摘要
为建立基于Nrf2、HO-1启动子活性的抗病毒药物筛选方法,实验以胖头鱥上皮细胞系(FHM)为材料,构建Nrf2、HO-1启动子重组质粒,利用双荧光素酶报告系统检测不同浓度(0、3.1、6.3、12.5、25.0、50.0μg/mL)的白藜芦醇、水飞蓟宾、穿心莲内酯及姜黄素对启动子活性的影响,并利用鲤春病毒血症病毒(spring viremia of carp virus,SVCV)和蛙虹彩病毒(rana grylio iridovirus,RGV)验证阳性药物的抗病毒效果。结果显示,Nrf2、HO-1启动子序列中存在FOX家族、IRF家族等多种转录因子的结合位点,并分别存在1个和3个与甲基化相关的CpG岛。双荧光素酶报告实验显示,水飞蓟宾、穿心莲内酯及姜黄素可激活Nrf2、HO-1启动子。药物浓度梯度实验显示,6.3μg/mL的姜黄素和穿心莲内酯可显著上调Nrf2和HO-1的启动子活性。病毒感染后的细胞病变效应、病毒的复制及病毒滴度结果均表明姜黄素和穿心莲内酯可抑制SVCV和RGV的感染。综上,本实验建立的pGL3-Nrf2和pGL3-HO-1启动子报告质粒,可用于抗水生病毒药物的筛选,也为Nrf2、HO-1转录调控机制的研究提供了工具。
In order to establish an antiviral drug screening method targeting to Nrf2/HO-1 pathway,the recombinant plasmids of Nrf2 and HO-1 promoter were constructed,respectively.The effects of different concentrations(0.0,3.1,6.3,12.5,25.0 and 50.0μg/mL)of resveratrol,silybin,andrographolide and curcumin on promoter activity were detected by the dual luciferase reporter system in fathead minnow cells(FHM).Further,spring viremia of carp virus(SVCV)and rana grylio iridovirus(RGV)were used to verify the antiviral effect of positive drugs in FHM cells.The results showed that a variety of binding sites for transcription factors were found in the Nrf2 and HO-1 promoter sequences,including FOX family and IRF family.As well,1 and 3 methylation-related CpG islands were found,respectively.The dual luciferase reporter assay showed that silybin,andrographolide and curcumin could activate the Nrf2 and HO-1 promoters.Drug concentration gradient experiments showed that curcumin and andrographolide at a concentration of 6.3μg/mL could significantly up-regulate the promoter activities of Nrf2 and HO-1.The cytopathic effect observation,virus replication tests and virus titration assay confirmed that curcumin and andrographolide can inhibit the infection of SVCV and RGV significantly.In conclusion,the pGL3-Nrf2 and pGL3-HO-1 promoter reporter plasmids established in this experiment can be used for the screening of anti-aquatic virus drugs,and also provide tools for the study of the transcriptional regulation mechanism of Nrf2 and HO-1.
作者
王靖雯
吴苗苗
李莉娟
顾泽茂
袁军法
WANG Jingwen;WU Miaomiao;LI Lijuan;GU Zemao;YUAN Junfa(College of Fisheries,Huazhong Agricultural University,Wuhan 430070,China;National Aquatic Animal Diseases Para-reference laboratory,Huazhong Agricultural University,Wuhan 430070,China)
出处
《水产学报》
CAS
CSCD
北大核心
2024年第2期141-152,共12页
Journal of Fisheries of China
基金
国家自然科学基金(31872598)。
