白蔹乙醇提取物体外抗登革病毒的作用研究  

Study on anti⁃dengue virus effect of ethanol extracts from Ampelopsis Radix in vitro

作　　者：方小川 刘俊珊 田春阳 郑远茹 卢子滨 曹惠慧 余林中 FANG Xiaochuan;LIU Junshan;TIAN Chunyang;ZHENG Yuanru;LU Zibin;CAO Huihui;YU Linzhong(Guangdong Provincial Key Laboratory of Chinese Medicine Pharmaceutics,Third-Level Research Laboratory of National Administration of Traditional Chinese Medicine for traditional Chinese Medicine Pharmacology Laboratory,School of Traditional Chinese Medicine,Southern Medical University,Guangzhou 510515,China)

机构地区：[1]南方医科大学中医药学院,广东省中药制剂重点实验室,广东省中西医结合防治情志病基础研究卓越中心,广东广州510515

出　　处：《上海中医药大学学报》2025年第1期65-73,共9页Academic Journal of Shanghai University of Traditional Chinese Medicine

基　　金：国家自然科学基金资助项目(81730110)。

摘　　要：目的:探讨白蔹乙醇提取物体外抗登革病毒(DENV)的活性及机制。方法:通过噻唑蓝(MTT)法明确白蔹乙醇提取物对BHK-21细胞的细胞毒性,确定药物的非毒浓度;采用分时给药法筛选白蔹乙醇提取物中具有抗DENV活性的部位并探究药物对DENV的吸附进入过程的影响;细胞病变效应(CPE)法评估药物干预时病毒诱导的细胞病变情况;采用实时荧光定量-聚合酶链式反应(Q-PCR)法、蛋白免疫印迹法以及免疫荧光法检测细胞中病毒E蛋白及NS1的mRNA及蛋白的表达。结果:成功筛选出12种具有抗DENV吸附进入细胞作用的白蔹乙醇提取馏分,且尤以B1~B5组分抗病毒活性最强;MTT结果显示,B4、B5组分的半数细胞毒性浓度(CC50)分别为(40.39±5.70)、(20.27±3.43)μg/mL;预给药及低温感染后给药时,对于DENV致细胞活力下降没有明显改善作用,而在低温感染同时给药时,BHK-21细胞活力得到明显提升,其中B4、B5的半数有效浓度(EC50)分别为(2.78±0.44)、(1.59±0.16)μg/mL,这表明B4、B5仅对病毒吸附阶段有抑制作用,此外,给予10μg/mL的B4或4μg/mL的B5时几乎完全抑制DENV诱导的CPE;Q-PCR及免疫荧光结果显示B4、B5干预后DENV的E蛋白及NS1蛋白显著降低,表明B4、B5对病毒RNA及蛋白合成阶段具有显著抑制作用。结论:白蔹乙醇提取物B1~B5具有较好的抗DENV活性,且该作用可能与阻断DENV吸附至细胞表面有关。Objective:To explore the activity and mechanism of ethanol extracts from Ampelopsis Radix on anti-dengue virus(DENV)in vitro.Methods:The cytotoxicity of ethanol extracts from Ampelopsis Radix on BHK-21 cells were determined by thiazolyl blue tetrazolium bromide(MTT)assay,and the non-toxic concentration of the drugs was determined.Time-sharing administration method was used to screen the anti-dengue active parts of ethanol extracts from Ampelopsis Radix,and to explore the influence of the drugs on the adsorption and entry process of DENV.Virus-induced cytopathic changes during drug intervention were evaluated by cytopathic effect(CPE).Real-time quantitative Polymerase Chain Reaction(Q-PCR),Western blotting and immunofluorescence were used to detect the expressions of virus E protein and NS1 mRNA and protein in cells.Results:Twelve ethanol extract fractions from Ampelopsis Radix with the effect of anti-DENV adsorption on and entry into cell were successfully screened and fractions B1~B5 had the best antiviral activity.MTT results showed that the median cytotoxic concentrations(CC50)of fractions B4 and B5 were(40.39±5.70)mg/L and(20.27±3.43)mg/L respectively.The decrease of cell viability caused by DENV was not ameliorated when pre-administration or administration after lowtemperature infection,but BHK-21 cell viability was significantly increased when simultaneous administration during low-temperature infection,with the median effective concentrations(EC50)of B4 and B5(2.78±0.44)mg/L and(1.59±0.16)mg/L respectively,indicating that B4 and B5 only had an inhibitory effect on the virus adsorption stage.In addtion,DENV-induced CPE was almost completely inhibited after 10 mg/L B4 or 4 mg/L B5 treatment.The results of Q-PCR and immunofluorescence showed that E protein and NS1 protein of DENV were significantly decreased after B4 and B5 intervention,indicating that B4 and B5 had a significant inhibitory effect on the viral RNA and protein synthesis stage.Conclusion:Ethanol extracts B1~B5 from Ampelopsis Radix have goo

关 键 词：白蔹 乙醇提取物 登革热 登革病毒 作用机制 

分 类 号：R285[医药卫生—中药学]