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

Kuang Tianyin; Yin Shuangqin; Dai Weihong; Luo Li; Chen Tao; Liang Xinghe; Wang Rixing; Liang Huaping; Zhu Junyu

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

Volume 39 Issue 7

Jul. 2023

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Article Navigation > CHINESE JOURNAL OF BURNS AND WOUNDS > 2023 > 39(7): 633-640

Kuang TY,Yin SQ,Dai WH,et al.Effects of enhancing the expression of aryl hydrocarbon receptor in post-traumatic mice macrophages on the inflammatory cytokine level and bactericidal ability[J].Chin J Burns Wounds,2023,39(7):633-640.DOI: 10.3760/cma.j.cn501225-20230210-00040.

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

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

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

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Corresponding author: Zhu Junyu, Email: zjykent@sina.com
Received Date: 2023-02-10

Abstract

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

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References

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

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

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

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