清开灵注射液体外对Ⅰ型登革热病毒的抗病毒作用研究  

The antiviral effect of Qingkailing injection against Dengue virus type 1 in vitro

作　　者：纪洵敏 梁宇恒 何炽明 周莹 肖微 蒲俏虹 许瑛 赖文辉 张吉凯 张磊 彭拓华 JI Xun-min;LIANG Yu-heng;HE Chi-ming;ZHOU Ying;XIAO Wei;PU Qiao-hong;XU Ying;LAI Wen-hui;ZHANG Ji-kai;ZHANG Lei;PENG Tuo-hua(Guangdong Provincial Institute of Biological Products and Materia,Guangzhou,Guangdong 510440,China)

机构地区：[1]广东省生物制品与药物研究所,广东广州510440

出　　处：《中国热带医学》2021年第2期107-111,共5页China Tropical Medicine

基　　金：广东省中医药局科研项目(No.20182013);广东省卫健委岭南中药干预重点传染病研究委托专项(No.174-2020-XMZC-4716);广东省医学科研基金项目(No.B2020139);广东省重点领域研发计划项目(No.2019B111103001)。

摘　　要：目的探讨清开灵注射液体外抗Ⅰ型登革热病毒(DENV-1)的作用。方法通过噻唑蓝(MTT)法,测定清开灵注射液对C6/36细胞的最大无毒剂量以及清开灵注射液抗DENV-1的作用;利用蛋白质印迹试验(Western Blot),以非结构蛋白NS1为目的蛋白,检测DENV-1经不同稀释度的清开灵注射液预处理后的感染能力;荧光定量PCR法检测清开灵注射液对DENV-1拷贝数的影响。采用方差分析比较不同稀释度清开灵注射液对DENV-1的影响。结果 MTT结果显示,清开灵注射液对C6/36细胞的最大无毒剂量为1/64;经过稀释度为1/64、1/128和1/256的清开灵注射液处理后,感染病毒的细胞存活率增加至(76.9±3.5)%、(72.5±2.5)%和(63.0±1.5)%,表明清开灵注射液能明显减弱DENV-1引起的细胞病变效应;Q-PCR结果显示稀释度为1/64、1/128的清开灵注射液对DENV-1拷贝数的抑制率达到(43.8±3.1)%和(27.4±1.4)%,表明其对病毒RNA复制产生明显的抑制作用;蛋白质印迹试验结果显示,清开灵注射液能有效地抑制病毒NS1蛋白的表达。结论清开灵注射液体外预处理病毒的给药方式对Ⅰ型登革热病毒具有明显的抑制作用。Objective To evaluate the antiviral effects of the Qingkailing injection against Dengue virus type 1(DENV-1) in vitro.MethodsThe maximum non-toxic dose and anti-DENV-1 effect of Qingkailing injection on the C6/36 cells was determined by MTT. The effect of Qingkailing injection on DENV-1 was determined by MTT. Using NS1 protein as the target protein, the infectivity of DENV-1 pretreated with different dilution of Qingkailing injection was detected by Western Blot. The effect of Qingkailing injection on DENV-1 copy number was determined by Q-PCR. Variance analysis was used to compare the effects of Qingkailing injection with different dilution on the virus.ResultsMTT results showed that the maximum nontoxic dose of Qingkailing injection on C6/36 cells was 1/64. MTT results showed that cell viability had been increased to(76.9±3.5)%,(72.5±2.5)% and(63.0±1.5)% by Qingkailing injection with dilutions of 1/64, 1/128 and 1/256. This result indicated that Qingkailing injection could significantly reduce the cytopathic effect of DENV-1. Q-PCR results showed that the copy numbers of DENV-1 was reduced by(43.8±3.1)% and(27.4±1.4)% with Qingkailing injection at the dilutions of 1/64 and 1/128, which showed that Qingkailing injection had significantly inhibitory effect on RNA replication of DENV-1. According to the Western blot results, the expression of NS1 protein was effectively reduced with Qingkailing injection after pretreatment.ConclusionQingkailing injection has obvious antivirus activity in vitro after pretreatment.

关 键 词：清开灵注射液 C6/36细胞 细胞毒性 登革热病毒 

分 类 号：R373.3+3[医药卫生—病原生物学]