干扰素基因刺激蛋白（STING）通路对乙型肝炎肝硬化患者外周血单核细胞炎症因子分泌及吞噬功能的影响

杨逸帆; 杜万威; 王霞; 杨广德; 李丽; 傅涓涓; 潘修成

doi:10.3969/j.issn.1001-5256.2023.09.013

干扰素基因刺激蛋白（STING）通路对乙型肝炎肝硬化患者外周血单核细胞炎症因子分泌及吞噬功能的影响

DOI: 10.3969/j.issn.1001-5256.2023.09.013

徐州医科大学附属医院感染性疾病科，江苏徐州 221002

基金项目:

国家“十三五”科技重大专项课题 (2018ZX10302－206)

伦理学声明：本研究方案于2020年7月2日经由徐州医科大学附属医院伦理委员会审批，批号XYFY2020－KL075－01，入组患者均采取自愿原则，并签署知情同意书。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：杨逸帆、杜万威、潘修成负责课题设计；杨逸帆、杜万威、李丽负责实验操作，资料分析和撰写论文；杨逸帆、李丽、王霞、杨广德、傅涓涓参与收集数据，修改论文；潘修成负责拟定写作思路，指导撰写文章并最后定稿。

详细信息

通信作者:
潘修成， xzpxc68@126.com （ORCID： 0000－0001－9706－9458）

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出版历程
- 收稿日期: 2022-12-13
- 录用日期: 2023-02-02
- 出版日期: 2023-09-19

Effect of the STING pathway on inflammatory cytokine secretion and phagocytosis by peripheral blood monocytes in patients with hepatitis B cirrhosis

Department of Infectious Diseases，The Affiliated Hospital of Xuzhou Medical University，Xuzhou，Jiangsu 221002，China

Research funding:

National Science and Technology Major Project during the 13th Five－Year Plan Period (2018ZX10302－206)

More Information

Corresponding author: PAN Xiucheng， xzpxc68@126.com （ORCID： 0000－0001－9706－9458）

摘要

摘要: 目的研究干扰素基因刺激蛋白（STING）通路对乙型肝炎肝硬化患者外周血单核细胞生成炎症细胞因子及吞噬功能的影响。方法收集2020年5月—10月于徐州医科大学附属医院感染性疾病科住院的35例乙型肝炎肝硬化患者和10例体检中心健康体检者的外周静脉全血，分离并提取外周血单核细胞；另将乙型肝炎肝硬化患者分为环鸟苷酸-腺苷酸（cGAMP）（n=12）、cGAMP+CCCP（n=12）和Control（n=11）三组。体外以STING激活剂cGAMP和/或STING抑制剂CCCP加入单核细胞培养液中，ELISA法检测上清液IFN－α、IFN－β、IL－6和TNF－α水平。此外，将含cGAMP和/或CCCP的单核细胞培养体系与荧光标记的E.coli共同孵育后，采用流式细胞仪检测单核细胞吞噬功能的改变。计量资料两组间比较采用成组t检验；多组间比较采用单因素方差分析，进一步两两比较采用LSD－t检验。结果与加入抑制剂CCCP组相比，乙型肝炎肝硬化患者外周血单核细胞经cGAMP刺激后分泌IFN－α、IFN－β、IL－6和TNF－α水平显著升高（P值均<0.05）。乙型肝炎肝硬化患者外周血单核细胞在有或无cGAMP/cGAMP+CCCP处理下，吞噬E.coli能力均较健康组明显降低（t值分别为4.647、2.790、2.504，P值均<0.05），且有或无cGAMP与cGAMP+CCCP刺激，对乙型肝炎肝硬化患者单核细胞吞噬E.coli的能力没有显著影响（P>0.05）。结论外周血单核细胞STING通路活化参与了乙型肝炎肝硬化状态下全身性炎症反应的发生，STING通路的激活与否不影响乙型肝炎肝硬化外周血单核细胞的吞噬细菌能力。
- 肝硬化 /
- 细胞因子类 /
- 干扰素基因刺激蛋白
Abstract: Objective To investigate the effect of the STING pathway on the inflammatory cytokine secretion and phagocytosis by peripheral blood monocytes in patients with hepatitis B cirrhosis. Methods Peripheral venous whole blood samples were collected from 35 patients with hepatitis B cirrhosis who were hospitalized in department of infectious diseases and 10 individuals who underwent physical examination in physical examination center in the Affiliated Hospital of Xuzhou Medical University from May to October 2020， and peripheral blood monocytes were isolated and extracted. Patients with hepatitis B cirrhosis were divided into three groups： cyclic GMP－AMP （cGAMP）（n=12）， cGAMP+CCCP （n=12） and Control （n=11）. In vitro， the STING activator cGAMP and/or the STING inhibitor CCCP were added to monocyte culture medium， and ELISA was used to measure the levels of interferon－α （IFN－α）， interferon－β （IFN－β）， interleukin－6 （IL－6）， and tumor necrosis factor－α （TNF－α） in supernatant. In addition， the monocyte culture system containing cGAMP and/or CCCP was co－cultured with fluorescein－labeled Escherichia coli， and then flow cytometry was used to observe changes in the phagocytosis of monocytes. The independent－samples t test was used for comparison of continuous data between two groups； a one－way analysis of variance was used for comparison between multiple groups， and the least significant difference t－test was used for further comparison between two groups. Results Compared with the peripheral blood monocytes treated by the inhibitor CCCP， the peripheral blood monocytes stimulated by cGAMP secreted significantly higher levels of IFN－α， IFN－β， IL－6， and TNF－α （all P<0.05）. Compared with the healthy group， the hepatitis B cirrhosis group had a significant reduction in the phagocytosis of E. coli by peripheral blood monocytes， with or without cGAMP/cGAMP+CCCP treatment （t=4.647， 2.790， and 2.504， all P<0.05）， and cGAMP or cGAMP+CCCP stimulation had no significant effect on the phagocytosis of E. coli by peripheral blood monocytes in the patients with hepatitis B cirrhosis （P>0.05）. Conclusion The activation of the STING pathway in peripheral blood monocytes is involved in the development of systemic inflammatory response in hepatitis B cirrhosis， and the activation of the STING pathway has no significant effect on the phagocytosis of bacteria by peripheral blood monocytes in hepatitis B cirrhosis.
- Liver Cirrhosis /
- Cytokines /
- Stimulator of Interferon Genes

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图 1 不同处理情况下乙型肝炎肝硬化组和健康组外周血单核细胞吞噬细菌能力比较

Figure 1. Phagocytic ability of peripheral blood mononuclear cells in patients with hepatitis B cirrhosis and healthy people under different treatments

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图 2 不同处理情况下乙型肝炎肝硬化患者外周血单核细胞吞噬细菌能力比较

Figure 2. Phagocytic ability of peripheral blood mononuclear cells in patients with hepatitis B cirrhosis under different treatments

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表 1 cGAMP作用下乙型肝炎肝硬化患者和健康成人外周血单核细胞炎症因子分泌水平比较

Table 1. Comparison of inflammatory factor secretion levels in peripheral blood mononuclear cells of patients with hepatitis B cirrhosis and healthy adults under the effect of cGAMP

组别	例数	IFN－α （pg/mL）	IFN－β （pg/mL）	IL－6 （pg/mL）	TNF－α （pg/mL）
乙型肝炎肝硬化组	35	31.31 ± 3.02	908.03 ± 109.98	47.02 ± 3.89	56.32 ± 4.92
健康组	10	21.69 ± 1.41	690.35 ± 52.90	41.07 ± 2.04	44.44 ± 4.11
t值		7.315	4.475	3.467	5.072
P值		<0.001	<0.001	0.003	<0.001

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表 2 cGAMP作用下乙型肝炎肝硬化患者外周血单核细胞炎症因子分泌水平

Table 2. Inflammatory factor secretion levels in peripheral blood mononuclear cells of patients with hepatitis B cirrhosis under the influence of cGAMP

组别	例数	IFN－α （pg/mL）	IFN－β （pg/mL）	IL－6 （pg/mL）	TNF－α （pg/mL）
cGAMP组	12	31.31 ± 3.02	908.03 ± 109.98	47.02 ± 3.89	56.32 ± 4.92
cGAMP+CCCP组	12	21.13 ± 2.47^1）	714.60 ± 151.26^1）	35.29 ± 4.12^1）	30.47 ± 6.89^1）
Control组	11	24.46 ± 2.55^1）	597.50 ± 99.56^1）	37.72 ± 4.28^1）	32.69 ± 7.94^1）
F值		46.006	14.823	25.138	54.784
P值		<0.001	<0.001	<0.001	<0.001
注：与cGAMP组比较，1）P<0.05。

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