双氢青蒿素体外抗猪繁殖与呼吸综合征病毒的作用研究

doi:10.16431/j.cnki.1671-7236.2025.09.034

摘要/Abstract

摘要： 【目的】探讨双氢青蒿素(dihydroartemisinin,DHA)体外抗猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus，PRRSV)作用。【方法】试验首先建立体外抗PRRSV的Marc-145细胞模型，采用MTT法检测DHA对Marc-145细胞毒性，确定其最大安全浓度。将Marc-145细胞分为4组，分别为空白对照组(Control)、阳性对照组(利巴韦林)、模型组(PRRSV)和药物组(DHA)，检测阻断、抑制和直接灭活作用方式下DHA抗PRRSV的作用效果。通过实时荧光定量PCR和Western blotting分别检测不同作用方式下、不同时间点PRRSV N的mRNA和蛋白表达量，利用Annexin/PI双染法检测PRRSV感染诱导的细胞凋亡情况。【结果】 DHA和阳性药物利巴韦林的最大安全浓度分别为3.13和15.63 μg/mL。在阻断、抑制和直接灭活作用下，DHA对PRRSV的的最大抑制率分别为66.2%、88.9%和62.1%；在抑制作用下细胞基本保持单层完好，在阻断和直接灭活作用下细胞出现了圆缩、团聚的现象。抑制作用下，利巴韦林对PRRSV的抑制率最大，抑制率为68.9%，细胞出现了圆缩、团聚的现象。实时荧光定量PCR检测结果显示，在阻断和抑制作用下，与模型组相比，利巴韦林和DHA组细胞中PRRSV N基因拷贝数显著降低(P＜0.05)；在直接灭活作用下，与模型组相比，DHA组细胞中PRRSV N基因拷贝数显著降低(P＜0.05)。Western blotting结果显示，不同作用方式下，与模型组相比，不同时间点DHA组细胞中PRRSV N蛋白表达量均显著降低(P＜0.05)。细胞凋亡检测结果显示，阻断作用下，与模型组相比，早期凋亡48 h时，DHA组细胞凋亡率显著降低(P＜0.05)；晚期凋亡不同时间点，利巴韦林和DHA组细胞凋亡率均显著降低(P＜0.05)。抑制作用下，与模型组相比，在早期凋亡和晚期凋亡72 h时，DHA组细胞凋亡率均显著降低(P＜0.05)。直接灭活作用下，与模型组相比，早期凋亡24、48 h时，DHA组细胞凋亡率显著降低(P＜0.05)；晚期凋亡24、72 h时，DHA组细胞凋亡率显著降低(P＜0.05)。【结论】 DHA体外抗PRRSV作用显著，主要通过抑制PRRSV N基因表达及N蛋白合成发挥作用，同时对细胞凋亡具有抑制效果。

关键词: 双氢青蒿素(DHA); 猪繁殖与呼吸综合征病毒(PRRSV); 抗病毒作用; 细胞凋亡

Abstract: 【Objective】 The purpose of this experiment was to explore the anti-Porcine reproductive and respiratory syndrome virus (PRRSV) effect of dihydroartemisinin (DHA) in vitro. 【Method】 The anti-PRRSV Marc-145 cell model was established in vitro.The cytotoxicity of DHA to Marc-145 cells was detected by MTT method to determine its maximum safe concentration.Marc-145 cells were divided into 4 groups,namely blank control group (Control),positive control group (ribavirin),model group (PRRSV) and drug group (DHA).The effects of DHA against PRRSV under the modes of blocking,inhibition and direct inactivation were detected.The mRNA and protein expression levels of PRRSV N under different modes of action and at different time points were detected by Real-time quantitative PCR and Western blotting,respectively.The apoptosis induced by PRRSV infection was detected by Annexin/PI double staining method. 【Result】 The maximum safe concentrations of DHA and the positive drug ribavirin were 3.13 and 15.63 μg/mL,respectively.Under the effects of blocking,inhibition and direct inactivation,the maximum inhibition rates of DHA on PRRSV were 66.2%,88.9% and 62.1% respectively.Under the inhibitory effect,the cells basically remained monolayer intact.Under the blocking and direct inactivation effects,the cells showed the phenomenon of roundness and agglomeration. Under the inhibitory effect,ribavirin had the highest inhibition rate of PRRSV,with an inhibition rate of 68.9%,and the cells showed the phenomenon of roundness and agglomeration.The results of Real-time quantitative PCR showed that under the effects of blocking and inhibition,compared with model group,the copy number of PRRSV N gene in cells of ribavirin and DHA groups was significantly decreased (P＜0.05).Under direct inactivation,compared with model group,the copy number of PRRSV N gene in cells of DHA group was significantly decreased (P＜0.05).The results of Western blotting showed that under different modes of action,compared with model group,the expression level of PRRSV N protein in DHA group at different time points was significantly decreased (P＜0.05).The results of apoptosis detection showed that under the blocking effect,compared with model group,at 48 hours of early apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).At different time points of late apoptosis,the apoptosis rates of both ribavirin and DHA groups were significantly decreased (P＜0.05).Under the inhibitory effect,compared with model group,at 72 hours of early apoptosis and late apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).Under the direct inactivation,compared with model group,at 24 and 48 hours of early apoptosis,the apoptosis rate of DHA group was significantly decreased (P＜0.05).At 24 and 72 hours of late apoptosis,the apoptosis rate of DHA group decreased significantly (P＜0.05). 【Conclusion】 DHA had a significant anti-PRRSV effect in vitro,mainly by inhibiting the expression of PRRSV N gene and the synthesis of N protein,and at the same time had an inhibitory effect on cell apoptosis.

Key words: dihydroartemisinin (DHA); Porcine reproductive and respiratory syndrome virus (PRRSV); antiviral effect; cell apoptosis

中图分类号:

S853.7

秦枫, 刘云, 唐楠楠, 吴植, 李勇军, 于生兰, 朱善元. 双氢青蒿素体外抗猪繁殖与呼吸综合征病毒的作用研究[J]. 中国畜牧兽医, 2025, 52(9): 4379-4393.

QIN Feng, LIU Yun, TANG Nannan, WU Zhi, LI Yongjun, YU Shenglan, ZHU Shanyuan. Study on the Anti-Porcine Reproductive and Respiratory Syndrome Virus of Dihydroartemisinin in vitro[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(9): 4379-4393.

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