# Improvement Effect of Membrane-Free Stem Cell Extract on
## 4 Discussion
Although DEX treatment also strongly inhibited AD-like symptoms, the body weight of the DEX group mice decreased significantly with repeated application of DEX. [PERSON] et al. [27] reported that DEX treatment (3 mg/kg body weight) reduced the body weight of 24-dinitrochlorobenzene-exposured NC/Nga mice in each experiment group. In addition, [PERSON] et al. [28] found that DEX application (10 mg/ear) significantly decreased the body weight of oxazoline-induced Balb/c mice compared to that of healthy mice. In our study, repeated topical application of MFSCE tends to show a therapeutic effect compared to the VEH group, without any side effects, including a decrease in body weight. Elevated serum IgE levels are a major characteristic of AD, and approximately 80% of patients with AD showed an increased concentration of IgE [20]. [PERSON] et al. [29] reported that systemic IgE elevation in serum is increased by inflammatory cytokines after repeated application of HDM. In the present study, repeated sensitization by HDM in the dorsal skin of NC/Nga mice stimulated the production of serum IgE, consequently increasing the concentration of serum inflammatory cytokines, including IL-4, IL-10, IFN-\(\gamma\), and TNF-\(\alpha\). However, treatment of the dorsal skin of NC/Nga mice with MFSCE inhibited both IgE and inflammatory cytokine levels in the serum. Interestingly, we found that the serum concentration of IFN-\(\gamma\) was markedly stimulated by HDM sensitization, and MFSCE downregulated this level in a dose-dependent manner. Evidence has shown that IFN-\(\gamma\) is an important factor in the severity and chronicity of skin inflammation in AD [30]. Moreover, the spontaneous production of IFN-\(\gamma\) is related to AD-like skin lesions in NC/Nga mice [31]. These data suggested that MFSCE suppressed chronic AD in part through systemic downregulation of IFN-\(\gamma\). Skin infiltration by mast cells is a common clinical phenomenon associated with AD-like skin lesions [32]. Elevated IgE antibodies in patients with AD have a high affinity for the specific receptor Fc epsilon RI on mast cells and stimulate mast cell degranulation, thereby releasing allergic mediators such as histamine, \(\beta\)-hexosaminidase, prostaglandandins, and leukotrienes, which are also associated with an increase in inflammatory cytokines (IL-4, IL-10, IFN-\(\gamma\), and TNF-\(\alpha\)) and chemokines (TARC) [33; 34]. TARC is expressed in the skin lesions of patients with AD, and its levels correlate with disease severity [35]. The expression of TARC is regulated by IFN-\(\gamma\) and various cytokines, including IL-4, IL-10, and TNF-\(\alpha\), which are produced by activated immune cells in the skin of AD patients [36; 37]. These cytokines bind to their specific receptors on keratinocytes, the predominant cell type in the epidermis, and activate a cascade of intracellular signaling pathways that lead to TARC expression [38]. Previous studies have verified these cytokines and chemokines overexpression in the skin of an allergic AD-like murine model and were used as markers of pathogen-stimulated allergic inflammation in an experimental murine model. In present study, the release and expression of IFN-\(\gamma\), especially when compared with the other inflammatory cytokines and chemokine, was remarkably inhibited by the MFSCE. These findings suggested that the MFSCE had the regulatory effect on IFN-\(\gamma\), consequently showing the protective effect on AD-related dysfunctions of HDM-sensitized NC/Nga mice. This cascade plays a significant role in the pathogenesis and aggravation of AD; therefore, targeting the production of cytokines and chemokines is a potential treatment strategy for AD. In our study, topical application of MFSCE in the dorsal skin decreased the expression levels of IL-4, IL-10, IFN-\(\gamma\), TNF-\(\alpha\), and TARC, as well as reduced mast cell infiltration and epidermal thickness.