表没食子儿茶素没食子酸酯衍生物及过硫酸氢钾复合物粉体外抑制Ⅰ型草鱼呼肠孤病毒的研究

Inhibition of genotype I GCRV by EGCG derivatives and compound potassium peroxymonosulfate powder in vitro

摘要:

【目的】为了将表没食子儿茶素没食子酸酯(epigallocatechin gallate, EGCG)的2种衍生物和过硫酸氢钾复合物粉(compound potassium peroxymonosulfate powder, KMPS)运用于体外细胞实验中，以评估这3种药物对I型草鱼呼肠孤病毒(grass carp reovirus, GCRV)的抑制和杀灭效果。【方法】利用MUSE法和CCK-8法评估表没食子儿茶素没食子酸酯棕榈酸酯(epigallocatechin gallate palmitate, EGCG-P)、乙酰化表没食子儿茶素没食子酸酯(peracetylated epigallocatechin gallate, AcEGCG)和过硫酸氢钾复合物粉3种药物对细胞的安全浓度，利用体外细胞感染病毒模型，使用不同浓度测试物处理病毒或细胞后感染病毒，通过实时荧光定量聚合酶链反应(quantitative real-time polymerase chain reaction, qRT-PCR)法分析不同测试物对病毒的抑制和杀灭效果。使用不同浓度测试物处理细胞后，利用qRT-PCR法检测宿主免疫相关基因变化情况。【结果】EGCG-P和AcEGCG对GCRV-JX01感染细胞的抑制作用呈剂量依赖性，且EGCG-P的抑制效果优于AcEGCG，EGCG-P浓度为10 μg/mL和40 μg/mL时病毒量相对于对照组上清中病毒的滴度分别降低了10² copies/μL和10³ copies/μL，而AcEGCG浓度为60 μg/mL和120 μg/mL时才能达到相同抑制效果。杀灭实验结果表明，EGCG-P、AcEGCG和KMPS对GCRV-I毒株均有杀灭效果，最低有效杀灭剂量KMPS (5 μg/mL)<EGCG-P (120 μg/mL)<AcEGCG (180 μg/mL)。草鱼白细胞介素-1β (interleukin-1β, IL-1β)、白细胞介素-8 (interleukin-8, IL-8)、肿瘤坏死因子α (tumor necrosis factor α, TNFα)和髓样分化因子(medullary differentiation factor, MyD88)相对于对照组在测试物中均有不同程度上调，其中EGCG-P (50 μg/mL)和AcEGCG (10 μg/mL)处理组分别在8 h和24 h上调较为显著。【结论】EGCG-P、AcEGCG和KMPS对GCRV-I均有良好的抑制效果和杀灭效果，其中EGCG-P和KMPS能够在40 μg/mL较低浓度下对GCRV发挥显著性的抑制和杀灭效果，且在24 h后不会引起机体产生炎症反应。本研究为拮抗草鱼呼肠孤病毒环境友好型消毒产品的开发提供参考。

关键词:草鱼出血病;草鱼呼肠孤病毒;表没食子儿茶素没食子酸酯棕榈酸酯;乙酰化表没食子儿茶素没食子酸酯;过硫酸氢钾复合物粉

Abstract:

[Objective] To evaluate the inhibitory and killing effects of two epigallocatechin gallate (EGCG) derivatives and compound potassium peroxymonosulfate powder (KMPS) on grass carp reovirus genotype I (GCRV) by in vitro cell experiments. [Methods] The safe concentrations of epigallocatechin gallate palmitate (EGCG-P), peracetylated epigallocatechin gallate (AcEGCG), and KMPS were assessed by MUSE and CCK-8 assays. Different concentrations of EGCG-P, AcEGCG, and KMPS were used to treat the cells infected with GCRV-JX01, and the inhibitory and killing effects of the three substances on the virus were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Furthermore, qRT-PCR was employed to determine the expression levels of the genes involved in host immune responses in the cells treated with the three substances and then infected with the virus. [Results] EGCG-P and AcEGCG suppressed the viral proliferation in a concentration- dependent manner, and EGCG-P outperformed AcEGCG. The EGCG-P of 10 μg/mL and 40 μg/mL decreased the titer of GCRV-JX01 by 10² copies/μL and 10³ copies/μL, respectively, compared with the control group, while AcEGCG achieved the same inhibitory effect at the concentrations of 60 μg/mL and 120 μg/mL, respectively. EGCG-P, AcEGCG, and KMPS all had killing effect on GCRV-JX01, and the lowest dose for effective killing followed the trend of KMPS (5 μg/mL)<EGCG-P (120 μg/mL)<AcEGCG (180 μg/mL). Moreover, EGCG-P, AcEGCG, and KMPS up-regulated the expression of interleukin-1β (IL-1β), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), and myeloid differentiation factor (MyD88) compared with the control group. In particular, the up-regulation was more significant after the treatment with EGCG-P (50 μg/mL) for 8 h and AcEGCG (10 μg/mL) for 24 h. [Conclusion] EGCG-P, AcEGCG, and KMPS have good inhibitory and killing effects on GCRV-I. EGCG-P and KMPS at a concentration of 40 μg/mL can significantly inhibit GCRV replication without evoking an inflammatory response in 24 h. This study provides a reference for the development of environmentally friendly disinfection products for GCRV.

Key words:grass carp hemorrhagic disease;grass carp reovirus (GCRV);epigallocatechin gallate palmitate (EGCG-P);peracetylated epigallocatechin gallate (AcEGCG);compound potassium peroxymonosulfate powder