副猪嗜血杆菌OmpP2刺激猪肺泡巨噬细胞炎性因子mRNA转录及致炎机制初步分析

doi:10.11843/j.issn.0366-6964.2016.07.016

畜牧兽医学报 ›› 2016, Vol. 47 ›› Issue (7): 1428-1434.doi: 10.11843/j.issn.0366-6964.2016.07.016

副猪嗜血杆菌OmpP2刺激猪肺泡巨噬细胞炎性因子mRNA转录及致炎机制初步分析

何欢，陈新诺，曾泽，任玉鹏，汤承，张斌^*，岳华^*

（西南民族大学生命科学与技术学院，成都 610041）

收稿日期:2015-12-25 出版日期:2016-07-23 发布日期:2016-07-23
通讯作者: 张斌，E-mail:binovy@sina.com；岳华，E-mail:yhua900@163.com
作者简介:何欢（1991-），女，蒙古族，辽宁康平人，硕士生，主要从事动物病原分子生物学研究，E-mail:1252469322@qq.com
基金资助:
国家自然科学基金（31302119）；四川省教育厅创新项目（14ZB046）；公益性行业（农业）科研专项（201303034-1）；西南民族大学研究生创新科研项目（CX2015SZ074）

The Initial Research of OmpP2 in Haemophilus parasuis Induces Pro-inflammatory Cytokine mRNA Transcription and Inflammatory Mechanism in Porcine Alveolar Macrophages

HE Huan，CHEN Xin-nuo，ZENG Ze，REN Yu-peng，TANG Cheng，ZHANG Bin^*，YUE Hua ^*

(College of Life Science and Technology，Southwest University for Nationalities，Chengdu 610041，China)

Received:2015-12-25 Online:2016-07-23 Published:2016-07-23

摘要/Abstract

摘要：

为了研究副猪嗜血杆菌（Haemophilus parasuis，HPS）弱毒株11型H465株OmpP2刺激猪肺泡巨噬细胞（PAMs）炎性因子mRNA的转录和对NF-κB和MAPKs信号通路的影响，提取并纯化HPS血清11型H465株的OmpP2，分别用5、10 μg•mL^－1 OmpP2刺激PAMs 6、12 h后收集细胞，提取总RNA和蛋白质。将提取的RNA反转录成cDNA，运用RT-PCR检测炎性因子（IL-1α、IL-1β、IL-6、IL-8和TNF-α）mRNA转录水平。利用Western blot方法检测ERK1/2、JNK、P38、P65和IκBα蛋白表达量的变化。结果表明：HPS H465株的OmpP2能够显著地诱导IL-1α、IL-1β、IL-6、IL-8和TNF-α的mRNA转录水平上调(P<0.05)，同时能够引起JNK、P65蛋白表达水平显著升高和 IκBα 蛋白表达水平显著降低 (P<0.05)。上述结果证实了HPS 血清11型H465株的OmpP2能够通过调节MAPKs信号通路中JNK蛋白以及NF-κB信号通路中P65蛋白和IκBα蛋白降解促进炎性因子IL-1α、IL-1β、IL-6、IL-8和TNF-α的转录。

Abstract:

The aim of this study was to explore the role of out membrane protein P2 (OmpP2) in Haemophilus parasuis serovar 11 reference H465 strain to induce pro-inflammatory cytokine mRNA transcription，and NF-κB and MAPKs signaling pathways in porcine alveolar macrophages (PAMs).The OmpP2 was extracted from the H.parasuis H465 and stimulated in PAMs with 5 and 10 μg•mL－1 for 6 and 12 hours.The total RNA and cells protein were extracted and the RNA was reversed into cDNA.The mRNA transcription levels of IL-1α，IL-β，IL-6，IL-8 and TNF-α were detected by Real-time PCR，and the protein of NF-κB P65，IκBα，ERK，JNK and P38 were detected by Western blot.The result showed that the mRNA transcription of IL-1α，IL-β，IL-6，IL-8 and TNF-α in PAMs induced by the OmpP2 from H.parasuis were significantly up-regulated (P<0.05)，and the protein of JNK，P65 in PAMs induced by the OmpP2 from H.parasuisis significantly increased and the protein of IκBα was significantly declined(P<0.05).The above results exhibit that the OmpP2 could effectively activate the JNK and P65 proteins and increase the degradation of IκBα in NF-κB and MAPKs signaling pathways to promote the pro-inflammatory cytokines expression.

中图分类号:

S852.613

何欢，陈新诺，曾泽，任玉鹏，汤承，张斌，岳华. 副猪嗜血杆菌OmpP2刺激猪肺泡巨噬细胞炎性因子mRNA转录及致炎机制初步分析[J]. 畜牧兽医学报, 2016, 47(7): 1428-1434.

HE Huan，CHEN Xin-nuo，ZENG Ze，REN Yu-peng，TANG Cheng，ZHANG Bin，YUE Hua. The Initial Research of OmpP2 in Haemophilus parasuis Induces Pro-inflammatory Cytokine mRNA Transcription and Inflammatory Mechanism in Porcine Alveolar Macrophages[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(7): 1428-1434.

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参考文献

［1］CAI X，CHEN H，BLACKALL P J，et al.Serological characterization of Haemophilus parasuis isolates from China［J］.Vet Microbiol，2005，111(3-4)：231-236.
［2］RAPP-GABRIELSON V J，GABRIELSON D A.Prevalence of Haemophilus parasuis serovars among isolates from swine［J］. Am J Vet Res，1992，53(5)：659-664.
［3］PAGÈS J M，JAMES C E，WINTERHALTER M.The porin and the permeating antibiotic：a selective diffusion barrier in Gram-negative bacteria［J］.Nat Rev Microbiol，2008，6(12)：893-903.
［4］赵倩，汤承，杨发龙，等.副猪嗜血杆菌ompP2基因的结构特征及其与毒力的联系［J］.中国科学：生命科学，2010，40(6)：522-532.
ZHAO Q，TANG C，YANG F L，et al.The correlation between structural characterization of ompP2 gene and virulence of Haemophilus parasuis［J］.Scientia Sinica：Vitae，2010，40(6)：522-532.(in Chinese)
［5］ZHANG B，XU C，ZHANG L，et al.Enhanced adherence to and invasion of PUVEC and PK-15 cells due to the overexpression of RfaD，ThyA and Mip in the ΔompP2 mutant of Haemophilus parasuis SC096 strain［J］.Vet Microbiol，2013，162(2-4)：713-723.
［6］ZHOU S，HE X，XU C，et al.The outer membrane protein P2 (OmpP2) of Haemophilus parasuis induces proinflammatory cytokine mRNA expression in porcine alveolar macrophages［J］.Vet J，2014，199(3)：461-464.
［7］CINAR M U，ISLAM M A，UDDIN M J，et al.Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA［J］.BMC Res Notes，2012，5：107.
［8］LIVAK K，SCHMITTGEN T D.Analysis of relative gene expression date using real-time quantitative PCR and the 2-ΔΔCT method［J］.Methods，2001，25(4)：402-408.
［9］RUIZ A，OLIVEIRA S，TORREMORELL M，et al.Outer membrane proteins and DNA profiles in strains of Haemophilus parasuis recovered from systemic and respiratory sites［J］.J Clin Microbiol，2001，39(5)：1757-1762.
［10］陆承平.兽医微生物学［M］.北京：中国农业出版社，2007：144-146.
LU C P.Veterinary microbiology［M］.Beijing：China Agriculture Press，2007：144-146.(in Chinese)
［11］OLIVEIRA S，PIJOAN C.Haemophilus parasuis：new trends on diagnosis，epidemiology and control［J］.Vet Microbiol，2004，99(1)：1-12.
［12］VAHLE J L，HAYNES J S，ANDREWS J J.Experimental reproduction of Haemophilus parasuis infection in swine：clinical，bacteriological，and morphologic findings［J］.J Vet Diagn Invest，1995，7(4)：476-480.
［13］GALDIERO M，D’AMICO M，GORGA F，et al. Haemophilus influenzae porin contributes to signaling of the inflammatory cascade in rat brain［J］.Infect Immun，2001，69(1)：221-227.
［14］RAY A，CHATTERJEE N S，BHATTACHARYA S K，et al.Porin of Shigella dysenteriae enhances mRNA levels for Toll-like receptor 2 and MyD88，up-regulates CD80 of murine macrophage，and induces the release of interleukin-12［J］.FEMS Immunol Med Microbiol，2003，39(3)：213-219.
［15］MASSARI P，GUNAWARDANA J，LIU X，et al.Meningococcal porin PorB prevents cellular apoptosis in a toll-like receptor 2- and NF-kappaB-independent manner［J］.Infect Immun，2010，78(3)：994-1003.
［16］KIM H J，LEE H S，CHONG Y H，et al.p38 mitogen-activated protein kinas up-regulates LPS-induced NF-κB activation in the development of lung injury and RAW 264.7 macrophages［J］.Toxicology，2006，225(1)：36-47.
［17］RAVANTI L，TORISEVA M，PENTTINEN R，et al.Expression of human collagenase-3(MMP-13)by fetal skin fibroblasts is induced by transforming growth factor β via p38 mitogen-activated protein kinase［J］.FASEB J，2001，15(6)：1098-1100.
［18］LIU Y W，CHEN C C，TSENG H P，et al.Lipopolysaccharide-induced transcriptional activation of interleukin-10 is mediated by MAPK- and NF-kappaB-induced CCAAT /enhancer-binding protein delta in mouse macrophages［J］. Cell Signal，2006，18(9)：1492- 1500.
［19］LEE J I，BURCKART G J.Nuclear factor kappa B：important transcription factor and therapeutic target［J］.J Clin Pharmacol，1998，38(11)：981-993.
［20］CHANG L，KARIN M.Mammalian MAP kinase signalling cascades［J］.Nature，2001，410(6824)：37-40.

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副猪嗜血杆菌OmpP2刺激猪肺泡巨噬细胞炎性因子mRNA转录及致炎机制初步分析

The Initial Research of OmpP2 in Haemophilus parasuis Induces Pro-inflammatory Cytokine mRNA Transcription and Inflammatory Mechanism in Porcine Alveolar Macrophages

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