丁酸抑制猪流行性腹泻病毒体外复制的分子机制

doi:10.3969/j.issn.1004-1524.20240224

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (3): 579-590.DOI: 10.3969/j.issn.1004-1524.20240224

丁酸抑制猪流行性腹泻病毒体外复制的分子机制

张楚妮¹(), 徐计东¹^,², 高钦¹, 单颖¹^,², 方维焕¹^,², 李肖梁¹^,²^,^*()

1.浙江大学动物科学学院,浙江省动物医学重点实验室,农业农村部动物病毒学重点实验室,浙江杭州 310058
2.浙江大学-新昌联合创新中心(天姥实验室),浙江新昌 312532

收稿日期:2024-03-08 出版日期:2025-03-25 发布日期:2025-04-02
作者简介:张楚妮(1999—),女,四川成都人,硕士研究生,研究方向为动物病原学与免疫学。E-mail:22117117@zju.edu.cn
通讯作者: * 李肖梁,E-mail:xlli@zju.edu.cn
基金资助:
国家自然科学基金(32372978);浙江省“尖兵”“领雁”研发攻关计划(2023C02036);浙江省“高层次人才特殊支持计划”科技创新领军人才项目(2021R52041)

The molecular mechanism of inhibition of butyric acid to inhibit porcine epidemic diarrhea virus replication in vitro

ZHANG Chuni¹(), XU Jidong¹^,², GAO Qin¹, SHAN Ying¹^,², FANG Weihuan¹^,², LI Xiaoliang¹^,²^,^*()

1. Zhejiang Province Key Laboratory of Veterinary Medicine, MOA Key Laboratory of Animal Virology, College of Animal Sciences, Zhejiang University, Hangzhou 310058, China
2. ZJU-Xinchang Joint Innovation Centre (Tianmu Laboratory), Xinchang 312532, Zhejiang, China

Received:2024-03-08 Online:2025-03-25 Published:2025-04-02

摘要/Abstract

摘要：

猪流行性腹泻病毒(porcine epidemic diarrhea virus, PEDV)是一种引起高度接触性急性肠道传染病的冠状病毒,严重威胁生猪产业的健康发展。丁酸作为饲料添加剂,在生猪养殖中广泛应用,其不仅作为能量物质,还参与调控基因表达、抗炎和细胞周期等生命活动,但丁酸在PEDV感染中的作用机制尚不明确。本研究旨在探讨丁酸对PEDV复制的抑制作用及其潜在机制。通过实时荧光定量PCR(qRT-PCR)、免疫印迹和免疫荧光等技术检测丁酸处理对PEDV复制的影响,利用细胞周期相关蛋白表达分析和流式细胞术,研究PEDV感染对细胞周期的影响与丁酸的调控作用。结果表明:丁酸处理显著抑制了PEDV在猪肠道上皮细胞(IPEC-J2细胞)中的复制;PEDV感染可以上调细胞周期相关蛋白Cyclin A2的表达,同时诱导细胞S期(DNA合成期)阻滞帮助自身复制。丁酸通过下调Cyclin A2蛋白表达,缓解PEDV诱导的S期阻滞,从而发挥抗病毒作用。综上所述,丁酸通过调控细胞周期,缓解PEDV感染诱导的细胞周期S期阻滞,进而抑制病毒复制。本研究为丁酸在动物生产中作为抗病毒策略的应用提供了理论依据。

关键词: 猪流行性腹泻病毒, 丁酸, 细胞周期, Cyclin A2, S期阻滞, 抗病毒作用, IPEC-J2细胞

Abstract:

Porcine epidemic diarrhea virus (PEDV) is a coronavirus that causes highly contagious acute intestinal infectious diseases and seriously threatens the healthy development of pig industry. Butyric acid, as a feed additive, is widely used in pig farming. It not only serves as an energy source but also participates in regulating gene expression, anti-inflammatory responses, cell cycle and other vital movements. However, the mechanism of butyric acid in PEDV infection remains unclear. This study aims to investigate the inhibitory effect of butyric acid on PEDV replication and its underlying mechanisms. Using techniques such as real-time fluorescent quantitative PCR (qRT-PCR), Western blot, and immunofluorescence, the impact of butyric acid treatment on PEDV replication was assessed. Additionally, cell cycle-related protein expression analysis and flow cytometry were used to study the effects of PEDV infection on the cell cycle and the regulatory role of butyric acid. The results demonstrated that butyric acid treatment significantly inhibited PEDV replication in IPEC-J2 cells. PEDV infection upregulated the expression of the cell cycle-related protein Cyclin A2 and induced S-phase (DNA synthesis phase) arrest to facilitate its own replication. Butyric acid alleviated PEDV-induced S-phase arrest by downregulating Cyclin A2 expression, thereby exerting its antiviral effects. In conclusion, butyric acid inhibits viral replication by modulating the cell cycle and mitigating PEDV-induced S-phase arrest. This study provides a theoretical basis for the application of butyric acid as an antiviral strategy in animal production.

Key words: porcine epidemic diarrhea virus, butyric acid, cell cycle, Cyclin A2, S-phase cell cycle arrest, antiviral effect, IPEC-J2 cell

中图分类号:

S855.3

张楚妮, 徐计东, 高钦, 单颖, 方维焕, 李肖梁. 丁酸抑制猪流行性腹泻病毒体外复制的分子机制[J]. 浙江农业学报, 2025, 37(3): 579-590.

ZHANG Chuni, XU Jidong, GAO Qin, SHAN Ying, FANG Weihuan, LI Xiaoliang. The molecular mechanism of inhibition of butyric acid to inhibit porcine epidemic diarrhea virus replication in vitro[J]. Acta Agriculturae Zhejiangensis, 2025, 37(3): 579-590.

图/表 5

表1 引物序列

Table 1 The sequences of primers

基因Gene	正向引物序列Forward primer sequence (5'→3')	反向引物序列Reverse primer sequence(5'→3')
PEDV N	CGGAACAGGACCTCACGCC	ACAATCTCAACTACGCTGGGAAG
GAPDH	CACTGAGGACCAGGTTGTGTCCTGTGAC	TCCACCACCCTGTTGCTGTAGCCAAATTC

图1 BA在IPEC-J2细胞中抑制PEDV复制 A,CCK-8法检测细胞活性;B、C,蛋白质免疫印迹法和灰度分析;D,qRT-PCR检测mRNA水平;E,病毒毒价测定;F,免疫荧光实验。数据以3个独立试验的平均值±标准差表示;ns,差异不显著;**,P<0.01;***,P<0.001。“+”代表PEDV感染,“-”代表Mock感染或者不添加丁酸处理。

Fig.1 BA inhibited PEDV replication in IPEC-J2 cells A, CCK-8 assay was used to detect cell activity; B and C, Western blotting and densitometric analysis of PEDV-N/β-actin ratio; D, mRNA levels were detected by qRT-PCR; E, 50% tissue culture infective dose; F, Immunofluorescence analysis. Data were expressed as the mean ±standard deviation of three independent experiments; ns, The difference was not significant; **, P<0.01; ***, P<0.001. The ‘+’ represented PEDV infection, while the ‘-’ represented Mock infection or treatment without adding butyric acid.

图2 BA对细胞周期相关蛋白表达的调节 A、B,用3 mmol·L-1 BA处理PEDV感染的IPEC-J2细胞,之后添加1 μmol·L-1蛋白酶体抑制剂MG132,24 h后收获细胞并提取总蛋白质,用Western blot检测PEDV N蛋白水平和H3K9的乙酰化水平,用qRT-PCR检测PEDV N基因的转录水平。C,用3 mmol·L-1 BA处理PEDV感染的IPEC-J2细胞,24 h后收获细胞并提取总蛋白质,用Western blot检测内质网应激相关蛋白PERK的磷酸化水平。D,用3 mmol·L-1 BA或NaB处理PEDV感染的IPEC-J2细胞,24 h后收获细胞并提取总蛋白质,用Western blot检测细胞周期相关蛋白。“+”代表抑制剂处理/PEDV感染,“-”代表等量DMSO处理/Mock感染。ns,差异不显著;***,P<0.001。

Fig.2 The regulation of cell cycle-related protein expression by BA A and B, PEDV-infected IPEC-J2 cells were treated with 3 mmol·L-1 BA, followed by the addition of 1 μmol·L-1 proteasome inhibitor MG132, after 24 hours, the cells were harvested, and total protein was extracted, Western blot was used to detect the levels of PEDV N protein and H3K9 acetylation, while qRT-PCR was employed to measure the transcriptional level of the PEDV N gene. C, PEDV-infected IPEC-J2 cells were treated with 3 mmol·L-1 BA, after 24 hours, the cells were harvested, and total protein was extracted, Western blot was used to assess the phosphorylation level of the endoplasmic reticulum stress-related protein PERK. D, PEDV-infected IPEC-J2 cells were treated with either 3 mmol·L-1 BA or NaB, after 24 hours, the cells were harvested, and total protein was extracted, Western blot was used to detect cell cycle-related proteins. The ‘+’ represented inhibitor treatment/PEDV infection, while the ‘-’ represented an equivalent amount of DMSO treatment/Mock infection. ns, The difference was not significant; ***, P<0.001.

图3 PEDV感染诱导细胞周期S期阻滞 A,PEDV感染不同时间点细胞周期相关蛋白的表达;B,流式细胞术检测细胞周期;C,流式结果的统计学分析;D,S期抑制剂对PEDV N蛋白表达的影响。“+”代表抑制剂处理/PEDV感染,“-”代表等量DMSO处理/Mock感染。*,P<0.05;**,P<0.01。

Fig.3 PEDV induced cell cycle arrest in S-phase A, Expression of cell cycle-related proteins at different time post-PEDV infection; B, Cell cycle analysis by flow cytometry; C, Statistical analysis of flow cytometry results; D, The impact of S-phase inhibitors on the expression of PEDV N protein. The ‘+’ represented inhibitor treatment/PEDV infection, while the ‘-’ represented an equivalent amount of DMSO treatment/Mock infection. *, P<0.05; **, P<0.01.

图4 BA减缓PEDV诱导的细胞周期S期阻滞 A,BA对细胞周期相关蛋白表达的影响;B,统计学分析;C,流式细胞术检测细胞周期。“+”代表PEDV感染, “-”代表Mock感染或者不添加丁酸处理。

Fig.4 BA mitigated PEDV-induced S-phase arrest of cell cycle A, The effect of BA on the expression of cell cycle-related proteins; B, Statistical analysis; C, Cell cycle was detected by flow cytometry. The ‘+’ represented PEDV infection, while the ‘-’ represented Mock infection or treatment without adding butyric acid.

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丁酸抑制猪流行性腹泻病毒体外复制的分子机制

The molecular mechanism of inhibition of butyric acid to inhibit porcine epidemic diarrhea virus replication in vitro

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