翻白草油对RSV感染宿主HeLa细胞Fas/FasL蛋白表达的影响

The effect of Potentilla discolor oil on the expression of Fas and FasL proteins in Hela cells infected with respiratory syncytial virus in vitro

目的通过检测呼吸道合胞病毒(RSV)感染宿主HeLa细胞Fas蛋白和FasL蛋白的变化,探讨翻白草油的抗RSV作用。方法用RSV感染宿主HeLa细胞,采用Reed-Muench法求出RSV对细胞半数感染量(CCID50);将翻白草油与HeLa细胞共孵育,MTT法检测翻白草油对Hela细胞的毒性作用;选取最大无毒浓度的翻白草油和100CCID50的RSV病毒进行抗病毒试验,在药物4种作用方式下(对RSV病毒的直接灭活作用,在生物合成阶段对RSV病毒的作用,对RSV病毒吸附的作用,药物预处理作用),采用免疫荧光和Western blot方法检测宿主HeLa细胞Fas/FasL蛋白的表达。结果 RSV培养液对HeLa细胞中的毒力为105.8 CCID50/L;翻白草油对HeLa细胞的最小毒性浓度是0.10mg/L。翻白草油(除药物预处理作用方式外)Fas蛋白和FasL蛋白表达的荧光强度分别为1.851±0.022、1.830±0.044、1.801±0.038和1.832±0.034、1.767±0.022、1.816±0.024,RSV病毒对照组荧光强度分别为2.112±0.048和1.904±0.025,翻白草油(除药物预处理作用方式)组与病毒组比较,宿主HeLa细胞Fas蛋白和FasL蛋白的表达显著降低(P<0.05);翻白草预处理组Fas蛋白和FasL蛋白表达的荧光强度分别为2.098±0.075和1.882±0.055,与RSV病毒对照组比较差异无统计学意义(P>0.05)。结论翻白草油在直接灭活阶段、病毒复制阶段、病毒吸附阶段的抗RSV作用与调节宿主HeLa细胞Fas/FasL蛋白的表达有关。

Objective To investigate the inhibitory effect of Potentilla discolor oil on respiratory syncytial virus （RSV） in vitro by detecting the expression of Fas and FasL proteins in Hela cells infected with RSV. Methods After Helm cells were infected with RSV, viral toxicity was assessed via the 50% cell culture infective dose （CCID50） according to the Reed Muench method. The conventional MTT （3 （4,5-dimethylthiazol-2-yl） 2,5 diphenyltetrazolium bromide） method was used to determine the effect of Potentilla discolor oil toxicity on HeLa cells. The maximum nontoxic concen- tration of Potentilla discolor oil and 100 CCIDs0 of the RSV virus were also used to perform antiviral experiments. The ex- pression of Fas and Fasl. proteins was detected using Western blotting and an immunofluorescence assay in four different modes of drug action （action to directly inactivate RSV, action on RSV during biosynthesis, action on the adsorption of RSV, and action as a pretreatment）. Results The CCID50 of RSV was l0s. 8 and the minimum toxic concentration （TC0） of Potentilla discolor oil was 0.10 mg/I. in HeLa cells. In three different modes of drug action （action as a pretreatment was excluded）, the fluorescence intensity of Fas expression was 1. 851±0. 022, 1. 830±0. 044, and 1. 801±0.038 versus 2. 098±0. 075 in untreated cells, and the fluorescence intensity of FasI. expression was 1. 832!0. 034, 1.767±0.022, and 1.816±0.024 versus 1.882±0.055 in untreated cells. When cells were treated withPotentilladiscol or oil （action as a pretreatment was excluded）, expression of Fas and FasL proteins was significantly lower （P〈0.05） than in untreated cells. However, the expression of Fas and FasL proteins did not differ significantly for cells pretreated with Potentilla discolor oil and untreated cells（P〉0.05）. Conclusion Potentilla discolor oil has action against RSV by directly inactivating the virus, inhibiting its adsorption, and blocking its replication. The anti-RSV action of Potentilla discolor oil may be related to regulation of expression of Fas and FasL proteins.