当归多糖对点带石斑鱼Toll样受体22信号通路相关基因表达的影响
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摘要
为研究当归多糖Angelica sinensis polysaccharide(ASP)对点带石斑鱼Epinephelus malabaricus Toll样受体22(Toll-like receptor 22,TLR22)信号通路相关基因表达的影响,以当归多糖体外孵育点带石斑鱼头肾白细胞,采用实时荧光定量PCR(q PCR)方法检测了不同时间点(3、6、12、24、48 h)TLR22信号通路相关基因m RNA相对表达量的变化。结果表明:TLR22及与其接头的Toll样接头蛋白1(TIR-domaincontaining adaptor inducing interferon-β,TRIF)的表达量均随时间的延长逐渐升高,分别于24、12 h达到峰值;TRIF下游的干扰素调节因子(Interferon regulatory factor 3,IRF3)在24 h表达量达到最大值,此时主要组织相容性复合体(Major histocompatibility complex,MHC)与肿瘤坏死因子(Tumor necrosis factor-α,TNF-α)的表达量也开始到达峰值;白介素-8(Interleukin-8,IL-8)的表达量在48 h达到最高。研究表明:当归多糖可激活TLR22及其下游相关基因,并在免疫调节过程中起到重要作用。
Head kidney leucocytes of malabar grouper Epinephelus malabaricus were cultured in Angelica sinensis polysaccharides( ASP) in vitro for 3,6,12,24 and 48 h,and changes in m RNA levels of Toll-like receptor 22(TLR22) pathway-related genes were analyzed by quantitative real-time PCR to investigate effects of ASP on the expression of TLR22 signal pathway- related genes in malabar grouper. The results showed that m RNA levels of TLR22 and TIR-domain-containing adaptor inducing interferon-β( TRIF) were up-regulated gradually with time,with the maximum TLR22 value in 24 h and the maximum TRIF value in 12 h. The peak expression of downstream transcription factor IRF3( interferon regulatory factor 3) was observed in 24 h,and the peak expression of major histocompatibility complex( MHC) and tumor necrosis factor-α( TNF-α) genes was found in 24 h. The maximal expression of interferon- 8( IL- 8) m RNA appeared in 48 h. These findings indicate that ASP leads to activate TLR22 and its downstream genes which play an important role in malabar grouper immune regulation.
Head kidney leucocytes of malabar grouper Epinephelus malabaricus were cultured in Angelica sinensis polysaccharides( ASP) in vitro for 3,6,12,24 and 48 h,and changes in m RNA levels of Toll-like receptor 22(TLR22) pathway-related genes were analyzed by quantitative real-time PCR to investigate effects of ASP on the expression of TLR22 signal pathway- related genes in malabar grouper. The results showed that m RNA levels of TLR22 and TIR-domain-containing adaptor inducing interferon-β( TRIF) were up-regulated gradually with time,with the maximum TLR22 value in 24 h and the maximum TRIF value in 12 h. The peak expression of downstream transcription factor IRF3( interferon regulatory factor 3) was observed in 24 h,and the peak expression of major histocompatibility complex( MHC) and tumor necrosis factor-α( TNF-α) genes was found in 24 h. The maximal expression of interferon- 8( IL- 8) m RNA appeared in 48 h. These findings indicate that ASP leads to activate TLR22 and its downstream genes which play an important role in malabar grouper immune regulation.
引文
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[2]罗正全.中草药在鱼类无公害养殖中的应用[J].中国水产,2014(1):64-65.
[3]戈贤平,缪凌鸿,刘波.中草药增强水生动物免疫和抗病能力的研究进展[J].中国渔业质量与标准,2015,5(6):1-7.
[4]吴旋.四种中草药多糖对黄颡鱼生长、体成分及部分生理生化指标的影响[D].天津:天津农学院,2011.
[5]张伟妮,林旋,王寿昆,等.黄芪多糖对罗非鱼非特异性免疫和胃肠内分泌功能的影响[J].动物营养学报,2010,22(2):401-409.
[6]刘红柏,张颖,杨雨辉,等.5种中草药作为饲料添加剂对鲤肠内细菌及生长的影响[J].大连水产学院学报,2004,19(1):16-20.
[7]王庆奎.当归多糖对点带石斑鱼非特异性免疫力的影响[D].青岛:中国海洋大学,2012.
[8]丛方地,王庆奎,周文礼,等.水产养殖中多糖的非特异性免疫研究[J].饲料工业,2014,35(19):65-69.
[9]杨兴斌.当归多糖的组成分析及其激活腹腔巨噬细胞的免疫机制[D].西安:第四军医大学,2004.
[10]程安玮,金征宇,王家启.功能性多糖免疫调节机理的研究进展[J].食品科技,2006,31(12):4-8.
[11]邹云,谢红兵,贺建华.中草药多糖的免疫调节作用及其受体的研究[J].中国饲料,2013(5):22-25.
[12]Leung M Y K,Liu C,Koon J C M,et al.Polysaccharide biological response modifiers[J].Immunology Letters,2006,105(2):101-114.
[13]Patel M V,Kumar P,Das H,et al.Evaluation of the constitutive expression levels of ch-TLR 3,ch-TLR 4,ch-TLR 15 and chTLR 21 genes in the peripheral blood mononuclear cells of native Indian poultry breeds,Aseel and Kadaknath[J].Veterinary World,2013,6(8):568-572.
[14]王轶南,于卓,刘洋,等.虾夷马粪海胆TLR基因c DNA克隆及表达分析[J].大连海洋大学学报,2014,29(4):329-335.
[15]隗黎丽,吴华东,熊六凤.柱状黄杆菌对草鱼TLRs基因表达水平的影响[J].大连海洋大学学报,2013,28(4):378-382.
[16]Hirono I,Takami M,Miyata M,et al.Characterization of gene structure and expression of two Toll-like receptors from Japanese flounder,Paralichthys olivaceus[J].Immunogenetics,2004,56(1):38-46.
[17]Rebl A,Siegl E,Kllner B,et al.Characterization of twin Tolllike receptors from rainbow trout(Oncorhynchus mykiss):evolutionary relationship and induced expression by Aeromonas salmonicid salmonicida[J].Developmental&Comparative Immunology,2007,31(5):499-510.
[18]Xiao Xiaoqiang,Qin Qiwei,Chen Xinhua,et al.Molecular characterization of a Toll-like receptor 22 homologue in large yellow croaker(Pseudosciaena crocea)and promoter activity analysis of its 5’-flanking sequence[J].Fish&Shellfish Immunology,2011,30(1):224-233.
[19]Sundaram A Y M,Consuegra S,Kiron V,et al.Positive selection pressure within teleost Toll-like receptors tlr21 and tlr22 subfamilies and their response to temperature stress and microbial components in zebrafish[J].Molecular Biology Reports,2012,39(9):8965-8975.
[20]Sahoo B R,Dikhit M R,Bhoi G K,et al.Understanding the distinguishable structural and functional features in zebrafish TLR3 and TLR22,and their binding modes with fish ds RNA viruses:an exploratory structural model analysis[J].Amino Acids,2015,47(2):381-400.
[21]李言伟.石斑鱼TLRs功能及刺激隐核虫感染后免疫相关基因表达分析[D].广州:中山大学,2012.
[22]Kawai T,Akira S.The role of pattern-recognition receptors in innate immunity:update on Toll-like receptors[J].Nature Immunology,2010,11(5):373-384.
[23]Brickey W J,Neuringer I P,Walton W,et al.My D88 provides a protective role in long-term radiation-induced lung injury[J].International Journal of Radiation Biology,2012,88(4):335-347.
[24]Into T,Inomata M,Niida S,et al.Regulation of My D88 aggregation and the My D88-dependent signaling pathway by sequestosome 1and histone deacetylase 6[J].The Journal of Biological Chemistry,2010,285(46):35759-35769.
[25]Piao Wenji,Ru L W,Piepenbrink K H,et al.Recruitment of TLR adapter TRIF to TLR4 signaling complex is mediated by the second helical region of TRIF TIR domain[J].Proceedings of the National Academy of Sciences of the United States of America,2013,110(47):19036-19041.
[26]Choi Y J,Im E,Chung H K,et al.TRIF mediates Toll-like receptor 5-induced signaling in intestinal epithelial cells[J].The Journal of Biological Chemistry,2010,285(48):37570-37578.
[27]丁旭.斜带石斑鱼Toll样受体22基因的c DNA克隆、表达模式分析及其信号通路的初步探讨[D].海口:海南大学,2012.
[28]张守锋.大菱鲆Toll样受体22(TLR22)全长基因的克隆及表达分析[D].青岛:中国海洋大学,2014.