基于抗氧化反应元件高通量药物筛选细胞模型的建立及应用  

Establishment and application of a cell model for high-througut screening of secondary metabolites of mangrove fungi based on ARE fluorescence expression

作　　者：魏明月 田银 吴科锋 汤熙翔 WEI Mingyue;TIAN Yin;WU Kefeng;TANG Xixiang(Institute of Marine Medicine,Guangdong Medical University,Zhanjiang 524023,China;Third Institute of Oceanography,Ministry of Natural Resources,Xiamen 361005,China;Southern Marine Science and Engineering Guangdong Laboratory(Zhanjiang),Zhanjiang 524000,China)

机构地区：[1]广东医科大学海洋医药研究院,广东湛江524023 [2]自然资源部第三海洋研究所,福建厦门361005 [3]南方海洋科学与工程广东省实验室(湛江),广东湛江524000

出　　处：《中国海洋药物》2025年第1期39-48,共10页Chinese Journal of Marine Drugs

基　　金：亚洲海洋合作基金项目(99950410);北京大学天然药物及仿生药物国家重点实验室开放基金项目(K202201);厦门市海洋与渔业发展专项资金项目(21CYY004HJ16)资助。

摘　　要：目的利用抗氧化反应元件(antioxidant response element,ARE)调控萤火虫荧光素酶报告基因的表达,建立高通量药物筛选细胞模型,筛选出具有潜在抗氧化活性的红树林真菌来源微生物次级代谢产物。方法构建ARE调控萤火虫荧光素酶表达的重组质粒pGL6-TA-2×ARE,并与内参质粒p RL-SV40-C瞬时共转染小鼠精母细胞GC-2spd(ts),富马酸二甲酯(dimethyl fumarate,DMF)为激活剂,建立基于双荧光素酶报告基因的体外药物筛选体系。对107个福建漳江口红树林沉积物来源真菌的发酵粗提物进行活性筛选;2,2-偶氮二(2-甲基丙基咪)二盐酸盐(AAPH)刺激GC-2spd(ts)细胞建立体外氧化应激模型;CCK-8法检测粗提物对GC-2spd(ts)的细胞活力和AAPH氧化应激处理后粗提物对细胞活力的影响。结果成功建立ARE调控萤火虫荧光素酶表达的细胞高通量药物筛选细胞模型,从107份红树林真菌次级代谢产物中发现3个菌株次级代谢产物在25、50和100μg/mL时显著激活ARE水平表达且呈剂量依赖性,并抑制了AAPH诱导的细胞损伤。结论基于ARE筛选体系及氧化应激细胞模型发现菌株编号分别为米H1509干、米H366干、米H346干的红树林真菌次级代谢产物可增强ARE荧光表达水平,发挥潜在抗氧化活性。Objective A cellular screening model for antioxidant response element-regulated luciferase expression was constructed to high-throughput screening of mangrove fungus-derived secondary metabolites with potential antioxidant activity.Methods The antioxidant response element was cloned into the firefly luciferase reporter gene vector pGL6-TA and the recombinant plasmid pGL6-TA-2×ARE was constructed,which transiently cotransfected with the endogenous reference plasmid pRL-SV40-C into GC-2spd(ts)cells.With dimethyl fumarate(DMF)as an activator,a dual-luciferase reporter gene screening system was constructed.107 samples of secondary metabolites from mangrove sediment in Zhangjiangkou,Fujian,were screened for activity;GC-2spd(ts)cells were stimulated by 2,2'-Azobis(2-methylpropionamidine)dihydrochlor--ide(AAPH)to establish an in vitro oxidative stress model.Effect of crude extracts on cell viability of GC-2spd(ts)and effect of crude extracts on cell viability after AAPH stimulation were detected by CCK-8 assay.Results A cellular high-throughput drug screening cell model based on ARE-regulated luciferase expression was successfully established.From 107 mangrove fungal secondary metabolites,three strains of secondary metabolites were found to significantly activate the expression level of ARE at 25,50,and 100μg/mL in a dose-dependent manner,and inhibited AAPH-induced cell damage.Conclusion Based on the ARE screening system and oxidative stress cell model,it was found that the secondary metabolites of mangrove fungi with strain numbers H1509,H366,and H346,respectively,could enhance the fluorescence expression level of ARE to exert potential antioxidant activities.

关 键 词：抗氧化反应元件 药物筛选模型 红树林真菌 抗氧化 

分 类 号：R931.77[医药卫生—生药学]