6种钩藤生物碱对P-gp介导开放血脑屏障的作用研究及活性成分属内分布

Cerebral diseases and cardiovascular diseases are the world's leading preventable cause of death. Due to the blood-brain barrier, many brain diseases treatment drugs can not pass and the loss efficacy. The P-glycoprotein is firstly involved on the efflux of drugs in blood-brain barrier, and can be over-expressed in the focus. P-glycoprotein is the key target of promoting drug cross blood brain barrier, and reversing treatment resistance. Uncaria owns clearing heat, calming liver function, sedation effects, and can be used as monarch drug in many prescriptions. It possesses obvious curative effects on brain diseases. In our preliminary study, it was found that rhynchophylline in Uncaria has inhibitory activity on P-glycoprotein in the high expression of P-glycoprotein cell model. Therefore, this project focus on screening P-glycoprotein inhibiting active alkaloids by using MCF-7/ADR cell model and verifying the activities in SD rats by immunohistochemistry, Western blotting etc. Furthermore, by means of LC-MS simultaneous multicomponent quantitative technique in complex system, the variation of Uncaria chemical ingredients that cross the blood brain barrier is monitored. Finally, active ingredients that can enhance the permeability of blood-brain barrier by regulating P-glycoprotein can be determined, and content of active ingredients in 11 species of Uncaria plants are assessed. The result of this project will give a glimpse of pharmacodynamic mechanism of Uncaria in a new perspective, and will provide theory basis for selecting evaluation indexes in genus resource assessment.

脑源性疾病与心血管疾病一同位居世界各种死因首位。由于人体血脑屏障的存在，使得诸多治疗药物无法通过而丧失疗效。P-糖蛋白首要参与了血脑屏障中对药物的外排、且尤其在病灶部位可过度表达，是增加药物通过血脑屏障、逆转治疗抵抗的关键靶点。钩藤自有清热平肝，息风定惊之功效，常在脑部疾病方剂中作君药使用且疗效显著。申请人用P-糖蛋白高表达细胞模型对钩藤碱的P-糖蛋白抑制活性进行初步研究，发现其特异性抑制效果。因此，本项目使用MCF-7/ADR模型筛选钩藤6种入脑生物碱的P-糖蛋白抑制活性，利用免疫组化、免疫印迹等方法在大鼠体内加以验证。并借助液质联用复杂体系多组分定量技术，监测活性成分对钩藤中同行组分通过血脑屏障的改变。最终确定可通过抑制P-糖蛋白改善血脑屏障对药物通透性的活性成分，并对我国钩藤属11种植物中活性成分含量加以评估。从新的角度揭示钩藤的药效机制，为资源品质评价指标选取提供理论依据。

本项目拟从8种钩藤入脑生物碱成分入手，借助于P-糖蛋白（P-gp）高表达细胞模型，快速筛选具有P-gp抑制效果的活性成分，并对其在生物体内的P-gp抑制作用机制进行探究；进而结合对P-gp表达的抑制活性，分别对八种中国钩藤属（Uncaria Shreb.）药用植物进行基础资源评价。项目首先通过HPLC法构建钩藤生物碱成分指纹图谱，并建立8种生物碱成分同时定量方法，从药材性状、显微性状以及化学成分剖析等多方面对钩藤属8种植物资源进行全面评价；随后采用实时荧光定量PCR和罗丹明123蓄积实验等技术手段，在已构建的MCF-7/ADR细胞中，进行8种生物碱对P-gp表达的抑制作用的比较；进一步选取抑制活性最强的2种生物碱成分，通过流式检测细胞膜蛋白MDR1实验、WB实验等方法，研究活性成分对NF-kB信号通路、STAT3信号通路的影响 的影响；最后以D-半乳糖腹腔注射法构建小鼠衰老模型，并通过被动趋暗回避反射实验初步验证常规药物治疗时辅以P-gp表达抑制成分，对抗衰老药物在模型小鼠体内的作用效果。.本项目研究结果显示，钩藤生物碱提取液指纹图谱显示6种钩藤药材生物碱成分相似度高，分别为钩藤、大叶钩藤、白钩藤、攀茎钩藤、平滑钩藤、华钩藤；不同种钩藤中钩藤碱、异去氢钩藤碱、异钩藤碱、缝籽木嗪甲醚、毛钩藤碱、去氢钩藤碱、去氢毛钩藤碱和四氢鸭脚木碱的含量范围分别为3.8243~263.6342 μg/g、99.1730~1784.2548 μg/g、50.8636~1281.9185 μg/g、0~172.5024 μg/g、0~201.5926 μg/g、0~298.2324 μg/g、0~1793.9941 μg/g和0~11.4952 μg/g，含量差异较大；其中倒挂金钩在生物碱数目及含量上均与其他种钩藤存在显著性差异。8种生物碱的P-gp抑制活性比较显示，毛钩藤碱、去氢钩藤碱活性最强；在胞质中，毛钩藤碱抑制IκBα磷酸化，从而减少了NF-κB亚基p65磷酸化并入核；转录因子p65入核的减少，最终导致P-gp表达下降；去氢毛钩藤碱可以抑制转录因子STAT3磷酸化激活，虽然其不影响总STAT3蛋白的表达，但可以显著抑制STAT3入核和细胞核内STAT3磷酸化，此外化合物还能抑制P-gp的表达。以上研究结果对倒挂金钩化学分类学研究、P-gp抑制活性成分的中药配伍研究有重要意义。