摘要
目的 探究中性粒细胞自噬、凋亡和胞外诱捕网(NET)形成在系统性红斑狼疮(SLE)中的作用。方法 收集36例女性SLE患者(SLE组)和同期32例健康体检者为对照,Western blot法检测SLE患者和健康体检者中性粒细胞微管相关蛋白1轻链3B(LC3B)、自噬相关基因5(ATG5)、 P62、 B细胞淋巴瘤因子2(Bcl2)、 Bcl2相关X蛋白(BAX)的蛋白表达;流式细胞术检测中性粒细胞LC3B的蛋白表达;ELISA检测血浆髓过氧化物酶(MPO)水平。体外分离培养中性粒细胞,分别用100 nmol/L雷帕霉素和10μg/mL脂多糖(LPS)处理6 h, Western blot法检测中性粒细胞LC3B、 ATG5、 P62、 Bcl2和BAX的蛋白表达,ELISA检测培养上清中MPO水平,SytoxTMGreen染色结合荧光光度法检测培养上清中NET形成的荧光强度变化。结果 与健康对照组比较,SLE组中性粒细胞自噬、凋亡水平和NET形成均显著增加,中性粒细胞自噬与凋亡、 NET分别正相关;雷帕霉素诱导中性粒细胞自噬活化,但对凋亡和NET形成无明显影响;LPS能诱导中性粒细胞自噬激活并促进NET形成,但对凋亡无明显影响。结论 SLE患者中性粒细胞自噬、凋亡和NET形成均增加,SLE患者中性粒细胞自噬过度激活和NET形成增多可能是炎性内环境长期作用的结果。
Objective To explore the role of autophagy,apoptosis of neutrophils and neutrophils extracellular traps(NET)formation in systemic lupus erythematosus(SLE).Methods Thirty-six patients with SLE were recruited as research subjects,and 32 healthy controls matched accordingly were enrolled as control subjects.The expression levels of microtubule associated protein 1 light chain 3B(LC3B),autophagy-related gene5(ATG5),P62,B-cell lymphoma 2(Bcl2),Bcl2-related X protein(BAX)in neutrophils were detected by Western blot analysis.Flow cytometry was employed to analyze the expression of LC3B on neutrophils.The expression level of myeloperoxidase(MPO)in plasma was estimated by ELISA.Furthermore,neutrophils were cultured in vitro and stimulated by 100 nmol/L rapamycin and 10μg/mL lipopolysaccharide(LPs)for 6 hours,respectively.And then,the expression levels of LC3B,ATG5,P62,Bcl2 and BAX in neutrophils were detected by Western blot analysis.The level of MPO in culture supernatant was detected by ELiSA.The change of fluorescence intensity of NET in culture supernatant was assayed by Sytox^(TM)Green staining combined with fluorescence spectrophotometry.Results Compared with healthy controls,the levels of autophagy and apoptosis of neutrophils and NET formation in SLE patients were increased.The level of apoptosis and NET formation was positively associated with neutrophil autophagy.The level of autophagy showed an increase but had no effect on apoptosis and NET formation for neutrophil stimulated by rapamycin.The levels of autophagy and NET formation also increased with no significant effect on apoptosis for neutrophil induced by LPS.Conclusion The autophagy,apoptosis and NET formation of neutrophils increase in SLE patients.The activation of autophagy and NET in neutrophils possibly result from the inflammatory internal environment in SLE patients.
作者
杨冬梅
祝静
肖剑波
何仁栋
邢艳
YANG Dongmei;ZHU Jing;XIAO Jianbo;HE Rendong;XING Yan(Clinic Laboratory in the Afiliated Hospital of North Sichuan Medical College,Nanchong 637000;Clinic Laboratory in Second People's Hospital of Yibin,Yibin 644600;Department of Laboratory Medicine in North of Sichuan Medical College,Nanchong 637000,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2023年第4期356-362,共7页
Chinese Journal of Cellular and Molecular Immunology
基金
四川省卫建委普及应用项目(18PJ107)
川北医学院校级青年项目(CBY21-QA08)。
