髓过氧化物酶抗体相关性血管炎患者外周血中性粒细胞胞内髓过氧化物酶表达及其意义

The expression and clinical significance of neutrophil myeloperoxidase in patients with myeloperoxidase-antineutrophil cytoplasmic antibody associated vasculitis

目的初步探讨髓过氧化物酶(MPO)抗体相关性血管炎(AAV)患者外周血中性粒细胞胞内MPO表达及其临床意义。方法选2018年7月至2021年6月安徽医科大学第一附属医院住院的初诊未治的MPO-AAV患者36例(MPO-AAV组), 另选年龄、性别与MPO-AAV组匹配的健康志愿者36例(健康对照组)。流式细胞术(FCM)和实时荧光定量聚合酶链式反应(RT-qPCR)分别检测所有受试者中性粒细胞胞内MPO水平和MPO mRNA表达。酶联免疫吸附试验(ELISA)法检测所有受试者外周血MPO、补体C5活化片段a(C5a)水平及MPO-AAV组患者MPO-ANCA。采用修订的伯明翰血管炎活动度评分(BVAS-V3)对MPO-AAV组患者的疾病活动度进行评估。结果与健康对照组比, MPO-AAV组中性粒细胞胞内MPO mRNA表达水平、血清MPO和血清补体C5a水平显著升高[MPO mRNA:30.2±11.5 比 1.9±0.6, P<0.001;MPO:(112.0±68.7)IU/L比(87.4±22.9)IU/L, P=0.01;补体C5a:(187.3±90.3)ng/ml比(107.3±31.1)ng/ml, P<0.001], 而中性粒细胞胞内MPO水平显著降低(平均荧光强度1 343.3±723.4比2 868.0±1 136.5, P<0.001)。相关分析结果显示, 中性粒细胞胞内MPO mRNA表达水平与MPO-ANCA阳性、血清MPO水平呈正相关(r=0.537, P=0.001;r=0.358, P=0.032), 多元线性回归分析显示, 中性粒细胞胞内MPO mRNA表达水平与MPO-ANCA阳性呈正相关(β=0.695, P=0.006);中性粒细胞胞内MPO水平与MPO-ANCA阳性、血清MPO、血清补体C5a水平呈负相关(r=-0.335, P=0.046;r=-0.372, P=0.026;r=-0.577, P<0.001), 多元线性回归分析显示, 中性粒细胞胞内MPO水平与血清补体C5a水平呈负相关(β=-0.374, P=0.043);BVAS-V3与中性粒细胞胞内MPO mRNA、MPO-ANCA阳性、血清MPO、血清补体C5a呈正相关(r=0.598, P<0.001;r=0.599, P<0.001;r=0.537, P=0.001;r=0.415, P=0.012), 与中性粒细胞胞内MPO水平呈负相关(r=-0.342, P=0.041), 多元线性回归分析显示, BVAS-V3与中性粒细胞胞内MPO mRNA表达水平呈正相关(β=0.511, P=0.002)。结论 MPO-AAV中性粒细胞胞内MPO的合成和释放活性均显著增强, MPO-ANCA阳性和补体C5a可能分别干预中性粒细胞胞内MPO的合成与释放, 中性粒细胞胞内MPO的合成活性是影响血管炎病情活动度的独立因素。

Objective To investigate the expression and clinical significance of neutrophil myeloperoxidase(MPO)in patients with MPO-antibody associated vasculitis(AAV).Methods Thirty-six newly diagnosed MPO-AAV patients who were hospitalized in the First Affiliated Hospital,Anhui Medical University from July 2018 to June 2021 were enrolled,and 36 age and sex matched healthy subjects were selected as controls.Neutrophil MPO level was detected by flow cytometry(FCM)and MPO mRNA was tested by real time quantitative polymerase chain reaction(RT-qPCR)in all subjects.Serum complement fragment C5(C5a)and MPO in both groups and serum MPO-anti-antineutrophilic cytoplasmic antibody(ANCA)in MPO-AAV group were measured by enzyme linked immunosorbent assay(ELISA),while the disease activity was evaluated by Birmingham vasculitis activity score-V3(BVAS-V3).Results Compared with the heathy control group,the expression of MPO mRNA in neutrophils,serum MPO and complement C5a in MPO-AAV group were significantly higher[MPO mRNA:30.2±11.5 vs.1.9±0.6,P<0.001;MPO:(112.0±68.7)IU/L vs.(87.4±22.9)IU/L,P=0.01;C5a:(187.3±90.3)ng/ml vs.(107.3±31.1)ng/ml,P<0.001;respectively],while the mean fluorescence intensity(MFI)of MPO in neutrophils were significantly lower[1343.3±723.4 vs.2868.0±1136.5,P<0.001].In MPO-AAV group,the expression of neutrophil MPO mRNA was positively correlated with serum MPO-ANCA and MPO levels(r=0.537,P=0.001 and r=0.358,P=0.032;respectively).Multiple regression analysis suggested that neutrophil MPO mRNA expression was positively correlated with serum MPO-ANCA level(β=0.695,P=0.006);neutrophil MPO level was negatively correlated with serum MPO-ANCA,MPO and complement C5a levels(r=-0.335,P=0.046;r=-0.372,P=0.026;r=-0.577,P<0.001;respectively).Further,neutrophil MPO level was negatively correlated with serum complement C5a level(β=-0.374,P=0.043).BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils,serum MPO-ANCA,MPO and complement C5a(r=0.598,P<0.001;r=0.599,P<0.001;r=0.537,P=0.001;r=0.415,P=0.012;respectively)and negatively correlated with MPO level in neutrophils(r=-0.342,P=0.041).In multiple regression analysis it suggested that BVAS-V3 was positively correlated with MPO mRNA expression in neutrophils(β=0.511,P=0.002).Conclusion In MPO-AAV patients,MPO synthesis and release in neutrophils are both significantly increased,which might be influenced by serum MPO-ANCA and C5a,respectively.Furthermore,MPO synthesis activity in neutrophils is an independent factor related to disease activity.