基于中医药化学生物信息学探讨归芪白术方抑制葡萄膜黑色素瘤细胞增殖的物质基础  

Study on the Material Basis of Guiqi Baizhu Prescription Inhibiting the Proliferation of Uveal Melanoma Cells Based on Traditional Chinese Medicine Chemical Bioinformatics

作　　者：王锐峰 靳晓杰 刘昊 李程豪 张敏 李咪 李浩田 张宇 马欢欢 张月梅[3] WANG Ruifeng;JIN Xiaojie;LIU Hao;LI Chenghao;ZHANG Min;LI Mi;LI Haotian;ZHANG Yu;MA Huanhuan;ZHANG Yuemei(Gansu University of Chinese Medicine,Gansu University Key Laboratory for Molecular Medicine&Chinese Medicine Prevention and Treatment of Major Diseases,Lanzhou 730000,China;Gansu University of Chinese Medicine,School of Pharmacy,Lanzhou 730000,China;Department of Ophthalmology,the First Hospital of Lanzhou University,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学甘肃省高校重大疾病分子医学与中医药防治研究重点实验室,兰州730000 [2]甘肃中医药大学药学院,兰州730000 [3]兰州大学第一医院眼科,兰州730000

出　　处：《中国现代应用药学》2024年第14期1900-1912,共13页Chinese Journal of Modern Applied Pharmacy

基　　金：国家自然科学基金项目(82160857);甘肃省科技计划项目(21JR7RA352);甘肃省高等学校产业支撑计划项目(2022CYZC-54)。

摘　　要：目的利用药效团模型-分子对接-结合自由能计算的虚拟筛选策略结合细胞生物学实验的中医药化学生物信息学方法,挖掘归芪白术方中靶向磷脂酰肌醇3-激酶(phosphatidylinositide 3-kinases,PI3K)抑制葡萄膜黑色素瘤(uveal melanoma,UM)细胞增殖的成分。方法构建PI3K抑制剂药效团模型,并对归芪白术方化合物进行虚拟筛选,对符合药效团模型的成分进行分子对接和结合自由能计算,选择潜在抑制成分进行生物学实验评价。利用CCK-8和克隆形成试验检测潜在抑制成分对UM细胞增殖的影响;流式细胞术检测UM细胞周期和凋亡情况;JC-10染色法检测UM细胞线粒体膜电位;Western blotting检测PI3K及下游通路蛋白表达。结果药效团模型包括2个氢键受体、2个芳环中心以及排除体积。CCK-8结果显示10、20、40、80μmol·L^(-1)槲皮素、川陈皮素、桔皮素,20、40、80μmol·L^(-1)桑辛素可抑制UM细胞的增殖。克隆形成试验显示不同浓度的槲皮素、川陈皮素、桔皮素、桑辛素均可明显抑制细胞的克隆增殖。流式细胞术结果显示桔皮素、槲皮素将细胞阻滞在G0/G1期,川陈皮素、桑辛素将UM细胞阻滞在G2/M期。JC-10染色法结果显示槲皮素、川陈皮素、桔皮素、桑辛素均可使UM细胞的线粒体膜电位降低,Western blotting结果显示4种化合物均可靶向PI3K发挥作用,但其影响的下游通路不同。结论本研究基于中医药化学生物信息学的方法挖掘了归芪白术方抑制UM细胞增殖的物质基础,为中药复方的现代化开发提供参考。OBJECTIVE To utilize the pharmacophore model-molecular docking combined with the virtual screening strategy of free energy calculation and the chemical bioinformatics method of traditional Chinese medicine in cell biology experiments to investigate the components of Guiqi Baizhu prescription that target phosphatidylinositol 3-kinase(PI3K)and inhibit the proliferation of uveal melanoma(UM)cells.METHODS The pharmacophore model of PI3K inhibitor was constructed,and the compounds of Guiqi Baizhu prescription were virtual screened.The components that fit the pharmacophore model were calculated by molecular docking and binding free energy,and the potential inhibitory components were selected for biological experimental evaluation.The effects of potential inhibitory components on UM cell proliferation were detected by CCK-8 and clonal formation assay.Flow cytometry was used to detect the cell cycle and apoptosis of UM cells.The mitochondrial membrane potential of UM cells was detected using JC-10 staining.The expressions of PI3K and downstream pathway proteins were detected by Western blotting.RESULTS The pharmacophore model included 2 hydrogen bond receptors,2 aromatic ring centers,and exclusion volumes.The results of the CCK-8 experiment showed that quercetin,tangerine,and nobiletin at concentrations of 10,20,40,80μmol·L^(-1),and cyrtin at concentrations of 20,40,80μmol·L^(-1),were able to inhibit the proliferation of UM cells.The clonal formation experiment showed that quercetin,tangerine,nobiletin,and morusin,at different concentrations,could significantly inhibit the clonal Chin J Mod Appl Pharm,2024 July,Vol.41,No.14 proliferation of UM cells.Flow cytometry showed that UM cells were arrested in the G0/G1 phase by tangeretin and quercetin,while UM cells were arrested in the G2/M phase by nobiletin and morusin.The results of JC-10 staining showed that quercetin,nobiletin,tangeretin,and morusin could reduce the mitochondrial membrane potential of UM cells.Western blotting results showed that 4 compounds could tar

关 键 词：葡萄膜黑色素瘤 归芪白术方 中医药化学生物信息学 药效团模型 

分 类 号：R285.5[医药卫生—中药学]