基于ADME/Tox研究钩吻畏玉叶金花的配伍减毒机制

Gelsemium elegans has significant anti-tumor activity, as well as non-addictive analgesic efficacy. However, the clinical application of Gelsemium elegans is limited for its highly toxic to human. The combining use of Gelsemium elegans and Mussaenda pubescens is based on the record of past dynasties of Chinese medicine and the theory of phase fear of traditional Chinese medicine that synergy can reduce toxicity. The experimental data have already shown that Mussaenda pubescens can reduce toxicity of Gelsemium elegans,and CYP2E1 can metabolize koumine. The study try to explore the decreased toxicity mechanism of Gelsemium elegans combining with Mussaenda pubescens by ADME/Tox. The study may clarify the transport mechanism of toxic substances before and after compatibility by in vitro Caco-2 cells method、expression of P-glycoprotein（P-gp）with SP method、in situ intestine perfusion in rat and component analysis of LC-MS; It may clarify the transformation mechanism of toxic substances before and after compatibility by gene mRNA expression of rat liver CYP2E1 with RT-PCR、protein expression of rat liver CYP2E1 with ELISA and component analysis of LC-MS; It may also determinate the equilibrium point of toxicity/efficacy by correlation index of toxicity/efficacy. Thus, The study will clarify the decreased toxicity mechanism of Gelsemium elegans combining with Mussaenda pubescens, and establish methodology of ADME/Tox to evaluate the decreased toxicity of synergy of traditional Chinese medicine. The study will provide experimental basis for the safety use of Gelsemium elegans and develop the theory of phase fear of traditional Chinese medicine.

钩吻治疗肿瘤有确切疗效，且还具无成瘾性的高效镇痛优势，但因毒性大，限制了临床的应用。钩吻配伍玉叶金花是根据历代本草所记载、中药七情配伍之相畏减毒理论，且前期实验表明：玉叶金花能降低钩吻的毒性，肝微粒体CYP2E1能代谢钩吻素子。本项目拟将ADME/Tox技术平台用于探讨钩吻畏玉叶金花的解毒机制，采用体外Caco-2细胞跨膜转运实验、免疫细胞化学SP法检测P-gp蛋白表达、在体大鼠原位肠灌流吸收实验，结合LC-MS成分分析，阐明配伍前后毒性物质的转运机制；采用RT-PCR法测定大鼠肝脏CYP2E1基因mRNA表达、采用免疫酶间接法检测大鼠肝脏CYP2E1蛋白表达，结合LC-MS分析成分变化，阐明配伍前后毒性物质的转化机制；采用毒/效相关指标，确定毒/效平衡点。该研究将阐明钩吻畏玉叶金花的减毒机制，建立ADME/ Tox评价中药配伍减毒的方法学，又可为钩吻安全应用提供依据，并丰富相畏配伍理论。

目的 探讨钩吻配伍玉叶金花减毒机制，为临床安全合理应用提供依据。.方法 1.毒效实验：筛选钩吻的最佳配比和相畏部位；测定配伍前后的LD50及靶器官细胞形态学变化；考察醋酸扭体、热板、福尔马林致痛实验及在体荷瘤实验。.2.转运研究：考察配伍前后双向跨膜转运及对P-gp及MRP2的影响；建立钩吻毒/效部位指纹图谱，并运用UPLC-MS/MS分析鉴定，考察吸收情况。.3.转化研究：考察配伍前后对Ⅰ相代谢中CYP2E1、1A2基因与蛋白表达的影响；对Ⅱ相代谢GST活力，GST-pi、GSTm1基因与蛋白表达的影响。.结果 1.毒效实验：①钩吻二氯甲烷部位配伍玉叶金花水部位（1:40）后平均死亡率大幅降低（P＜0.05）；钩吻LD50为0.32g•kg-1，配伍后LD50为0.68g•kg-1；配伍后未见肝坏死、水肿，损伤明显减轻。②配伍后能显著提高小鼠热板痛阈值（P＜0.05）；能显著减少醋酸所致小鼠扭体次数（P＜0.01）；能使第Ⅰ、Ⅱ时相内出现的疼痛反应持续时间显著减少（P＜0.01）；对裸鼠结肠癌有显著抑瘤作用。.2.对转运的影响：①配伍后3种生物碱外排比率γ均大于2。②维拉帕米（或环孢素）与配伍后的钩吻毒/效部位合用后，6种吲哚类生物碱的吸收量、外排量均明显增加，但生物碱外排比均减少了，其中4种生物碱减少显著。③钩吻毒/效部位48h内基本稳定；指纹图谱相似度大于0.93，具有很好的相关性；UPLC-MS／MS鉴定出6种吲哚类生物碱，6种吲哚类生物碱吸收良好，但配伍后吸收率降低了。.3.对转化的影响：①与空白组比，各剂量组CYP2E1 mRNA/蛋白表达量均极显著下降（P＜0.01），配伍后表达量较高剂量上升（P＜0.05）；免疫组化法显示阳性染色细胞（CYP2E1、CYP1A2）均呈棕黄色或棕褐色，配伍后阳性细胞面密度、数密度较高剂量组上升（P＜0.01或P＜0.05）。②GSTm1 mRNA/蛋白表达量结果与2E1一致；与空白组比，各剂量组GST酶活力均明显下降（P＜0.01或P＜0.05），配伍后酶活力较高剂量上升（P＜0.05）。.结论 钩吻配伍后能减毒存效；降低吲哚类生物碱的吸收；上调了CYP2E1、CYP1A2的表达，增强GST的活性，达到减毒目的。