猪流行性腹泻病毒感染对仔猪肠道损伤及肠道细胞巨泡式死亡的影响

doi:10.11843/j.issn.0366-6964.2025.12.028

摘要/Abstract

摘要： 为了探讨猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)新型野毒株对初生仔猪肠道损伤及肠道细胞巨泡式死亡的影响，选取8头5日龄的健康仔猪，随机分为空白对照组(Mock组)和PEDV感染组，病毒感染后记录临床症状并在2 d后实施安乐死，之后分别采集十二指肠、空肠、回肠、盲肠、结肠和直肠的组织样品。采用苏木精-伊红(HE)染色、免疫组织化学(IHC)染色、RT-qPCR和Western blot技术检测PEDV感染仔猪后各肠段的组织病理学变化、病毒在肠道中的定位及对巨泡式死亡的影响。HE染色结果显示，PEDV对仔猪各肠段黏膜结构均有不同程度的损伤，其中对空肠的损伤最为严重。RT-qPCR结果证实PEDV感染仔猪后，空肠中病毒载量最高。IHC染色发现PEDV主要分布在小肠黏膜的上皮层中，其中在空肠中阳性信号最多，与RT-qPCR结果一致。深入研究揭示PEDV感染组的空肠组织中巨泡式死亡相关基因Rac1(Ras相关C3肉毒菌毒素底物1)、Rab7(Ras相关蛋白7)和LAMP1(溶酶体相关膜蛋白1)的表达量显著上调，Western blot试验也进一步证实了这一发现。以上结果表明，PEDV新型野毒株感染仔猪后主要定位于小肠，对空肠的损伤最严重，并使空肠发生巨泡式死亡。本研究可为深入理解PEDV的感染发病机制提供可靠的参考依据。

关键词: 猪流行性腹泻病毒, 空肠, 病理损伤, 巨泡式死亡

Abstract: To investigate the effects of new wild-type PEDV infection on the intestinal mucosal injury and intestinal cell methuosis of piglets, 8 healthy piglets aged 5 days were randomly divided into Mock and PEDV-infected groups. The clinical symptoms were monitored. After 2 days of virus infection, the piglets were euthanized and the duodenum, jejunum, ileum, cecum, colon, and rectum were collected. Hematoxylin-eosin (HE) staining, immunohistochemistry (IHC) staining, RT-qPCR and Western blot were used to detect the pathological changes of the intestinal tissues, the viral location in the intestines, and its effect on methuosis. HE staining revealed that PEDV-infected group had different degree of damage to the intestinal mucosa, and the jejunum showed more obvious pathological damage. Compared with other intestinal segments, the viral load was the highest in jejunum after PEDV infection using RT-qPCR. IHC staining showed that PEDV mainly distributed in the upper cortex of small intestinal mucosa, and the most positive signals were observed in jejunum segment, which was consistent with the result of RT-qPCR. Further study revealed that the expression levels of megavesicular death-related genes Rac1(Ras-related C3 sarcoma virus oncogene 1), Rab7(Ras-related protein 7) and LAMP1(lysosomal-related membrane protein 1) in PEDV infection group increased significantly in jejunum, which was further confirmed by Western blot. The above results indicate that the new wild-type PEDV mainly located in the small intestine, caused the most serious pathological damage to jejunum and induced methuosis of the jejunal cells. This study could provide a reliable basis for further understanding of the pathogenesis of PEDV infection.

Key words: porcine epidemic diarrhea virus, jejunum, pathological injury, methuosis

中图分类号:

S852.659.6

陈琳琳, 著龙祥, 祖少坡, 陈红英, 李庆豪, 金鑫, 李艺雷, 马世杰, 魏战勇. 猪流行性腹泻病毒感染对仔猪肠道损伤及肠道细胞巨泡式死亡的影响[J]. 畜牧兽医学报, 2025, 56(12): 6271-6280.

CHEN Linlin, ZHU Longxiang, ZU Shaopo, CHEN Hongying, LI Qinghao, JIN Xin, LI Yilei, MA Shijie, WEI Zhanyong. Effects of Porcine Epidemic Diarrhea Virus Infection on Intestinal Injury and Intestinal Cell Methuosis in Piglet[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(12): 6271-6280.

参考文献

[1] KOCHERHANS R, BRIDGEN A, ACKERMANN M, et al. Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence[J]. Virus Gene, 2001, 23(2): 137-144.
[2] LI W, LI H, LIU Y, et al. New variants of porcine epidemic diarrhea virus, China, 2011[J]. Emerg Infect Dis, 2012, 18(8): 1350-1353.
[3] 高改林, 王晨星.猪流行性腹泻流行趋势与防控[J].北方牧业,2023(19):34.
GAO G L, WANG C X. Epidemic trend and prevention of porcine epidemic diarrhea[J]. Northern Animal Husbandry, 2023(19): 34.(in Chinese)
[4] JAYARAMAN B, NYACHOTI C M. Husbandry practices and gut health outcomes in weaned piglets: A review[J]. Anim Nutr, 2017, 3(3): 205-211.
[5] ZHENG X, ZHU D, XIANG Q, et al. Ginsenoside Rb1 inhibits porcine epidemic diarrhea virus replication through suppressing S1 protein mediated the MAPK/ERK pathway and reducing apoptosis[J]. Int J Biol Macromol, 2025(Pt2): 140937.
[6] 阚先进.猪流行性腹泻病毒诱导细胞自噬的分子机制[D].扬州：扬州大学,2018.
KAN X J. Mechanisms of porcine epidemic diarrhea virus-induced autophagy[D]. Yangzhou: Yangzhou University, 2018.(in Chinese)
[7] 闫清源,周书亭,谢洋洋,等.猪流行性腹泻病毒感染Vero细胞的细胞病理学研究[J].上海交通大学学报(农业科学版),2018,36(2):39-42.
YAN Q Y, ZHOU S T, XIE Y Y, et al. Cytopathology study on porcine epidemic diarrhea virus(PEDV) infected vero cells[J]. Journal of Shanghai Jiaotong University(Agricultural Science), 2018, 36(2): 39-42.(in Chinese)
[8] 蔡红兵,刘金坤,范钦,等.巨泡式死亡:一种新的细胞死亡方式[J].南方医科大学学报,2013,33(12):1844-1847.
CAI H B, LIU J K, FAN Q, et al. Methuosis-a novel type of cell death[J]. Journal of Southern Medical University, 2013, 33(12): 1844-1847.(in Chinese)
[9] RITTER M, BRESGEN N, KERSCHBAUM H H. From pinocytosis to methuosis-fluid consumption as a risk factor for cell death[J]. Front Cell Dev Biol, 2021, 9: 651982.
[10] OVERMEYER J H, KAUL A, JOHNSON E E, et al. Active ras triggers death in glioblastoma cells through hyperstimulation of macropinocytosis[J]. Mol Cancer Res, 2008, 6(6): 965-977.
[11] BHANOT H, YOUNG A M, OVERMEYER J H, et al. Induction of nonapoptotic cell death by activated ras requires inverse regulation of Rac1 and Arf6[J]. Mol Cancer Res, 2010, 8(10): 1358-1374.
A. Mock组十二指肠免疫组化染色；B. Mock组空肠免疫组化染色；C. Mock组回肠免疫组化染色；D. PEDV组十二指肠免疫组化染色；E. PEDV组空肠免疫组化染色；F. PEDV组回肠免疫组化染色；G. Mock组盲肠免疫组化染色；H. Mock组结肠免疫组化染色；I. Mock组直肠免疫组化染色；J. PEDV组盲肠免疫组化染色；K. PEDV组结肠免疫组化染色；L. PEDV组盲肠免疫组化染色(图4 Fig.4) (转下页 Carried forward) (续图4 Continued) A. Immunohistochemical staining of duodenum in the Mock group; B. Immunohistochemical staining of jejunum in the Mock group; C. Immunohistochemical staining of ileum in the Mock group; D. Immunohistochemical staining of duodenum in the PEDV-infected group; E. Immunohistochemical staining of jejunum in the PEDV-infected group; F. Immunohistochemical staining of ileum in the PEDV-infected group； G. Immunohistochemical staining of cecum in the Mock group; H. Immunohistochemical staining of colon in the Mock group; I. Immunohistochemical staining of rectum in the Mock group; J. Immunohistochemical staining of cecum in the PEDV-infected group; K. Immunohistochemical staining of colon in the PEDV-infected group; L. Immunohistochemical staining of rectum in the PEDV-infected group 图4 肠道免疫组织化学变化 Fig.4 Intestinal immunohistochemical changes A. Rac1在空肠中mRNA相对表达；B. Rab7在空肠中mRNA相对表达；C. LAMP1在空肠中mRNA相对表达。*. P<0.05；**. P<0.01 A. Relative mRNA expression of Rac1 in jejunum；B. Relative mRNA expression of Rab7 in jejunum；C. Relative mRNA expression of LAMP1 in jejunum. *. P<0.05；**. P<0.01 图5 荧光定量PCR检测空肠中Rac1、Rab7和LAMP1的mRNA相对表达 Fig.5 Relative mRNA expression of Rac1, Rab7 and LAMP1 in jejunum detected by fluorescence quantitative PCR A. 检测空肠中Rac1、Rab7和LAMP1蛋白的表达；B. Rac1、Rab7和LAMP1灰度分析。*. P<0.05；**. P<0.01 A. Detection of Rac1, Rab7and LAMP1 protein expression in different immune organs; B. Grayscale analysis of Rac1, Rab7and LAMP1. *. P<0.05；**. P<0.01 图6 Western blot检测PEDV仔猪感染空肠中Rac1、Rab7和LAMP1的蛋白表达 Fig.6 Western blot analysis of Rac1, Rab7 and LAMP1 protein expression in jejunum of PEDV-infected piglets
[12] OVERMEYER J H, YOUNG A M, BHANOT H, et al. A chalcone-related small molecule that induces methuosis, a novel form of non-apoptotic cell death, in glioblastoma cells[J]. Mol Cancer, 2011, 10(1): 1-17.
[13] BHATTACHARYA S S, JIN L, ROY D, et al. Abstract 2658: PFKFB3 inhibition reprograms malignant pleural mesothelioma to glycolytic stress-induced macropinocytosis and ER stress as independent binary adaptive responses[J]. Cell Death Dis, 2019, 10(10): 725.
[14] HAN Q F, LI W J, HU K S, et al. Exosome biogenesis: machinery, regulation, and therapeutic implications in cancer[J]. Mol Cancer, 2022, 21(1): 207.
[15] LUO Y, GUAN B, DENG X, et al. Methuosis inducer SGI-1027 cooperates with everolimus to promote apoptosis and pyroptosis by triggering lysosomal membrane permeability in renal cancer[J]. Adv Sci (Weinh), 2024, 11(38): e2404693.
[16] MULLIGAN R J, MAGAJ M M, DIGILIO L, et al. Collapse of late endosomal pH elicits a rapid Rab7 response via the V-ATPase and RILP[J]. J Cell Sci, 2024, 137(9): jcs261765.
[17] ZHANG Y H, LI H X, CHEN X M, et al. Genetic characteristics and pathogenicity of a novel porcine epidemic diarrhea virus with a naturally occurring truncated ORF3 Gene[J]. Viruses, 2022, 14(3): 487.
[18] 李洪炫,滑宇鹏,彭琳颖,等.猪流行性腹泻病毒和猪圆环病毒4型双重SYBR GreenⅠ实时荧光定量PCR方法的建立[J].中国兽医学报,2023,43(1):23-28.
LI H X, HUA Y P, PENG L Y, et al. Development of a SYBR Green Ⅰ-based real-time PCR assay for detection of virus and porcine circovirus 4[J]. Chinese Journal of Veterinary Science, 2023, 43(1): 23-28.(in Chinese)
[19] 倪艳秀,林继煌,何孔旺,等.猪流行性腹泻研究概况[J].畜牧与兽医,2001(1):38-40.
NI Y X, LIN J H, HE K W, et al. Research overview of porcine epidemic diarrhea[J]. Animal Husbandry & Veterinary Medicine, 2001(1): 38-40.(in Chinese)
[20] 卓秀萍,朱玲,乔小改,等.人工感染猪流行性腹泻病毒的哺乳仔猪的病理学观察[J].中国兽医科学,2015,45(2):202-207.
ZHUO X P, ZHU L, QIAO X G,et al. Pathological observation of suckling piglets infected with porcine epidemic diarrhea virus artificially[J]. Chinese Veterinary Science, 2015, 45(2): 202-207.(in Chinese)
[21] JUNG K, MIYAZAKI A, SAIF L J. Immunohistochemical detection of the vomiting-inducing monoamine neurotransmitter serotonin and enterochromaffin cells in the intestines of conventional or gnotobiotic (Gn) pigs infected with porcine epidemic diarrhea virus (PEDV) and serum cytokine responses of Gn pigs to acute PEDV infection[J]. Res Vet Sci, 2018, 119: 99-108.
[22] JUNG K, SAIF L J. Goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus[J]. Res Vet Sci, 2017, 110: 12-15.
[23] 肖静,董斌,王俊棋,等.细胞骨架与细胞膜损伤介导药物引起心肌细胞巨泡式死亡的作用与机制[C].厦门:中国毒理学会第九次全国青年科技大会暨第二届生物技术药物毒理与安全评价委员会学术会议论文集,2023:46-47.
XIAO J, DONG B, WANG J Q, et al. The role and mechanism of cytoskeleton and cell membrane damage mediating drug induced cardiomyocyte methuosis[C]. Xiamen: Proceedings of the 9th National Youth Science and Technology Conference of the Chinese Society of Toxicology and the 2nd Academic Conference of the Committee for Toxicology and Safety Evaluation of Biotechnology Drugs, 2023: 46-47.
[24] LUISONI S, SUOMALAINEN M, BOUCKE K, et al. Co-option of membrane wounding enables virus penetration into cells[J]. Cell Host Microbe, 2015, 18(1): 75-85.