肉桂酸衍生物抗PRV及其作用机制研究

Cinnamic acid derivative Against Pseudorabies Virus and Its Mechanisms In Vitro

关键词:  肉桂酸衍生物  PRV  PK-15  抗病毒  天然化合物

Key words:  Cinnamic Acid Derivative  PRV  PK-15  antiviral  natural compounds

摘要:

研究以猪伪狂犬病病毒(PRV)感染PK-15细胞,对27种天然化合物的抗PRV作用进行了筛查,并探讨了抗PRV活性物质的作用机制。分别将27种天然化合物以不同方式作用于感染细胞,采用细胞病变法(CPE)和噻唑蓝比色法(MTT)测定其细胞毒性及对病毒吸附、复制和直接杀灭的作用。在药物与PRV共同作用于PK-15细胞24 h、48 h、72 h后,RT-PCR检测PRV的代表基因IE180、UL30和UL22在胞内的表达量。结果显示,肉桂酸衍生物(cinnamic acid derivative,CAD)具有抗PRV的活性,其EC50为(8.443±0.216)μg/mL,SI为6.6,最高抑制率为76%。3个时间点胞内IE180、UL30和UL22基因的拷贝数显著降低(P <0.01),且呈剂量依赖性。结果表明,CAD对PRV有直接灭活作用,其作用机制是阻断PRV在细胞内的生物合成过程,但CAD对PRV的吸附过程没有阻断作用。因此,CAD可做为抗PRV的候选药物。

Abstract:

27 kinds of natural compounds were tested for their antiviral activity against pseudorabies virus(PRV)in PRV infected PK-15 cells and determined their antiviral mechanisms. Visualization with the cytopathologic effect (CPE) assay and the 3-(4, 5-dimethyithiazol-2-yl)-2,5-diphenyltetrazolium bromide test (MTT) were used to assess the cytotoxic concentrations of compounds and the antiviral effects on viral adsorption, replication and virucidal activity after each compound incubating infected cells in different ways. RT-PCR is adopted to detect the influence of compounds on RPV representative genes (IE180,UL30 and UL22) expression when the compound and PRV simultaneously acted on the cells in 24 h, 48 h and 72 h.SThe results showed that cinnamic acid derivative(CAD) has anti-PRV activity, the EC50 value was (8.443±0.216) μg/mL, the selectivity indexes was 6.6 and the maximum inhibition ratio was 76%.The copy numbers of IE180, UL30 and UL22 in cytoplasm are decreased apparently(P <0.01) in a dose-dependent manner at three various time-points after CAD and PRV acting simultaneously on the cells.SIt indicated that CAD could directly inactivate PRV in vitro and the mechanism is inhibiting biosynthesis of PRV in cells rather than inhibiting PRV adsorption. CAD could be developed as new anti-PRV drugs for clinical application.