猴头菇多糖对MDRV感染RAW264.7细胞TLR3/TRIF诱导表达及病毒复制的影响

doi:10.16431/j.cnki.1671-7236.2021.09.033

摘要/Abstract

摘要： 试验旨在探究猴头菇多糖（HEP）预处理RAW264.7细胞对番鸭呼肠孤病毒（MDRV）复制的影响及其机制，从Toll样受体3/β干扰素TIR结构域衔接蛋白（TLR3/TRIF）信号通路解析HEP在调节MDRV所致的雏番鸭机体免疫抑制中的作用机制并提供体外试验参考。试验设空白对照组（BCG）、病毒感染对照组（VCG）、HEP对照组（HCG）和HEP预防病毒感染组（HPG），RAW264.7细胞在MDRV感染12和24 h后，通过Western blotting检测各组细胞中TLR3、TRIF和肿瘤坏死因子受体相关因子6（TRAF6）的蛋白表达量；病毒感染24 h后，用ELISA法检测各组细胞培养液中肿瘤坏死因子-α（TNF-α）、白介素-10（IL-10）、IL-6、IL-1β、干扰素-β（IFN-β）的含量。TCID₅₀检测结果表明，MDRV病毒的TCID₅₀为10^3.46。病毒感染后12 h，细胞未出现明显变化；病毒感染后24 h，细胞变圆；病毒感染后36 h，细胞大量死亡。MDRV在RAW264.7细胞中的复制结果显示，在感染病毒12~24 h内，σNS的表达量呈现上升趋势；在24~36 h，σNS的表达量维持在较高水平。Western blotting结果显示，与空白对照组相比，VCG组细胞TLR3、TRIF和TRAF6蛋白的表达量在感染后12和24 h均显著升高（P<0.05），HCG组TRIF蛋白的表达量均显著升高（P<0.05）；与感染组相比，HPG组的σNS、TLR3、TRIF和TRAF6蛋白表达量在病毒感染12和24 h均显著降低（P<0.05）。ELISA检测结果显示，与空白对照组相比，VCG组细胞培养液中TNF-α、IL-6、IL-1β和IFN-β的含量均显著升高（P<0.05）；与感染组相比，HPG组细胞培养液中TNF-α、IL-6、IL-1β含量均显著降低（P<0.05），IFN-β的含量显著升高（P<0.05）。结果表明，HEP可调节MDRV感染诱导RAW264.7细胞TLR3信号转导通路活化，抑制TLR3信号转导通路下游产物TNF-α、IL-10、IL-6和IL-1β的过度表达，同时上调IFN-β的表达，从而抑制MDRV在RAW264.7细胞中的复制。

关键词: 番鸭呼肠孤病毒; 猴头菇多糖; Toll样受体3; 信号转导; 病毒复制

Abstract: The aim of this study was to investigate the effect of Hericium erinaceus polysaccharide (HEP) pretreatment of RAW264.7 cells on the replication of Muscovy duck reovirus (MDRV) and its mechanism, and dissect the mechanism of HEP in regulating MDRV-induced immunosuppression in Muscovy ducks from the Toll-like receptor 3/interferon-β TIR domain adaptor protein (TLR3/TRIF) signaling pathway. The trial included blank control group (BCG), viral infection control group (VCG), HEP control group (HCG) and HEP prevention viral infection group (HPG). The protein expression levels of TLR3, TRIF and tumor necrosis factor receptor-associated factor 6 (TRAF6) in RAW264.7 cell at 12 and 24 h of MDRV infection was detected by Western blotting. After virus infection for 24 h, the contents of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), IL-6, IL-1β and interferon β (IFN-β) in the cell supernatant culture medium of each group were detected by ELISA. The TCID₅₀ detection results showed that the TCID₅₀ of MDRV was 10^3.46. At 12 h after infection, the cells did not change significantly, 24 h after infection, the cells became round, and 36 h after infection, a large number of cells died. The replication results of MDRV in RAW264.7 cells showed that within 12-24 h of virus infection, the expression of σNS increased, and at 24-36 h, the expression of σNS was maintained at a high level. Western blotting results showed that compared with blank control group, the expression levels of TLR3, TRIF and TRAF6 proteins in VCG group were significantly increased (P<0.05) after 12 and 24 h infection, and TRIF protein in HCG group were significantly increased (P<0.05) after 24 h infection. When compared with infected group, the expression of σNS, TLR3, TRIF and TRAF6 proteins were significantly decreased (P<0.05) after 12 and 24 h infection in the HPG group. ELISA results showed that compared with blank control group, the contents of TNF-α, IL-6, IL-1β and IFN-β in the culture medium of VCG group were significantly increased (P<0.05). When compared with infected group, the contents of TNF-α, IL-6 and IL-1β in the cell culture medium of HPG group were significantly decreased (P<0.05), while the content of IFN-β was significantly increased (P<0.05). The results showed that HEP could regulate MDRV infection to induce TLR3 signal transduction pathway activation, inhibit the overexpression of TNF-α, IL-10, IL-6 and IL-1β of downstream products of TLR3 signal transduction pathway, and up-regulate the expression of IFN-β in RAW264.7 cells, thereby inhibiting the replication of MDRV in RAW264.7 cells.

Key words: Muscovy duck reovirus; Hericium erinaceus polysaccharide; Toll-like receptor 3; signal transduction; virus replication

中图分类号:

S858.32

严萍, 林绍青, 李明慧, 孙雪宁, 李健, 黄一帆, 吴异健. 猴头菇多糖对MDRV感染RAW264.7细胞TLR3/TRIF诱导表达及病毒复制的影响[J]. 中国畜牧兽医, 2021, 48(9): 3415-3422.

YAN Ping, LIN Shaoqing, LI Minghui, SUN Xuening, LI Jian, HUANG Yifan, WU Yijian. Effects of Hericium erinaceus Polysaccharide on TLR3/TRIF-induced Expression and Viral Replication in MDRV-infected RAW264.7 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(9): 3415-3422.

参考文献

[1] 吴异健.两种中药多糖对呼肠孤病毒感染番鸭的免疫调节作用[D].福州:福建农林大学, 2010. WU Y J.Immunoregulating effects of two Chinese herbal polysaceharideson Museovy duck infecting of MDRV[D].Fuzhou:Fujian Agriculture and Forestry University, 2010.(in Chinese)
[2] 黄丽娜, 姜慧慧, 王洁, 等.猴头菇多糖对呼肠孤病毒感染番鸭肠道黏膜免疫及抗氧化能力的影响[J]. 畜牧兽医学报, 2015, 46(9):1671-1677. HUANG L N, JIANG H H, WANG J, et al. Effect of Hericium erinaceus polysaccharide on intestinal mucosal immunity and antioxidant capacity of reovirus infected Muscovy duck[J]. Chinese Journal of Animal and Veterinary Sciences, 2015, 46(9):1671-1677.(in Chinese)
[3] 姚金水, 吴宝成, 陈文列, 等.禽(番鸭)呼肠孤病毒诱导脾脏单核-巨噬细胞和淋巴细胞凋亡的超微观察[J]. 福建农业大学学报(自然科学版):2004, 33(4):501-504. YAO J S, WU B C, CHEN W L, et al. Ultrastructural observation on spleen cell, mononuclear macrophage and lymphocytes incurring apoptosis induced by Avian (Muscovy duck) reovirus[J]. Journal of Fujian Agriculture and Forestry University (Natural Science Edition), 2004, 33(4):501-504.(in Chinese)
[4] 祁保民, 陈晓燕, 吴宝成, 等.番鸭呼肠孤病毒诱导的细胞凋亡观察[J]. 畜牧兽医学报, 2010, 41(4):495-499. QI B M, CHEN X Y, WU B C, et al. The observation of the apoptosis induced by Muscovy duck reovirus[J]. Chinese Journal of Animal and Veterinary Sciences, 2010, 41(4):495-499.(in Chinese)
[5] 卿柯香, 袁远华, 郭霄峰, 等.新型鸭呼肠孤病毒S组基因克隆及序列分析[J]. 中国兽医杂志, 2015, 51(6):17-19. QING K X, YUAN Y H, GUO X F, et al. Cloning and sequence analysis S-class genomes of New-type duck reovirus isolated from Muscovy duck[J]. Chinese Journal of Veterinary Medicine, 2015, 51(6):17-19.(in Chinese)
[6] 刘燕飞, 时飞.Toll样受体信号转导通路及对炎性因子调控的研究进展[J]. 国际麻醉学与复苏杂志, 2016, 37(1):86-91. LIU Y F, SHI F.Research progress about Toll-like receptors signaling pathways and the regulation on inflammatory cytokines[J]. International Journal of Anesthesiology and Resuscitation, 2016, 37(1):86-91.(in Chinese)
[7] 杨季云, 张思仲, 郭红, 等.肿瘤坏死因子α通过激活NF-κB信号通路加快肝细胞周期进程[J]. 生物化学与生物物理进展, 2007, 34(6):604-610. YANG J Y, ZHANG S Z, GUO H, et al. TNF-α promotes cell cycle progression by activating NF-κB signaling pathway in hepatic cell line L-02[J]. Progress in Biochemistry and Biophysics, 2007, 34(6):604-610.(in Chinese)
[8] 邢飞跃, 赵克森, 姜勇.NF-κB的信号通路与阻断策略[J]. 中国病理生理杂志, 2003, 19(6):849-855. XING F Y, ZHAO K S, JIANG Y.Signal transduction pathway and blocking strategies of NF-κB[J]. Chinese Journal of Pathophysiology, 2003, 19(6):849-855.(in Chinese)
[9] HUSEBYE H, AUNE MH, STENVIK J, et al. The Rab11a GTPase controls Toll-like receptor 4-induced activation of interferon regulatory factor-3 on phagosomes[J]. Immunity, 2010, 33(4):583-596.
[10] PALSSON-MCDERMOTT E M, DOYLE S L, MCGETTRICK A F, et al. TAG, a splice variant of the adaptor TRAM, negatively regulates the adaptor MyD88-independent TLR4 pathway[J]. Nature Immunology, 2009, 10(6):579-586.
[11] 孙冰, 韩代书.Toll样受体信号通路的负调控[J]. 生物化学与生物物理进展, 2009, 369(12):1516-1522. SUN B, HAN D S.Negative regulation of Toll-like receptors signaling pathways[J]. Progress in Biochemistry and Biophysics, 2009, 36(12):1516-1522.(in Chinese)
[12] KARIN M, BEN-NERIAH Y.Phosphorylation meets ubiquitination:The control of NF-(kappa)B activity[J]. Annual Review of Immunology, 2000, 18(1):621-663.
[13] SHYANG-CHWEN S, YING L, MENG-SHIOU L, et al. Immunomodulatory effects of polysaccharides isolated from Hericium erinaceus on dendritic cells[J]. Process Biochemistry, 2013, 48(9):1402-1408.
[14] LEE J S, CHOJ Y, HONG E K.Study on macrophage activation and structural characteristics of purified polysaccharides from the liquid culture broth of Hericium erinaceus[J]. Carbohydrate Polymers, 2009, 78(1):162-168.
[15] WU Y J, JIANG H H, ZHU E P, et al. Hericium erinaceus polysaccharide facilitates restoration of injured intestinal mucosal immunity in Muscovy duck reovirus-infected Muscovy ducklings[J]. International Journal of Biological Macromolecules, 2018, 107(Pt A):1151-1161.
[16] 林绍青, 骆钰, 李明慧, 等.猴头菇多糖对MDRV感染RAW264.7细胞后病毒复制及TLR3诱导表达的影响[J]. 中国兽医科学, 2019, 49(6):773-780. LIN S Q, LUO Y, LI M H, et al. Effect of Hericium erinaceus polysaccharide on viral replication and TLR3 induced expression of MDRV-infected RAW264.7 cells[J]. Chinese Veterinary Science, 2019, 49(6):773-780.(in Chinese)
[17] 吴宝成, 陈家祥, 姚金水, 等.番鸭呼肠孤病毒的分离与鉴定[J]. 福建农业大学学报(自然科学版), 2001, 30(2):227-230. WU B C, CHEN J X, YAO J S, et al. Isolation and identification of Muscovy duck reovirus[J]. Journal of Fujian Agriculture and Forestry University(Natural Science Edition), 2001, 30(2):227-230.(in Chinese)
[18] 梁英, 吴异健, 李文迹, 等.禽呼肠孤病毒鸡源分离株的鉴定与人工感染试验[J]. 中国兽医科学, 2007, 4:295-300. LIANG Y, WU Y J, LI W J, et al. Isolation and identification of a strain of Avian reovirus and experimental infection with the strain[J]. Chinese Veterinary Science, 2007, 4:295-300.(in Chinese)
[19] 赵赫.TLR3与RIG-Ⅰ相关信号分子在鸭呼肠孤病毒感染雏鸭免疫器官中表达变化研究[D].武汉:华中农业大学, 2015. ZHAO H. Research on expression of TLR3 and RIG-Ⅰ related factors in immune organs of duckling infected by DRV[D].Wuhan:Huazhong Agricultural University, 2015.(in Chinese)
[20] 陆英杰, 王舍玉, 马春全, 等.番鸭呼肠孤病毒致病机理研究进展[J]. 湖北农业科学, 2010, 49(8):1993-1994. LU Y J, WANG S Y, MA C Q, et al. Research progress in Muscovy duck reovirus pathogenesis[J]. Hubei Agricultural Sciences, 2010, 49(8):1993-1994. (in Chinese)
[21] CHEN Z L, LUO G, WANG Q, et al. Muscovy duck reovirus infection rapidly activates host innate immune signaling and induces an effective antiviral immune response involving critical interferons[J]. Veterinary Microbiology, 2014, 175(2-4):232-243.
[22] 孙雪宁.猴头菇多糖对MDRV感染RAW264.7细胞TLR3-TRIF-TRAF6信号转导通路的影响[D].福州:福建农林大学, 2016. SUN X N.The impact of Hericium erinaceus polysaccharide on the TLR3-TRIF-TRAF6 signaling pathway in MDRV-infected RAW264.7 cells[D].Fuzhou:Fujian Agriculture and Forestry University, 2016.(in Chinese)
[23] 闫静.抗病毒活性糖苷的筛选及其对TLR3及相关基因的调控[D].长春:吉林大学, 2014. YAN J.Screening and studying its regulation mechanism on TLR3 of antiviral glycoside compounds[D].Changchun:Jilin University, 2014.(in Chinese)
[24] 赵保胜.桂枝汤等解表/清热方和相关成分对TLRs及其信号转导通路的影响[D].北京:中国中医科学院, 2007. ZHAO B S.Effects of diaphoretic recipe, heat-clearing recipe and their related components on TLRs and downstream signaling transduction pathway[D].Beijing:China Academy of Chinese Medical Sciences, 2007.(in Chinese)
[25] 孙秀宁.TLR3在HSV-1感染小鼠NSCs中的作用研究[D].青岛:青岛大学, 2013. SUN X N.The effect of TLR3 on mouse NSCs after HSV-1 infection[D].Qingdao:Qingdao University, 2013.(in Chinese)
[26] TAKAOKA A, TANIGUCHI T.New aspects of IFN-alpha/beta signalling in immunity, oncogenesis and bone metabolism[J]. Cancer Science, 2003, 94(5):405-411.