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

Objective To evaluate the antiviral effects of the Qingkailing injection against Dengue virus type 1 (DENV- 1) in vitro. Methods The 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. Results MTT 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. Conclusion Qingkailing injection has obvious antivirus activity in vitro after pretreatment. 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、/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。