Penicilazaphilone C抑制NETosis诱导哮喘炎症的机制研究

Mechanism of inhibition of NETosis-induced asthma inflammation by Penicilazaphilone C

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、DNaseⅠ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/mL PAC组结果分别为(0.72±0.17)RFU、(0.35±0.09)RFU、(0.16±0.02)RFU、(0.11±0.03)RFU)],差异有统计学意义(P<0.05);在体内实验中,小鼠BALF中的dsDNA与对照组(165.40±6.99)ng/mL相比,哮喘组(300.00±8.27)ng/mL的含量明显增加,差异有统计学意义(P<0.05);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(235.50±21.03)×10^(4)cells/mL,明显低于哮喘组的(796.50±12.47)×10^(4)cells/mL,差异有统计学意义(P<0.05);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(1.17±0.41)分,明显低于哮喘组的(3.17±0.75)分,差异有统计学意义(P<0.05);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。

Objective To investigate the effect of Penicilazaphilone C(PAC)on the inflammatory response of asthmatic mice and the possible mechanism of the formation of Neutrophil extracellular traps(NETosis)in the development of asthma.Methods In vitro,NETs were induced in neutrophils of healthy mice with phorbol 12-myristate-13-acetate(PMA).Four groups of different doses of PAC and control groups were set up to understand inhibitory effect by measuring the production of dsDNA.In vivo,a asthma mouse model was established by inducing lipopolysaccharide(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 lavage fluid(BALF)were measured.Lung tissues were stained with HE staining,PAS staining,and immunofluorescence.Citrullinated histone H3 in lung tissue was detected by western blotting.NETs production in asthmatic mice was verified by fluorescence microscopy.Results In vitro,1.2 mg/mL PAC could significantly inhibit the formation of NETs:the levels of NETs in the 0.3 mg/mL,0.6 mg/mL,0.9 mg/mL,1.2 mg/mL PAC group were(0.72±0.17)RFU,(0.35±0.09)RFU,(0.16±0.02)RFU,(0.11±0.03)RFU,respectively,with statistically significant differences(P<0.05).In vivo,the dsDNA of BALF in the asthma group was significantly higher than that of the control group:(300.00±8.27)ng/mL vs(165.40±6.99)ng/mL(P<0.05).There were a large number of cytokines and inflammatory cells in BALF,and neutrophils accounted for about 62%.In addition,lung tissue was obviously damaged.The level of neutrophils in PAC group was significantly lower than that in asthma group:(235.50±21.03)×10^(4) cells/mL vs(796.50±12.47)×10^(4) cells/mL(P<0.05).The inflammation score was(1.17±0.41)points in the PAC group,significantly lower than(3.17±0.75)points in the asthma group(P<0.05).The expression level of citrullinated histone H3 was significantly decreased in the PAC group compared to the asthma group.Conclusion The 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 citrullinated histone H3 and NETosis.