佳提取物、桂皮醛、桂皮酸作为研究对象，通过Caco-2/HT29-MTX共培养细胞模型，考察桂枝对小肠上皮细胞屏障、紧密连接以及小肠黏液屏障的突破作用及相关机制，明确桂枝中促进肠吸收的主要成分。本课题的开展将有助于探索桂枝促进药物肠吸收的作用机制，揭示桂枝“宜导诸药”的科学内涵，丰富中药“君臣佐使”中“使药”的科学意义，也为从中医药宝库中筛选吸收促进剂提供新思路。

桂枝为樟科植物肉桂的干燥嫩枝，用于风寒感冒、脘腹冷痛、血寒经闭、关节痹痛等。基于目前对中药“使药”的研究，再结合现代药物的肠道吸收理论，我们推测桂枝“宜导诸药”的原因是突破了某种肠道屏障，从而提高生物利用度。利用UHPLC-Q-TOF-MS、GC-MS及HPLC分析桂枝汤的有效成分，发现与青藤碱共煎时，化合物的提取率和种类略有不同，其中8种主要成分的含量较单味药材相比都有所减少。成功建立Caco-2/HT29共培养细胞模型，考察药物的摄取及转运，发现桂皮醛主要以被动扩散的形式在细胞内转运，其纳米粒和自微乳的转运不仅存在被动扩散且还有内吞机制参与。此外研究桂皮醛对肠黏液的影响，桂皮醛自微乳及微乳的渗透行为表明，以天然黏液和黏蛋白溶液分别用作屏障时，桂皮醛自微乳的渗透率增加 5.18倍和 2.80 倍，桂皮醛微乳的渗透率增加 1.20倍和1.47倍。流变学研究发现，天然黏液及黏蛋白溶液均是假塑性流体。桂皮醛及其微粒制剂能增加肠黏液的黏度，延长药物在肠道的滞留时间，从而增加药物的渗透。药动学结果表明：桂皮醛自微乳的AUC0-∞是其溶液的 2.42倍，桂皮醛微乳的AUC0-∞是其溶液的2.55倍。我们还构建了体外尤斯灌流模型，发现桂皮醛微乳能促进桂皮醛在不同肠段的吸收，尤其是回肠的吸收，转运方式为被动转运。结合体内药动学分析发现桂皮醛本身为全肠道吸收的药物，而桂皮醛促进其他药物吸收的机制可能是延长在肠道的滞留时间，与各种外排抑制剂无关。本项目成功构建的Caco-2/HT29细胞共培养模型，与单独Caco-2模型相比，不仅能够分泌黏液而且还能改善Caco-2细胞过度的紧密连接，更加接近真实的体内肠道情况。以天然黏液为屏障，Transwell法研究药物的渗透能更准确预测药物在体内的吸收，且操作简单，重现性好。药物对肠黏液流变学性质的作用也是影响药物体内吸收的重要因素。从Caco-2/HT29细胞共培养模型及肠黏液屏障出发考察药物的摄取、转运及渗透等，为研究药物的体内吸收提供新角度，也为科学解释中药的体内过程提供新策略，为筛选口服吸收促进剂提供理论基础和参考依据。

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