白藜芦醇对轮状病毒感染猪肠上皮细胞IPEC-J2的抑制效应

doi:10.11843/j.issn.0366-6964.2024.09.045

摘要/Abstract

摘要：

旨在研究白藜芦醇对轮状病毒(porcine rotavirus, PoRV)感染猪肠上皮细胞的抗病毒作用。本试验以猪肠上皮细胞(IPEC-J2)为研究对象, 先测定不同白藜芦醇浓度对IPEC-J2细胞活力的影响, 再根据是否用PoRV或白藜芦醇(100 μmol·L^-1)处理IPEC-J2细胞, 分别设置阴性对照组、PoRV感染组和不同时间段添加白藜芦醇+PoRV感染组。通过实时荧光定量PCR技术、病毒滴度测定法和蛋白免疫印迹法检测PoRV在IPEC-J2细胞的复制与增殖情况。结果表明: 1)在PoRV感染IPEC-J2的后期和全期添加白藜芦醇(100 μmol·L^-1)可显著抑制PoRV的感染和复制(P < 0.05);2)与PoRV感染组相比, 在PoRV感染IPEC-J2的全期添加白藜芦醇(100 μmol·L^-1)可极显著抑制IPEC-J2细胞上清液中炎症细胞因子IL-1β、IL-10、TNF-α和IFN-β的含量(P < 0.01), 后期添加白藜芦醇可显著抑制IL-10和IFN-β的含量(P < 0.05);后期和全期添加白藜芦醇极显著抑制了IPEC-J2细胞中的免疫相关因子MDA5、RIG-I、TRIF、MAVS、LGALS9、EIF2AK2、IRF9和IFI44L的mRNA相对表达量(P < 0.01), 极显著抑制了IPEC-J2细胞中可变剪接因子SRPK1、SRPK2、HNRNPC和HNRNPR的mRNA相对表达量(P < 0.01);3)与阴性对照组相比, PoRV感染显著促进了IPEC-J2细胞中LGALS9的5号外显子和EF2AK2的2号外显子发生外显子跳跃, 而在PoRV感染IPEC-J2的后期和全期添加白藜芦醇(100 μmol·L^-1)可显著抑制靶基因发生外显子跳跃。综上证实, 本研究添加100 μmol·L^-1白藜芦醇可抑制PoRV对IPEC-J2细胞的感染和复制, 且在PoRV感染后的维持阶段发挥作用, 可为预防和治疗仔猪病毒性腹泻提供新的依据和参考。

关键词: 白藜芦醇, 猪轮状病毒, 抗病毒, 可变剪接

Abstract:

The aim of this study was to investigate the antiviral effect of resveratrol on porcine rotavirus (PoRV) infected porcine intestinal epithelial cells(IPEC-J2). In this experiment, IPEC-J2 cells were used as research object. Firstly, the effect of different concentration of resveratrol on the cell viability of IPEC-J2 was determined. Then experimental groups including negative control group, PoRV-infected group and PoRV-infected group with the addition of resveratrol at different time points were set up depending on whether the IPEC-J2 cells were treated with PoRV or resveratrol (100 μmol·L^-1). The replication and proliferation of PoRV in IPEC-J2 cells were detected by real-time fluorescence quantitative PCR, viral titer assay and Western blot. The results showed that: 1) The addition of resveratrol (100 μmol·L^-1) significantly inhibited PoRV replication and infection at the late and full phases of viral infection of PoRV infection with IPEC-J2 (P < 0.05); 2) Compared with the PoRV-infected group, the addition of resveratrol (100 μmol·L^-1) during the full phase of PoRV-infected IPEC-J2 extremely significantly suppressed the levels of inflammatory cytokines IL-1β, IL-10, TNF-α and IFN-β in the supernatant fluid of IPEC-J2 cells (P < 0.01), while significantly suppressed the IL-10 and IFN-β levels during the late phase (P < 0.05); Extremely significantly suppressed the relative expression of immune-related factors MDA5, RIG-I, TRIF, MAVS, LGALS9, EIF2AK2, IRF9 and IFI44L in IPEC-J2 cells (P < 0.01) during late and full phases; Extremely significantly suppressed the relative mRNA expression of variable splicing factors SRPK1, SRPK2, HNRNPC and HNRNPR in IPEC-J2 cells (P < 0.01) during late and full phases; 3) Compared with the negative control group, PoRV infection significantly promoted exon jumps in exon 5 of LGALS9 and exon 2 of EF2AK2 in IPEC-J2 cells, while the addition of resveratrol (100 μmol·L^-1) significantly inhibited the exon skipping of target genes in the late and full phases of PoRV infection IPEC-J2 cells. In conclusion, this study confirmed that the addition of 100 μmol·L^-1 resveratrol could inhibit the infection and replication of PoRV in IPEC-J2 cells and play a role in the late phases of PoRV replication, which can provide a new basis and reference for the prevention and treatment of viral diarrhea in piglets.

Key words: resveratrol, porcine rotavirus, antiviral, alternative splicing

中图分类号:

S828.9

彭宁, 梁雅旭, 龙菲, 余东明, 钟翔. 白藜芦醇对轮状病毒感染猪肠上皮细胞IPEC-J2的抑制效应[J]. 畜牧兽医学报, 2024, 55(9): 4213-4225.

Ning PENG, Yaxu LIANG, Fei LONG, Dongming YU, Xiang ZHONG. Inhibitory Effect of Resveratrol on Rotavirus-infected Porcine Intestinal Epithelial Cells IPEC-J2[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(9): 4213-4225.

图/表 8

表 1

表 2

图 1

图 2

图 3

图 4

图 5

图 6

参考文献 35

1	沈思思. 猪轮状病毒的研究进展[J]. 中国畜牧业, 2022, (14): 48- 49. doi: 10.3969/j.issn.2095-2473.2022.14.023
	SHEN S S . Research progress of porcine rotavirus[J]. China Animal Industry, 2022, (14): 48- 49. doi: 10.3969/j.issn.2095-2473.2022.14.023
2	VLASOVA A N , AMIMO J O , SAIF L J . Porcine rotaviruses: epidemiology, immune responses and control strategies[J]. Viruses, 2017, 9 (3): 48. doi: 10.3390/v9030048
3	杨志刚, 汤德元, 张森, 等. 猪轮状病毒检测技术研究进展[J]. 动物医学进展, 2021, 42 (7): 96- 100. doi: 10.3969/j.issn.1007-5038.2021.07.018
	YANG Z G , TANG D Y , ZHANG S , et al. Progress on detection techniques of porcine rotavirus[J]. Progress in Veterinary Medicine, 2021, 42 (7): 96- 100. doi: 10.3969/j.issn.1007-5038.2021.07.018
4	卓儒浩, 吴嘉敏, 许梦慧, 等. 姜黄素抑制轮状病毒感染猪肠上皮细胞的作用研究[J]. 动物营养学报, 2022, 34 (2): 1285- 1295. doi: 10.3969/j.issn.1006-267x.2022.02.060
	ZHUO R H , WU J M , XU M H , et al. Inhibitory effect of curcumin against rotavirus infection in intestinal porcine epithelial cells[J]. Chinese Journal of Animal Nutrition, 2022, 34 (2): 1285- 1295. doi: 10.3969/j.issn.1006-267x.2022.02.060
5	REN X L , SALEEM W , HAES R , et al. Milk lactose protects against porcine group A rotavirus infection[J]. Front Microbiol, 2022, 13, 989242. doi: 10.3389/fmicb.2022.989242
6	MANN J T , RILEY B A , BAKER S F . All differential on the splicing front: host alternative splicing alters the landscape of virus-host conflict[J]. Semin Cell Dev Biol, 2023, 146, 40- 56. doi: 10.1016/j.semcdb.2023.01.013
7	EMERY A , SWANSTROM R . HIV-1:to splice or not to splice, that is the question[J]. Viruses, 2021, 13 (2): 181. doi: 10.3390/v13020181
8	MEYER F . Viral interactions with components of the splicing machinery[J]. Prog Mol Biol Transl Sci, 2016, 142, 241- 268.
9	AJIRO M , ZHENG Z M . Oncogenes and RNA splicing of human tumor viruses[J]. Emerg Microbes Infect, 2014, 3 (9): e63.
10	ZHENG Z M . Viral oncogenes, noncoding RNAs, and RNA splicing in human tumor viruses[J]. Int J Biol Sci, 2010, 6 (7): 730- 755.
11	LI R X , GAO S Y , CHEN H Y , et al. Virus usurps alternative splicing to clear the decks for infection[J]. Virol J, 2023, 20 (1): 131. doi: 10.1186/s12985-023-02098-9
12	BOUDREAULT S , ROY P , LEMAY G , et al. Viral modulation of cellular RNA alternative splicing: a new key player in virus-host interactions?[J]. Wiley Interdiscip Rev RNA, 2019, 10 (5): e1543. doi: 10.1002/wrna.1543
13	FRANCIES F Z , DLAMINI Z . Aberrant splicing events and epigenetics in viral oncogenomics: current therapeutic strategies[J]. Cells, 2021, 10 (2): 239. doi: 10.3390/cells10020239
14	LYU M Y , LAI H L , WANG Y L , et al. Roles of alternative splicing in infectious diseases: from hosts, pathogens to their interactions[J]. Chin Med J (Engl), 2023, 136 (7): 767- 779.
15	BEEMON K L . Retroviral RNA processing[J]. Viruses, 2022, 14 (5): 1113. doi: 10.3390/v14051113
16	ESPARZA M , BHAT P , FONTOURA B M . Viral-host interactions during splicing and nuclear export of influenza virus mRNAs[J]. Curr Opin Virol, 2022, 55, 101254. doi: 10.1016/j.coviro.2022.101254
17	GAN Z D , WEI W Y , WU J M , et al. Resveratrol and curcumin improve intestinal mucosal integrity and decrease m⁶A RNA methylation in the intestine of weaning piglets[J]. ACS Omega, 2019, 4 (17): 17438- 17446. doi: 10.1021/acsomega.9b02236
18	PASQUARIELLO R , VERDILE N , BREVINI T A L , et al. The role of resveratrol in mammalian reproduction[J]. Molecules, 2020, 25 (19): 4554. doi: 10.3390/molecules25194554
19	MENG T T , XIAO D F , MUHAMMED A , et al. Anti-inflammatory action and mechanisms of resveratrol[J]. Molecules, 2021, 26 (1): 229. doi: 10.3390/molecules26010229
20	ZHOU D D , LUO M , HUANG S Y , et al. Effects and mechanisms of resveratrol on aging and age-related diseases[J]. Oxid Med Cell Longev, 2021, 2021, 9932218. doi: 10.1155/2021/9932218
21	DOCHERTY J J , FU M M H , STIFFLER B S , et al. Resveratrol inhibition of herpes simplex virus replication[J]. Antiviral Res, 1999, 43 (3): 145- 155. doi: 10.1016/S0166-3542(99)00042-X
22	ZHAO X H , CUI Q K , FU Q T , et al. Antiviral properties of resveratrol against pseudorabies virus are associated with the inhibition of IκB kinase activation[J]. Sci Rep, 2017, 7 (1): 8782. doi: 10.1038/s41598-017-09365-0
23	MOHD A , ZAINAL N , TAN K K , et al. Resveratrol affects Zika virus replication in vitro[J]. Sci Rep, 2019, 9 (1): 14336. doi: 10.1038/s41598-019-50674-3
24	CHEN X X , SONG X , ZHAO X H , et al. Insights into the anti-inflammatory and antiviral mechanisms of resveratrol[J]. Mediators Inflamm, 2022, 2022, 7138756.
25	ALESCI A , NICOSIA N , FUMIA A , et al. Resveratrol and immune cells: a link to improve human health[J]. Molecules, 2022, 27 (2): 424. doi: 10.3390/molecules27020424
26	OTSUKA K , YAMAMOTO Y , OCHIYA T . Regulatory role of resveratrol, a microRNA-controlling compound, in HNRNPA1 expression, which is associated with poor prognosis in breast cancer[J]. Oncotarget, 2018, 9 (37): 24718- 24730. doi: 10.18632/oncotarget.25339
27	QIAN S , GU J L , DAI W F , et al. Sirt1 enhances tau exon 10 inclusion and improves spatial memory of Htau mice[J]. Aging (Albany NY), 2018, 10 (9): 2498- 2510.
28	ZHOU X , NIU J W , ZHANG J F , et al. Commentary: identification of pulmonary infections with porcine Rotavirus A in pigs with respiratory disease[J]. Front Vet Sci, 2023, 10, 1102602. doi: 10.3389/fvets.2023.1102602
29	LI W , LEI M K , LI Z F , et al. Development of a genetically engineered bivalent vaccine against porcine epidemic diarrhea virus and porcine rotavirus[J]. Viruses, 2022, 14 (8): 1746. doi: 10.3390/v14081746
30	WANG A M , TAO W , TONG J Y , et al. m6A modifications regulate intestinal immunity and rotavirus infection[J]. eLife, 2022, 11, e73628. doi: 10.7554/eLife.73628
31	李萍, 程晓馨. 白藜芦醇抗菌抗病毒作用的研究进展[J]. 中国微生态学杂志, 2014, 26 (10): 1215- 1219.
	LI P , CHENG X X . The effects of resveratrol on antibacterial and antiviral properties[J]. Chinese Journal of Microecology, 2014, 26 (10): 1215- 1219.
32	RUTKOWSKI A J , ERHARD F , L'HERNAULT A , et al. Widespread disruption of host transcription termination in HSV-1 infection[J]. Nat Commun, 2015, 6, 7126. doi: 10.1038/ncomms8126
33	ASHRAF U , BENOIT-PILVEN C , NAVRATIL V , et al. Influenza virus infection induces widespread alterations of host cell splicing[J]. NAR Genom Bioinform, 2020, 2 (4): lqaa095. doi: 10.1093/nargab/lqaa095
34	BATRA R , STARK T J , CLARK A E , et al. RNA-binding protein CPEB1 remodels host and viral RNA landscapes[J]. Nat Struct Mol Biol, 2016, 23 (12): 1101- 1110. doi: 10.1038/nsmb.3310
35	BUSCH A , HERTEL K J . Evolution of SR protein and hnRNP splicing regulatory factors[J]. Wiley Interdiscip Rev RNA, 2012, 3 (1): 1- 12. doi: 10.1002/wrna.100

基因 Gene name		引物序列(5′→3′) Primer sequence (5′→3′)	登录号 GenBank number
NSP5	Forward	CTGCTTCAAACGATCCACTCAC	MG451386
	Reverse	TGAATCCATAGACACGCC
MDA5	Forward	GCCACAGATCAGCCAAGTCT	MF358967
	Reverse	AGCTGACACTTCCTTCTGCC
RIG-I	Forward	GAATTTCCGGTCTGCCCTGA	NM_213770
	Reverse	GTCTGGCTCAGGAAAGTCCC
TRIF	Forward	TGACGTCACAGACCAGAAGC	MK302497
	Reverse	GGGGAGCTGAAGAAAGGGTC
MAVS	Forward	CTACTGCCTCCTCCTCCACT	MK302496
	Reverse	CAAATGCTGAGTTGGTGGGC
IRF9	Forward	GTTACCACTGAGGCCCCTTT	NM_001078670
	Reverse	GCAGCAGCGAGTAGTCTGAAT
IFI44L	Forward	GGCCCTATGCAGACTTGGTT	XM_021096421
	Reverse	CTATGGCCTGGCGAGTCAAA C
SRPK1	Forward	CATGGAGCGGAAAGTGCTTG	XM_021098048
	Reverse	CCTCGGTGCTGAGTTTCAGA
SRPK2	Forward	ATGAGCTCCCGGAAAGTGC	XM_021102583
	Reverse	TGTTGAGGCTCCGGCTTTTT
HNRNPC	Forward	GTCCCCTCTACTCAGCTCCT	XM_021098264
	Reverse	AACACGTGCTGGGTAACTGT
HNRNPR	Forward	GCCCTGGTAAACACCTTGGA	XM_021098239
	Reverse	ACCCTCTGTTAAACCTGTGACT
GAPDH	Forward	GGAGAACGGGAAGCTTGTCA	NM_001206359
	Reverse	GCCTTCTCCATGGTCGTGAA
LGALS9	Forward	GGACTGGACCCTGCACTTC	NM_214319
	Reverse	AGAAACACGGTGAACGACCA
EIF2AK2	Forward	GACAACCAGGAGCCACAAGG	XM_02108095
	Reverse	AAACACGGTGAACGACCACT

抗体 Antibodies	编号 Identifier	来源 Source	宿主 Host	稀释比例 Dilution rate
VP6	定制	Abconal	兔	1∶5 000
GAPDH	60004-1-Ig	Proteintech	兔	1∶10 000
山羊抗兔IgG	ab205718	Abcam	兔	1∶10 000

[1]	高力国, 申翰钦, 陈诒全, 陈胜, 蔺文成, 陈峰. 猪轮状病毒重组VP6*蛋白的原核表达及间接ELISA检测方法的建立[J]. 畜牧兽医学报, 2024, 55(9): 4021-4028.
[2]	呙会会, 张浩, 杨丹, 旷燕, 李亚菲, 刘绍蒙, 刘青芸, 王湘如. 伪狂犬病病毒荧光标签毒株构建及其在抗病毒药物筛选中的初步应用[J]. 畜牧兽医学报, 2024, 55(8): 3600-3611.
[3]	徐红, 商红旗, 张雪, 钱嘉莉, 王传红, 宋旭, 宝梅英, 刘诗雨, 张格格, 郭容利, 赵永祥, 范宝超, 李彬. C8orf4基因编码蛋白对猪流行性腹泻病毒体外复制的抑制效应[J]. 畜牧兽医学报, 2024, 55(5): 2100-2108.
[4]	翁雅娟, 李蓓, 芒来, 薛嘉宁, 特日格乐, 宋代玲, 王国庆, 蔺雅楠. 剪接因子hnRNPF对蒙古马精子生成的影响[J]. 畜牧兽医学报, 2024, 55(2): 598-606.
[5]	陈姝宇, 朱雪蛟, 周金柱, 陶然, 张雪寒, 索朗斯珠, 贡嘎, 李彬. 鸟苷酸结合蛋白GBP1和GBP2抑制猪轮状病毒体外复制[J]. 畜牧兽医学报, 2024, 55(1): 245-257.
[6]	王姿月, 张子卉, 吴文学, 彭辰. 猴痘的诊断、预防和治疗[J]. 畜牧兽医学报, 2023, 54(8): 3195-3205.
[7]	李倬伟, 王方, 王君君, 陈祯涵, 李焕荣, 周双海, 刘雪威. 双香豆素对猪繁殖与呼吸综合征病毒的体外抑制作用[J]. 畜牧兽医学报, 2023, 54(3): 1160-1168.
[8]	刘广, 龚成燕, 牛凯敏, 张硕, 陶灯, 马军, 吴信, 印遇龙, 唐宇龙. 连花清瘟药渣及发酵产物的生物活性研究[J]. 畜牧兽医学报, 2023, 54(3): 1281-1299.
[9]	卓儒浩, 柳清扬, 钟翔. 姜黄素调控肠道菌群及抗病毒作用研究进展[J]. 畜牧兽医学报, 2023, 54(2): 473-483.
[10]	孙敏, 郝飞, 张纹纹, 李文良, 杨蕾蕾, 毛立, 程子龙, 刘茂军. IFN-α对山羊副流感病毒3型的抗病毒活性[J]. 畜牧兽医学报, 2023, 54(2): 736-743.
[11]	宫浩阳, 吴佳鑫, 杨晓钰, 解伟纯, 王雪莹, 李佳璇, 姜艳平, 崔文, 李一经, 唐丽杰. 肠道菌群抗病毒机制研究进展[J]. 畜牧兽医学报, 2023, 54(12): 4910-4919.
[12]	范锦全, 张宇航, 唐午阳, 赵欣宇, 李丕顺, 郑晓峰. 地西他滨对猪圆环病毒2型的体外抑制作用[J]. 畜牧兽医学报, 2023, 54(12): 5134-5142.
[13]	陈文哲, 张向乐, 顾峰幸, 赵振翔, 李康丽, 薛钊宁, 郑海学, 张小丽, 朱紫祥. 猪GRK2蛋白抗口蹄疫病毒作用分析[J]. 畜牧兽医学报, 2023, 54(10): 4350-4361.
[14]	周婉婷, 杨晨, 彭翠甜, 付新亮, 钟焯华, 许丹宁, 黄运茂, 田允波, 刘文俊. 白藜芦醇对急性热应激条件下鸭肝抗氧化能力和细胞凋亡的影响[J]. 畜牧兽医学报, 2023, 54(1): 239-251.
[15]	姚晨, 郭萌, 胡慧, 史晨曦, 杨国宇. 苯基吡啶酮衍生物影响猪δ冠状病毒复制的作用分析[J]. 畜牧兽医学报, 2022, 53(9): 3190-3198.