关键词: 皮炎, 特应性, 疾病模型, 动物, 二硝基氯苯, 棒杆菌属, 皮肤菌群, 皮肤屏障

Abstract: 【Abstract】 Objective To investigate characteristics and changes of skin microbiota in atopic dermatitis-like mouse models induced by different concentrations of 2,4-dinitrochlorobenzene （DNCB）. Methods Totally, 30 male specific-pathogen-free BALB/c mice were randomly divided into 3 groups by using a random number table: negative control group topically treated with 200 μl of mixture of acetone and olive oil at a volume ratio of 3∶1 on the back twice a week for 6 consecutive weeks; high- and low-concentration DNCB groups both topically treated with 200 μl of 1% DNCB on the first and third day at the first week, followed by topical application of 200 μl of 0.5% and 0.1% DNCB, respectively, twice a week for 5 weeks from the second week. Twenty-four hours after the last treatment, the severity of skin lesions was evaluated, and the transepidermal water loss and stratum corneum hydration were measured. After the experiment, the mice were sacrificed, and skin tissues were resected from the back of the mice for histopathological examination. Full-thickness skin tissue samples were obtained from the back of 3 mice in each group. Illumina Miseq PE300 high-throughput sequencing was performed to sequence the V3 -V4 variable region of 16S rRNA gene of skin microbiota on the back of the mice, and the composition and structure of the skin microbiota and changes in the relative abundance of different genera were analyzed. One-way analysis of variance was used to analyze differences in indices among the 3 groups, and the Games-Howell method was used for multiple comparisons. Results The severity scores of skin lesions were significantly higher in the high- and low-concentration DNCB groups （9.83 ± 2.45 points, 2.71 ± 0.56 points, respectively） than in the negative control group （0.51 ± 0.12 points, t = -7.19, -2.85, respectively, both P < 0.05）. Compared with the negative control group, the high- and low-concentration DNCB groups showed significantly increased transepidermal water loss （t = -7.72, -2.68, respectively, both P < 0.05）, but significantly decreased stratum corneum hydration （t = 6.77, 5.99, respectively, both P < 0.05）; the transepidermal water loss was significantly higher in the high-concentration DNCB group than in the low-concentration DNCB group （t = 2.76, P < 0.05）, while no significant difference in the stratum corneum hydration was observed between the high- and low-concentration DNCB groups （P > 0.05）. There was a significant difference in the relative abundance of Corynebacterium among the 3 groups （F = 249.85, P < 0.001）, which was highest in the high-concentration DNCB group. No significant differences in the observed species and Chao1 index of the skin samples were observed among the 3 groups （both P > 0.05）, and the Shannon index was significantly lower in the high-concentration DNCB group than in the low-concentration DNCB group and negative control group （t = 6.96, -6.37, respectively, both P < 0.05）. Conclusion DNCB could induce atopic dermatitis-like dermatitis in mice, and the severity of skin lesions and degree of barrier function impairment were related to the concentration of DNCB; the species diversity of skin microbiota markedly decreased in the high-concentration DNCB group, indicating that high-concentration DNCB modeling has more advantages in studying microbiological changes associated with atopic dermatitis.

Key words: Dermatitis, atopic, Disease models, animal, Dinitrochlorobenzene, Corynebacterium, Skin bacterial flora, Skin barrier