1、Hainan Med J,Apr.2023,Vol.34,No.8海南医学2023年4月第34卷第8期Penicilazaphilone C抑制NETosis诱导哮喘炎症的机制研究黄俊敏，王才春海南医学院第一附属医院呼吸内科，海南海口571199【摘要】目的探讨Penicilazaphilone C(PAC)对哮喘小鼠炎症反应的影响，以及中性粒细胞胞外诱捕网的形成(NETosis)介导哮喘发生发展的可能机制。方法在体外，采用佛波醇12-十四酸酯13-乙酸酯(PMA)诱导健康小鼠的中性粒细胞产生中性粒细胞胞外诱网(NETs)。设置四组PAC的不同剂量组及对照组，通过测定dsDNA的产生了解抑制效
2、果。在体内，采用脂多糖(LPS)诱导建立哮喘小鼠模型，将30只小鼠随机分成对照组、哮喘组、地塞米松组、DNase 组及PAC组。测定支气管肺泡灌洗液(BALF)中细胞因子和炎性细胞水平；对肺组织采用 HE染色、PAS染色及免疫荧光；通过Western blotting检测肺组织中的瓜氨酸化组蛋白；通过荧光镜检验证哮喘小鼠是否产生了NETs。结果在体外，1.2 mg/mL的PAC可以显著抑制NETs的形成0.3 mg/mL、0.6 mg/mL、0.9 mg/mL、1.2 mg/mLPAC组结果分别为(0.720.17)RFU、(0.350.09)RFU、(0.160.02)RFU、(0.110.
3、03)RFU)，差异有统计学意义(P0.05)；在体内实验中，小鼠BALF中的dsDNA与对照组(165.406.99)ng/mL相比，哮喘组(300.008.27)ng/mL的含量明显增加，差异有统计学意义(P0.05)；BALF中有大量细胞因子及炎性细胞，中性粒细胞约占62%，肺组织明显受损。PAC组小鼠的中性粒细胞水平为(235.5021.03)104cells/mL，明显低于哮喘组的(796.5012.47)104cells/mL，差异有统计学意义(P0.05)；PAC组小鼠肺组织病理改变较哮喘组明显减少，PAC组小鼠的炎症评分为(1.170.41)分，明显低于哮喘组的(3.170.7
4、5)分，差异有统计学意义(P0.05)；与哮喘组对比，PAC组Western blotting检测出的瓜氨酸化组蛋白水平显著下降。结论利用LPS成功建立中性粒细胞型哮喘小鼠模型；PAC可以显著缓解哮喘小鼠炎症，其机制与抑制瓜氨酸化组蛋白和NETosis有关。【关键词】小鼠；哮喘；炎症；中性粒细胞；中性粒细胞胞外诱捕网；组蛋白；机制【中图分类号】R-332【文献标识码】A【文章编号】10036350（2023）08106507Mechanism of inhibition of NETosis-induced asthma inflammation by Penicilazaphilone C.
5、HUANG Jun-min,WANG Cai-chun.Department of Respiratory Medicine,the First Affiliated Hospital of Hainan Medical University,Haikou571199,Hainan,CHINA【Abstract】ObjectiveTo investigate the effect of Penicilazaphilone C(PAC)on the inflammatory response ofasthmatic mice and the possible mechanism of the f
6、ormation of Neutrophil extracellular traps(NETosis)in the develop-ment of asthma.MethodsIn vitro,NETs were induced in neutrophils of healthy mice with phorbol 12-my-ristate-13-acetate(PMA).Four groups of different doses of PAC and control groups were set up to understand inhibitoryeffect by measurin
7、g the production of dsDNA.In vivo,a asthma mouse model was established by inducing lipopolysac-charide(LPS),and 30 mice were randomly divided into 5 groups:control group,asthma group,dexamethasone group,DNaseI group,and PAC group.The levels of cytokines and inflammatory cells in bronchoalveolar lava
8、ge fluid(BALF)were measured.Lung tissues were stained with HE staining,PAS staining,and immunofluorescence.Citrullinated his-tone H3 in lung tissue was detected by western blotting.NETs production in asthmatic mice was verified by fluores-cence microscopy.ResultsIn vitro,1.2 mg/mL PAC could signific
9、antly inhibit the formation of NETs:the levels ofNETs in the 0.3 mg/mL,0.6 mg/mL,0.9 mg/mL,1.2 mg/mL PAC group were(0.720.17)RFU,(0.350.09)RFU,(0.160.02)RFU,(0.110.03)RFU,respectively,with statistically significant differences(P0.05).In vivo,the dsDNAof BALF in the asthma group was significantly hig
10、her than that of the control group:(300.008.27)ng/mL vs(165.406.99)ng/mL(P0.05).There were a large number of cytokines and inflammatory cells in BALF,and neutrophils account-ed for about 62%.In addition,lung tissue was obviously damaged.The level of neutrophils in PAC group was signifi-cantly lower
11、than that in asthma group:(235.5021.03)104cells/mL vs(796.5012.47)104cells/mL(P0.05).The in-flammation score was(1.170.41)points in the PAC group,significantly lower than(3.170.75)points in the asthmagroup(P0.05).The expression level of citrullinated histone H3 was significantly decreased in the PAC
12、 group com-pared to the asthma group.ConclusionThe neutrophilic asthma mouse model was successfully established by LPS.PAC can significantly alleviate inflammation in asthmatic mice,and the mechanism is related to the inhibition of citrulli-nated histone H3 and NETosis.【Key words】Mice;Asthma;Inflamm
13、ation;Neutrophil;Neutrophil extracellular traps;Histone;Mechanism 论著 doi:10.3969/j.issn.1003-6350.2023.08.001基金项目：国家自然科学基金(编号：81660004)。第一作者：黄俊敏(1996)，女，住院医师，硕士，主要研究方向为呼吸系统疾病。通讯作者：王才春(1966)，男，主任医师，研究生导师，主要研究方向为呼吸系统疾病，E-mail：S。哮喘是一类常见的慢性呼吸道疾病，全球约有3.34亿哮喘患者1。中性粒细胞胞外诱捕网(neutro-phil extracellular traps，
14、NETs)是中性粒细胞衍生的胞外支架，由颗粒蛋白装饰的解聚染色质组成。NETs不1065海南医学2023年4月第34卷第8期Hainan Med J,Apr.2023,Vol.34,No.8仅有抗感染的作用，而且对非感染性疾病也有重要影响2。Toussaint等3发现NETs释放的DNA促进鼻病毒诱导的过敏性哮喘的恶化。Pham等4指出NETs通过损伤气道的上皮细胞，从而导致多种细胞(包括中性粒细胞、肥大细胞等)参与炎症的发生，加快哮喘的进展。多数研究表明NETs与哮喘的炎症进程相关。但针对NETosis诱发哮喘的详细机制及治疗研究较少。Penicilazaphilone C是从中国海南省海口
15、市海岸的腐烂树叶中生长的真菌Penicillium sclerotiorum分离并进行结构鉴定为新的嗜氮酮类化合物。由于该类化合物的基本结构和文献报道的Penicilazaphilone A和B相同5-6，故将其命名为 Penicilazaphilone C(PAC)。PAC对金黄色葡萄球菌、铜绿假单胞菌、肺炎克雷伯菌等细菌有较强的抑制作用7。PAC具有很多生物活性，包括抗细菌、抗真菌、抗病毒、抗炎、抗氧化、细胞毒作用等8。在目前的国内外文献中，针对PAC的研究为数不多。本研究在PAC抗氧化及抗炎作用的基础上推测PAC通过抑制中性粒细胞胞外诱捕网形成(NETosis)的产生来达到其预防或治疗哮
16、喘的作用。1材料与方法1.1材料Penicilazaphilone C由本课题研究团队提供，化学结构见图1。BCA蛋白定量检测试剂盒、脂多糖、DNase 均购自 Servicebio(货号分别为：G2026-200T、G5032-10MG、G3342-200U)。ELISA试剂盒、Triton X-100、DAPI均购自Sigma(货号分别为：RAB0263-1KT、T8787-50ML、D9542-1MG)。1.2实验方法1.2.1小鼠体外诱导NETs所有实验均严格按照申请内容进行。实验所用的30只68周龄健康雄性BALB/c小鼠从广州市赛柏诺生物科技公司购买，提前送到海南医学院实验室进行饲养。取健康小鼠新鲜外周血，用10 nmol/L的PMA刺激中性粒细胞。4%甲醛固定细胞，0.1%Triton X-100 通透，再用 5%BSA 阻断 1 h，细胞与抗髓过氧化物酶(Myeloperoxi-dase，MPO)抗体孵育，接着与Alexa Fluor 488标记的山羊抗兔抗体孵育。对中性粒细胞培养上清液中的双链DNA(dsDNA)的水平进行定量，用酶标仪来测定dsDNA的荧光强度。PA
