Casticin inhibits interleukin-1β-induced ICAM-1 and MUC5AC expression by blocking NF-κB, PI3K-Akt, and MAPK signaling in human lung epithelial cells

Abstract

The compound casticin, isolated from Vitex rotundifolia, exerts anti-inflammatory effects and causes apoptosis of cancer cells. In this study, we explored the anti-inflammatory effects of casticin and modulation of cyclooxygenase (COX)-2, intercellular adhesion molecule 1 (ICAM-1), and mucin 5AC (MUC5AC) expression in interleukin-1β (IL-1β)-activated A549 human pulmonary epithelial cells. A549 cells were treated with various concentrations of casticin (5-20 μM), and an inflammatory response was triggered with interleukin (IL)-1β cytokines. Casticin decreased levels of IL-6, tumor necrosis factor α, and IL-8 and suppressed COX-2 expression and prostaglandin E2 production. It also reduced MUC5AC, proinflammatory cytokine, and chemokine gene expression and inhibited ICAM-1 expression for monocyte adhesion in IL-1β-stimulated A549 cells. In addition, casticin inhibited phosphorylation of Akt, phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) and blocked nuclear transcription factor kappa-B (NF-κB) subunit p65 protein translocation into the nucleus. Co-culture of NF-κB, MAPK, and PI3K inhibitors with casticin also led to more significantly suppressed ICAM-1 expression in inflammatory A549 cells. These results provide evidence that casticin has an anti-inflammatory effect by blocking proinflammatory cytokine, chemokine, and ICAM-1 expression via suppression of the PI3K/Akt, NF-κB, and MAPK signaling pathways in IL-1β-stimulated inflammatory pulmonary epithelial cells.

Keywords: ICAM-1; Interleukin-1β; MAPK; NF-κB; casticin.

Figure 1. The effects of casticin (CAS) on IL-1β–induced production of IL-6, IL-8, TNF-α, CCL5, and MCP-1

A549 cells (10⁶ cells/well) were pretreated with CAS for 1 h and then stimulated with IL-1β (1 ng/ml) for 24 h. The presented data are mean ± SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 2. Effects of casticin (CAS) on IL-1β–induced gene expression

A549 cells (10⁶ cells/ml) were pretreated with CAS for 1 h and then stimulated with IL-1β (1 ng/ml) for 4 h to assay gene expression levels, determined using real-time RT-PCR. The presented data are mean±SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 3. Effects of casticin (CAS) on IL-1β–induced production of COX-2 and PGE₂

A549 cells (10⁶ cells/ml) were pretreated with CAS for 1 h and then stimulated with IL-1β (1 ng/ml) for 24 h. COX-2 proteins were detected using β-actin as an internal control (A), and COX-2 protein expressions were measured relative to the expression of β-actin (internal control) (B). COX-2 gene expression was measured by real-time PCR (C), and levels of PGE₂ were evaluated by ELISA (D). Data are presented as mean ± SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 4. Effects of casticin (CAS) on IL-1β–induced production of ICAM-1

A549 cells (10⁶ cells/ml) were pretreated with CAS for 1 h and then stimulated with IL-1β (1 ng/ml) for 24 h to assay ICAM-1 protein expression by western blot (A), and ICAM-1 protein expressions were measured relative to the expression of β-actin (internal control) (B). ICAM-1 of the culture medium by ELISA (C). ICAM-1 gene expression levels, determined using real-time RT-PCR (D), and promoter assay were used to evaluate ICAM-1 promoter activity (E). THP-1 cells were labeled with calcein AM and co-cultured with A549 cells, followed by observation using fluorescence microscopy (F). Fluorescence intensity of THP-1 cell adhesion to A549 cells (G). The presented data are mean ± SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 5. Effects of casticin (CAS) or interleukin-1 receptor antagonist (IL-1RA) on the IL-1β–induced production of ICAM-1 and MUC5AC

A549 cells (10⁶ cells/ml) were pretreated with casticin (CAS) or (20 ng/ml) IL-1RA for 1 h and then stimulated with IL-1β (1 ng/ml) for 4 h to assay MUC5AC (A) and ICAM-1 (B) gene expression, determined using real-time RT-PCR. THP-1 cells were labeled with calcein AM and co-cultured with A549 cells, followed by observation using fluorescence microscopy (C). Fluorescence intensity of THP-1 cell adhesion to A549 cells (D). The presented data are mean±SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group. ^#p < 0.05, ^#p < 0.01, compared with the IL-1β/IL-1RA group.

Figure 6. Effect of casticin (CAS) on IL-1β–induced activation of PI3K-Akt, and MAPK signaling in A549 cells

A549 cells were pretreated with CAS for 1 h and then incubated with IL-1β (1 ng/ml) for 30 min. Phospho-specific proteins were detected using western blot, and total MAPK (A, B) and PI3K/Akt (C, D) levels were used as internal controls. The presented data are mean ± SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 7. Effect of casticin (CAS) on IL-1β–induced activation of NF-κB signaling in A549 cells

A549 cells were pretreated with CAS for 1 h and then incubated with IL-1β (1 ng/ml) for 30 min. Phospho-specific proteins were detected using western blot, and total IκB-α (A, B) levels were used as internal controls. For the nuclear translocation of NF-κB, cells were pretreated with CAS for 1 h and then incubated with IL-1β (1 ng/ml) for 1 h (B, C). The internal controls were Lamin B1 in the nucleus and β-actin in the cytosol. A promoter assay was used to evaluate NF-κB promoter activity (D). The presented data are mean ± SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group.

Figure 8. Inhibitory effects of MAPK, PI3K, Akt, and NF-κB inhibitors or casticin (CAS) on IL-1β–induced ICAM-1 protein expression in A549 cells

A549 cells were pretreated with ERK1/2 inhibitors (PD: 10 μM PD98059), p38 inhibitors (SB: 10 μM SB203580), JNK inhibitors (SP: 10 μM SP600165), PI3K inhibitor (PI3Ki: 10 μM AS604850), AKT inhibitor (AKTi: 10 μM Akt1/2 kinase inhibitor), and NF-κB inhibitor (NFi: 10 μM BAY11-7085) with or without 20 μM CAS for 1 h, followed by IL-1β stimulation for 4 h. ICAM-1 gene expression was detected by real-time PCR. The presented data are mean±SEM; ^*p < 0.05, ^**p < 0.01, compared with the IL-1β–treated group. ^#p < 0.05, ^##p < 0.01, compared with the inhibitor / IL-1β group.

Figure 9. Model explaining the mechanism for the anti-inflammatory effects of casticin (CAS) in IL-1β–induced A549 cells