title: [Effects of exogenous carbon monoxide-releasing molecule 2 intervention in vitro on formation of human neutrophil extracellular traps stimulated by endotoxin/lipopolysaccharide and its mechanism].
abstract: OBJECTIVE
  To explore the effects of exogenous carbon monoxide-releasing molecule 2 (CORM-2) on formation of human neutrophil extracellular traps (NETs) stimulated by endotoxin/lipopolysaccharide (LPS) and its relevant mechanism.
  
  
  METHODS
  Venous blood samples were collected from a healthy adult volunteer to isolate neutrophils. The neutrophils were divided into normal control (NC) group, LPS group, LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ inactive CORM-2 (iCORM-2) group according to the random number table. No treatment was given to the neutrophils in NC group. The neutrophils in LPS group underwent LPS stimulation (1 μL, 1 μg/mL). The neutrophils in LPS+ 10 μmol/L CORM-2 group, LPS+ 50 μmol/L CORM-2 group, and LPS+ iCORM-2 group underwent the same LPS stimulation as that in LPS group and treatment of 10 μmol/L CORM-2, 50 μmol/L CORM-2, and 50 μmol/L iCORM-2, respectively, with the volune of 1 μL. After conventional culture for 1 h, the number of NETs was determined with propidium iodide staining method; the early cell apoptosis rate was determined with flow cytometer; the generation level of reactive oxygen species (ROS) was assessed with dihydrogenrhodamine 123 fluorescent probe staining method (denoted as mean fluorescence intensity); the expression level of phosphorylated extracellular regulated kinase 1/2 (p-ERK1/2) was determined by Western blotting. The sample numbers of each group in the 4 experiments were all 5. Data were processed with one-way analysis of variance and SNK test.
  
  
  RESULTS
  (1) The numbers of NETs per 400-time visual field in cells of LPS and LPS+ iCORM-2 groups were close to the number in NC group (with P values above 0.05). The number of NETs per 400-time visual field was significantly larger in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in NC and LPS groups (with P values below 0.05). The number of NETs per 400-time visual field in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (2) The early cell apoptosis rate was significantly increased in LPS, LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The early cell apoptosis rates in LPS+ 10 μmol/L CORM-2, LPS+ 50 μmol/L CORM-2, and LPS+ iCORM-2 groups were close to the rate in LPS group (with P values above 0.05). (3) The generation level of ROS was significantly higher in cells of LPS, LPS+ 10 μmol/L CORM-2, and LPS+ iCORM-2 groups than in NC group (with P values below 0.05). The generation level of ROS in cells of LPS+ 50 μmol/L CORM-2 group was close to that of NC group (P>0.05). The generation level of ROS was lower in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05), while the generation level of ROS in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05). (4) The expression levels of p-ERK1/2 in cells of LPS and LPS+ iCORM-2 groups (respectively 0.0311±0.001 and 0.0309±0.0018) were close to the level in NC group (0.0304±0.0046, with P values above 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups (respectively 0.7891±0.0201 and 1.2970±0.0056) than in NC group (with P values below 0.05). The expression level of p-ERK1/2 was significantly higher in cells of LPS+ 10 μmol/L CORM-2 and LPS+ 50 μmol/L CORM-2 groups than in LPS group (with P values below 0.05). The expression level of p-ERK1/2 in cells of LPS+ iCORM-2 group was close to that of LPS group (P>0.05).
  
  
  CONCLUSIONS
  CORM-2 can obviously increase the production of NETs in LPS-induced neutrophils, and it might be attributable to the promotion of inhibition of ROS generation and phosphorylation of ERK1/2.
authors.affiliation: Department of Burns and Plastic Surgery, the Affiliated Hospital of Jiangsu University, Zhenjiang 212001, China.
authors.forename: Mingming
authors.initials: M
authors.lastname: Song
authors.forename: Xu
authors.initials: X
authors.lastname: Wang
authors.forename: Weiting
authors.initials: W
authors.lastname: Qin
authors.forename: Mingfeng
authors.initials: M
authors.lastname: Zhuang
authors.forename: Xiaohan
authors.initials: X
authors.lastname: Xu
authors.forename: Yisen
authors.initials: Y
authors.lastname: Zhang
authors.forename: Bingwei
authors.initials: B
authors.lastname: Sun
mesh_terms: D017209:Apoptosis; D002248:Carbon Monoxide / Q000378:metabolism*; D065206:Extracellular Traps; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D009942:Organometallic Compounds / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects
chemical_list: D008070:Lipopolysaccharides; D009942:Organometallic Compounds; C447082:tricarbonyldichlororuthenium (II) dimer; D002248:Carbon Monoxide