The Skin Histopathology of Pro- and Parabiotics in a Mouse Model of Atopic Dermatitis

Abstract

As it has been revealed that the activation of human immune cells through the activity of intestinal microorganisms such as pro- and prebiotics plays a vital role, controlling the proliferation of beneficial bacteria and suppressing harmful bacteria in the intestine has become essential. The importance of probiotics, especially for skin health and the immune system, has led to the emergence of products in various forms, including probiotics, prebiotics, and parabiotics. In particular, atopic dermatitis (AD) produces hypersensitive immunosuppressive substances by promoting the differentiation and activity of immune regulatory T cells. As a result, it has been in the Th1 and Th2 immune balance through a mechanism that suppresses skin inflammation or allergic immune responses caused by bacteria. Furthermore, an immune mechanism has recently emerged that simultaneously controls the expression of IL-17 produced by Th17. Therefore, the anti-atopic effect was investigated by administering doses of anti-atopic candidate substances (Lactobacilus sakei CVL-001, Lactobacilus casei MCL, and Lactobacilus sakei CVL-001 Lactobacilus casei MCL mixed at a ratio of 4:3) in an atopy model using 2,4-dinitrochlorobenzene and observing symptom changes for 2 weeks to confirm the effect of pro-, para-, and mixed biotics on AD. First, the body weight and feed intake of the experimental animals were investigated, and total IgG and IgM were confirmed through blood biochemical tests. Afterward, histopathological staining was performed using H&E staining, Toluidine blue staining, Filaggrin staining, and CD8 antibody staining. In the treatment group, the hyperproliferation of the epidermal layer, the inflammatory cell infiltration of the dermal layer, the expression of CD8, the expression of filaggrin, and the secretion of mast cells were confirmed to be significantly reduced. Lastly, small intestine villi were observed through a scanning microscope, and scoring evaluation was performed through skin damage. Through these results, it was confirmed that AD was reduced when treated with pro-, para-, and mixed biotics containing probiotics and parabiotics.

Keywords: Lactobacilus casei MCL; Lactobacilus sakei CVL-001; anti-inflammation; atopic dermatitis (AD); parabiotics; probiotics.

Figure 1

Histologic features of DNCB-induced AD-like skin damage were evaluated using H&E staining. (A) H&E staining of experimental animal skin for anti-atopic effects of pro-, para-, and mixed biotics (H&E × 200). (B) Histopathologic score in AD-like skin lesions. H&E staining showed that pro-, para-, and mixed biotics all alleviated DNCB-induced inflammation in the epidermal layer, with a histologically significant reduction in the T3 group. The red line in the figure is the relative width of the tissue stain. The data represent the mean ± SD of three independent experiments. (## p < 0.01 vs. VC, * p < 0.05 vs. NC, ** p < 0.01 vs. NC).

Figure 2

Histologic features of CD8 antibody staining and Filaggrin staining of AD-like skin lesions with DNCB. (A) CD8 antibody staining of experimental animal skin for anti-atopic effects of pro-, para-, and mixed biotics. Increased expression of CD8 in the epidermal and dermal layers was observed in the DNCB-induced NC, but relatively decreased CD8 expression was observed in the T1, T2, and T3 groups. Similarly, (B) Filaggrin staining in AD showed an increase in the thickness of the epithelial layer in the DNCB-induced NC, but a decrease in T1, T2, and T3. The data represent the mean ± SD of three independent experiments. (# p < 0.05 vs. VC, ## p < 0.01 vs. VC, * p < 0.05 vs. NC, ** p < 0.01 vs. NC, *** p < 0.001 vs. NC).

Figure 3

Toluidine blue staining histologic features of AD-like skin lesions with DNCB. (A) Toluidine blue staining of experimental animal skin for anti-atopic effects of pro-, para-, and mixed biotics. (B) Mast cell count in AD-like skin lesions. Mast cells increased dramatically in the DNCB-induced group, but decreased in the T1, T2, and T3 groups, with a significant decrease in the T3 group. The red arrow is the estimated number of mast cells. Data represent the mean ± SD of three independent experiments. (## p < 0.01 vs. VC, * p < 0.05 vs. NC, ** p < 0.01 vs. NC).

Figure 4

(A) Visual assessment and average atopic score for each symptom between days 1–15 of atopy-induced skin damage with DNCB and scratching behavior scoring results. (B) Enlarged the condition of atopic dermatitis in each group. (C) Each item is scored as no symptoms (0), mild (1), moderate (2), or severe (3). Means with different superscripts in the same row are significantly different at p < 0.05 via Duncan’s multiple range tests. The data represent the mean ± SD of three independent experiments. (# p < 0.05, ## p < 0.01 vs. VC).