摘要
目的探讨虎杖Polygonum cuspidatum富集活性组分体外对人类免疫缺陷病毒1型(HIV-1)的抑制作用及作用靶点。方法运用表面等离子共振HIV-1多靶点筛选系统对虎杖不同工艺提取物进行筛选,整合酶氨基耦联柱靶向富集提取物中高活性成分,在TZM-bl细胞和PBMC细胞中对富集产物进行抗HIV-1病毒活性评价;采用荧光共振能量转移分析法测定药物对HIV-1整合酶3’加工的抑制作用;运用高通量ELISA法测定虎杖60%乙醇提取物的整合酶靶向富集产物(HZ60-IN)对整合酶链转移的影响;试剂盒检测HZ60-IN对逆转录酶和蛋白酶的影响。结果 HZ60-IN对整合酶有高亲和性。细胞水平病毒感染实验表明,在TZM-bl细胞中,HZ60-IN对HIV-1 NL4.3和1084i的半数抑制浓度(IC50)分别为(31.94±8.96)、(38.07±11.25)μg/mL;在2株PBMC细胞中,HZ60-IN对HIV-1 NL4.3病毒均显示显著的抑制活性。HZ60-IN对整合酶的3’加工和链转移均有抑制作用,其中对3’加工的IC50为(6.54±1.69)μg/mL,对链转移的IC50为(2.56±0.97)μg/mL,其不作用于HIV感染的进入阶段,对逆转录酶抑制活性较弱,对蛋白酶活性没有影响。结论 HZ60-IN有抗HIV-1病毒活性,主要通过影响HIV-1的整合酶活性发挥作用。
Objective To investigate the inhibitory effect and targets of enriched components from Polygonum cuspidatum on HIV-1 in vitro. Methods Four extracts of P. cuspidatum were screened by HIV-1 multi-target screening system based on the surface plasmon resonance. The highly active components were obtained by NHS-activated HiTrap conjugated with integrase. The anti-HIV activity of the sample was determined with TZM-bl infection assay and PBMCs infection assay. The inhibition of HZ60-IN on integrase 3’ processing was detected by fluorescence resonance energy transfer analysis. High-throughput ELISA was used to determine the effect of enriched active ingredients of P. cuspidatum(HZ60-IN) on integrase chain transfer;Effects of HZ60-IN on reverse transcriptase and protease were detected by kit. Results HZ60-IN displayed higher affinity with integrase. HZ60-IN demonstrated potent antiviral activity against NL4.3 and 1084i strains in TZM-bl cells with the IC50 of(31.94 ± 8.96) and(38.07 ± 11.25) μg/mL, respectively. HZ60-IN showed significant inhibition on HIV-1 NL4.3 strains in PBMCs from two donors. HZ60-IN acted on the integrase with the IC50 of(6.54 ± 1.69) μg/mL for 3’ processing and(2.56 ± 0.97) μg/mL for strand transfer activity. It showed weak effects on the entry stage of HIV infection, with weak inhibitory activity on reverse transcriptase and no effect on protease activity. Conclusion HZ60-IN showed significant inhibitive effect on HIV-1 replication and might specifically interfere with integrase activities.
作者
王小利
胡秦
刘伟
艾萍
沈思嗣
马洪涛
杨怡姝
曾毅
WANG Xiao-li;HU Qin;LIU Wei;AI Ping;SHEN Si-si;MA Hong-tao;YANG Yi-shu;ZENG Yi(College of Life Science and Bio-Engineering,Beijing University of Technology,Beijing 100124,China;Beijing International Science and Technology Cooperation Base of Antivirus Drug,Beijing 100124,China)
出处
《中草药》
CAS
CSCD
北大核心
2020年第3期717-725,共9页
Chinese Traditional and Herbal Drugs
基金
国家科技重大专项(2014ZX10005-002)
国家自然科学基金资助项目(81760783)
北京市自然科学基金(7182012).
关键词
虎杖
HIV-1
整合酶
逆转录酶
蛋白酶
靶向富集
Polygonum cuspidatum Sieb.et Zucc.
HIV-1
integrase
reverse transcriptase
protease
targeted enrichment
