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

• 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
• 2:贵州医科大学实验动物中心
• 3:贵州医科大学附属医院临床检验中心微生物免疫科

摘要(Abstract)：

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×TCID_(50) H1N1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 human β-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-γ at the doses of 12.5, 25.0, 50.0 and 100.0 μg/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(25.0 μg/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(25.0 μg/L), HBD3(1.00 mg/L) and IFN-γ neutralizing 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-γ or 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-γ expression(P<0.01). IFN-γ alone 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-γ neutralizing antibody did not remarkably influence HBD3-inhibited H1N1 NP expression(P>0.05). Conclusion HBD3 and IFN-γ mutually 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(KeyWords)： 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
influenza A virus(IAV);human β-defensin-3(HBD3);interferon-γ(IFN-γ);mitogen-activated protein kinase(MAPK)

Abstract:

Keywords:

基金项目(Foundation): 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)

作者(Author): 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

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