rfaE基因通过MAPKs/NF-κB信号通路对副猪嗜血杆菌LOS诱导猪肺泡巨噬细胞炎症反应中作用的研究

doi:10.11843/j.issn.0366-6964.2016.05.015

摘要/Abstract

摘要：

作者拟研究rfaE基因在副猪嗜血杆菌（Haemophilus parasuis，HPS）脂寡糖（LOS）刺激猪肺泡巨噬细胞（PAMs）信号通路分子的转录表达和MAPKs/ NF-κB信号通路中的作用。提取HPS SC096株及其rfaE基因缺失株（ΔrfaE）和互补株（cΔrfaE）的LOS，分别用5和10 μg HPS-LOS、ΔrfaE-LOS和cΔrfaE-LOS刺激PAMs，分别在不同时间点收集细胞，提取RNA和蛋白质。将提取的RNA反转录成cDNA，运用RT-PCR检测TLR4、MD2、NF-κB、MAP2K2、ERK、P38和JNK的mRNA转录水平。测定提取蛋白质的浓度，利用Western blot方法检测NF-κB p65/phospho-NF-κB p65、IκBα、ERK1/2、JNK和p38/phospho-p38蛋白的表达量。结果表明用5和10 μg HPS-LOS刺激细胞6、12和24 h后，TLR4、MD2、MAP2K2、ERK、P38和JNK的mRNA转录水平均显著高于ΔrfaE-LOS刺激细胞后的以上转录因子的mRNA水平（P<0.05），但 NF-κB的mRNA转录水平无显著差异。另外，用5和10 μg HPS-LOS刺激细胞6和12 h后，IκBα蛋白的表达量显著低于ΔrfaE-LOS刺激细胞后的IκBα蛋白的表达量（P<0.05），NF-κB p65和p38的磷酸化水平及ERK1/2和JNK蛋白的表达量显著高于ΔrfaE-LOS刺激细胞后NF-κB p65和p38的磷酸化水平及ERK1/2和JNK蛋白的表达量（P<0.05）。同时cΔrfaE-LOS刺激PAMs后TLR4、MD2、MAP2K2、ERK、P38和JNK的mRNA转录水平以及NF-κB p65和p38的磷酸化水平和IκBα、ERK1/2和JNK蛋白水平能够恢复到HPS-LOS水平。以上试验结果证实在HPS-LOS诱导的炎症反应中，缺失rfaE基因后通过阻断MAPKs/ NF-κB信号通路以减轻炎症反应。

Abstract:

The aim of this study was to study the role of rfaE gene in Haemophilus parasuis lipooligosaccharide (LOS) which induced signal molecules mRNA transcription and MAPKs/ NF-κB signaling pathways in porcine alveolar macrophages (PAMs).The LOS of H.parasuis SC096 strain，rfaE mutant and its complementation was extracted.The PAMs were stimulated with LOS (5 and 10 μg) from H.parasuis SC096，ΔrfaE mutant (ΔrfaE) and its complementation (cΔrfaE) for different time points.The RNA and protein was extracted from the collected cells.The extracted RNA was reversed into cDNA.The mRNA transcription of TLR4，MD2，NF-κB，MAP2K2，ERK，P38 and JNK were then detected by Real-time PCR.The protein of NF-κB p65/ Phospho-NF-κB p65，IκBα，ERK，JNK and p38/ Phospho-p38 were detected by western blot.The results showed that the mRNA transcription of TLR4，MD2，MAP2K2，ERK，P38 and JNK in PAMs induced by 5 and 10 μg HPS-LOS for 6，12 and 24 h were up-regulated，and significantly higher than that in PAMs induced by 5 and 10 μg ΔrfaE-LOS (P<0.05).The mRNA transcription of NF-κB in PAMs induced by 5 and 10 μg HPS-LOS for 6，12 and 24 h were higher than that in PAMs induced by 5 and 10 μg ΔrfaE-LOS，but no significant difference was observed.The protein of IκBα in PAMs induced by 5 and 10 μg HPS-LOS for 6 and 12 h were significantly lower than that in PAMs induced by 5 and 10 μg ΔrfaE-LOS (P<0.05).The p65 phosphorylation，p38 phosphorylation，ERK and JNK in PAMs induced by 5 and 10 μg HPS-LOS for 6 and 12 h were significantly higher than that in PAMs induced by 5 and 10 μg ΔrfaE-LOS (P<0.05).At the same time，the signal molecules mRNA transcription level and the protein level in PAMs induced by cΔrfaE-LOS can restore to the level in PAMs induced by HPS-LOS.The date confirmed that the loss of rfaE gene could effectively reduced the inflammatory process in PAMs induced by HPS-LOS by blocking the MAPKs/ NF-κB signaling pathways.

中图分类号:

S852.613

曾泽，何欢，任玉鹏，岳华，张斌. rfaE基因通过MAPKs/NF-κB信号通路对副猪嗜血杆菌LOS诱导猪肺泡巨噬细胞炎症反应中作用的研究[J]. 畜牧兽医学报, 2016, 47(5): 978-984.

ZENG Ze，HE Huan，REN Yu-peng，YUE Hua，ZHANG Bin. The Effect of rfaE Gene in Haemophilus parasuis Lipooligosaccharide Induced Inflammation via the MAPKs/ NF-κB Signaling Pathways in Porcine Alveolar Macrophages[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(5): 978-984.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2016.05.015

https://www.xmsyxb.com/CN/Y2016/V47/I5/978

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］OLIVEIRA S，PIJOAN C.Haemophilus parasuis：new trends on diagnosis，epidemiology and control［J］.Vet Microbiol，2004，99(1)：1-12.
［3］KIELSTEIN P，RAPP-GABRIELSON V J.Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts［J］.J Clin Microbiol，1992，30(4)：862-865.
［4］HO D K，RAM S，NELSON K L，et al.lgtC expression modulates resistance to C4b deposition on an invasive nontypeable Haemophilus influenzae［J］.J Immunol，2007，178(2)：1002-1012.
［5］KANIPES M I，TAN X，AKELAITIS A，et al.Genetic analysis of lipooligosaccharide core biosynthesis in Campylobacter jejuni 81-176［J］.J Bacteriol，2008，190(5)：1568-1574.
［6］SWORDS W E，BUSCHER B A，VER STEEG LI K，et al.Non-typeable Haemophilus influenzae adhere to and invade human bronchial epithelial cells via an interaction of lipooligosaccharide with the PAF receptor［J］.Mol Microbiol，2000，37(1)：13-27.
［7］GOLEC M.Cathelicidin LL-37：LPS-neutralizing，pleiotropic peptide［J］.Ann Agric Environ Med，2007，14(1)：1-4.
［8］COHEN J.The immunopathogenesis of sepsis［J］.Nature，2002，420(6917)：885-891.
［9］KIM H J，LEE H S，CHONG Y H，et al.p38 mitogen-activated protein kinase 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.
［10］GHOSH S，MAY M J，KOPP E B.NF-kappa B and Rel proteins：Evolutionarily conserved mediators of immune responses［J］.Annu Rev Immunol，1998，16：225-260.
［11］SCHMECK B，ZAHLEN J，MOOG K，et al.Streptococcus pneumoniae-induced p38 MAPK-dependent phosphorylation of RelA at the interleukin-8 promotor［J］.J Biol Chem，2004，279(51)：53241-53247.
［12］SCHERLE P A，JONES E A，FAVATA M F，et al.Inhibition of MAP kinase kinase prevents cytokine and prostaglandin E2 production in lipopolysaccharide-stimulated monocytes［J］.J Immunol，1998，161(10)：5681-5686.
［13］CRAIG R，LARKIN A，MINGO A M，et al.p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release.Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system［J］.J Biol Chem，2000，275(31)：23814-23824.
［14］曾泽，岳华，冯晓辉，等.rfaE基因在副猪嗜血杆菌LOS刺激猪肺泡巨噬细胞炎性因子mRNA转录中的作用［J］.畜牧兽医学报，2015，46(7)：1232-1237.
ZENG Z，YUE H，FENG X H，et al.The effect of rfaE gene in Haemophilus parasuis LOS induces pro-inflammatory cytokine mRNA transcription in porcine alveolar macrophages［J］.Acta Veterinaria et Zootechnica Sinica，2015，46(7)：1232-1237.(in Chinese)
［15］ZHANG B，YU Y，ZENG Z，et al.Deletion of the rfaE gene in Haemophilus parasuis SC096 strain attenuates serum resistance，adhesion and invasion［J］.Microb Pathog，2014，74：33-37.
［16］HITCHCOCK P J，BROWN T M.Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels［J］.J Bacteriol，1983，154(1)：269-277.
［17］ZHOU S M，HE X H，XU C G，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.
［18］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.
［19］WU Y，SINGER M，THOURON F，et al.Effect of surfactant on pulmonary expression of type IIA PLA(2) in an animal model of acute lung injury［J］.Am J Physiol Lung Cell Mol Physiol，2002，282(4)：L743-L750.
［20］BOUCHET B，VANIER G，JACQUES M，et al.Studies on the interactions of Haemophilus parasuis with porcine epithelial tracheal cells：limited role of LOS in apoptosis and pro-inflammatory cytokine release［J］.Microb Pathog，2009，46(2)：108-113.
［21］BOUCHET B，VANIER G，JACQUES M，et al.Interactions of Haemophilus parasuis and its LOS with porcine brain microvascular endothelial cells［J］.Vet Res，2008，39(5)：42.
［22］BAKER R G，HAYDEN M S，GHOSH S.NF-κB，inflammation，and metabolic disease［J］.Cell Metab，2011，13(1)：11-22.
［23］ZOU J，CREWS F.Induction of innate immune gene expression cascades in brain slice cultures by ethanol：key role of NF-κB and proinflammatory cytokines［J］.Alcohol Clin Exp Res，2010，34(5)：777-789.
［24］CRONIN J G，TURNER M L，GOETZE L，et al.Toll-like receptor 4 and MYD88-dependent signaling mechanisms of the innate immune system are essential for the response to lipopolysaccharide by epithelial and stromal cells of the bovine endometrium［J］.Biol Reprod，2012，86(2)：51.
［25］TURNER M L，CRONIN J G，HEALEY G D，et al.Epithelial and stromal cells of bovine endometrium have roles in innate immunity and initiate inflammatory responses to bacterial lipopeptides in vitro via Toll-like receptors TLR2，TLR1，and TLR6［J］.Endocrinology，2014，155(4)：1453-1465.
［26］KIM C H.A Salmonella typhimurium rfaE mutant recovers invasiveness for human epithelial cells when complemented by wild type rfaE (controlling biosynthesis of ADP-L-glycero-D-mannoheptose-containing lipopolysaccharide)［J］. Mol Cells，2003，15(2)：226-232.
［27］VALVANO M A，MAROLDA C L，BITTNER M，et al.The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose［J］.J Bacteriol，2000，182(2)：488-497.

[1]	肖静, 肖坤雪, 王翘楚, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Flp-FRT双基因敲除方法的建立与优化[J]. 畜牧兽医学报, 2022, 53(1): 219-230.
[2]	田杨, 刘云宝, 马辉, 潘其聪, 肖静, 陈焕春, 蔡旭旺, 徐晓娟. 副猪嗜血杆菌Apd-ELISA抗体检测试剂盒的制备和初步应用[J]. 畜牧兽医学报, 2020, 51(9): 2227-2237.
[3]	唐梦君, 周倩, 张小燕, 张静, 唐修君, 陆俊贤, 周生, 蒲俊华, 高玉时. 江苏部分地区鸡源与猪源弯曲菌耐药性分析与毒力基因检测[J]. 畜牧兽医学报, 2020, 51(9): 2284-2292.
[4]	沈懿娟, 周妮妮, 张健淞, 王美芬, 李玉峰, 姜平. TGF-β1信号通路在副猪嗜血杆菌侵入3D4/21细胞中的作用[J]. 畜牧兽医学报, 2018, 49(8): 1720-1726.
[5]	程蕾, 陈洪波. 巨噬细胞-γδT细胞免疫轴在猪格拉瑟氏病发病机制中的作用[J]. 畜牧兽医学报, 2018, 49(5): 879-886.
[6]	杨开杰, 冯賽祥, 周琪, 张建民, 廖明, 樊惠英. 副猪嗜血杆菌qseC基因缺失突变对生物被膜形成的影响[J]. 畜牧兽医学报, 2017, 48(8): 1499-1504.
[7]	谨瑾，张禄滑，文心田，李英，代科，周鹏，赵玉佳，曹三杰，黄小波，伍锐，赵勤，文翼平. 副猪嗜血杆菌potD基因缺失株的构建及其部分生物学特性[J]. 畜牧兽医学报, 2016, 47(11): 2274-2279.
[8]	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.
[9]	曾泽，何欢，岳华，汤承，张斌. 副猪嗜血杆菌lgtF基因缺失株的构建及其部分生物学特性[J]. 畜牧兽医学报, 2015, 46(11): 2056-2062.
[10]	曾泽，岳华，冯晓辉，何欢，张斌. rfaE基因在副猪嗜血杆菌LOS刺激猪肺泡巨噬细胞炎性因子mRNA转录中的作用[J]. 畜牧兽医学报, 2015, 46(7): 1232-1237.
[11]	白挨泉，郭建超，覃宗华，蒲文珺，马春全，李国清，张浩吉. 胞内劳森菌TaqMan荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2014, 45(10): 1733-1738.
[12]	匡学谦，岳华，陈小飞，汤承. 畜禽空肠弯曲杆菌cmp基因结构特点及ST型研究[J]. 畜牧兽医学报, 2013, 44(4): 657-664.