探讨穿心莲内酯对HIV-1感染辅助受体CXCR4启动子作用机制的研究  

Investigation of the Effect of Andrographolide on CXCR4 Promoter in HIV-1 Infection

作　　者：冯龙[1] 郭文涛 李志慧 郑文锦 耿宇轩 FENG Long;GUO Wentao;LI Zhihui;ZHENG Wenjin;GENG Yuxuan(Henan University of Traditional Chinese Medicine,Zhengzhou 450046,China;Guangdong Medicial Universily,Shenzhen 524023,China)

机构地区：[1]河南中医药大学,郑州450046 [2]广东医科大学,深圳523808

出　　处：《病毒学报》2022年第2期363-370,共8页Chinese Journal of Virology

基　　金：国家自然科学基金(项目号:81503677),题目:针对HIV-1和CXCR4/CCR5启动子为靶点的抗HIV/AIDS药物筛选系统的建立;河南省青年骨干教师资助项目(项目号:2015GGJS-095),题目:穿心莲提取物对外周血CD4+T淋巴细胞表面CXCR4和CCR5的影响及其作用机制的研究;河南省高校重点科研项目(项目号:20A310008),题目:姜黄素调控Foxp3干预人趋化性细胞因子CCR5启动子机制的研究。

摘　　要：穿心莲内酯具有明显的抗病毒作用,对HIV-1(Human immunodeficiency virus type 1)具有明显的抑制作用,本研究探讨了穿心莲内酯影响CXCR4启动子活性的作用机制。首先构建双荧光素酶报告基因载体pFireRlucCXCR4(C-X-C chemokine receptor 4),并转染入人HEK293T细胞;利用CCK8法检测穿心莲内酯对人HEK293T细胞细胞毒性作用;双荧光素酶报告基因技术检测穿心莲内酯对CXCR4启动子活性的影响;MTT法检测穿心莲内酯对人T淋巴细胞Jurkat细胞活性影响;实时荧光定量PCR检测穿心莲内酯对人T淋巴细胞Jurkat细胞表面CXCR4 mRNA和蛋白表达的影响。PCR分别扩增了CXCR4启动子(777 bp)和Rluc(1 997 bp)表达单元,通过测序和酶切鉴定双荧光素酶报告基因载体插入正确;穿心莲内酯作用于转染pFireRluc-CXCR4HEK293T细胞,双荧光素酶结果显示:穿心莲内酯能够下调CXCR4启动子活性,差异具有显著性(P<0.05)。穿心莲内酯作用于人T淋巴细胞Jurkat细胞后,qPCR和Western Blot结果显示:Jurkat细胞表面CXCR4 mRNA和蛋白表达降低。因此,穿心莲内酯能够影响CXCR4启动子活性,具有潜在的抗HIV-1的作用。Andrographolide showed obvious antiviral effect and inhibitory effect on HIV-1. This study explored the mechanism of andrographolide affecting CXCR4 promoter activity. Double luciferase reporter gene expression vector pFIRerluc-CXCR4 was constructed and transfected into human HEK293 T cells. The cytotoxic effect of andrographolide on human HEK293 T cells was detected by CCK8 method. The effect of andrographolide on the activity of CXCR4 promoter was detected by dual-luciferase reporter technique. The cytotoxic effect of andrographolide on human T lymphocytes was determined by MTT assay. The effect of andrographolide on the expression of CXCR4 mRNA and protein on the surface of human T lymphocyte Jurkat cells were detected by real-time fluorescence quantitative PCR. The CXCR4 promoter(777 bp) and Rluc(1,997 bp)expression units were amplified by PCR,respectively. The expression vector of the dual-luciferase reporter gene was confirmed by enzyme digestion and sequencing. Andrographolide was applied on human HEK293 T cells transfected with PFIRerluc-CXCR4,and results showed that the CXCR4 promoter activity was significantly down-regulated(P<0.05),suggesting that andrographolide could significantly inhibit the CXCR4 promoter activity. qPCR and Western Blot results showed that CXCR4 mRNA and protein expression were decreased after andrographolide was applied to human T lymphocyte Jurkat cells. This study suggested that andrographolide can affect the activity of CXCR4 promoter and has potential anti-HIV-1 effect.

关 键 词：穿心莲内酯 CXCR4 启动子 HIV-1 

分 类 号：R373.9[医药卫生—病原生物学]