DNCB was bought from Sigma-Aldrich (St

DNCB was bought from Sigma-Aldrich (St. in skin lesions. Moreover, MMB reduced the expression of CD80, CD86, MHCII and mRNA of OX40L in TSLP-activated dendritic cells. In general, our study suggests that MMB can improve the symptoms of AD and topical application of MMB can become a promising new therapy strategy for AD. 0.001) and the 0.1% MMB group decreased significantly from the 24th day ( 0.001), compared with vehicle group. Open in a separate window Figure 1 Momelotinib (MMB) ointment macroscopically alleviated the symptom of AD mice. (A) The skin lesion manifestation of groups on 28th day: (a) The normal group; (b) the vehicle group; (c) the 0.1% MMB group; (d) the 0.2% Iloprost MMB group; (e) the 0.5% MMB group; and (f) the tacrolimus group. (B) The severity scores of skin lesion changes over time during the experiments. (C)The weight of mice on the 28th day of the experiment. Values are mean SD (= 6). 0.05, 0.01, versus the vehicle group. Meanwhile, we weighed the mice on the 28th day of the experiment (Figure 1C), and found that the 0.5% MMB group Iloprost was significantly higher than that of the vehicle group ( 0.05) while the tacrolimus group was significantly lower than that of the vehicle group ( 0.01). 2.2. The Histological Effect of MMB on AD HematoxylinCeosin (H&E) stains of skin lesion tissue showed that the reduction of epidermal thickness and inflammatory cells were observed in all treatment groups, most significantly in the 0.5% MMB group (Figure 2A,B). Toluidine blue stains of skin lesion tissue showed that reduced mast cells were observed in MMB groups. However, tacrolimus failed to inhibit the number of mast cells (Figure 2C,D). Above results revealed the topical application of MMB can improve the symptoms of AD histologically. Open in a separate window Figure 2 Skin lesions were stained with H&E and toluidine blue (100 magnification, A,C) to represent the thickness of epidermis and number of mast cells respectively (200 magnification, B,D). (a) The normal group, (b) the vehicle group, (c) the 0.1% MMB group, (d) the 0.2% MMB group, (e) the 0.5% MMB group, and (f) the tacrolimus group. Values are mean SD (= 6). 0.01, 0.001 versus the vehicle group. 2.3. MMB Suppresses IgE Generation Total serum IgE was measured by ELISA. The result displayed that the IgE concentration in vehicle group increased by six times compared Iloprost with normal group, showing obvious AD-like symptoms. Meanwhile, the MMB groups resulted in a marked decrease of serum IgE. However, no dose effect was shown. Unexpectedly, tacrolimus failed to inhibit the elevation of serum IgE levels. The reason may be that in hapten-induced AD model, tacrolimus cooperated with hapten to increase RAB25 serum IgE, and this is also demonstrated by other studies [17,18] (Figure 3). Open in a separate window Figure 3 The total IgE levels in mice serum of different groups. Values are mean SD (= 6). 0.01, 0.001 versus the vehicle group. 2.4. MMB Suppresses the Increased mRNA Expression of Cytokines in Skin Lesion The effect of MMB on the mRNA expression of cytokines in lesion tissues was detected by RT-qPCR in the work. The mRNA expression of IL-4, IL-5, and IFN- (Figure 4ACC) were increased in the vehicle group and decreased expression was observed in all MMB and tacrolimus groups in a dose-dependent manner. Furthermore, the mRNA expression of TSLP (Figure 4D) was notably decreased in all MMB and tacrolimus groups, compared with the vehicle group. However, no dose dependence was observed in MMB groups. Open in a separate window Figure 4 The relative mRNA expression of IL-4 (A), IL-5 (B), IFN- (C), and TSLP (D) in different groups. Values are mean SD (= 6). 0.01, 0.001 versus the vehicle group. 2.5. MMB Suppressed Expression of p-STAT1, p-STAT3, and p-STAT5 Proteins in Skin Lesions Western blot results exhibited that the expression of p-STAT1, p-STAT3, and p-STAT5 proteins were observably increased in the vehicle group. In the 0.5% MMB group, the expressions of p-STAT1, Iloprost p-STAT3, and p-STAT5 were markedly inhibited (Figure 5). These results suggest that the JAK/STAT signaling pathway may be participated in the stimulation of DNCB-induced atopic dermatitis mice. Moreover, the effectiveness of MMB on AD mice may contribute to the down-regulation of p-STAT1, p-STAT3, and p-STAT5 proteins. Open in a separate window Figure 5 The protein levels of p-STAT1; p-STAT3; p-STAT5; and STAT-1, STAT-3, and STAT5 in normal, vehicle, and 0.5% MMB groups, respectively. (A) Western blot and (B) quantitative analysis of dot intensity. Actin was used as a reference protein. Values are mean SD (= 3). * 0.01, 0.001, n.s.: no statistical significance. 2.6. MMB Suppressed the Costimulatory Molecules, and.