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. 2022 Jan 26;13:817526. doi: 10.3389/fphar.2022.817526

FIGURE 2.

FIGURE 2

Punicalagin suppresses the TNF-α and IL-17A-induced IL-1β upregulation via inhibiting NF-κB activation and cleaved caspase-1 expression in vitro. (A) The relative mRNA level of IL-1β in different groups. Here we used IL-17A (25 ng/ml) and TNF-α (25 ng/ml) to stimulate HaCaT cells. Simultaneously, we also added 2.5/5/10/20 μM PUN into these IL-17A and TNF-α-stimulated HaCaT cells. The total RNA was subsequently extracted after treatment with IL-17A (25 ng/ml) + TNF-α (25 ng/ml) and 2.5/5/10/20 μM PUN for 24 h. (B) The pro- and mature expression of IL-1β in different groups. The total protein was collected after treatment with IL-17A (25 ng/ml) + TNF-α (25 ng/ml) and 2.5/5/10/20 μM PUN for 48 h. (C) The expression of phosphorylation (Ser536) and total p65 in the cytoplasm and nucleus after exposure of IL-17A (25 ng/ml) + TNF-α (25 ng/ml) and 2.5/5/10/20 μM PUN for 24 h. (D) Immunostaining with an anti-p65 antibody showed that 2.5/5/10/20 μM PUN functionally blocks TNF-α and IL-17A-induced nuclear translocation of p65. Scale bar, 50 μm. (E) The expression of cleaved and total caspase-1 after exposure of IL-17A (25 ng/ml) + TNF-α (25 ng/ml) and 2.5/5/10/20 μM PUN for 48 h. (F) The relative mRNA level of CXCL-1 (left) and CCL-20 (right) in HaCaT cells after treatment with IL-1β and IL-1β + PUN for 48 h *p < 0.05, **p < 0.01, ***p < 0.001. One-way ANOVA with Student-Newman-Keuls method for (A, F). Data represent the mean ± S.E.M.