Fig 2: The impact of polyinosinic:polycytidylic acid [poly (I:C)] treatment on serum IL-4, TSLP, IL-13, and IFN-γ was assessed in a murine model of MC903-induced atopic dermatitis (AD)-like disease (A-D). Tissue TSLP and IL-13 levels were additionally measured using enzyme-linked immunosorbent assay (ELISA) kits with normalization to total protein concentrations as quantified by bicinchoninic acid (BCA) assay (E,F). Western blotting analyses of TLR3, TSLP, and IL-13Rα1 levels (G-J). Densitometric analyses are representative of 3 independent experiments shown in Figure S1. Immunohistochemical analyses of TSLP+ and IL-13Rα1+ cells in tissue sections. Scale bar = 100 µm (K). Data are expressed as means ± SD, in (A-F), n=10/group, in (G-J), n=3/group. *, P<0.05, **, P<0.01, ***, P<0.001 vs. MC903 + vehicle treatment group.

Fig 3: Isoliquiritin downregulates the expression of proinflammatory factors in an AD model using NC/Nga mice. Concentrations of IL-4, IgE, TSLP, and IL-6 in serum were determined by ELISA. Results are the mean ± SD; one-way ANOVA; data in bar charts are the mean ± SEM of 6 mice per group (***P<0.001, ****P<0.0001 vs. control group; ####P<0.0001 vs. model group). ISO, isoliquiritin; DEX, dexamethasone; AD, atopic dermatitis; TSLP, thymic stromal lymphopoietin; DNCB, 1-chloro-2,4-dinitrochlorobenzene; SD, standard deviation; ANOVA, Analysis of Variance; SEM, standard error of mean.

Fig 4: Activation of NLRP3 partially averted the alleviating effect of CQ on type 2 inflammatory response in AD mice. (A) Western blot to test the expression levels of NLRP3, ASC and cleaved caspase-1 proteins; (B) the severity of ear skin lesions; (C) dermatitis severity score; (D) left ear thickness; (E) H&E staining; (F) TB staining; (G) ELISA detection of serum TSLP, IL-4, IL-13, IgE, IL-1β and IL-18 levels; (H) flow cytometry to assess the content of Th2 cells (CD3+CD4+CD193+). N = 6. Data were represented as mean ± standard deviation, and independent sample t-test was employed for comparisons in panels A-B/E-H. Two-way ANOVA was used in panels C-D, and Šídák’s multiple comparisons test was conducted for post hoc test. *P < 0.05, **P < 0.01.

Fig 5: CQ abated type 2 inflammatory response in AD mice by inactivating TLR3. (A) Western blot detection of TLR3 protein expression; (B) the severity of ear skin lesions; (C) dermatitis severity score; (D) left ear thickness; (E) H&E staining; (F) TB staining; (G) ELISA detections of serum TSLP, IL-4, IL-13, IFN-γ, and IgE levels; (H) flow cytometry to assess the content of Th2 cells (CD3+CD4+CD193+). N = 6. Data were represented as mean ± standard deviation. Data in panel A were tested by one-way ANOVA, followed by post hoc testing using Tukey’s test. Two-way ANOVA was used for panels (C and D), and Šídák’s multiple comparisons test was used for post hoc test. Data in panels B/E-H were examined by independent sample t-test. ns represented P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001.