粗壮女贞提取物抗甲型流感病毒的作用及机制初探  

The anti-influenza A virus effect of Ligustrum robustum extract and the preliminary study on its mechanism

作　　者：李晶晶 徐佳伊 左浩江 罗姝菡 潘美伶 洪泽慧 杨慧萍[3] 裴晓方 LI Jing-jing;XU Jia-yi;ZUO Hao-jiang;LUO Shu-han;PAN Mei-ling;HONG Ze-hui;YANG Hui-ping;PEI Xiao-fang(West China School of Public Health and West China Fourth Hospital,Sichuan University^Chengdu y Sichuan 610041,China;不详)

机构地区：[1]四川大学华西公共卫生学院/华西第四医院,四川成都610041 [2]食品安全监测与风险评估四川省重点实验室 [3]四川省疾病预防控制中心

出　　处：《现代预防医学》2022年第3期503-507,共5页Modern Preventive Medicine

基　　金：四川省科技厅应用基础研究项目(2020YJ0233);成都市科技局技术创新研发项目(2019-YF05-01247-SN-6)。

摘　　要：目的探讨粗壮女贞提取物体外不同作用方式下抗甲型流感病毒的作用及机制。方法以狗肾上皮(Madin-Darby Canine Kidney,MDCK)细胞建立甲型H1N1流感病毒感染模型,采用粗壮女贞提取物对MDCK细胞最大无毒浓度(0.8 mg/ml)进行实验,CCK-8法测定提取物预防式、治疗式和共同式干预下MDCK细胞存活率;荧光定量PCR法、间接免疫荧光法检测预防式干预下细胞内病毒核蛋白(nucleoprotein,NP)基因表达、蛋白定位;荧光法检测病毒神经氨酸酶(neuraminidase,NA)荧光强度;ELISA法测定细胞内炎症因子IL-6、IL-8、IFN-γ、IP-10表达水平。结果与病毒对照组相比,治疗式、共同式干预下实验组细胞存活率差异无统计学意义(q=2.658,P>0.05;Z=1.547,P>0.05);预防式干预下,实验组中细胞存活率上升(Z=4.195,P<0.001),病毒NP多定位于细胞核内,且基因表达下降(Z=-2.662,P<0.05);NA活性降低(Z=-2.405,P<0.05);IL-6(Z=-2.772,P<0.05)、IL-8(q=8.493,P<0.001)和IFN-γ(Z=-2.863,P<0.05)蛋白表达量下降。结论预防式干预下粗壮女贞提取物具有抗甲型H1N1流感病毒作用,其机制可能与干扰病毒NP功能、抑制NA活性以及减轻炎症反应有关。Objective To explore the anti-influenza A virus(IAV)effect of the Ligustrum robustum extract in vitro under different intervention methods and its mechanism.Methods Establishing the IAV infection model with madin-darby canine kidney(MDCK)cells.Using the maximum non-toxic concentration(0.8 mg/ml)of Ligustrum robustum extract on MDCK cells for experiments.The activity of MDCK cells under the intervention of prophylactic,therapeutic and premixed of Ligustrum robustum extract was determined by cell counting kit-8(CCK-8).Intervened by prophylactic administration,real-time quantitative polymerase chain reaction and indirect immunofluorescence method were used to assess the expression of viral nucleoprotein(NP)gene and its localization.Fluorescence assay was used to detect neuraminidase(NA)fluorescence intensity.The expression of inflammatory factors IL-6,IL-8,IP-10 and IFN-γwere determined by enzyme linked immunosorbent assay(ELISA).Results Compared with the virus control group,there was no statistically significant difference in the cell viability rate of the experimental group under therapeutic and premixed administration(q=2.658,P>0.05;Z=1.547,P>0.05).Ligustrum robustum extract could improve the activity of MDCK cells infected with influenza A virus under the prophylactic administration(Z=4.195,P<0.001).In the experimental group,the expression level of viral NP was decreased(Z=-2.662,P<0.05)and NP mostly existed in the nucleus.The NA activity in experimental group was also decreased(Z=-2.405,P<0.05).Meanwhile,the levels of inflammatory factor IL-6(Z=-2.772,P<0.05),IL-8(q=8.493,P<0.001)and IFN-γ(Z=-2.863,P<0.05)were decreased.Conclusion Ligustrum robustum extract has the function of anti-influenza A virus under the prophylactic administration.Its mechanism may be related to the ability of Ligustrum robustum extract to inhibit viral NA activity,interfere with NP functionality and reduce the expression levels of inflammatory factors.

关 键 词：粗壮女贞 甲型流感病毒 核蛋白 神经氨酸酶 

分 类 号：R285[医药卫生—中药学]