生物利用度屏障的双重性在调控有毒中药附子药效和毒性中的作用及分子机制

Aconiti Radix (Chinese name: Fuzi), a toxic Chinese herb, showed great effectiveness without toxicity in Chinese medicinal practice. Actually, the effects and toxicity of orally-administrated drug is dominantly modulated by bioavailability barrier (BB), a physiological barrier in the liver and intestine. Our previous studies showed that efflux transporters in BB such as p-glycoprotein, breast cancer resistance protein and multi-drug resistance protein 2, partly blocked the absorption of Aconitum alkaloids, the effective/toxic ingredients in Fuzi. More importantly, diester-diterpene alkaloids were hydrolyzed into low toxicity monoester-diterpene alkaloids by CYP enzymes and hydrolases in the liver and intestine. All of these results indicate that the safe use of Fuzi in clinic is due to the bidirectional driving behavior of BB to obtain the optimized plasma concentrations of Aconitum alkaloids in the body, resulting in producing the better effects and low toxicity of Fuzi. Therefore, this project would consequently conduct bidirectional driver of BB with a focus on the interplay of efflux transporters and drug metabolizing enzymes for modulating the effectiveness and toxicity of Fuzi. The advanced models of pharmacokineitcs (PK) such as knock-out/in animals and cell lines combined pharmacodynamics and toxicity models will be employed to deeply explore the molecular mechanism of BB on bidirectional modulation for the effective/toxic ingredients of Fuzi. The expected results of this study would provide a new principle and basic evidence for the usage, dosing, processing, and compatibility, etc. of Fuzi. On the other hand, a new PK-PD molecular mode in this project would be established to investigate relationship between the effects and toxicity for other Chinese herbs and its resulting formula.

中医临床合理使用有毒中药附子时无毒性反应，同时也获得较好的药效，其机制尚不清晰。生物利用度屏障（BB）是机体肝肠器官中的自身防御体系，对外源性药物的药效和毒性具有双重调控作用。我们发现BB中的外排转运蛋白如P-gp、BCRP和MRP2能阻抗附子毒效成分乌头生物碱吸收入血，同时，BB中的CYPs酶和肝肠水解酶也能使毒性大的双酯型生物碱变成单酯型而降低毒性。因此，我们认为附子之所以能安全有效使用，是因为BB的双重调控作用，使附子的毒效成分入血浓度达到最适度而产生最合适的药效和最小的毒性。在前期基础上，课题将以BB中关键元素外排转运蛋白和药物代谢酶的相互作用为重点，采用药代动力学前沿技术如基因敲除/入细胞与动物模型，配合毒效评价，深入阐明BB对附子毒效成分的双重调控作用及分子机制，为附子安全有效的使用、剂量、炮制和配伍等提供新依据。同时，亦能建立PK-PD分子模式研究中药及复方毒效关系的新方法。

附子在中医临床应用中时有毒性发生，但其毒性成分的体内调控机制不清楚。本课题以生物利用度屏障为核心，采用基因敲除动物和细胞模型，研究附子中主要毒效成分乌头生物碱体内暴露（PK）以及毒效的调控及其机制，以及乌头生物碱对外排转运蛋白和药物代谢酶的影响进行了研究，主要得到以下三方面结论：1. 附子毒效成分乌头碱（AC），中乌头碱（MA），次乌头碱（HA），苯甲酰乌头原碱（BAC），苯甲酰中乌头原碱（BMA）和苯甲酰次乌头原碱（BHA）在体内被CYP酶广泛代谢，外排转运蛋白P-gp、BCRP和MRP2 外排 AC， MA， HA， BAC， BMA和BHA，阻抗其入血，且毒性越大，外排转运蛋白的阻抗作用越强。2. 外排转运蛋白敲除后，显著改变乌头生物碱的体内暴露及毒效。具体表现在，P-gp、BCRP和MRP2缺失显著增加AC， MA和HA在小鼠脑、心脏、肝脏和肾脏中的分布，乌头生物碱的中枢镇痛作用显著增加，同时心脏和脑神经毒性亦显著增加，其中，P-gp对乌头生物碱PK及毒效的影响最大，其次是BCRP和MRP2；CYP3A缺失后，乌头碱在血中的暴露显著上升。3. 核受体（PXR和CAR）介导AC、BMA和乌头原碱（aconine）上调P-gp的表达和活性，同时亦可上调BCRP和MRP2的基因和蛋白表达；AC、BMA和aconine在体内外均可上调细胞核中Nrf2的表达；生乌头和制乌头水提物抑制大鼠体内CYP3A活性。这些研究结果提示，附子体内暴露和毒效被外排转运蛋白和CYP酶调控，其毒效成分亦能诱导外排转运蛋白的表达和活性。这为附子的临床合理应用提供了依据。课题共发表SCI论文15篇，培养硕/博研究生共9名，博士后1名，培养出广东省杰青等优秀青年教师4名。获2018年高等学校科学研究优秀成果奖。

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