增强创伤后小鼠巨噬细胞的芳香烃受体表达对炎症因子水平和杀菌能力的影响

旷田垠; 殷双琴; 代伟宏; 罗莉; 陈涛; 梁星河; 王日兴; 梁华平; 朱俊宇

doi:10.3760/cma.j.cn501225-20230210-00040

增强创伤后小鼠巨噬细胞的芳香烃受体表达对炎症因子水平和杀菌能力的影响

doi: 10.3760/cma.j.cn501225-20230210-00040

1.
陆军军医大学大坪医院战伤感染与特需药品研究室，创伤、烧伤与复合伤国家重点实验室，重庆　　400042
2.
海南医学院第二附属医院急诊科，海南省急危重症临床医学研究中心，海口　　570216
3.
陆军军医大学基础医学院学员旅17队，重庆　　400038

基金项目:

重庆市自然科学基金面上项目 cstc2021jcyj-msxmX0234

重庆市博士“直通车”科研项目 CSTB2022BSXM-JCX0009

中央引导地方科技发展专项资金 ZY2021HN19

国家级大学生创新创业训练计划 202190035010

详细信息

通讯作者:
朱俊宇，Email：zjykent@sina.com

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出版历程
- 收稿日期: 2023-02-10

Effects of enhancing the expression of aryl hydrocarbon receptor in post-traumatic mice macrophages on the inflammatory cytokine level and bactericidal ability

1.
State Key Laboratory of Trauma, Burns and Combined Injury, Department of Wound Infection and Drug, Daping Hospital, Army Medical University, Chongqing 400042, China
2.
Emergency Department of the Second Affiliated Hospital of Hainan Medical University, the Emergency and Critical Care Clinical Medicine Research Center of Hainan, Haikou 570216, China
3.
The 17th Team of Cadet Brigade, College of Basic Medical Sciences, Army Medical University, Chongqing 400038, China

Funds:

General Program of Chongqing Natural Science Foundation cstc2021jcyj-msxmX0234

Doctoral "Fast-service Channel" Research Program of Chongqing CSTB2022BSXM-JCX0009

The Central Government Guides Local Special Funds for Scientific and Technological Development ZY2021HN19

National College Students' Innovation and Entrepreneurship Training Program 202190035010

More Information

Corresponding author: Zhu Junyu, Email: zjykent@sina.com

摘要

摘要: 目的探讨严重创伤后小鼠腹腔巨噬细胞（PM）中芳香烃受体（AhR）的表达规律并分析增强创伤后PM的AhR表达对炎症因子水平和杀菌能力的影响。方法采用实验研究方法。取40只6~8周龄雄性C57BL/6J小鼠（小鼠周龄、性别、品系下同），按随机数字表法（分组方法下同）分为对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组，每组10只。将后3组小鼠构建骨折+失血的严重创伤模型，对照组小鼠不做处理。分别在未创伤和创伤后2、6、12 h时，提取对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组小鼠原代PM（提取细胞下同），分别采用蛋白质印迹法和实时荧光定量反转录PCR法检测AhR的蛋白和mRNA表达，采用转录组测序分析AhR信号通路相关分子的基因表达。取20只小鼠分为对照组、创伤后6 h组，每组10只，提取PM后，采用免疫沉淀法检测AhR泛素化水平。取12只小鼠，分为单纯二甲基亚砜（DMSO）组、创伤后6 h+DMSO组、单纯MG-132组、创伤后6 h+MG-132组，每组3只，并行相应处理后，提取PM，采用蛋白质印迹法检测AhR蛋白的表达。取20只小鼠构建创伤后6 h模型并提取PM，分为空载腺病毒（Ad-NC）组和AhR过表达腺病毒（Ad-AhR）组，部分细胞转染相应腺病毒36 h后，采用蛋白质印迹法检测AhR的蛋白表达；将剩余Ad-NC组细胞分为单纯Ad-NC组、Ad-NC+内毒素/脂多糖（LPS）组，将剩余Ad-AhR组细胞分为单纯Ad-AhR组和Ad-AhR+LPS组，并行相应处理12 h后，采用酶联免疫吸附测定法检测细胞上清液中白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）的表达（样本数为6）。取20只小鼠提取PM并分为对照+Ad-NC组、创伤后6 h+Ad-NC组、对照+Ad-AhR组、创伤后6 h+Ad-AhR组，并行相应处理后，采用平板涂布法检测细胞内载菌量（样本数为6）。对数据行单因素方差分析、LSD检验、析因设计方差分析、独立样本 t检验。结果与对照组（1.16±0.28）比较，创伤后2 h组（0.59±0.14）、创伤后6 h组（0.72±0.16）、创伤后12 h组（0.71±0.17）PM中AhR的蛋白表达水平均明显降低（ P<0.05）。对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组PM中AhR的mRNA表达量组间总体比较，差异无统计学意义（ P>0.05）。AhR信号通路相关分子包括 AhR、AhR抑制因子、细胞色素P450家族成员1b1、细胞色素P450家族成员11a1 、热休克蛋白90、芳香烃受体-相互作用蛋白、热休克蛋白70相互作用蛋白。除创伤后2 h组PM的热休克蛋白90表达水平较对照组高，其他分子的表达水平在创伤后变化不明显。与对照组比较，创伤后6 h组PM中AhR泛素化水平升高。与单纯DMSO组比较，创伤后6 h+DMSO组PM中AhR的蛋白表达量下降，单纯MG-132组PM中AhR的蛋白表达量无明显变化。与创伤后6 h+DMSO组比较，创伤后6 h+MG-132组PM中AhR的蛋白表达量上调。转染36 h，与Ad-NC组比较，Ad-AhR组PM中AhR的蛋白表达水平明显升高。处理12 h，与Ad-NC+LPS组比较，Ad-AhR+LPS组PM上清液中IL-6、TNF-α的表达水平均明显降低（ t值分别为4.80、3.82， P<0.05）。对照+Ad-NC组、创伤后6 h+Ad-NC组、对照+Ad-AhR组、创伤后6 h+Ad-AhR组1×10 ⁶个PM的胞内载菌量分别为（3.0±1.8）、（41.8±10.2）、（1.8±1.2）、（24.2±6.3）集落形成单位。与创伤后6 h+Ad-NC组比较，创伤后6 h+Ad-AhR组PM的胞内载菌量明显减少（ t=3.61， P<0.05）。结论严重创伤后小鼠PM中AhR发生泛素化降解导致其蛋白表达降低，增强创伤后巨噬细胞AhR的表达可降低LPS诱导的炎症因子IL-6和TNF-α的表达，且提升创伤后巨噬细胞的杀菌能力。
- 巨噬细胞 /
- 创伤和损伤 /
- 芳香烃受体核转位子 /
- 炎症因子 /
- 杀菌能力
Abstract: Objective To explore the expression pattern of aryl hydrocarbon receptor (AhR) in mice peritoneal macrophages (PMs) after major trauma and analyze the effects of enhanced AhR expression on the inflammatory cytokine level and bactericidal ability after trauma. Methods The experimental study method was used. Forty 6-8-week-old male C57BL/6J mice (the same mouse age, sex, and strain below) were divided into control group, post trauma hour (PTH) 2 group, PTH 6 group, and PTH 12 group according to the random number table (the same grouping method below), with 10 mice in each group. Mice in the latter 3 groups were constructed as severe trauma model with fracture+blood loss, while mice in control group were left untreated. The primary PMs (the same cells below) were extracted from the mice in control group, PTH 2 group, PTH 6 group, and PTH 12 group when uninjured or at PTH 2, 6, and 12, respectively. Then the protein and mRNA expressions of AhR were detected by Western blotting and real-time fluorescence quantitative reverse transcription polymerase chain reaction, respectively, and the gene expressions of AhR signaling pathway related molecules were analyzed by transcriptome sequencing. Twenty mice were divided into control group and PTH 6 group, with 10 mice in each group, and the PMs were extracted. The level of ubiquitin of AhR was detected by immunoprecipitation. Twelve mice were divided into dimethyl sulfoxide (DMSO) alone group, PTH 6+DMSO group, MG-132 alone group, and PTH 6+MG-132 group, with 3 mice in each group. After the corresponding treatment, PMs were extracted, and the protein expression of AhR was detected by Western blotting. Twenty mice were constructed as PTH 6 model. Then, the PMs were extracted and divided into empty negative control adenovirus (Ad-NC) group and AhR overexpression adenovirus (Ad-AhR) group. The protein expression of AhR was detected by Western blotting at 36 h after some PMs were transfected with the corresponding adenovirus. The rest cells in Ad-NC group were divided into Ad-NC alone group and Ad-NC+endotoxin/lipopolysaccharide (LPS) group, and the rest cells in Ad-AhR group were divided into Ad-AhR alone group and Ad-AhR+LPS group. The expressions of interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α) in the cell supernatant were detected by enzyme-linked immunosorbent assay at 12 h after the corresponding treatment ( n=6). Twenty mice were obtained to extract PMs. The cells were divided into control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group, and the intracellular bacterial load was detected by plate spread method after the corresponding treatment ( n=6). Data were statistically analyzed with one-way analysis of variance, least significant difference test, analysis of variance for factorial design, and independent sample ttest. Results Compared with 1.16±0.28 of control group, the protein expressions of AhR in PMs in PTH 2 group (0.59±0.14), PTH 6 group (0.72±0.16), and PTH 12 group (0.71±0.17) were all significantly decreased ( P<0.05). The overall comparison of the difference of AhR mRNA expression in PMs among control group, PTH 2 group, PTH 6 group, and PTH 12 group showed no statistical significance ( P>0.05). The AhR signaling pathway related molecules included AhR, AhR inhibitor, cytochrome P450 family member 1b1, cytochrome P450 family member 11a1, heat shock protein 90, aryl hydrocarbon receptor-interaction protein, and heat shock protein 70 interaction protein. The heat shock protein 90 expression of PMs in PTH 2 group was higher than that in control group, while the expressions of other molecules did not change significantly after trauma. Compared with that in control group, the level of ubiquitin of AhR in PMs in PTH 6 group was increased. Compared with that in DMSO alone group, the protein expression of AhR in PMs in PTH 6+DMSO group was decreased, while that in PMs in MG-132 alone group had no significant change. Compared with that in PTH 6+DMSO group, the protein expression of AhR in PMs in PTH 6+MG-132 group was up-regulated. At transfection hour 36, compared with that in Ad-NC group, the protein expression of AhR in PMs in Ad-AhR group was increased. At treatment hour 12, compared with those in Ad-NC+LPS group, the expressions of IL-6 and TNF-α in PM supernatant of Ad-AhR+LPS group were significantly decreased (with tvalues of 4.80 and 3.82, respectively, P<0.05). The number of intracellular bacteria of 1×10 ⁶ PMs in control+Ad-NC group, PTH 6+Ad-NC group, control+Ad-AhR group, and PTH 6+Ad-AhR group was (3.0±1.8), (41.8±10.2), (1.8±1.2), and (24.2±6.3) colony forming unit, respectively. Compared with that in PTH 6+Ad-NC group, the number of intracellular bacteria of PMs in PTH 6+Ad-AhR group was significantly decreased ( t=3.61, P<0.05). Conclusions Ubiquitin degradation of AhR in PMs of mice after major trauma results in decreased protein expression of AhR. Increasing the expression of AhR in post-traumatic macrophages can reduce the expressions of LPS-induced inflammatory cytokines IL-6 and TNF-α, and improve the bactericidal ability of macrophages after trauma.
- Macrophages /
- Wounds and injuries /
- Aryl hydrocarbon receptor nuclear translocator /
- Inflammatory cytokine /
- Bactericidal ability
旷田垠、罗莉：实验操作、论文撰写；殷双琴、代伟宏：实验操作；陈涛、梁星河：数据整理、统计学分析；王日兴、梁华平：研究指导、论文修改以及经费支持；朱俊宇：实验操作、论文撰写、研究指导、论文修改以及经费支持

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1 蛋白质印迹法检测的对照组与3组创伤小鼠腹腔巨噬细胞AhR的蛋白表达

注：AhR为芳香烃受体；条带上方1、2、3、4分别指示未致创伤的对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组

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2 对照组与3组创伤小鼠腹腔巨噬细胞AhR信号通路相关分子的表达水平热图

注：左侧缩写均为基因名称，AhR为芳香烃受体，Ahrr为芳香烃受体抑制因子，Cyp1b1为细胞色素P450家族成员1b1，Cyp11a1为细胞色素P450家族成员11a1，Hsp90为热休克蛋白90，Aip为芳香烃受体-相互作用蛋白，Chip为热休克蛋白70相互作用蛋白；热图上方1、2、3、4分别指示未致创伤的对照组、创伤后2 h组、创伤后6 h组、创伤后12 h组

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3 免疫沉淀法检测的对照组和创伤后6 h组小鼠腹腔巨噬细胞中AhR泛素化水平

注：AhR为芳香烃受体；条带上方1、2分别指示未致创伤的对照组和创伤后6 h组

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4 蛋白质印迹法检测的4组小鼠腹腔巨噬细胞中AhR的蛋白表达

注：AhR为芳香烃受体，DMSO为二甲基亚砜；条带上方1、2、3、4分别指示单纯DMSO组、创伤后6 h+DMSO组、单纯MG-132组、创伤后6 h+MG-132组

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5 蛋白质印迹法检测的2组创伤后小鼠腹腔巨噬细胞转染腺病毒36 h的AhR蛋白表达

注：AhR为芳香烃受体，Ad-NC为空载腺病毒，Ad-AhR为AhR过表达腺病毒；条带上方1、2分别指示Ad-NC组、Ad-AhR组

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表1 4组创伤后小鼠腹腔巨噬细胞处理12 h后上清液中IL-6和TNF-α表达水平比较（pg/mL， x ¯ ± s ）

组别	样本数	IL-6	TNF-α
单纯Ad-NC组	6	451±207	379±181
Ad-NC+LPS组	6	5 580±779	4 534±642
单纯Ad-AhR组	6	399±200	345±190
Ad-AhR+LPS组	6	3 397±797	3 063±689
t值		4.80	3.82
P值		<0.001	0.003
注：Ad-NC为空载腺病毒，LPS为内毒素/脂多糖，AhR为芳香烃受体，Ad-AhR为AhR过表达腺病毒，IL-6为白细胞介素6，TNF-α为肿瘤坏死因子α；IL-6、TNF-α表达的感染腺病毒类型主效应， F值分别为22.63、14.20， P值均<0.001；刺激类型主效应， F值分别为299.20、296.70， P值均<0.001；两者交互作用， F值分别为20.57、12.97， P值均<0.001； t值、 P值为Ad-NC+LPS组和Ad-AhR+LPS组各指标比较所得