抗菌肽BNBD5调节肺脏先天免疫抵御胸膜肺炎放线杆菌感染的研究

Study on Antibacterial Peptide BNBD5 Regulating the Innate Immune Response of Lung Against Actinobacillus pleuropneumoniae

【目的】探究抗菌肽牛中性粒细胞β防御素5(bovine neutrophilβ-defensins 5,BNBD5)调节肺脏先天免疫抵御胸膜肺炎放线杆菌(Actinobacillus pleuropneumoniae,APP)感染的作用,为防控猪胸膜肺炎提供试验基础和理论支持。【方法】将30只雌性BALB/c小鼠随机分为3组,即正常对照组(Con)、APP感染对照组(APP)、抗菌肽BNBD5预处理后感染APP组(B5),每组10只,Con组小鼠鼻内给予2次PBS(20μL/只),每次间隔2 d;APP组鼻内给予2次PBS(20μL/只),间隔2 d,3 d后感染APP(107 CFU/只);B5组小鼠鼻内给予2次抗菌肽BNBD5(20μg/只),间隔2 d,3 d后鼻内感染APP(107 CFU/只),感染6 h后剖杀所有小鼠,观察肺脏和脾脏的病变,测定脏器系数和肺脏中的细菌载量;HE染色观察肺脏组织病理变化;ELISA检测肺脏组织匀浆液中的细胞因子肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素-1β(interleukin 1β,IL-1β)、IL-23、IL-17和IL-22水平;流式细胞术检测肺脏中的中性粒细胞数量。【结果】与Con组相比,APP组小鼠被毛粗乱、精神萎靡,肺脏肿大,可见明显斑块状或点状出血,镜下可见肺脏组织有明显出血和大量炎性细胞浸润,肺脏中TNF-α、IL-1β、IL-17、IL-22和IL-23细胞因子水平变化不显著(P>0.05),但均呈不同程度下降,中性粒细胞数量减少;与APP组相比,B5组小鼠精神状态较好,肺脏肿胀程度减轻,肺脏指数显著降低(P<0.05),未见明显斑块状出血,肺脏中的细菌载量极显著减少(P<0.01),肺脏中炎性细胞明显减少,肺脏组织中TNF-α、IL-1β、IL-17和IL-22水平显著升高(P<0.05),肺脏组织中中性粒细胞数量极显著增多(P<0.01)。【结论】抗菌肽BNBD5能在感染早期改善APP引起的肺脏免疫抑制,增强肺脏先天免疫杀伤胸膜肺炎放线杆菌。

【Objective】This study was aimed to investigate the role of antimicrobial peptide bovine neutrophilβ-defensins 5(BNBD5)in regulating the innate immune response of lung against Actinobacillus pleuropneumoniae(APP)infection,and provide experimental basis and theoretical support for the prevention and control of porcine pleuropneumonia.【Method】30 BALB/c mice were randomly divided into three groups,which were normal control group(Con),APP infection group(APP),antibacterial peptide BNBD5 pretreatment and APP infection group(B5),10 mice in each group.Mice in Con group were treated with PBS(20μL per mouse)two times at two-day intervals.Mice in APP group were treated with PBS(20μL per mouse)two times at two-day intervals and intranasally challenged with 107 CFU of APP 3 d after treatment.Mice in B5 group were treated with antibacterial peptide BNBD5(20μg per mouse)two times at two-day intervals and intranasally challenged with 107 CFU of APP 3 d after treatment.Mice were euthanized 6 h after APP challenge.The general pathological changes of lung and spleen were observed,and the organ coefficient and bacterial load in lung were measured.The pathological changes of lung were observed by HE staining.Levels of cytokines tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),IL-23,IL-17 and IL-22 in lung were tested using ELISA.The number of neutrophils in lung was measured using flow cytometry.【Result】Compared with Con group,mice in APP group had rough and disordered hair,decreased mental state,enlarged lungs,and obvious patchy or punctate bleeding.Microscopically,there was significant bleeding and a large amount of inflammatory cell infiltration in lung.The cytokine levels of TNF-α,IL-1β,IL-17,IL-22 and IL-23 in lung were not significantly changed(P>0.05),but they all decreased to different degrees,and the number of neutrophils decreased.Compared with APP group,the mental state of mice in B5 group was better,the degree of lung swelling was reduced,the lung index was significantly decreased(P<0.05),no obvious plaque bleeding was observed,the bacterial load in lung was extremely significantly decreased(P<0.01),and the inflammatory cells in lung were significantly decreased.The levels of TNF-α,IL-1β,IL-17 and IL-22 in lung were significantly increased(P<0.05),and the number of neutrophils in lung was extremely significantly increased(P<0.01).【Conclusion】Antimicrobial peptide BNBD5 could ameliorate APP-caused immune suppression of lung and enhance the innate immunity of lung against APP in the early stages of infection.