人β防御素3和γ干扰素在MAPK信号通路中的相互诱导及联合抗甲型流感病毒的机制  

Mutually induction between humanβ-defensin-3 and interferon-γ in MAPK signaling pathway and mechanism of their combination in anti-influenza A virus

作　　者：唐源 祝洁 杨小余 张乙进 罗红 江滟 TANG Yuan;ZHU Jie;YANG Xiaoyu;ZHANG Yijin;LUO Hong;JIANG Yan(Department of Microbiology and Immunology,School of Clinical Laboratory,Guizhou Medical University,Guiyang 550004,Guizhou,China;Laboratory Animal Center,Guizhou Medical University,Guiyang 550025,Guizhou,China;Department of Microbiology and Immunology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China)

机构地区：[1]贵州医科大学医学检验学院临床微生物与免疫学教研室,贵州贵阳550004 [2]贵州医科大学实验动物中心,贵州贵阳550025 [3]贵州医科大学附属医院临床检验中心微生物免疫科,贵州贵阳550004

出　　处：《贵州医科大学学报》2023年第4期373-382,共10页Journal of Guizhou Medical University

基　　金：国家自然科学基金项目(81260249);贵州省科技创新人才团队项目(黔科合人才团队[2015]4025);贵州省科技计划项目(黔科合平台人才[2019]5610)。

摘　　要：目的 探讨人β防御素3(HBD3)和γ干扰素(IFN-γ)在丝裂源活化蛋白激酶(MAPK)信号通路中相互诱导作用及体外联合抗甲型流感病毒(IAV)H1N1的作用。方法 不同剂量IFN-γ(12.5、25.0、50.0、100.0μg/L)或HBD3(0.25、0.50、1.00、2.00 mg/L)分别作用人支气管上皮细胞BEAS-2B 4、12、24、48 h,另设立p38 MAPK、ERK和JNK通路抑制剂预处理后、用IFN-γ(25.0μg/L)或HBD3(1.00 mg/L)处理BEAS-2B细胞4 h, qRT-PCR和ELISA检测HBD3及IFN-γ基因和蛋白表达,Western blot检测p-p38 MAPK、p-ERK、p-JNK蛋白表达;25.0μg/L IFN-γ、1.00 mg/L HBD3、HBD3 siRNA转染及IFN-γ中和抗体(0.50 mg/L)单独或联合作用BEAS-2B细胞、用5×TCID50H1N1感染,qRT-PCR和Western blot检测IAV核蛋白(NP)基因和蛋白的表达情况。结果 在BEAS-2B细胞中,不同剂量的IFN-γ与HBD3可互相诱导表达(P<0.05或P<0.01),且均可诱导p-p38 MAPK、p-ERK和p-JNK的表达上调(P<0.01);p38 MAPK、ERK和JNK通路抑制剂预处理,均明显下调IFN-γ诱导的HBD3表达、及HBD3诱导的IFN-γ表达(P<0.01);IFN-γ和HBD3单独或联合使用均可明显抑制BEAS-2B细胞中H1N1 NP的表达(P<0.05或P<0.01),转染HBD3 siRNA减弱了IFN-γ抑制H1N1 NP表达的作用(P<0.01),IFN-γ中和抗体介入对HBD3抑制H1N1 NP表达的作用无显著影响(P>0.05)。结论 在BEAS-2B细胞中,HBD3和IFN-γ可通过MAPK通路相互诱导表达,且2者联合使用可增强抗IAV H1N1的作用。Objective To investigate mutually induction between humanβ-defensin-3(HBD3)and interferon-γ(IFN-γ)in MAPK signaling pathway and mechanism of their combination in anti-influenza A virus(IAV)H1N1.Methods Human bronchial epithelial cells BEAS-2B were treated with IFN-γat the doses of 12.5,25.0,50.0 and 100.0μg/L for 4 h,respectively,or HBD3 at the doses of 0.25,0.50,1.00,and 2.00 mg/L for 4 h,respectively.In addition,BEAS-2B cells were pretreated with the inhibitors of p38 MAPK,ERK,and JNK pathways,respectively,then treated with IFN-γ(25.0μg/L)or HBD3(1.00 mg/L)for 4 h.qRT-PCR and ELISA were performed to detect the mRNA and protein expression of HBD3 and IFN-γ,respectively.Western blot was applied to detect the protein expression of p-p38 MAPK,p-ERK,and p-JNK.BEAS-2B cells were treated with IFN-γ(25.0μg/L),HBD3(1.00 mg/L)and IFN-γneutralizing antibody(0.50 mg/L),or transfected with HBD3 siRNA,respectively,or treated with their combination,then infected with 5×TCID 50 H1N1.The expression of IAV nucleoprotein(NP)was detected by qRT-PCR and Western blot.Results In BEAS-2B cells,different doses of IFN-γor HBD3 induced the expression of each other(P<0.05 or P<0.01),and both upregulated the expression levels of p-p38 MAPK,p-ERK,and p-JNK(P<0.01).The pretreatment with the inhibitors of p38 MAPK,ERK,and JNK pathways obviously downregulated IFN-γ-induced HBD3 expression and HBD3-induced IFN-γexpression(P<0.01).IFN-γalone and HBD3 alone or their combination significantly inhibited H1N1 NP expression in BEAS-2B cells(P<0.05 or P<0.01).HBD3 siRNA transfection impaired IFN-γ-inhibited H1N1 NP expression(P<0.01).IFN-γneutralizing antibody did not remarkably influence HBD3-inhibited H1N1 NP expression(P>0.05).Conclusion HBD3 and IFN-γmutually induce the expression of each other through MAPK signaling pathway in BEAS-2B cells,and their combination enhances anti-IAV H1N1 effect.

关 键 词：甲型流感病毒 人Β防御素3 Γ干扰素 丝裂原活化蛋白激酶 

分 类 号：R511.7[医药卫生—内科学]