虎杖水提取物对猪流行性腹泻病毒在IPEC-J2细胞中复制的影响

doi:10.7668/hbnxb.20195708

华北农学报 ›› 2025, Vol. 40 ›› Issue (3): 215-224. doi: 10.7668/hbnxb.20195708

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虎杖水提取物对猪流行性腹泻病毒在IPEC-J2细胞中复制的影响

王煜轩¹^,², 杨植¹^,², 刘昊阳¹^,², 王兴海¹^,², 杨富琳¹^,², 芦烘德¹^,², 何至远¹^,²^,, 董虹¹^,²^,

¹ 北京农学院,兽医学(中医药)北京市重点实验室,北京 102206
² 北京农学院动物科学技术学院,北京 102206

收稿日期:2024-12-22 出版日期:2025-07-04
通讯作者:
董虹(1978—),女,四川绵竹人,教授,博士,主要从事提高机体免疫和抗菌中药筛选及分子机制研究。
何至远(1989—),男,江西赣州人,副教授,博士,主要从事中药替抗防控动物疫病研究。
作者简介:
王煜轩(1996—),男,河北石家庄人,在读硕士,主要从事中药对生猪疫病治疗的相关研究。
王煜轩、杨植为同等贡献作者。
基金资助:
“十四五”重点研发计划项目(2022YFD1801103); 国家自然科学基金项目(32373058); 国家自然科学基金项目(32302916); 市属高校分类发展-都市农林特色教师队伍建设项目(11000024T000002961733); 北京市教育委员会科学研究计划项目(KM202310020006); 北京市教育委员会科技计划一般项目(KM202410020007); 北京农学院青年教师科研创新能力提升计划资助项目(QJKC-2023011); 北京农学院科技创新“火花行动”支持计划项目(BUA-HHXD2023001)

Effect of Polygonum cuspidatum Aqueous Extract on Porcine epidemic diarrhoea virus Replication in IPEC-J2 Cells

WANG Yuxuan¹^,², YANG Zhi¹^,², LIU Haoyang¹^,², WANG Xinghai¹^,², YANG Fulin¹^,², LU Hongde¹^,², HE Zhiyuan¹^,²^,, DONG Hong¹^,²^,

¹ Beijing Key Laboratory of Traditional Chinese Veterinary Medicine,Beijing University of Agriculture,Beijing 102206,China
² Animal Science and Technology College,Beijing University of Agriculture,Beijing 102206,China

Received:2024-12-22 Published:2025-07-04

摘要/Abstract

摘要：

为探究猪流行性腹泻病毒(PEDV)在猪小肠上皮细胞(IPEC-J2)中的繁殖适应性,并评估虎杖水提取物对其体外抑制作用,通过细胞毒性试验和Western Blot试验,评估不同胰酶浓度、不同时间对IPEC-J2细胞活性以及PEDV N蛋白在细胞内表达的影响;再通过TCID₅₀、实时荧光定量PCR和Western Blot等方法检测病毒滴度、PEDV N基因转录水平及N蛋白的表达量;使用CCK-8法检测虎杖水提取物对IPEC-J2细胞增殖活力的影响;进一步通过实时荧光定量PCR和Western Blot试验,检测虎杖水提取物对PEDV N基因和N蛋白表达的抑制作用,以及探究虎杖水提取物对PEDV生命周期各个阶段的影响。结果表明,当胰酶浓度为2 μg/mL时,胰酶对细胞无影响,此时PEDV展示出最佳的繁殖适应性,且36 h后达到最高毒力值;虎杖水提取物浓度低于100 μg/mL时在12 h,24 h无细胞毒性。虎杖水提取物在All-treatment Co-treatment、Post-treatment 3种加药方式下显著抑制N基因表达,并呈剂量依赖显著抑制PEDV N基因mRNA和N蛋白的表达;虎杖水提取物主要在病毒生命周期中复制增殖阶段对PEDV N基因有显著抑制效果,在其他阶段无明显抑制作用。综上,2 μg/mL胰酶浓度对PEDV繁殖适应性最佳,并且虎杖水提取物在体外有显著的抑制PEDV效果,主要作用于PEDV生命周期的复制增殖阶段。

关键词: 虎杖水提取物, PEDV, IPEC-J2, 细胞适应性, 体外抑制

Abstract:

The aim of this study was to explore the reproductive adaptability of PEDV in porcine intestinal epithelial cells(IPEC-J2)and to evaluate the inhibitory effect of Polygonum cuspidatum water extract on it in vitro.The effects of different trypsin concentrations and different time on the activity of IPEC-J2 cells and the expression of PEDV N protein in cells were evaluated by cytotoxicity assay and Western Blot assay.The virus titer,PEDV N gene transcription level and N protein expression were detected by TCID₅₀,Real-time Fluorescence Quantitative PCR and Western Blot.The effect of water extract of P.cuspidatum on the proliferation activity of IPEC-J2 cells was detected by CCK-8 method.Further,the inhibitory effect of P.cuspidatum water extract on the expression of PEDV N gene and N protein was detected by Real-time Fluorescence Quantitative PCR and Western Blot experiments,and the effect of P.cuspidatum water extract on each stage of PEDV life cycle was explored.The results showed that when the concentration of trypsin was 2 μg/mL,trypsin had no effect on the cells.At this time,PEDV showed the best reproductive adaptability and reached the highest virulence value after 36 h.When the concentration of P.cuspidatum water extract was lower than 100 μg/mL,there was no cytotoxicity at 12,24 h.The water extract of P.cuspidatum significantly inhibited the expression of N gene under Co-treatment and Post-treatment,and significantly inhibited the expression of PEDV N gene mRNA and N protein in a dose-dependent manner.The water extract of P.cuspidatum had a significant inhibitory effect on PEDV N gene mainly in the replication and proliferation stage of the virus life cycle,and had no significant inhibitory effect in other stages.In summary,2 μg/mL trypsin had the best adaptability to PEDV reproduction,and the water extract of P.cuspidatum had a significant inhibitory effect on PEDV in vitro,which mainly acted on the replication and value-added stage of PEDV life cycle.

Key words: Polygonum cuspidatum water extract, PEDV, IPEC-J2, Adaptability, Inhibition in vitro

中图分类号:

S858.28

王煜轩, 杨植, 刘昊阳, 王兴海, 杨富琳, 芦烘德, 何至远, 董虹. 虎杖水提取物对猪流行性腹泻病毒在IPEC-J2细胞中复制的影响[J]. 华北农学报, 2025, 40(3): 215-224. doi: 10.7668/hbnxb.20195708.

WANG Yuxuan, YANG Zhi, LIU Haoyang, WANG Xinghai, YANG Fulin, LU Hongde, HE Zhiyuan, DONG Hong. Effect of Polygonum cuspidatum Aqueous Extract on Porcine epidemic diarrhoea virus Replication in IPEC-J2 Cells[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3): 215-224. doi: 10.7668/hbnxb.20195708.

http://www.hbnxb.net/CN/Y2025/V40/I3/215

图/表 9

表1 RT-qPCR引物信息

Tab.1 RT-qPCR primer information

引物名称 Primer names	序列(5'—3') Sequences(5'—3')
PEDV N	Forward:ACCAGTCCAAGAACAGAAACCAGTC
	Reverse:CATCCTTGACAGCAGCCACCAG
GAPDH	Forward:AAGGAGTAAGAGCCCCTGGA
	Reverse:TCTGGGATGGAAACTGGAA

图1 不同浓度胰酶在不同时间对PEDV N蛋白表达水平的影响不同字母表示差异显著(P<0.05)。图2,4同。

Fig.1 The effects of different concentrations of trypsin on the expression level of PEDV N protein at different time Different letters indicated significant difference(P<0.05).The same as Fig.2,4.

图2 不同浓度胰酶在不同时间对细胞活性的影响

Fig.2 The effects of different concentrations of trypsin on the cell activity at different time

图3 细胞病变观察 A、C、E、G.对照组;B、D、F、H.PEDV感染细胞。

Fig.3 Cytopathic observation chart A,C,E,G.Control group;B,D,F,H.PEDV infected cells.

图4 PEDV在IPEC-J2细胞中的生长特性 A.PEDV LW/L株在IPEC-J2细胞中的生长曲线;B.不同时间PEDV N基因mRNA表达量的变化;C.Western Blot检测PEDV N蛋白表达结果;D.灰度值分析。

Fig.4 Growth characteristics of PEDV in IPEC-J2 cells A.The growth curve of PEDV LW/L strain in IPEC-J2 cells;B.Changes in mRNA expression of PEDV N gene; C.The PEDV N protein expression was detected by Western Blot;D.Gray value analysis.

图5 虎杖水提取物对IPEC-J2细胞毒性的影响

Fig.5 The effect after aclding P.cuspidatum water extract on cytotoxicity of IPEC-J2 cells

图6 IPEC-J2细胞中不同方式加入虎杖水提取物对PEDV N基因表达的影响 A.全过程治疗组;B.预处理治疗组;C.共孵育组;D.治疗组;E.直接治疗组。

Fig.6 The effect of adding P.cuspidatum water extract in IPEC-J2 cells in different ways on the expression of PEDV N gene A.All-treatment group;B.Pre-treatment group;C.Co-treatment group;D.Post-treatment group;E.Direct-treatment group.

图7 不同浓度虎杖水提取物对PEDV N基因和N蛋白水平表达的影响 A.PEDV N基因表达水平;B.Western Blot检测PEDV N蛋白表达水平;C.灰度值分析。

Fig.7 Effects of different concentrations of P.cuspidatum water extract on the expression of PEDV N gene and N protein levels A.PEDV N gene expression level;B.The PEDV N protein expression was detected by Western Blot;C.Gray value analysis.

图8 虎杖水提取物在IPEC-J2细胞中对PEDV生命周期中N基因表达的影响 A.吸附阶段(PEDV和Pol.c在4 ℃ 2 h);B.侵入阶段(PEDV 4 ℃ 2 h,Pol.c 37 ℃ 3 h);C.复制阶段(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 12 h);D.释放阶段(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 2 h)。

Fig.8 The expression of N gene in PEDV life cycle in IPEC-J2 cells affected by P.cuspidatum water extract A.Adsorption stage(PEDV and Pol.c 4 ℃ 2 h);B.Penetration stage(PEDV 4 ℃ 2 h,Pol.c 37 ℃ 3 h);C.Replicative stage(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 12 h);D.Release stage(PEDV 37 ℃ 1 h,Pol.c 37 ℃ 2 h).

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Effect of Polygonum cuspidatum Aqueous Extract on Porcine epidemic diarrhoea virus Replication in IPEC-J2 Cells

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虎杖水提取物对猪流行性腹泻病毒在IPEC-J2细胞中复制的影响

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