川芎调控脑血瘤屏障上转运蛋白“引药入瘤”的作用及机制研究

Because of the blood brain barrier/blood brain tumor barrier (BBB/BBTB), it is difficult for anticancer drugs to distribute into the brain or into the tumor. The result is that the treatment of glioblastoma has become the difficult problem in biological medicine field. Many studies showed that the message drug could regulate the permeability of BBB to promote other drugs into the brain, but the mechanism of the message drugs regulate the BBTB to promote other drugs into the tumor is still unknown. On the basis of our research that Ligusticum chuanxiong Hort. could regulate the permeability of BBB to promote other drugs into the brain, the hypothesis that chuanxiong may promote the distribution of drugs to the tumor by regulating the key transport proteins in BBTB has been proposed. Temozolomide, Bis-chloroethyltrosourea and cis-Dichlorodiamineplatinum, which are clinically used as chemotherapeutic drug on brain glioma, are adopted as model drugs. The BBTB promoting part of chuanxiong will be found through the effects of them on the distribution of model drugs in blood, brain and tumor, then the promoting ingredient of chuanxiong will continue to be found by high-throughput screening approaches on BBTB cell model in vitro. The result will be further proved by the whole animal testing in vivo. The changed levels of key transport proteins (GLU-1, GLU-3 and Caveolin-1) and normal efflux proteins caused by chuanxiong, will be showed the promoting mechanism by Immunohistochemistry and Western blotting. The Material basis and the mechanism of chuanxiong promoting the distribution of drugs to the tumor will be clarified in our studies from the level of cells, tissues and organs, and it will provide a new way to solve the problem of drugs through BBTB.

抗肿瘤药物因血脑屏障/脑血瘤屏障（BBB/BBTB）而难“入脑、入瘤”，使得脑胶质瘤的治疗成为医药学界的难题。研究发现引经药能调节BBB初步解决“入脑”难题，但较少针对BBTB特点研究其“入瘤”的机制。本课题结合川芎可调节BBB引药入脑的工作基础，提出“川芎可通过调节BBTB上多种转运蛋白引药入瘤”假说。针对该假说，以治疗脑胶质瘤的临床用药替莫唑胺、卡莫司汀及顺铂为模型药，微透析法研究川芎对模型药在C6胶质瘤大鼠的血、瘤内分布，找出川芎具有促透BBTB的活性部位；体外BBTB细胞跨膜转运试验进行高通量筛选，找出促透活性成分并进行体内整体动物试验验证；免疫组化、分子印迹法研究川芎促透的活性成分对关键转运蛋白（GLU-1、GLU-3、Caveolin-1）、常规转运蛋白水平的调节，从细胞、组织及器官水平阐明川芎调节BBTB引药“入瘤”物质基础和作用机制，为解决药物透BBTB难题提供新思路。

抗肿瘤药物因血脑屏障/脑血瘤屏障（BBB/BBTB）而难“入脑、入瘤”，使得脑胶质瘤的治疗成为医药学界的难题。研究发现引经药能调节BBB初步解决“入脑”难题，但较少针对BBTB特点研究其“入瘤”的机制。本课题以引药上行的川芎为研究对象，通过C6胶质瘤大鼠体内药动学研究发现，川芎中的挥发油、总酚酸、总生物碱能增加替莫唑胺脑内蓄积，同时挥发油还能增加替莫唑胺以及顺铂胶质瘤内蓄积，具有引药入脑、入瘤的作用，western blot试验结果表明该作用机制与下调P-gp、GLU-1，紧密连接蛋白claudin-5、occludin蛋白水平有关。C6胶质瘤大鼠体药效学结果表明川芎挥发油下调 P-gp 蛋白水平，促进替莫唑胺进入胶质瘤细胞内，发挥协同抗肿瘤作用，同时还阻止胶质瘤诱导的NF-kB通路激活，减少体内炎症反应发挥抗胶质瘤的协同作用。hCMEC/D3和U251共培养建立的体外BBTB细胞模型试验结果表明，挥发油中正丁基苯酞、新蛇床内酯、丁烯基苯酞、藁本内酯、洋川芎内酯A和洋川芎内酯H通过下调GLU-1、GLU-3、caveolin-1，进而正相关的下调紧密连接蛋白occludin、claudin-5和ZO-1水平，增加BBTB膜通透性，促进配伍药物细胞旁路转运。同时，正丁基苯酞、新蛇床内酯、藁本内酯和洋川芎内酯A还能增加P-gp介导的药物跨细胞转运，是川芎中引药入脑、入瘤的物质基础。然而这几个成分体内药动学结果表明，口服生物利用度较差（均低于25%），可能会影响配伍药物“入脑、入瘤”的效果，因此本课题将挥发油成分采用固体分散体及环糊精包合技术固化，均显著提高了口服生物利用度。本课题从细胞、组织及器官水平阐明川芎调节BBTB引药“入瘤”物质基础和作用机制，为解决药物透BBTB难题提供新思路，研究成果可用于指导临床上胶质瘤治疗药物配伍，以及脑肿瘤靶向制剂的产品设计和研究开发。