外源性一氧化碳释放分子2对脂多糖刺激人中性粒细胞杀菌功能的调控作用及其机制

doi:10.3877/cma.j.issn.2096-1537.2018.04.012

中华重症医学电子杂志 ›› 2018, Vol. 04 ›› Issue (04) : 354 -362. doi: 10.3877/cma.j.issn.2096-1537.2018.04.012

所属专题：文献；

基础研究

外源性一氧化碳释放分子2对脂多糖刺激人中性粒细胞杀菌功能的调控作用及其机制

宋明明¹^,^†(

), 孙炳伟¹, 李平², 丁盛¹

^1. 215002　江苏苏州，南京医科大学附属苏州医院烧伤整形科
^2. 215002　江苏苏州，南京医科大学附属苏州医院消化营养中心

收稿日期:2017-12-13 出版日期:2018-11-28
通信作者: 宋明明
基金资助:
国家自然科学基金(81471903，81272148); 苏州科技计划项目(sys2018086)

Regulation effect and the relevant mechanism of CORM-2 on bactericidal function in LPS-induced neutrophils

Mingming Song¹^,^†(), Bingwei Sun¹, Ping Li², Sheng Ding¹

^1. Department of Burns and Plastic Surgery, Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou 215002, China
^2. Digestive Nutrition Center, Suzhou Hospital Affiliated to Nanjing Medical University, Suzhou 215002, China

Received:2017-12-13 Published:2018-11-28
Corresponding author: Mingming Song
About author:
Corresponding author: Song Mingming, Email: mingming536@126.com

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引用本文：

宋明明, 孙炳伟, 李平, 丁盛. 外源性一氧化碳释放分子2对脂多糖刺激人中性粒细胞杀菌功能的调控作用及其机制[J/OL]. 中华重症医学电子杂志, 2018, 04(04): 354-362.

Mingming Song, Bingwei Sun, Ping Li, Sheng Ding. Regulation effect and the relevant mechanism of CORM-2 on bactericidal function in LPS-induced neutrophils[J/OL]. Chinese Journal of Critical Care & Intensive Care Medicine(Electronic Edition), 2018, 04(04): 354-362.

目的

探讨外源性一氧化碳释放分子2（CORM-2）对脂多糖（LPS）刺激人中性粒细胞杀菌功能的调控作用及其机制。

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 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进行干预，常规培养1 h后检测相关指标。用琼脂糖趋化模型检测中性粒细胞的趋化功能，流式细胞仪检测细胞颗粒释放及吞噬功能，酶联免疫吸附试验（ELISA）监测中性粒细胞颗粒释放功能的改变，蛋白质印迹法检测磷脂酰肌醇3激酶（PI3K）/蛋白激酶B（Akt）信号通路蛋白Akt磷酸化的表达水平。以上指标均重复测定3次，对数据行单因素方差分析、SNK检验。

结果

正常对照组、LPS组、LPS＋10 μmol/L CORM-2组、LPS＋50 μmol/L CORM-2组、LPS＋iCORM-2组细胞的趋化距离分别为（2241.33±67.30）、（919.00±55.02）、（1784.33±17.79）、（2202.33±91.69）、（1000.00±55.02）μm，LPS组细胞趋化距离较正常对照组明显降低（P<0.01）；LPS组和LPS＋iCORM-2组细胞的迁移距离较正常对照组明显降低（P<0.05）；LPS＋10 μmol/L CORM-2组和LPS＋50 μmol/L CORM-2组细胞迁移距离较LPS组明显增加（P<0.01）；LPS＋iCORM-2组细胞细胞迁移距离与LPS组相近（P>0.05）。对于中性粒细胞四级颗粒的释放研究，LPS刺激后中性粒细胞的颗粒释放均发生了明显的增加，而使用10 μmol/L与50 μmol/L CORM-2干预后，中性粒细胞的颗粒释放均得到明显抑制（P<0.01）；使用50 μmol/L iCORM-2干预后中性粒细胞颗粒释放与LPS组相近（P>0.05）。用LPS刺激后中性粒细胞Phagocytosis平均荧光强度较正常对照组升高（P<0.05）；而使用10 μmol/L与50 μmol/L CORM-2干预后中性粒细胞Phagocytosis平均荧光强度较LPS组明显增高（P<0.01）；使用50 μmol/L iCORM-2干预后中性粒细胞Phagocytosis平均荧光强度为与LPS组相近（P>0.05）。LPS刺激后中性粒细胞蛋白Akt磷酸化与总Akt比值灰度扫描值比值与正常对照组相近（P>0.05）；而使用10 μmol/L与50 μmol/L CORM-2干预后中性粒细胞蛋白Akt磷酸化与总Akt比值灰度扫描值比值较正常对照组、LPS组明显增高（P<0.05）；使用50 μmol/L iCORM-2干预后中性粒细胞蛋白Akt磷酸化与总Akt比值灰度扫描值比值与LPS组相近（P>0.05）。

结论

CORM-2干预可以明显增加LPS刺激后中性粒细胞的早期凋亡，恢复中性粒细胞的趋化功能，并促进中性粒细胞的吞噬功能。CORM-2调控LPS刺激后中性粒细胞凋亡与吞噬功能，其机制可能与CORM-2促进磷酸化Akt有关。

关键词: 脓毒症, 一氧化碳, 中性粒细胞, 吞噬, 凋亡

Objective

To explore the regulated effect of exogenous carbon monoxide releasing molecule 2 (CORM-2) on phagocytosis of LPS-chanllanged neutrophils and its relevant mechanism.

Methods

Venous blood of a healthy adult volunteer was collected and neutrophils were isolated. Then the neutrophils were divided into five groups according to the random number table, including control, LPS, LPS+ CORM-2 (10 and 50 μmol/L) and LPS+ iCORM-2 group. The control group did not receive any treatment. The LPS group treated with LPS (1 μg/ml) for 60 min. The CORM-2 group and iCORM-2 group underwent the same stimulation and immediate administrated indicated dosage of CORM-2 (10 and 50 μmol/L) and iCORM-2 (50 μmol/L). After neutrophils isolation and individual treatment, the chemotaxis was measured using agarosechemotaxis model. Besides, the early apoptosis rate and phagocytosis of neutrophils was determined by flow cytometry and the level of Akt phosphorylation in MAPK signaling were detected by Western blot. The parameters above were processed with 3 times repeated assayed and all values were analyzed using one-way analysis of variation, and SNK test.

Results

As shown in chemotaxis measured, compared with control group, there was a significant decrease of the chemotaxis distance when neutrophils stimulated with LPS and iCORM-2, (both P<0.05). Compared with LPS group, treated with CORM-2 (10 μmol/L and 50 μmol/L) sharply recovered the chemotaxis of neutrophil (both P< 0.01). The chemotaxis distanceof neutrophil of iCORM-2 group was similar with LPS group (P>0.05). As shown in neutrophil exocytosis, there was a slightly increase of exocytosis when neutrophil stimulate with LPS compared with control group (P>0.05). Whereas there was a significant decrease of exocytosis when treated with CORM-2 (10 μmol/L and 50 μmol/L) (both P<0.01). The exocytosis of neutrophil of iCORM-2 group was similar with LPS group (P>0.05). In terms of phagocytosis, compared with control group, there was a significant increase with LPS (P<0.05). Compared with LPS group, treated with CORM-2 (10 μmol/L and 50 μmol/L) sharply elevated the phagocytosis of neutrophil (both P<0.01). The phagocytosis of iCORM-2 group was similar with LPS group (both P>0.05). Compared with control group, it was similar of the level of Akt phosphorylation with LPS and iCORM-2 (all P>0.05). Compared with control group, the level of Akt phosphorylation was increased in CORM-2 (10 μmol/L and 50 μmol/L) groups (both P<0.05). Compared with LPS group, the level of Akt phosphorylation was increased in CORM-2 (10 μmol/L and 50 μmol/L) groups (both P<0.05).

Conclusion

CORM-2 can promote the apoptosis in LPS-chanllaged neutrophils, sharply recover the chemotaxis and promoted the phagocytosis. The possible mechanism of CORM-2 regulated the apoptosis and phagocytosis of LPS-induced neutrophil is related with the elevation of phosphorylation of Akt.

Key words: Sepsis, Carbon monoxide, Neutrophils, Phagocytosis, Apoptosis

图1 5组琼脂糖细胞迁移模型检测下中性粒细胞迁移距离（光镜×10物镜）。1a.正常对照组；1b. LPS组（10×物镜），较图1a明显降低；1c. LPS＋10 μmoL CORM-2组（10×物镜），较图1b增加；1d. LPS＋50 μmoL CORM-2组，较图1c进一步增加；1e. LPS＋iCORM-2组（10×物镜），与图1b相近；黑色箭头指示为中性粒细胞，左边空洞为细胞，右边为趋化物fMLP；CORM-2为外源性一氧化碳释放分子2，iCORM-2为无活性CORM-2

图2 5组中性粒细胞不同刺激时其特殊颗粒释放CD35释放MCF比较。2a.正常对照组；2b. LPS组；2c. LPS＋10 μmol/L CORM-2组；2d. LPS＋50 μmol/L CORM-2组；2e. LPS＋iCORM-2组

图3 5组中性粒细胞不同刺激时MMP-9释放比较

图4 5组中性粒细胞不同刺激时其初级颗粒释放CD63释放MCF比较。4a.正常对照组；4b. LPS组；4c. LPS＋10 μmol/L CORM-2组；4d. LPS＋50 μmol/L CORM-2组，较图4c进一步增加；4e. LPS＋iCORM-2组

图5 5组中性粒细胞不同刺激时其特殊颗粒释放CD66b释放MCF比较。5a.正常对照组；5b. LPS组；5c. LPS＋10 μmol/L CORM-2组；5d. LPS＋50 μmol/L CORM-2组，较图5c进一步增加；5e. LPS＋iCORM-2组

图6 5组流式细胞仪检测下中性粒细胞细胞吞噬功能改变。6a.正常对照组；6b. LPS组，较图6a稍增多；6c. LPS＋10 μmol/L CORM-2组，较图6a、6b明显增加；6d. LPS＋50 μmol/L CORM-2组，较图6c进一步增加；6e. LPS＋50 μmol/L iCORM-2组，较图6a增加，与图6b相近

图7 CORM-2对中性粒细胞蛋白Akt的影响

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Regulation effect and the relevant mechanism of CORM-2 on bactericidal function in LPS-induced neutrophils

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Regulation effect and the relevant mechanism of CORM-2 on bactericidal function in LPS-induced neutrophils