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

The role of inflammatory release from human eosinophils-induced by the antimicrobial peptide LL-37 in the pathogenesis of asthma

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,P<0.05]。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(P<0.05)。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(P<0.05)。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(P<0.05)。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。

Objective The antimicrobial peptide LL-37（ LL-37） is the mature form of Human Cationic Antimicrobial Peptide of 18 k D（ hCAP18） and play a certain regulation role in the pathogenesis of asthma. However,the mechanism is unclear. The aim of this study was to investigate the role of inflammatory release from human eosinophils induced by the antimicrobial peptide LL-37 in the pathogenesis of asthma and the underlying mechanisms. Methods Sixteen mild or medium allergic asthma patients from January2015 to January 2016 in Panzhihua college affiliated hospital were enrolled. Another 16 healthy volunteers were enrolled as control. Primary eosinophils were isolated from peripheral blood. The cells were divided into two groups： asthma group and healthy control group.Cells were divided into blank,PAF,LL37,single cytokine（ IL-5,GM-CSF） and cytokines combined with LL-37 group based on intervention（ cell treating factors） difference; Cells were divided into PTx,WRW4,suramin,and LL-37 combined with inhibitors group based on inhibitors difference; Cells were grouped into LTD4 and LTB4 treatment based on leukotrienen difference; ELISA was applied to analyze cysteinyl leukotrienes（ cys-LTs） level in various treatment groups; Western blot was used to detect change of cPLA2,p-cPLA2,ERK1/2,p-ERK1/2 in the cells from the control group after PTx and WRW4 treatment and the level of hCAP18 after leukotriene treatment. Results Compared with the control 15 μg/mL LL-37 sub group,the expression of Cys-LTs was increased in the control 30 μg/mL LL-37 sub group 15 and 30 minutes after the LL-37 treatment [（ 54.02± 7.15） pg/10^5vs（ 37. 86 ± 6. 33） pg/10^5,（ 53.30±6.99） pg/10^5vs（ 36.27±6.46） pg/10^5,P〈0.05]. Compared with the control IL-5 sub group（ 26.18±4.86） pg/10^5,the expression of Cys-LTs was increased in the control IL-5 ＋ 15 μg/mL LL-37 sub group（ 59.97±6.83） pg/10^5 and the control IL-5 ＋ 30μg/mL LL-37 sub group（ 81.44±13.70） pg/10^5（ P〈0.05）. Compared with the control sub group,the expression of Cys-LTs was increased in the asthma 15 μg/mL LL-37 sub group and the asthma 30 μg/mL LL-37 sub group（ P〈0.05）. Compared with the control LL-37 sub group,the expression of Cys-LTs was decreased in the control PTx sub group,control WRW4 sub group,control suramin sub group,control PTx ＋ LL-37 sub group,and control WRW4 ＋ LL-37 sub group（ P〈0.05）. Western blot results indicated that LL-37 treatment induced the activation and phosphorylation of ERK1/2 in eosinophil,and PTx and WRW4 blocked the upregulation of pERK1/2 induced by LL-37. Treatment with PD inhibited the phosphorylation of cPLA2 and the release of Cys-LTs induced by LL-37.hCAP18 was higher in the asthma groups than the healthy control. Conclusion LL-37 was identified as an eosinophil-activating peptide that could trigger the release of inflammatory mediators,which might be involved in occurrence and development of asthma through regulating ERK1/2 phosphorylation,inducing cPLA2 phosphorylation and finally initiate synthesis of cys-LTs. This suggests that LL-37/hCAP18 and its signaling pathway might be potential therapeutic targets for asthma.