卵清蛋白诱导的特应性皮炎小鼠模型中白细胞介素-25的作用及其调控意义

doi:10.19723/j.issn.1671-167X.2024.05.002

摘要/Abstract

摘要：

目的: 探讨白细胞介素-25(interleukin-25，IL-25)对卵清蛋白(ovalbumin，OVA)诱导的特应性皮炎小鼠模型的影响，以及调控IL-25的意义。方法: 将90只健康雄性6周龄无特定病原体(specific pathogen free, SPF)级BALB/c小鼠分为6组(每组15只)，分别为: ①皮下注射磷酸盐缓冲液(phosphate buffered saline, PBS)组(正常对照组); ②皮下注射小鼠IL-25组(IL-25组); ③皮下注射抗小鼠IL-25单克隆抗体组(anti-IL-25组)，每日皮下注射1次×1周，间隔2周，重复每日皮下注射1次×1周，间隔2周，再重复每日皮下注射1次×1周，总共7周; ④ OVA致敏组(模型组); ⑤ OVA致敏及IL-25皮下注射组(IL-25干预致敏组); ⑥ OVA致敏及anti-IL-25注射组(anti-IL-25干预致敏组)。⑤⑥组在致敏过程中给予IL-25或anti-IL-25的方式同②③组。致敏期间观察比较小鼠的搔抓行为和皮肤表现，致敏结束24 h后由小鼠心脏取血，分离血清，采用酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测总IgE、IL-4、IL-5、IL-13等。取致敏部位的皮肤进行苏木精-伊红(hematoxylin-eosin，HE)染色、免疫组织化学、实时PCR(real-time PCR)及Western blot检测。采用单因素(ANOVA)方差分析比较各组间各项指标的差异。结果: 实验小鼠末次处理24 h后，IL-25干预致敏组的搔抓次数高于模型组，anti-IL-25干预致敏组的搔抓行为显著低于模型组; IL-25干预致敏组特应性皮炎表现、表皮增厚及真皮炎细胞浸润程度均明显重于模型组及anti-IL-25干预致敏组; IL-25干预致敏组血清IgE、IL-4、IL-5、IL-13水平显著高于模型组及anti-IL-25干预致敏组; IL-25干预致敏组CD4⁺ T细胞在真皮层较anti-IL-25干预致敏组显著增多; IL-25干预致敏组的丝聚蛋白(filaggrin)及防御素β2(defensin β2)蛋白水平明显低于模型组或anti-IL-25干预致敏组。结论: 在OVA诱导的皮炎模型中，IL-25能够明显促进小鼠表皮屏障功能损害，加重OVA诱导的皮炎损害，拮抗IL-25可一定程度上缓解OVA诱导的皮炎损害。

关键词: 特应性皮炎, 白细胞介素-25, 卵清蛋白, 动物疾病模型

Abstract:

Objective: To investigate the effect of interleukin-25 (IL-25) on ovalbumin (OVA) induced atopic dermatitis of mice, and the significance of regulating IL-25. Methods: In this study, 90 healthy male 6-week-old specific pathogen free (SPF) BALB/c mice were divided into 6 groups (15 in each group): ① subcutaneous injection of phosphate buffered saline (PBS) group (normal control group); ② subcutaneous injection of mouse IL-25 group (IL-25 group); ③ subcutaneous injection of anti-mouse IL-25 monoclonal antibody (anti-IL-25 group), each group received subcutaneous injection once a day for 1 week, 2 weeks apart, repeated daily subcutaneous injections for 1 week, 2 weeks apart, and repeated daily subcutaneous injections for 1 week, for a total of 7 weeks; ④ OVA treated group (model group); ⑤ OVA treated and IL-25 subcutaneous injection group (IL-25 treated dermatitis group); ⑥ OVA treated and anti-mouse IL-25 monoclonal antibody injection group (anti-IL-25 treated dermatitis group). The ⑤ and ⑥ groups in the process of treatment with OVA, IL-25 or anti-IL-25 antibody were given in the same way as the ② and ③ groups. Scratching behavior and skin performance of the mice were recorded during the seven-week-treatment. Twenty four hours after the final treatment, blood was taken from the mouse heart, and the serum was separated to detect the total IgE, IL-4, IL-5, IL-13, etc. The skin samples of the treatment sites were used for hematoxylin-eosin (HE) staining, immunohistochemistry, real-time PCR and Western blot detections. A single factor (ANOVA) analysis of variance was used to compare the differences in various indicators between the groups. Results: The frequency of scratches in the IL-25 treated dermatitis group was higher than that in the model group, and the scratching behavior of the anti-IL-25 treated dermatitis group was significantly lower than that in the model group. The appearance of atopic dermatitis, thickening of the epidermis and the degree of dermal inflammation in the IL-25 treated dermatitis group were more serious than those in the model group and the anti-IL-25 treated dermatitis group. The levels of serum IgE, IL-4, IL-5, and IL-13 in the IL-25 treated dermatitis group were significantly higher than that in the model group and the anti-IL-25 treated dermatitis group. There were significantly more CD4⁺ T cells in the dermis of IL-25 treated dermatitis group than that in the anti-IL-25 treated dermatitis group. The expression levels of filaggrin and defensin β2 proteins in the IL-25 treated dermatitis group were significantly lower than those in the model group and the anti-IL-25 treated dermatitis group. Conclusion: In the OVA induced atopic dermatitis mice model, IL-25 can significantly promote the damage of the epidermal barrier function and aggravate the OVA-induced dermatitis. Antagonizing IL-25 can alleviate OVA induced dermatitis to a certain extent.

Key words: Atopic dermatitis, Interleukin-25, Ovalbumin, Animal disease models

中图分类号:

R758.2

金江, 陈雪, 赵琰, 贾军, 张建中. 卵清蛋白诱导的特应性皮炎小鼠模型中白细胞介素-25的作用及其调控意义[J]. 北京大学学报（医学版）, 2024, 56(5): 756-762.

Jiang JIN, Xue CHEN, Yan ZHAO, Jun JIA, Jianzhong ZHANG. The role and its regulatory significance of interleukin-25 in ovalbumin induced atopic dermatitis of mice[J]. Journal of Peking University (Health Sciences), 2024, 56(5): 756-762.

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Gene	Primer	Sequence
IL-25	Forward	5′-CAGCAAAGAGCAAGAACC-3′
IL-25	Reverse	5′-CCCTGTCCAACTCATAGC-3′
GAPDH	Forward	5′-AACAGGCGTCCCTTTCCGA-3′
GAPDH	Reverse	5′-GCCCAAGATGCCCTTCAGT-3′

Items	Normal	IL-25	Anti-IL-25	OVA	OVA (IL-25)	OVA (anti-IL-25)
IgE/(μg/L)	27.9±3.5	37.9±2.6^aa	28.2±2.5	42.6±6.2^c	66.3±8.3^d	39.0±6.0^b
IL-4/(ng/L)	26.2±3.6	37.0±11.1	27.9±6.3	47.1±7.7^c	68.1±8.5^d	42.8±15.9^bb
IL-5/(ng/L)	314.6±58.1	633.9±81.1^aa	325.7±72.9	784.2±86.7^cc	959.8±90.2^dd	696.7±84.3
IL-13/(ng/L)	334.2±29.7	424.9±66.9	349.0±48.0	507.0±24.9^cc	588.4±12.7^dd	473.7±24.7^bb

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