抗菌肽LL-37激活嗜酸性粒细胞释放炎性递质对哮喘的发病机制研究

发布时间：2018-03-26 11:16

本文选题：哮喘　切入点：抗菌肽　出处：《医学研究生学报》2017年01期

【摘要】：目的抗菌肽LL-37(LL-37)是人源性阳离子抗菌18 000多肽(h CAP18)的成熟形式,对哮喘的发病具有一定的调节作用,但具体机制尚不清楚。文中旨在研究LL-37在诱导人嗜酸性粒细胞释放炎性递质中的作用,并探讨其影响哮喘发病的可能机制。方法选取2015年1月至2016年1月攀枝花学院附属医院呼吸科就诊的16例轻度或中度过敏性哮喘患者,16名健康志愿者。从研究对象的外周血中提取原代嗜酸性粒细胞,将细胞分别分为哮喘组和对照组。依据干预嗜酸性粒细胞的方式将哮喘组和健康组细胞分别分为:哮喘a亚组、对照a亚组;哮喘血小板活化因子(PAF)亚组、对照PAF亚组;哮喘15μg/m L LL-37亚组、对照15μg/m L LL-37亚组;哮喘30μg/m L LL-37亚组、对照30μg/m L LL-37亚组;哮喘IL-5亚组、对照IL-5亚组;哮喘IL-5+15μg/m L LL-37亚组、对照IL-5+15μg/m L LL-37亚组;哮喘IL-5+30μg/m L LL-37亚组、对照IL-5+30μg/m L LL-37亚组。依据嗜酸性粒细胞中添加抑制剂种类及抗菌肽将哮喘组和健康组细胞分别分为:哮喘b亚组、对照b亚组;哮喘PTX亚组、对照PTX亚组;哮喘WRW4亚组、对照WRW4亚组;哮喘苏拉明亚组、对照苏拉明亚组;哮喘LL-37亚组、对照LL-37亚组;哮喘PTX+LL-37亚组、对照PTX+LL-37亚组;哮喘WRW4+LL-37亚组、对照WRW4+LL-37亚组;哮喘苏拉明+LL-37亚组、对照苏拉明+LL-37亚组。ELISA检测各组细胞上清的半胱氨酸白三烯(Cysteinyl leukotrienes,Cys-LTs)浓度;Western blot分析健康组细胞内加入PTX、WRW4后PLA2、磷酸化c PLA2、磷酸化ERK水平变化以及白三烯刺激后h CAP18水平。结果 LL-37诱导对照组嗜酸性粒细胞15、30 min后,对照30μg/m L LL-37亚组Cys-LTs表达量较对照15μg/m L LL-37亚组均显著升高[(54.02±7.15)pg/105vs(37.86±6.33)pg/105、(53.30±6.99)pg/105vs(36.27±6.46)pg/105,P0.05]。对照组嗜酸粒细胞中Cys-LTs表达量的比较,对照IL-5+15μg/m L LL-37亚组[(59.97±6.83)pg/105]、对照IL-5+30μg/m L LL-37亚组[(81.44±13.70)pg/105]较对照IL-5亚组[(26.18±4.86)pg/105]均明显升高(P0.05)。哮喘15μg/m L LL-37亚组、哮喘30μg/m L LL-37亚组Cys-LTs表达量较对照a亚组明显升高(P0.05)。与对照LL-37亚组Cys-LTs表达量比较,对照PTX亚组、对照WRW4亚组、对照苏拉明亚组、对照PTX+LL-37亚组、对照WRW4+LL-37亚组显著降低(P0.05)。Western blot结果显示,LL-37刺激嗜酸性粒细胞可促进ERK1/2的活化及磷酸化;当同时使用PTX或WRW4可抵消LL-37诱导的p ERK1/2的上调;抑制剂PD处理可阻断LL-37诱导的c PLA2的磷酸化及Cys-LTs的释放;嗜酸性粒细胞中h CAP18表达量比较,哮喘组较对照组升高。结论 LL-37可作为嗜酸性粒细胞激活肽触发炎症介质的释放,通过调节ERK1/2的磷酸化激活c PLA2磷酸化,进而引起Cys-LTs的合成,参与哮喘的发生发展,提示LL-37、h CAP18及其信号通路可作为临床治疗哮喘的潜在靶标。
[Abstract]:Objective the antimicrobial peptide LL-37 (LL-37) is a mature form of human cationic antibacterial peptide (18 000 polypeptide hCAP18) and has a certain regulatory effect on the pathogenesis of asthma. But the specific mechanism is not clear. The purpose of this paper is to study the role of LL-37 in inducing the release of inflammatory transmitters from human eosinophils. Methods from January 2015 to January 2016, 16 healthy volunteers with mild or moderate allergic asthma were selected from respiratory department of affiliated hospital of Panzhihua college. Primary eosinophilic granulocytes were extracted from the peripheral blood of the elephant. According to the intervention of eosinophil, the cells of asthmatic group and healthy group were divided into subgroup a of asthma, subgroup A of control, subgroup of platelet activating factor of asthma and subgroup of PAF, respectively. Asthma 15 渭 g / m L LL-37 subgroup, control 15 渭 g / m L LL-37 subgroup; asthma 30 渭 g / m L LL-37 subgroup, control 30 渭 g / m L LL-37 subgroup; asthma IL-5 subgroup, control IL-5 subgroup; asthma IL-5 15 渭 g / m L LL-37 subgroup, control IL-5 15 渭 g / m L LL-37 subgroup; asthma IL-5 30 渭 g / m L LL-37 subgroup, Control IL-5 30 渭 g / mL LL-37 subgroup. According to the type of eosinophilic granulocyte and antimicrobial peptide, the asthmatic group and the healthy group were divided into two groups: asthma B subgroup, control B subgroup, asthma PTX subgroup, control PTX subgroup, asthma WRW4 subgroup. Control WRW4 subgroup; asthmatic sulamine subgroup, control sulamine subgroup; asthmatic LL-37 subgroup, control LL-37 subgroup; asthma PTX LL-37 subgroup, control PTX LL-37 subgroup; asthma WRW4 LL-37 subgroup, control WRW4 LL-37 subgroup; asthmatic subgroup Salamine LL-37 subgroup, Detection of cysteinyl leukotrienes (cysteinyl leukotrienes) Cys-LTs in the cell supernatant of each group by Elisa and Western blot analysis of PLA2, phosphorylated PLA2, phosphorylated ERK and CAP18 after stimulation of leukotriene in healthy cells with PTX WRW4. Results the eosinophil of control group was induced by LL-37 for 1530 min. The expression of Cys-LTs in the control 30 渭 g / mL LL-37 subgroup was significantly higher than that in the control 15 渭 g / mL LL-37 subgroup [54.02 卤7.15)pg/105vs(37.86 卤6.33 渭 g / 105]. The expression of Cys-LTs in eosinophil was compared with that in the control group (53.30 卤6.99)pg/105vs(36.27 卤6.46 g / 105 P 0.05). The IL-5 15 渭 g / mL LL-37 subgroup [59.97 卤6.83)pg/105] and the control IL-5 30 渭 g / mL LL-37 subgroup [81.44 卤13.70)pg/105] were significantly higher than that of the control IL-5 subgroup [26.18 卤4.86)pg/105]. The Cys-LTs expression in the asthmatic 15 渭 g / mL LL-37 subgroup and asthma 30 渭 g / mL LL-37 subgroup was significantly higher than that in the control a subgroup. In the control PTX subgroup, the control WRW4 subgroup, the control sulamine subgroup and the control PTX LL-37 subgroup, the control WRW4 LL-37 subgroup significantly decreased the activity and phosphorylation of ERK1/2 in the control WRW4 LL-37 subgroup. The results showed that LL-37 stimulated eosinophil activation and phosphorylation. When PTX or WRW4 were used at the same time, the up-regulation of p ERK1/2 induced by LL-37 was counteracted; the phosphorylation of c PLA2 induced by LL-37 and the release of Cys-LTs were blocked by inhibitor PD; the expression of h CAP18 in eosinophilic granulocyte was compared with that in eosinophilic granulocyte. Conclusion LL-37 can trigger the release of inflammatory mediators as eosinophil activating peptide, and regulate the phosphorylation of ERK1/2 to activate c PLA2 phosphorylation, which leads to the synthesis of Cys-LTs and participates in the occurrence and development of asthma. These results suggest that LL-37 CAP18 and its signal pathway may be potential targets for the treatment of asthma.
【作者单位】：四川攀枝花学院附属医院呼吸科;四川攀枝花学院附属医院内分泌科;重庆市医疗急救中心呼吸科;
【基金】：重庆市卫生2014年医学科研计划项目(20142077)
【分类号】：R562.25

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