不可分型流感嗜血杆菌诱导14-3-3ε表达负向调控肺部炎症反应  

Non-Typeable Haemophilus influenzae Induces Expression of 14-3-3εto Negatively Regulate Pulmonary Inflammatory Response

作　　者：颜红霞 霍佳雯 魏大飞 谭雄 YAN Hongxia;HUO Jiawen;WEI Dafei;TAN Xiong(Department of Pediatrics,the Second Affiliated Hospital of University of South China,Hengyang 421001,China;Department of Vascular and Hernia Surgery,the Second Affiliated Hospital of University of South China,Hengyang 421001,China)

机构地区：[1]南华大学附属第二医院儿科,衡阳421001 [2]南华大学附属第二医院血管疝儿童普外科,衡阳421001

出　　处：《中国细胞生物学学报》2023年第9期1320-1330,共11页Chinese Journal of Cell Biology

基　　金：南华大学新冠肺炎疫情防控科研攻关应急专项(批准号:nk20200335);衡阳市科技局科技计划(批准号:2020jh042)资助的课题。

摘　　要：该文旨在观察14-3-3ε在不可分型流感嗜血杆菌(NTHi)诱导人支气管上皮细胞分泌促炎细胞因子中的作用,并研究其作用机制。分别采用感染复数(MOI)为5、10和20的NTHi感染BEAS-2B细胞,实时定量PCR或Western blot检测感染前后细胞内14-3-3ε的表达情况以及酪氨酸激酶c-Src在其中的作用。同时通过免疫共沉淀检测14-3-3ε与TLR2的结合水平。采用RNA干扰或使细胞内过表达14-3-3ε,免疫荧光和ELISA等方法测定其对NF-κB的活化以及培养上清中TNF-α和IL-6浓度的影响。最后通过动物实验观察抑制14-3-3ε的活性对NTHi感染后肺部炎症反应的影响。结果显示,NTHi感染上皮细胞后,14-3-3ε的蛋白和mRNA水平随着MOI的递增而增高,采用ActD和CHX预处理细胞后,14-3-3ε表达水平和对照组相比分别降低了71.05%和59.21%。NTHi感染15 min后BEAS-2B细胞中c-Src磷酸化水平增高了3.63倍,而抑制c-Src活性后14-3-3ε表达水平降低了50.60%。免疫共沉淀结果也显示不同MOI NTHi感染后,14-3-3ε与TLR2的结合分别增高了1.78、4.33和6.89倍。沉默14-3-3ε表达后,NF-κB的抑制因子IκB的含量降低了75.24%,其细胞核内p65亚基水平增加了36.9%,TNF-α和IL-6的分泌水平分别由(269.24±16.71)pg/mL和(116.08±5.61)pg/mL增高至(332.27±20.57)pg/mL和(172.32±9.78)pg/mL。而细胞内过表达14-3-3ε后,TNF-α和IL-1β水平分别降至(145.34±22.16)pg/mL和(65.22±11.74)pg/mL,以上差异均有统计学意义(P<0.05)。此外,NTHi感染C57BL/6小鼠后肺组织中14-3-3εmRNA表达水平也有所增高,预先采用14-3-3ε抑制剂R18腹腔注射后,小鼠肺组织病理评分由(6.42±1.52)增高至(11.86±1.63),肺泡灌洗液中TNF-α和IL-6的含量由(186.39±16.71)pg/mL和(76.21±12.63)pg/mL增高至(258.91±32.05)pg/mL和(151.25±23.87)pg/mL。以上结果表明,NTHi感染能诱导气道上皮细胞表达14-3-3ε,后者能下调TLR2/NF-κB通路活性,最终负向调控肺部炎症反应。This paper aimed to investigate the effect of 14-3-3εon the secretion of proinflammatory cytokines by human bronchial epithelial cells induced by NTHi(non-typeable Haemophilus influenzae)and itsmechanism.BEAS-2B cells were infected with NTHi with MOI(multiplicity of infection)of 5,10,and 20,respectively.Real-time quantitative PCR or Western blot were used to detect the expression of 14-3-3εin the cellsand the role of tyrosine kinase c-Src before and after infection.The binding levels of 14-3-3εand TLR2 weredetected by co-immunoprecipitation.The activation of NF-κB and the concentration of TNF-αand IL-6 in theculture supernatant after RNA interference or BEAS-2B cell overexpression of 14-3-3εwere measured by immunofluorescence and ELISA.Finally,the effects of 14-3-3εinhibitor treatment on pulmonary inflammation afterNTHi infection were observed in animal experiments.The results showed that following the infection of epithelial cells with NTHi,there was an observed elevation in the protein and mRNA levels of 14-3-3εconcomitantwith increasing MOI.However,upon pre-treatment of cells with ActD and CHX,the expression levels of 14-3-3εdecreased by 71.05%and 59.21%respectively,in comparison to the control group.At 15 minutes post NTHiinfection,the phosphorylation levels of c-Src in BEAS-2B cells surged by 3.63-fold.However,upon inhibitionof c-Src activity,the expression of 14-3-3εdecreased by 50.60%.Immunoprecipitation results also revealed thatfollowing infection with varying MOI of NTHi,the binding of 14-3-3εto TLR2 was augmented by 1.78,4.33,and 6.89-fold respectively.Silencing of 14-3-3εexpression led to a decrease in the inhibitor of NF-κB,IκB,by 75.24%and an intranuclear increase of the p65 subunit by 36.9%.The secretion levels of TNF-αand IL-6 wereelevated from(269.24±16.71)pg/mL and(116.08±5.61)pg/mL to(332.27±20.57)pg/mL and(172.32±9.78)pg/mLrespectively.Conversely,cellular overexpression of 14-3-3εresulted in reduced levels of TNF-αand IL-1βto(145.34±22.16)pg/mL and(65.22±11.74)pg/mL res

关 键 词：不可分型流感嗜血杆菌 14-3-3ε NF-ΚB 细胞因子 

分 类 号：R563[医药卫生—呼吸系统]