粒细胞活化、组蛋白去乙酰化酶2与重症哮喘关系的临床研究

Clinical study of relationship between granulocyte activation, HDAC2 and severe asthma

[摘要] 目的 了解粒细胞活化和组蛋白去乙酰化酶2 （histonedeacetylase2,HDAC2）表达与重症哮喘的关系,以探索早期预测和辨别重症哮喘的生物标志物。方法 采用 ELISA 法测定受试者血清粒 细 胞 活 化 标 志 物 髓 过 氧 化 物 酶 （myeloperoxidase, MPO）、 基 质 金 属 蛋 白 酶 9 （matrix metalloproteinase9,MMP-9）、中性粒细胞弹性蛋白酶 （neutrophilelastase,NE）及嗜酸粒细胞阳离子蛋 白 （eosinophilcationicprotein,ECP） 水 平,Western-blot测 定 外 周 血 单 个 核 细 胞 （peripheral bloodmononuclearcells,PBMCs）HDAC2活性,分别比较重症哮喘组、轻中度哮喘组及健康对照组各指标并进行统计学分析。结果 ①重症哮喘组受试者粒细胞活化指标 MPO [（102.43±28.19）μg/L]、 MMP-9 [（992.97±176.44）ng/L]、NE [（9.40±0.99）μg/L]及ECP [（232.85±48.37）μg/L]浓度值均显著高于轻中度哮喘组 [MPO、MMP-9、NE及 ECP分别为：（48.56±8.78）μg/L、（554.35± 99.03）ng/L、（8.01±1.27）μg/L、 （105.30±26.93）μg/L,P 值均＜0.05]和正常对照组 [MPO、 MMP-9、NE及 ECP分别为： （37.46±9.93）μg/L、 （374.00±79.08）ng/L、 （4.66±0.64）μg/L、 （40.87±15.35）μg/L,P ＜0.05];轻中度哮喘组受试者外周血 MMP-9、NE及 ECP浓度值显著高于正常对照组 （P ＜0.05）;轻中度哮喘组与正常对照组比较 MPO 浓度值差异无统计学意义 （P ＞ 0.05）;②重症哮喘组 PBMCsHDAC2活性 （HDAC2/GAPDH：0.19±0.09）较轻中度哮喘组 （0.60± 0.08）及正常对照组 （1.09±0.26）均显著降低,差异有统计学意义 （P ＜0.05）;轻中度哮喘组 PBMCsHDAC2活性较正常对照组低,差异有统计学意义 （P ＜0.05）;③ MPO、MMP-9、NE 及 ECP与 FEV1%pred之 间 呈 负 相 关,双 侧 Pearson 检 验 差 异 有 统 计 学 意 义 （r 值 分 别 为：-0.60、 -0.75、-0.43和-0.67,P ＜0.01）;与 FEV1/FVC之间呈负相关,双侧 Pearson检验差异有统计学意义 （r值 分 别 为：-0.52、-0.60、-0.42和 -0.50,P ＜0.05）;与 ACT 之 间 呈 负 相 关,双 侧 Pearson检 验 差 异 有 统 计 学 意 义 （r 值 分 别 为： -0.69、 -0.67、 -0.50和 -0.70,P ＜0.01）; ④HDAC2与 FEV1%pred及 FEV1/FVC之间呈正相关,双侧 Pearson检验差异有统计学意义 （r值分别为0.68和0.61,P ＜0.01）;与 ACT之间呈正相关,双侧Pearson检验差异有统计学意义 （r=0.68,P ＜ 0.01）。结论 粒细胞活化指标 MPO、MMP-9、NE和 ECP升高提示重症哮喘患者存在中性粒细胞和嗜酸粒细胞活化,其程度显著高于轻中度哮喘患者;PBMCsHDAC2在重症哮喘组明显降低提示该类患者的皮质激素敏感性降低,激素治疗的效果可能不佳。

Objective To investigate the relationships between granuloeyte activation markers, HDAC2 expression and severe asthma, and to explore the biomarkers for the early prediction and identification of severe asthma. Methods The myeloperoxidase （ MPO）, matrix metalloproteinase 9 （MMP 9）, neutruphil elastase （NE） and eosinophil cationicprotein（ECP） levels of serum granulocyte activation markers were detected by enzyme linked immunosorbent assay （ELISA）, the activity of peripheral blood mononuclear cells（PBMCs） HDAC2 was detected by the Western-blot. Compared each index of severe asthma group with the relevant index of mild-and-moderate asthma group and healthy control group and statistical analysis. Results （1） The concentration of granulocyte activation markers MPO [-（102,43±28.19） μg/L3 ,MMP-9[（992.97±176.44） ng/L] ,NE [-（9.40±0.99） μg/L3 and ECP [-（232.85± 48.37） μg/L] of severe asthma subjects were significantly higher than that in mild-and- moderate asthma subjects [-MPO, MMP-9, NE and ECP were as follows： （48.56!8.78） μg/L, （554.35± 99.03） ng/L, （8.01±1.27） μg/L, （ 105.30 ± 26.93） μg/L, P G 0. 053 and healthy control subjects EMPO, MMP-9, NE and ECP respectively as follows ： （ 37.46 ± 9.93） μg/L, （374.00 ± 79.08） ng/L, （4.66＋0.64） /Lg/L, （40.87±15.35） μg/L, P G0. 053. There was no significant difference in MPO level between the mild-and-moderate asthma group and the healthy control group （ P 〉0.05）. （2）The HDAC2 activity （HDAC2/GAPDH： 0.19±0.09） in severe asthma group was significantly lower than that in mild-and-moderate asthma group （0.60±0.08） and healthy control group （1.09 ± 0.26）, the difference was statistical significantly （ P 〈0.05）. HDAC2 activity in mild-and-moderate asthma group was lower than that in healthy control group, and the difference was statistical significantly （ P 〈0.05）. （3）The level of MPO, MMP-9, NE and ECP were significantly negatively correlated with FEV1 % pred, and the difference was significant （ r values were - 0.60, -0.75, - 0.43 and - 0.67, respectively, P 〈 0.01 ）. And a significantly negatively correlated with FEV1/FVC, the difference was significant （ r values were -0.52,-0.60,-0.42 and -0.50, respectively, P G0.05）. There was also a significant negative correlation between ACT and the concentration of MPO, MMP-9, NE and ECP （ r values were -0.69, -0.67, -0.50 and -0.70, respectively, P G 0.01 ）. （4） There was a significant positive correlation between FEVa % pred, FEV1/FVC and the expression of HDAC2, and the difference was marked in the bilateral Pearson test （ r values were 0.68 and 0.61, respectively, P 〈 0.01 ）. A significant positive correlation between HDAC2 activity and ACT was observed （ r = 0.68, P 〈0.01）. Conclusions The elevation of serum levels of the MPO, MMP-9, NE and ECP in patients with severe asthma indicated the activation in neutrophil and eosinophilie granulocytes, and was significantly higher than that in patients with mild-and-moderate asthma. The activity of PBMCs HDAC2 in severe asthma group was decreased markedly, implying the possibility of the decrease of glueoeortieoid sensitivity, and poor therapeutic effect in severe asthma.