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-1NL4.3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.

R285.5

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）