2,4-二硝基氟苯诱导特应性皮炎氧化应激模型的建立及评价

doi:10.12300/j.issn.1674-5817.2025.044

摘要/Abstract

摘要：

目的应用2，4-二硝基氟苯（2，4-dinitrofluorobenzene，DNFB）涂抹KM小鼠背部及耳后皮肤，建立特应性皮炎（atopic dermatitis，AD）氧化应激小鼠模型，以评价晚期糖基化终末产物受体-核苷酸结合寡聚化结构域样受体家族pyrin域包含蛋白3轴（receptor for advanced glycation end products-NOD-like receptor family，pyrin domain containing 3 axis，RAGE-NLRP3 axis）在AD氧化应激中的调控作用，从而为AD提供潜在的治疗靶点。方法选取20只SPF级雌性KM小鼠，随机分为对照组（Control）和实验组（DNFB），每组10只。Control组小鼠背部和耳后涂抹丙酮-橄榄油基质（丙酮∶橄榄油=3∶1），DNFB组小鼠背部和耳后涂抹0.5% DNFB（按100 mL丙酮-橄榄油基质加入0.5 g DNFB比例配制），每日1次，连续14 d。分别在处理后第2、4、6、9、12、14天时对小鼠的皮损症状严重程度进行评分，并在处理第14天时对小鼠的搔抓行为和耳缘厚度进行评价。实验结束当天，用游标卡尺测量小鼠双耳厚度，测量3次取平均值，以评估耳部肿胀程度。经HE染色来观测各组小鼠背部皮肤组织内的细胞形态和结构变化。通过实时荧光定量PCR、蛋白质印迹法和免疫组织化学染色法来检测各组小鼠皮肤组织中RAGE mRNA和蛋白的表达情况。并用实时荧光定量PCR法检测氧化应激相关分子NLRP3、半胱氨酸天冬氨酸蛋白酶-1（cysteine-dependent aspartate-specific protease 1，caspase-1）、白细胞介素-1β（interleukin-1 beta，IL-1β）的mRNA表达变化。结果处理第14天，Control组和DNFB组小鼠背部皮损评分分别为（0.20±0.42）分和（9.93±1.30）分（P<0.000 1），小鼠搔抓行为评分分别为（5.00±2.05）次和（49.26±8.49）次（P<0.000 1），耳缘厚度分别为（213.00±11.87）μm和（765.93±140.47）μm（P<0.000 1）；DNFB组小鼠背部处置部位皮肤出现明显干燥、脱屑、增厚等情况。HE染色结果显示，DNFB组小鼠皮肤炎症反应明显，符合AD的病理特征。实时荧光定量PCR和蛋白质印迹结果显示，与Control组相比，DNFB组小鼠皮肤组织中RAGE mRNA表达水平明显增加（P＜0.05），RAGE蛋白表达水平也明显增加（P＜0.01）。免疫组织化学染色结果显示，与Control组相比，DNFB组小鼠皮肤组织角质层增厚，间质内成纤维细胞纤维化增生，RAGE蛋白在小鼠皮肤组织中明显增加（P<0.01）。实时荧光定量PCR检测结果显示，DNFB组小鼠皮肤组织中NLRP3、caspase-1和IL-1β mRNA表达水平均明显增加（P＜0.01）。结论通过涂抹DNFB成功构建了AD小鼠氧化应激模型。该模型提示，RAGE可能通过调控NLRP3炎症小体与IL-1β释放，形成氧化-炎症级联反应，从而促进AD发生。这表明，RAGE可能是AD治疗的潜在靶点。

关键词: 2，4-二硝基氟苯, 特应性皮炎, 炎症反应, 氧化应激, KM小鼠

Abstract:

Objective To establish an oxidative stress mouse model of atopic dermatitis (AD) by applying 2,4-dinitrofluorobenzene (DNFB) to the back and post-auricular skin of KM mice, and to evaluate the regulatory role of the RAGE-NLRP3 axis (receptor for advanced glycation end products-NOD-like receptor family, pyrin domain containing 3 axis) in AD-related oxidative stress, thereby providing a potential therapeutic target for AD treatment. Methods Twenty SPF-grade female KM mice were randomly divided into a control group (Control group) and an experimental group (DNFB group), with 10 mice in each group. Mice in the Control group were treated with an acetone-olive oil vehicle (acetone: olive oil = 3:1) on their back and post-auricular skin. Mice in the DNFB group were treated with 0.5% DNFB (prepared by adding 0.5 g DNFB per 100 mL of acetone-olive oil vehicle) on the same areas, once daily for 14 consecutive days. The severity of skin lesions was scored on days 2, 4, 6, 9, 12, and 14 of treatment. On day 14, scratching behavior and ear thickness were evaluated. Ear swelling was evaluated on the final day by measuring bilateral ear thickness three times with a vernier caliper; the three measurements were averaged. HE staining was used to observe morphological and structural changes of cells in the back skin tissues. The mRNA and protein expression levels of RAGE (receptor for advanced glycation end products) in skin tissues were detected by quantitative real-time PCR, Western blot, and immunohistochemical staining. The mRNA expression levels of oxidative stress-related molecules, including NLRP3 (NOD-like receptor family, pyrin domain containing 3), caspase-1 (cysteine-dependent aspartate-specific protease 1), and IL-1β (Interleukin-1β), were detected by quantitative real-time PCR. Results On day 14, the back skin lesion scores of the Control group and DNFB group were (0.20±0.42) and (9.93±1.30) (P<0.000 1), respectively. Scratching behavior scores were (5.00±2.05) and (49.26±8.49) episodes, respectively (P<0.000 1), and ear thicknesses were (213.00±11.87) μm and (765.93±140.47) μm (P<0.000 1), respectively. The DNFB group exhibited marked skin dryness, desquamation, and thickening. HE staining results showed that skin inflammation was obvious in the DNFB group, consistent with the pathological features of AD. Quantitative real-time PCR and Western blot results showed that compared with the Control group, the mRNA expression level of RAGE in skin tissues of the DNFB group was significantly increased (P<0.05), and the protein expression level of RAGE was also significantly increased (P<0.01). Immunohistochemical staining results showed that compared with the Control group, skin tissue sections of the DNFB group exhibited thickened stratum corneum and fibrotic proliferation of fibroblasts in the interstitium under microscopic observation, with a significant increase in RAGE protein expression in the skin tissues (P<0.01). Quantitative real-time PCR results showed that the mRNA expression levels of NLRP3, caspase-1, and IL-1β in skin tissues of the DNFB group were all significantly increased (P<0.01). Conclusion The AD mouse oxidative stress model has been successfully established by topical DNFB application. RAGE may promote the development of AD by regulating the NLRP3 inflammasome and IL-1β release, forming an oxidative-inflammatory cascade, suggesting that it could be a potential therapeutic target for AD.

Key words: 2,4-dinitrofluorobenzene, Atopic dermatitis, Inflammatory response, Oxidative stress, KM mice

中图分类号:

Q95-33

刘畅,项雪松,贺辉煌,等. 2,4-二硝基氟苯诱导特应性皮炎氧化应激模型的建立及评价[J]. 实验动物与比较医学, 2026, 46(1): 46-54. DOI: 10.12300/j.issn.1674-5817.2025.044.

LIU Chang,XIANG Xuesong,HE Huihuang,et al. Establishment and Evaluation of an Oxidative Stress Model of Atopic Dermatitis Induced by 2,4-dinitrofluorobenzene[J]. Laboratory Animal and Comparative Medicine, 2026, 46(1): 46-54. DOI: 10.12300/j.issn.1674-5817.2025.044.

图/表 4

表1 实时荧光定量PCR引物序列

Table 1 Primer sequences for quantitative real-time PCR

引物名称 Primer names	引物序列 Sequences（5'→ 3'）
RAGE-F	ACCGAGTCCGTGTCTACCGTAAG
RAGE-R	ATGAGGCCAGTGGAAGTCAGAGG
NLRP3-F	CGGTGGAGTGTCGGAGAAGAG
NLRP3-R	GATGCTGTCATTGTCCTGGTGTC
Caspase-1-F	GATGGCGATACACTCAGAGAACAC
Caspase-1-R	CAAGGAGGCAACTGGAGGAATG
IL-1β-F	CGAGGCACAAGGCACAACAG
IL-1β-R	ACAAAGAGGCAGAGAGACAGAGAG
GAPDH-F	AATTCCATGGCACCGTCAAG
GAPDH-R	AGCATCGCCCCACTTGATTT

图1 DNFB诱导的特应性皮炎小鼠模型皮肤炎症损伤效果及评价注：A，造模结束后对照组和实验组背部皮肤状况；B，造模结束后，Control组和DNFB组10 min内小鼠搔抓次数评价（n＝10）；C，造模结束后，Control组和DNFB组小鼠耳朵肿胀程度评价（n＝10）；D，造模第2、4、6、9、12、14天两组小鼠皮损严重程度评分情况（n＝10）；E，苏木精-伊红染色检测小鼠背部皮肤组织病理学变化（比例尺为100 μm）。Control，对照组；DNFB，2，4-二硝基氟苯诱导实验组。与对照组比较，****P<0.000 1。

Figure 1 Assessment of skin inflammation and injury in a DNFB-induced mouse model of atopic dermatitisNote： A， skin conditions on the back of the control group and the DNFB group after modeling； B， scratching times of mice in the control group and DNFB group within 10 min after modeling （n=10）； C， evaluation of ear swelling degree in Control group and DNFB group after modeling （n=10）； D， on day 2， 4， 6， 9， 12， and 14 of modeling， the severity of skin lesions in the two groups was scored （n=10）. E， hematoxylin and eosin staining of the dorsal skin tissues of mice to evaluate histopathological changes（scale bar is 100 μm）. Control， the control group； DNFB， the 2，4-dinitrofluorobenzene-induced experimental group. Compared with the control group， ****P<0.000 1.

图2 DNFB诱导的特应性皮炎小鼠模型皮肤组织中RAGE表达水平的变化注：A，实时荧光定量PCR检测皮肤组织RAGE基因的mRNA表达水平（n＝10）；B~C，蛋白质印迹法检测皮肤组织RAGE蛋白表达及其定量分析（n＝10）；D，免疫组织化学染色法检测小鼠背部皮肤组织RAGE蛋白表达（比例尺为100 μm）。Control，对照组；DNFB，2，4-二硝基氟苯诱导实验组。与对照组相比，*P<0.05，**P<0.01。

Figure 2 Changes in RAGE expression levels in skin tissues of mice in the DNFB-induced atopic dermatitis mouse modelNote： A， quantitative real-time PCR analysis of RAGE mRNA expression levels in skin tissues（n=10）； B-C， Western blot analysis of RAGE protein expression and quantitative analysis in skin tissues（n=10）； D， immunohistochemical staining of RAGE protein expression in the dorsal skin tissues of mice （scale bar is 100 μm）. Control， the control group； DNFB， the 2，4-dinitrofluorobenzene-induced experimental group. Compared with the control group， *P<0.05， **P<0.01.

图3 DNFB诱导的特应性皮炎小鼠模型皮肤组织中IL-1β、NLRP3和caspase-1 mRNA水平变化注：A，实时荧光定量PCR检测皮肤组织IL-1β基因的mRNA表达水平（n＝10）；B，皮肤组织NLRP3基因的mRNA表达水平（n＝10）；C，皮肤组织caspase-1基因的mRNA表达水平（n＝10）。Control，对照组；DNFB，2，4-二硝基氟苯诱导实验组。与对照组相比，**P<0.01，****P<0.000 1。

Figure 3 Changes in mRNA levels of IL-1β，NLRP3， and caspase-1 in skin tissues of atopic dermatitis mice induced by DNFBNote： A， quantitative real-time PCR analysis of mRNA expression levels of IL-1β gene in skin tissues （n=10）； B， mRNA expression level of NLRP3 gene in skin tissues（n=10）； C， mRNA expression level of caspase-1 gene in skin tissues （n=10）. Control， the control group； DNFB， the 2，4-dinitro-fluorobenzene induced experimental group. Compared with the control group， **P<0.01， ****P<0.000 1.

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