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
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摘要: 目的 探讨外源性一氧化碳释放分子2(CORM-2)对LPS刺激人中性粒细胞胞外诱捕网(NET)形成的作用及其机制。 方法 采集1名健康成年志愿者的静脉血,分离出中性粒细胞后,按随机数字表法分为正常对照组、LPS组、LPS+10 μmol/L CORM-2组、LPS+50 μmol/L CORM-2组、LPS+无活性CORM-2(iCORM-2)组。正常对照组不进行任何处理;LPS组采用1 μL 1 μg/mL的LPS刺激;LPS+10 μmol/L CORM-2组、LPS+50 μmol/L CORM-2组、LPS+iCORM-2组在采用上述相同剂量LPS刺激的同时,分别采用10 μmol/L CORM-2、50 μmol/L CORM-2、50 μmol/L iCORM-2各1 μL进行干预,常规培养1 h后检测相关指标。用碘化丙啶染色标记法检测NET数量,流式细胞仪检测细胞早期凋亡率,二氢罗丹明123荧光探针法检测活性氧生成水平(结果以平均荧光强度表示),蛋白质印迹法检测磷酸化细胞外信号调节激酶1/2(ERK1/2)表达水平。每组以上4个实验样本数均为5。对数据行单因素方差分析、SNK检验。 结果 (1)LPS组和LPS+iCORM-2组细胞每400倍视野下NET数量与正常对照组相近(
P 值均大于0.05)。LPS+10 μmol/L CORM-2组和LPS+50 μmol/L CORM-2组细胞每400倍视野下NET数量较正常对照组和LPS组均明显增多(P 值均小于0.05)。LPS+iCORM-2组细胞每400倍视野下NET数量与LPS组相近(P >0.05)。(2)LPS组、LPS+10 μmol/L CORM-2组、LPS+50 μmol/L CORM-2组、LPS+iCORM-2组细胞早期凋亡率较正常对照组明显增加(P 值均小于0.05),LPS+10 μmol/L CORM-2组、LPS+50 μmol/L CORM-2组、LPS+iCORM-2组细胞早期凋亡率与LPS组相近(P 值均大于0.05)。(3)LPS组、LPS+10 μmol/L CORM-2组、LPS+iCORM-2组细胞活性氧生成水平高于正常对照组(P 值均小于0.05),LPS+50 μmol/L CORM-2组细胞活性氧生成水平与正常对照组相近(P >0.05)。LPS+10 μmol/L CORM-2组和LPS+50 μmol/L CORM-2组细胞活性氧生成水平明显低于LPS组(P 值均小于0.05),LPS+iCORM-2组细胞活性氧生成水平与LPS组相近(P >0.05)。(4)LPS组和LPS+iCORM-2组细胞磷酸化ERK1/2表达水平分别为0.031 1±0.001 4和0.030 9±0.001 8,与正常对照组的0.030 4±0.004 6相近(P 值均大于0.05);LPS+10 μmol/L CORM-2组和LPS+50 μmol/L CORM-2组细胞磷酸化ERK1/2表达水平分别为0.789 1±0.020 1和1.297 0±0.005 6,高于正常对照组(P 值均小于0.05)。LPS+10 μmol/L CORM-2组、LPS+50 μmol/L CORM-2组细胞磷酸化ERK1/2表达水平高于LPS组(P 值均小于0.05),LPS+iCORM-2组细胞磷酸化ERK1/2表达水平与LPS组相近(P >0.05)。 结论 CORM-2干预可以明显增加LPS刺激后NET的形成,其机制可能与CORM-2抑制活性氧生成和促进ERK1/2磷酸化有关。-
关键词:
- 脓毒症 /
- 脂多糖类 /
- 外源性一氧化碳释放分子2 /
- 中性粒细胞 /
- 中性粒细胞胞外诱捕网
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 (withP 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 (withP 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 (withP 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 (withP 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 (withP 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 (withP 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 0.031 1±0.001 4 and 0.030 9±0.001 8) were close to the level in NC group (0.030 4±0.004 6, withP 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 0.789 1±0.020 1 and 1.297 0±0.005 6) than in NC group (withP 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 (withP 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.
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