木犀草素抗汉滩病毒效果初步研究  

The effects of luteolin against Hantaan virus

作　　者：赵悦汐 罗璐璐 王彦博 强遵先 吕依雯 马宏炜 程林峰 张芳琳 ZHAO Yuexi;LUO Lulu;WANG Yanbo;QIANG Zunxian;LV Yiwen;MA Hongwei;CHENG Linfeng;ZHANG Fanglin(School of Medical Technology,Shaanxi University of Chinese Medicine,Xianyang,Shaanxi 712046,China;School of Basic Medicine,Air Force Medical University,Xi'an,Shaanxi 710032,China)

机构地区：[1]陕西中医药大学医学技术学院,陕西咸阳712046 [2]空军军医大学基础医学院,陕西西安710032

出　　处：《热带医学杂志》2024年第3期318-323,I0001,共7页Journal of Tropical Medicine

基　　金：陕西省重点研发计划项目(2022ZDLSF01-08)。

摘　　要：目的 探索木犀草素对汉滩病毒(HTNV)的抑制作用。方法 用0、3.125、6.25、12.5、25、50、100、150、200 μmol/L的木犀草素处理Huh-7细胞,用0、10、20、25、50、75、100、150、200 μmol/L的木犀草素处理A549细胞,培养48 h后,细胞计数试剂盒8(CCK-8)法检测细胞活力并计算木犀草素半数毒性浓度(CC_(50))。在HTNV感染同时Huh-7细胞分别加入0、8、10、14、16、22 μmol/L木犀草素,A549细胞分别加入0、4、6、8、10、12 μmol/L木犀草素,2 h后换液为含对应浓度药物的培养基,培养48 h后,实时荧光定量PCR检测细胞内HTNV RNA水平并计算半数抑制浓度(IC_(50))。在Huh-7细胞感染HTNV的不同时间阶段(感染前1~48 h、感染前1 h、感染0~2 h、感染后2~48 h、感染后6~48 h、感染后12~48 h、感染后24~48 h),给予25 μmol/L木犀草素处理,感染48 h后,通过蛋白质印迹、免疫荧光、实时荧光定量PCR检测细胞内HTNV蛋白和RNA水平,通过酶联斑块形成实验检测子代HTNV滴度。分别在HTNV入胞阶段(感染0~2 h)及复制阶段(感染后2~48 h),用高、中、低浓度(25、12.5、6.25 μmol/L)木犀草素处理感染HTNV的Huh-7细胞,感染48 h后检测细胞内HTNV蛋白和RNA水平。最后通过分子对接模拟木犀草素与HTNV的核衣壳蛋白(NP)和RNA依赖的RNA聚合酶(RdRp)的相互作用效果。结果 与0 μmol/L木犀草素处理比较,25 μmol/L木犀草素处理的Huh-7和A549细胞的细胞存活率差异无统计学意义(F=79.94、14.64,P均>0.05);后续实验选择25 μmol/L为木犀草素的最大安全浓度;木犀草素对于Huh-7、A549细胞的CC_(50)分别为141、83 μmol/L。Huh-7、A549细胞的木犀草素的IC_(50)分别为16.18、11.64 μmol/L。在HTNV感染后2~48 h、6~48 h、12~48 h、24~48 h给予木犀草素处理,HTNV蛋白、RNA及滴度水平降低,差异均有统计学意义(F=3.48、6.29、18.69,P均<0.05)。木犀草素抑制复制组的HTNV蛋白、RNA水平随着药物浓度的增加而降低,差异均有统�Objective To confirm the anti-Hantaan virus(HTNV)activity of luteolin.Methods Huh-7 cells were treated with luteolin at the concentrations of 0,3.125,6.25,12.5,25,50,100,150 and 200 μmol/L;A549 cells were treated with luteolin at the concentrations of 0,10,20,25,50,75,100,150,200 μmol/L.After 48 h of culture,the viability of Huh-7 and A549 cells was detected by cell counting kit-8(CCK-8).Then,the 50% cytotoxic concentration(CC_(50))of luteolin was calculated.Huh-7 cells infected with HTNV were treated with luteolin at 0,8,10,14,16 and 22 μmol/L;A549 cells infected with HTNV were treated with luteolin at 0,4,6,8,10 and 12 μmol/L.After 48 h of culture,the HTNV RNA level in the cells was detected by qRT-PCR and the half maximal inhibitory concentration(IC_(50)) was calculated.At different stages of HTNV infection(1-48 h before infection,1 h before infection,0-2 h after infection,2-48 h after infection,6-48 h after infection,12-48 h after infection,24-48 h after infection),Huh-7 cells were treated with 25 μmol/L luteolin.After 48 h infection,the levels of HTNV protein and RNA in cells were detected by Western blot,immunofluorescence and qRT-PCR,and the HTNV titer was detected by enzyme-linked focus formation assay test.Huh-7 cells infected with HTNV were treated with high,medium and low concentrations(25,12.5,6.25 μmol/L)of luteolin at the cell entry stage(0-2 h after infection)and replication stage(2~48 h after infection),and the HTNV protein and RNA levels in the cells were detected after 48 h of infection.To detect the interaction between luteolin and HTNV,luteolin is docked with Nucleocapsid protein(NP)and RNA dependent RNA polymerase(RdRp)of HTNV.Results Compared with 0 μmol/L luteolin-treated,there was no statistical significance in cell viability between 25 μmol/L luteolin-treated Huh-7 and A549 cells(F=79.94,14.64;both P>0.05).In the following experiments,25 μmol/L was selected as the maximum safe concentration of luteolin.The CC_(50)of luteolin to Huh-7 and A549 cells were 141 and 83 μmol/L respect

关 键 词：汉滩病毒 肾综合征出血热 木犀草素 抗病毒感染 

分 类 号：R373.3[医药卫生—病原生物学]