Establishment and evaluation of a TRPA1-knockout mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis
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Establishment and evaluation of a TRPA1-knockout mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis
Journal of Chongqing Medical UniversityVol. 45, Issue 8, (2020)
作者机构:
1. 重庆医科大学附属第一医院皮肤科,重庆,400016
2. 重庆大学生物工程学院,重庆,400044
3. 重庆市人民医院过敏反应科,重庆,400014
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Published:2020,
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Chen Chao, Zhou Weikang, Chen Aijun, et al. Establishment and evaluation of a TRPA1-knockout mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis. [J]. Journal of Chongqing Medical University 45(8).(2020)
DOI:
Chen Chao, Zhou Weikang, Chen Aijun, et al. Establishment and evaluation of a TRPA1-knockout mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis. [J]. Journal of Chongqing Medical University 45(8).(2020)DOI:
Establishment and evaluation of a TRPA1-knockout mouse model of 2,4-dinitrochlorobenzene-induced atopic dermatitis
平均光密度值差异有统计学意义(t=34.32,P=0.000);免疫组化显示,实验组TRPA1在表皮和真皮的表达均有不同程度的增加。进一步在TRPA1基因敲除小鼠上建立AD模型显示:与对照组相比,模型Ⅰ、Ⅱ组背部皮肤表现出明显的炎症性皮损,耳部明显肿胀。病理显示角化过度和(或)角化不全、棘细胞层增厚、真皮淋巴细胞为主的炎性细胞浸润,同时发现肥大细胞浸润增加。血清总IgE水平升高,且差异有统计学意义[(1.27±0.10) μg/mL vs.(14.82±0.26) μg/mL,t=19.27,P=0.000;(1.27±0.10) μg/mL vs. (8.63±0.28) μg/mL,t=25.07,P=0.000]。TH2细胞因子[白介素-4(interleukin-4,IL-4)和白介素-13(interleukin-13,IL-13)]相对表达升高,且差异有统计学意义(P<0.05)。与模型Ⅰ组相比,模型Ⅱ组小鼠炎症性皮损和耳部肿胀程度减轻,棘细胞层的肥厚程度及炎性细胞的浸润程度减轻,血清总IgE水平下降[(14.82±0.26) μg/mL vs. (8.63±0.28) μg/mL,t=16.34,P=0.000],TH2炎症因子相对表达下降。结论:在C57BL/6遗传背景下的野生型和TRPA1敲基因小鼠上成功建立DNCB诱导的AD模型,发现TRPA1表达上调及TRPA1基因敲除后抑制了DNCB诱导的AD炎症,说明TRPA1在DNCB诱导的AD中可能发挥重要的作用,但具体机制有待进一步研究。
Abstract
Objective:To establish a model of 2,4-dinitrochlorobenzene(DNCB)-induced atopic dermatitis(AD) in wild-type and TRPA1-knockout C57BL/6 mice,and to investigate the role of TRPA1 in this model. Methods:Wild-type male C57BL/6 mice were randomly divided into experimental group and control group,with 6 mice in each group. A model of AD was established. In the sensitization stage,the mice were given external applica-tion of 2% DNCB and acetone-olive oil solution(3∶1) on the back and acetone-olive oil solution alone on the ears on day 1,and in the challenge stage,the mice were given external application of 0.5% DNCB on the back and acetone-olive oil solution alone on the ears on days 5,8,11,14,17,and 20,with 6 times of treatment in total. HE staining was performed for skin lesions to identify whether an AD model was established successfully. Western blot and qRT-PCR were used to measure the protein and mRNA expression of TRPA1,and immunohistochemistry was used to identify the location of TRPA1 and measure its protein expression. A total of 6 TRPA1+/+(wild-type) mice were randomly selected as model group Ⅰ,6 TRPA1-/-(knockout genotype) mice were selected as model group Ⅱ,and 6 TRPA1+/+ mice were selected as control group. An AD model was established according to the method above. After modeling,serum samples and skin lesions at the ears and the back were collected to measure related pathophys-iological indices. Results:Skin lesions and HE staining showed that an AD model was successfully established in wild-type mice. As for the experimental group,the results of qRT-PCR showed a significant increase in the relative mRNA expression of TRPA1 in the back lesions(t=4.93,P=0.008);Western blot showed a significant increase in the protein expression of TRPA1 in the back lesions,with a significant change in mean optical density(t=34.32,P=0.000);immunohistochemistry showed varying degrees of increases in the expression of TRPA1 in the epidermis and the dermis. The AD model established in TRPA1-knockout mice showed that compared with the control group,the model groups Ⅰ and Ⅱ had marked inflammatory skin lesions on the back and marked ear swelling. Pathological examination showed hyperkeratosis and/or parakeratosis,thickening of the prickle cell layer,inflammatory cell infiltration dominated by lymphocytes in the dermis,and increased infiltration of mast cells in the model groups Ⅰ and Ⅱ. There was an in-crease in total serum IgE level and there was a significant difference between the control group and the model groups Ⅰ/Ⅱ[(1.27±0.10) μg/mL vs. (14.82±0.26) μg/mL,t=19.27,P=0.000;(1.27±0.10) μg/mL vs. (8.63±0.28) μg/mL,t=25.07,P=0.000]. There were increases in the relative expression of the TH2 cytokines interleukin-4 and interleukin-13,and there were significant differences between groups(P<0.05). Compared with the model group Ⅰ,the model group Ⅱ had significant reductions in the severity of inflam-matory skin lesions,the degree of ear swelling,the thickening of the prickle cell layer,the degree of inflammatory cell infiltration,and the total serum IgE level[(14.82±0.26) μg/mL vs. (8.63±0.28) μg/mL,t=16.34,P=0.000],as well as significant reductions in the relative expression of TH2 cytokines. Conclusion:A model of DNCB-induced AD has been successfully established in wild-type and TRPA1-knockout C57BL/6 mice. Upregulation of TRPA1 expression and TRPA1 knockout inhibit DNCB-induced inflammation in AD,suggesting that TRPA1 may play an important role in DNCB-induced AD,but further studies are needed to clarify the specific mechanism.
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