中华皮肤科杂志 ›› 2024, Vol. 57 ›› Issue (5): 435-444.doi: 10.35541/cjd.20230683

• 论著 • 上一篇    下一篇

角质形成细胞表达的S100A8/A9在3种常见皮肤炎症损伤模式中调控效应差异的研究

胡梦瑶1    李敏2    陈思涵2    魏雪翠1    陈宇杰2    徐松2    陈旭1，2   

  1. 1南京医科大学公共卫生学院，南京  211166；2中国医学科学院  北京协和医学院皮肤病研究所，南京  210042
  • 收稿日期:2023-11-21 修回日期:2024-03-11 发布日期:2024-04-30
  • 通讯作者: 陈旭 E-mail:chenx@pumcderm.cams.cn
  • 基金资助:
    国家自然科学基金（81972952、82173438、82273550）；江苏省高层次人才“六个一工程”拔尖人才科研项目（LGY2018095）；江苏省“六大人才高峰”高层次人才选拔培养资助计划项目（WSW-016）；中国医学科学院医学与健康科技创新工程项目（CIFMS-2021-I2M-1-059、CIFMS-2017-I2M-1-017）

Different regulatory effects of S100A8/A9 expressed by keratinocytes in three common inflammatory skin injury modes

Hu Mengyao1, Li Min2, Chen Sihan2, Wei Xuecui1, Chen Yujie2, Xu Song2, Chen Xu1,2   

  1. 1School of Public Health, Nanjing Medical University, Nanjing 211166, China; 2Institute of Dermatology, Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing 210042, China
  • Received:2023-11-21 Revised:2024-03-11 Published:2024-04-30
  • Contact: Chen Xu E-mail:chenx@pumcderm.cams.cn
  • Supported by:
    National Natural Science Foundation of China （81972952, 82173438, 82273550）; Jiangsu Provincial High-level Health Personnel "Six One Project" Top Talent Project （LGY2018095）; Six Talent Peaks Project of Jiangsu Province （WSW-016）; CAMS Innovation Fund for Medical Sciences （2021-I2M-1-059, 2017-I2M-1-017）

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摘要： 【摘要】 目的 研究皮肤光损伤、变应性接触性皮炎和银屑病这3种常见皮肤炎症损伤模式中角质形成细胞S100A8/A9表达的调控效应差异。方法 选取6 ~ 8周龄野生型C57BL/6JGpt小鼠分别进行以下几组实验：①使用单次中波紫外线（UVB）照射小鼠脱毛背部，构建皮肤光损伤模型（UVB组，n = 4），对照组不照射（n = 4）；②使用2，4-二硝基氯苯（DNCB）涂抹小鼠右耳构建变应性接触性皮炎模型（DNCB组，n = 4），小鼠左耳涂抹基质对照（对照组，n = 4）；③小鼠脱毛背部外用咪喹莫特乳膏构建银屑病样皮炎模型（咪喹莫特组，n = 4），对照组小鼠涂抹凡士林（n = 4）。使用苏木精-伊红（HE）染色检测各组小鼠皮肤样本组织病理变化，免疫组化及Western印迹法检测小鼠背部表皮或耳部皮损中S100A8和S100A9的表达。体外培养的HaCaT细胞分别进行以下几组实验：①UVB组细胞接受单次50 mJ/cm2 UVB照射，对照组不照射；②采用模拟变应性接触性皮炎中炎症环境的肿瘤坏死因子α（TNF-α）和γ干扰素（IFN-γ）（两者简称为TI）处理HaCaT细胞（TI组），对照组加入相应溶媒处理；③采用模拟银屑病样炎症环境的5种细胞因子［白细胞介素17A（IL-17A）、IL-22、IL-1α、抑瘤素M和TNF-α，简称为M5］处理HaCaT细胞（M5组），对照组加入相应溶媒。采用实时荧光定量PCR、Western印迹法和酶联免疫吸附试验分别测定S100A8和S100A9的转录、表达和胞外分泌水平。结果 免疫组化及Western印迹检测显示，在皮肤光损伤、变应性接触性皮炎和银屑病这3种炎症小鼠模型皮损组织中，S100A8、S100A9表达量均明显高于相应的对照组，且免疫组化结果显示，这两种蛋白在银屑病样皮炎模型中增多更为显著。体外细胞实验中，单次UVB、TI和M5分别处理HaCaT细胞12 h 后S100A8、S100A9转录水平明显高于相应对照组，处理24 h后亦均高于相应对照组［S100A8、S100A9 mRNA表达水平：UVB组比对照组分别为6.14 ± 0.60比1.00 ± 0.08，2.58 ± 0.06比1.02 ± 0.22，均P < 0.01；TI组比对照组分别为3.90 ± 0.75比1.00 ± 0.02，2.42 ± 0.30比1.01 ± 0.13，均P < 0.05；M5组比对照组分别为157.59 ± 9.30比1.00 ± 0.11，251.37 ± 6.63比1.00 ± 0.03，均P < 0.001］；24、48 h，UVB组、TI组和M5组的S100A8/A9分泌蛋白水平均显著高于相应的对照组（均P < 0.001），但其响应模式存在一些差异，在银屑病样皮炎模型中响应更为显著；同时，M5组HaCaT细胞中S100A8、S100A9胞内蛋白表达水平显著高于对照组（t = 4.66、4.63，均P < 0.01），而UVB组和TI组HaCaT细胞中S100A8、S100A9胞内蛋白表达水平低于对照组（UVB组比对照组：t = -3.75、-3.34，P = 0.020、0.029；TI组比对照组：t = -3.30、-4.50，P = 0.030、0.011）。结论 角质形成细胞在3种常见皮肤炎症损伤后响应活跃，其效应分子S100A8、S100A9作为机体的损伤相关分子密切参与UVB诱导的皮肤光损伤、变应性接触性皮炎和银屑病中炎症反应，尤其在银屑病样皮炎中响应可能更为显著。

关键词: 炎症, S100蛋白质类, 疾病模型, 动物, 皮炎, 变应性接触性, 银屑病, 中波紫外线, 皮肤光损伤, HaCaT细胞, S100A8/A9

Abstract: 【Abstract】 Objective To investigate different regulatory effects of S100A8/A9 expressed by keratinocytes in 3 common inflammatory skin injury modes: UVB-induced skin injury, allergic contact dermatitis, and psoriasis. Methods Wild-type C57BL/6JGpt mice aged 6 to 8 weeks were selected for the following experiments: （1） mouse models of UVB-induced skin injury were established by single exposure to ultraviolet B （UVB） radiation on the shaved dorsal skin of mice （UVB group, n = 4）, with the mice receiving no UVB radiation serving as a control group （n = 4）; （2） mouse models of allergic contact dermatitis were established by application of 2,4-dinitrochlorobenzene （DNCB） to the right ears of mice （DNCB group, n = 4）, with the left ears of mice treated with a vehicle control serving as a control group （n = 4）; （3） mouse models of psoriasis-like skin inflammation were established by topical application of imiquimod cream to the depilated dorsal skin of mice （imiquimod group, n = 4）, with the mice treated with vaseline serving as a control group （n = 4）. Hematoxylin-eosin （HE） staining was performed to assess histopathological changes in mouse skin tissues obtained from each group, and immunohistochemical study and Western blot analysis were performed to determine the expression of S100A8 and S100A9 in the mouse dorsal epidermis or ear skin lesions. In vitro cultured HaCaT cells were subjected to the following experiments: （1） cells in the UVB group were treated with a single UVB irradiation at a dose of 50 mJ/cm2, and cells in the control group received no irradiation; （2） some cells were treated with tumor necrosis factor-α （TNF-α） and interferon-γ （IFN-γ） （collectively referred to as TI）, and named as the TI group, which simulated the inflammatory environment in allergic contact dermatitis, while cells treated with corresponding solvents served as the control group; （3） cells were treated with 5 cytokines （interleukin 17A ［IL-17A］, IL-22, IL-1α, oncostatin M, and TNF-α, collectively referred to as M5）, and named as the M5 group, which simulated the inflammatory environment in psoriasis, while cells treated with corresponding solvents served as the control group. Real-time fluorescence-based quantitative PCR, Western blot analysis, and enzyme-linked immunosorbent assay were performed to determine the mRNA and protein expression of S100A8 and S100A9, and to detect the extracellular secretion level of S100A8/A9, respectively. Results Immunohistochemical study and Western blot analysis revealed that S100A8 and S100A9 expression levels were significantly higher in the skin lesions of mouse models of UVB-induced skin injury, allergic contact dermatitis, and psoriasis-like skin inflammation than in their corresponding control groups; immunohistochemical study further demonstrated that the increase in the expression of the two proteins was more pronounced in the mouse models of psoriasis-like skin inflammation. In the in vitro cell experiments, the mRNA expression of S100A8 and S100A9 in HaCaT cells at 12 and 24 hours were markedly higher in the UVB group （e.g., at 24 hours, 6.14 ± 0.60 vs. 1.00 ± 0.08, 2.58 ± 0.06 vs. 1.02 ± 0.22, respectively, both P < 0.01）, TI group （e.g., at 24 hours, 3.90 ± 0.75 vs. 1.00 ± 0.02, 2.42 ± 0.30 vs. 1.01 ± 0.13, respectively, both P < 0.05）, and M5 group （e.g., at 24 hours, 157.59 ± 9.30 vs. 1.00 ± 0.11, 251.37 ± 6.63 vs. 1.00 ± 0.03, both P < 0.001） than in the corresponding control groups, so were the extracellular secretion levels of S100A8/A9 at 24 and 48 hours （all P < 0.001）, with some differences observed in their response patterns; notably, the response was more pronounced in the mouse model of psoriasis-like skin inflammation. Additionally, the protein expression levels of S100A8 and S100A9 in HaCaT cells were significantly higher in the M5 group than in the control group （t = 4.66, 4.63, respectively, both P < 0.01）, but were significantly lower in the UVB group （t = -3.75, -3.34, P = 0.020, 0.029, respectively） and TI group （t = -3.30, -4.50, P = 0.030, 0.011, respectively） than in the control groups. Conclusion Keratinocytes exhibited active responses following 3 common inflammatory skin injuries, with their effector molecules S100A8 and S100A9, as damage-associated molecular patterns, playing crucial roles in UVB-induced skin injury, allergic contact dermatitis, and psoriasis, and the response seemed to be more pronounced in psoriasis-like dermatitis.

Key words: Inflammation, S100 proteins, Disease models, animal, Dermatitis, allergic contact, Psoriasis, UVB, UVB-induced skin injury, HaCaT cells, S100A8/A9

引用本文

胡梦瑶 李敏 陈思涵 魏雪翠 陈宇杰 徐松 陈旭. 角质形成细胞表达的S100A8/A9在3种常见皮肤炎症损伤模式中调控效应差异的研究[J]. 中华皮肤科杂志, 2024,57(5):435-444. doi:10.35541/cjd.20230683

Hu Mengyao, Li Min, Chen Sihan, Wei Xuecui, Chen Yujie, Xu Song, Chen Xu, . Different regulatory effects of S100A8/A9 expressed by keratinocytes in three common inflammatory skin injury modes[J]. Chinese Journal of Dermatology, 2024, 57(5): 435-444.doi:10.35541/cjd.20230683