Cicadidae Periostracum Attenuates Atopic Dermatitis Symptoms and Pathology via the Regulation of NLRP3 Inflammasome Activation

Figure 7

CP treatments modulate ROS production and NLRP3 inflammasome activation in vitro. Two different NLRP3 inflammasome activators, TNF-α/IFN-γ or MSU, were used in HaCaT keratinocyte and 3HSE. (a) Hematoxylin and eosin staining was used to identify structures in cells and tissues, in 3HSE stimulated with TNF-α/IFN-γ, in the presence or absence of CP (31.3 or 62.5 μg/mL). (b, c) ROS levels were measured by the DCFDA assay, in HaCaT cells stimulated with TNF-α/IFN-γ, in the presence or absence of CP (31.3 or 62.5 μg/mL). (d) TSLP and NLRP3 expression levels were measured by Western blot in HaCaT cells. Normalized values of (f) TSLP and (g) NLRP3 expression are represented. (e) NLRP3, ASC, and cleaved caspase-1 expression levels were also measured in HaCaT cells pretreated (or not) with CP (62.5 μg/mL) or MCC950 (10 μM) and stimulated with monosodium urate crystal (MSU). Western blot was performed to determine the effect of CP on the expression of NLRP3 inflammasome activation. Results were normalized and are represented for (h) NLRP3, (i) ASC, and (j) cleaved caspase-1 for (e). (k) TSLP and NLRP3 expression levels were measured by Western blot in 3HSE. Normalized values of (m) TSLP and (n) NLRP3 expression are represented. (l) NLRP3, ASC, and cleaved caspase-1 expression levels were also measured in 3HSE pretreated (or not) with CP (62.5 μg/mL) or MCC950 (10 μM) and stimulated with monosodium urate crystal (MSU). Western blot was performed to determine the effect of CP on the expression of NLRP3 inflammasome activation. Results were normalized and are represented for (o) NLRP3, (p) ASC, and (q) cleaved caspase-1 for (l). MCC950 is an inhibitor of NLRP3. All data are represented per group, as of three independent experiments. Statistical differences were evaluated using the one-way ANOVA test, with Dunnett’s post hoc analysis, and are represented as follows: ^## and ^### compared with nontreated cells; and compared with TNF-α/IFN-γ- or MSU-treated cells.