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

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.