支原体巨噬细胞活化脂肽2诱导单核细胞系THP-1表达血红素氧合酶1及其机制研究  

Macrophage-activating lipopeptide-2 induces the expression of hemeoxygenase-1 in THP-1 cells and its possible mechanism

作　　者：游晓星[1] 马小华[2] 刘良专[1] 曾焱华[1] 余敏君[1] 朱翠明[1] 何军[3] 蒋传好[1] 吴移谋[1] 

机构地区：[1]南华大学病原生物学研究所,衡阳421001 [2]南华大学附属南华医院 [3]长沙市中心医院

出　　处：《中华微生物学和免疫学杂志》2014年第6期453-458,共6页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金（31000091,81072418）;湖南省重点实验室资助项目（湘科计字[2014]5号,湘教通[2012]312号）;湖南省科研条件创新专项（2013TT2013）

摘　　要：目的：观察支原体巨噬细胞活化脂肽2（macrophage-activating lipopeptide-2， MALP-2）能否诱导人单核细胞系THP-1表达血红素氧合酶-1（hemeoxygenase，HO-1），并探讨其相应的调控机制，以明确机体抵抗支原体感染所致炎性损伤的自我防御机制。方法体外培养THP-1细胞，用不同浓度的MALP-2作用12 h， Western blot检测HO-1的表达；THP-1细胞经TLR2和TLR6中和抗体孵育，或构建TLR2和TLR6负显性突变体转染细胞，以明确TLR2和TLR6在介导HO-1表达中的作用；Western blot检测Akt磷酸化情况，并采用PI3K抑制剂LY294002处理细胞，以证实PI3K参与HO-1表达。同时，分别采用EMSA和免疫荧光观察核转录因子Nrf2的DNA结合活性和核转位，并采用Nrf2 siRNA干扰其表达后，Western blot检测HO-1表达。最后，采用siRNA干扰HO-1表达，或采用HO-1的激动剂钴原卟啉（ cobalt protoporphyrin ，CoPP）处理THP-1细胞，ELISA检测细胞因子TNF-α和IL-1β的分泌。结果 MALP-2能诱导THP-1细胞表达HO-1。且TLR2和TLR6中和抗体以及其负显性突变体转染后，HO-1表达水平显著降低；此外，MALP-2能激活PI3K，PI3K抑制剂能抑制HO-1表达；MALP-2能增强Nrf2的DNA结合活性及核转位，PI3K抑制剂处理后，Nrf2的DNA结合活性以及核转位水平进一步降低。 RNA干扰Nrf2后，HO-1表达显著降低。沉默HO-1表达后，TNF-α和IL-1β分泌增高，而CoPP处理能进一步降低TNF-α和IL-1β水平。结论 MALP-2能诱导THP-1细胞表达HO-1，其机制可能受TLR2、6/PI3K/Nrf2通路调控。 HO-1的表达可在一定程度上负调控细胞因子过度分泌。Objective To investigate whether macrophage-activating lipopeptide-2 （ MALP-2） induces the expression of hemoxygenase-1 （ HO-1 ） in THP-1 cells and to further elucidate its possible regulatory mechanism for a better understanding of protective response upon mycoplasma infection .Methods THP-1 cells were cultured in vitro and stimulated by MALP-2 at different concentrations for 12 h.THP-1 cells were incubated with TLR 2 or TLR6 neutralizing antibodies , or transfected with their dominant negative plasmids to evaluate the effects of TLR 2 and TLR6 on HO-1 expression .Phosphorylation of Akt was detected by Western blot.PI3K inhibitor LY294002 was used to investigate the role of PI3K in HO-1 expression.Immunofluorescence and electrophoretic mobility shift assay （ EMSA ） were performed to observe the nuclear translocation and DNA-binding activity of nuclear factor Nrf 2.Small interfering RNA （ siRNA） was used to silence the genes encoding Nrf2 and HO-1.Cobalt protoporphyrin （CoPP）, an inducer of HO-1, was used to treat THP-1 cells.The expression of HO-1 was detected by Western blot .The secretion of TNF-αand IL-1βby THP-1 cells were measured by ELISA .Results MALP-2 induced the expression of HO-1 in THP-1 cells.However, the expression of HO-1 was inhibited by TLR2 and TLR6 neutralizing antibodies and expression of their dominant negative plasmids .Moreover, PI3K pathway was activated by MALP-2, and with the use of PI3K inhibitor, the expression of HO-1 was decreased.The translocation of Nrf2 to the nucleus and itsDNA-binding activity were enhanced by MALP-2, but were inhibited by the treatment of PI3K inhibitor.The expression of HO-1 was significantly down-regulated upon the interference of Nrf2 gene expression with siRNA.Silenced expression of HO-1 increased the level of TNF-αand IL-1β, while CoPP treatment decreasedthe secretion of MALP-2-induced cytokines.Conclusion MALP-2 might induce the expression ofHO-1 in THP-1 cells through TLR2,6/PI3K/Nrf2 pathways.The expression of HO-1 could negativ

关 键 词：支原体巨噬细胞活化脂肽2 血红素氧合酶-1 单核细胞 

分 类 号：R375[医药卫生—病原生物学]