基于腺苷信号转导通路的芳香开窍类中药“开窍于脑”的机制研究

Borneol and grass-leaved sweetflag can modualate the permeability of blood-brain barrier, but the intrinsicmechanism is unknown. Our previous works found that the concentration of components of Nao De Sheng tablet was increased remarkably after co-administration with Borneol and Grassleaved Sweetflag Rhizome, which was reversible and irrelevant to transporters. In recent years, CD73(5'-ectonucleotidase) attracted popular attention. Adenosine was produced after activation of CD73 , which resulted in cellular changes in vitro including decreased transendothelial electrical resistance, increased actinomyosin stress fiber formation, and alterations in tight junction molecules and established an channel entering into BBB. Based on above-mentioned facts, we put forward the following hypothesis that adenosine receptor signaling mediated the process that Borneol and grass-leaved sweetflag promoted drugs entering into BBB, which makes it feasible that the drugs was accumulated in BBB. To validate those hypothesis, we will carry out the following studies:(1) Borneol and grass-leaved sweetflag induce the passage of components of Nao De Sheng tablet through the blood-brain barrier model in vitro ;(2)the effect of Borneol and grass-leaved sweetflag on transcription ,expression and function of CD73 and adenosine receptors. This study will illustrate the biopharmaceutical nature of traditional medicine for inducing resuscitation ,enrich the compatibility theory of traditional Chinese medicine.

芳香开窍药冰片和石菖蒲能够增加血脑屏障（BBB）通透性，但其机制尚不清楚。课题组前期研究证实配伍冰片及石菖蒲后能显著增加脑得生片成分脑内分布，其作用与转运蛋白无关，且呈现智能、可逆特征。近年研究表明CD73（胞外-5'-核苷酸酶）活化后，产生腺苷激活腺苷受体，使BBB细胞骨架和超微结构发生改变，导致细胞旁间隙增加，从而建立起一条进入BBB的“开关式”通道，这与课题组研究结果极其相似。据此提出以下假说：芳香开窍药可能是通过腺苷信号转导通路调节BBB通透性，实现配伍脑靶向。课题将通过冰片、石菖蒲：①体外促进脑得生有效成分透过BBB与腺苷受体信号通路的关系；②对CD73及腺苷受体活性、表达、转录的影响等两个方面验证该假说，以揭示芳香开窍类中药“开窍于脑”的机制及科学内涵，为中药复方通过配伍实现脑部疾病靶向治疗提供新的思路和方法。

芳香开窍药冰片和石菖蒲能够增加血脑屏障（BBB）通透性，但其机制尚不清楚。课题组前期研究证实配伍冰片及石菖蒲后能显著增加脑得生片有效成分脑内分布，其作用与转运蛋白无关，且呈现智能、可逆特征。近年研究表明CD73（胞外-5'-核苷酸酶）活化后，产生腺苷激活腺苷受体，使BBB细胞骨架和超微结构发生改变，导致细胞旁间隙增加，从而建立起一条进入BBB的“开关式”通道，这与课题组研究结果极其相似。据此提出以下假说：芳香开窍药可能是通过腺苷信号转导通路调节BBB通透性，实现配伍脑靶向。课题通过冰片、石菖蒲（有效成分 α-细辛醚）：①体外促进脑得生有效成分透过BBB与腺苷受体信号通路的关系；②对CD73及腺苷受体活性、表达、转录的影响等两个方面验证该假说，实验结果显示：50 μmol/L葛根素组（Puer），50 μmol/L川芎嗪组（TMP）药物透过率分别为(33.44±2.24)%,(13.53±2.6)%, 而Puer, TMP分别与冰片和 α-细辛醚联用组其药物透过率显著提高，冰片和 α-细辛醚的促透作用随着冰片和 α-细辛醚作用浓度的升高而增强，其中高浓度组分别为200μmol/L 冰片+50 μmol/L puer组，200μmol/L α-细辛醚+50 μmol/L puer组，200 μmol/L 冰片+50 μmol/L TMP组，200 μmol/L α-细辛醚+50 μmol/L TMP组，其药物透过率分别为(82.11±4.90)%，(85.51±2.20)%，(44.00±3.80)%，(44.79±0.41)%。在联用组前加入25μmol/L anti-CD73，anti-A1AR，anti-A2AAR干预后，Puer,TMP药物透过率显著降低，且与单独应用Puer,TMP组无显著性差异。Western blotting结果显示CD73,A1AR,A2AAR在各组差异与通透率一致。冰片，α-细辛醚联合Puer,TMP用药可以一定程度上促进Puer,TMP透过率，联用时加入与腺苷通路相关的抑制剂anti-CD73，anti-A1AR，anti-A2AAR后，冰片和α-细辛醚的促透作用被抑制，表明其开窍机制可能与腺苷受体信号通路相关，初步揭示了芳香开窍类中药“开窍于脑”的相关机制及科学内涵，为中药复方通过配伍实现脑部疾病靶向治疗提供了新的思路和方法。