基于“化瘀、开窍”治法的川芎-冰片脑区特异性调控缺血性脑卒中的PK-PD机制研究

Cerebral ischemic stroke (CIS) is related to “qi xu xue yu, feng yang shang rao, feng tan zu luo, xie bi qing qiao”, and “yu xue bi zu nao mai” is its basic pathogenesis. “Huo xue hua yu, kai qiao xing shen” is its basic rule of therapy, while ligusticum wallichii and borneol are just their representative drugs respectively. It has been confirmed that ligusticum wallichii and borneol have cooperative effect in CIS disorder in clinic and experiment studies. However, its mechanism is unclear till now. It has been shown that borneol can improve celebral ischemia except increase the delivery of other drugs in the brain. Yet there are different emphases between borneol and ligusticum wallichii. We found that the delivery of borneol was specific in different brain region and that although its delivery in cortex is much more than other regions, the opening of BBB in the region was lower than other regions. What’more, too high dose of borenol did not help to open the blood brain-barrier (BBB) in borneol and ligusticum wallichii combination. So, we assume that both the pharmacodynamics and pharmacokinetics factors were involved with the cooperation of the two drugs in specific pretection on brain regions. In addition, the best manner of their combination may be various in different brain regions, which may derive from various delivery of their effective ingredients. The present project will research the cooperative methanism of ligusticum wallichii and borneol in protecting neuron and vascular endothelial cell with PK-PD combination to extend the scientific basis of “huayu, kaiqiao” therapy in CIS disorder, and to supply a new strategy of specific brain region study when an investigation on the therapy and compatibility of Chinese medicine is done.

缺血性脑卒中（CIS）与气虚血瘀、风阳上扰、风痰阻络、邪闭清窍等有关，其中瘀血闭阻脑脉是其基本病机，活血化瘀、开窍醒神为其基本治则，而川芎和冰片分别是它们的代表药物。临床和实验均证实川芎、冰片对CIS的改善具有协同作用，但具体机制不详。资料显示冰片除了能促进药物脑内分布外，本身对脑缺血也有保护作用，与川芎比较各有侧重点。我们前期发现冰片的分布具有脑区特异性，且分布较多的皮层并不利于血脑屏障的开放，另外冰片剂量过大也不利于川芎-冰片的脑保护效应。因此我们推测川芎-冰片的脑区特异性保护有药效学和药动学的双重协同因素。对于不同脑区CIS损伤，它们的最佳配伍方式可能不同，这种不同来源于效应物质在各脑区的分布差异。本项目拟通过PK-PD关联研究川芎-冰片在不同脑区保护神经元和血管内皮细胞的协同机制，拓展“化瘀、开窍”治法用于CIS的科学依据，并提供中医治法和配伍的脑区特异性研究这一新策略。

（1）基于多指标综合权重评分，明确皮层和纹状体的最佳配伍形式是0.08g/kg的冰片+1.0g/kg的川芎，海马和下丘脑的最佳配伍形式是0.16g/kg的冰片+1.0g/kg的川芎。.（2）整体机制研究发现：①冰片仅降低Glu水平，川芎能升高GABA，降低Glu。配伍后还能升高下丘脑Gly含量。②抗氧化主要是川芎在皮层和海马贡献。③川芎调控自噬的pAMPK-LC3 II/I和Beclin1-LC3 II/I。冰片主要调控Beclin1。两药配伍后产生新的靶点，如皮层ULK1、海马ULK1、BNIP3和Beclin1、纹状体ULK1和mTOR。④川芎调控bFGF表达来促进血管生成。而冰片的促血管生成作用较弱。两药配伍不仅能产生新的靶点，如纹状体的FGFR，还可对皮层的FGFR、下丘脑的VEGF、FGFR和VEGFR2，以及纹状体的VEGFR2进一步增强调控。⑤川芎和冰片在皮层、海马对凋亡相关基因的调控能力较强。⑥川芎和冰片对四个脑区超微结构均有改善。.（3）离体神经元-胶质细胞共培养结果：①川芎在四个脑区抗氧化作用明显。②川芎可调节四个脑区的Glu、GABA，以及纹状体的Gly含量。而冰片仅减少Glu水平。它们配伍后能进一步抑制海马Glu释放。③川芎和冰片均能减少四个脑区钙超载的发生。④四个脑区的抗凋亡作用主要为川芎所贡献，其机制涉及对Bcl-2、Bax、p53和caspase-3调控。⑤自噬调控主要是川芎所介导，其机制与调控pAMPK、Beclin1和mTOR有关。冰片仅调控下丘脑pAMPK。联合应用后不仅新增对下丘脑BNIP3的调节，还进一步增强对皮层mTOR、海马pAMPK和LC3 II/I、下丘脑mTOR、ULK1和LC3 II/I的调控。.（4）脑毛细血管内皮细胞结果：川芎和冰片均可增加bFGF、VEGF和VEGFR2表达，减少VEGFR1表达。配伍后能进一步增加VEGF和VEGFR2。川芎抗凋亡与调控Bcl-2、Bax、p53、caspase-3有关。冰片仅调控Bcl-2、Bax。此外川芎还有较好的抗氧化效应。. （5）PK-PD拟合结果：在皮层和海马，冰片能减少洋川芎内酯I的消除，增加其在脑内的暴露程度。同时川芎能放大冰片改善脑微循环的效应。在下丘脑区，冰片促进洋川芎内酯I的脑内分布，并提高后者的内在活性。纹状体区未见两者配伍后发生参数的显著改变。