Anti-Inflammatory Effects of Compounds from Cudrania tricuspidata in HaCaT Human Keratinocytes

Abstract

The root bark of Cudrania tricuspidata has been reported to have anti-sclerotic, anti-inflammatory, antioxidant, neuroprotective, hepatoprotective, and cytotoxic activities. In the present study, the effect of 16 compounds from C. tricuspidata on tumor necrosis factor-α+interferon-γ-treated HaCaT cells were investigated. Among these 16 compounds, 11 decreased IL-6 production and 15 decreased IL-8 production. The six most effective compounds, namely, steppogenin (2), cudraflavone C (6), macluraxanthone B (12), 1,6,7-trihydroxy-2-(1,1-dimethyl-2-propenyl)-3- methoxyxanthone (13), cudraflavanone B (4), and cudratricusxanthone L (14), were selected for further experiments. These six compounds decreased the expression levels of chemokines, such as regulated on activation, normal T cell expressed and secreted (RANTES) and thymus and activation-regulated chemokine (TARC), and downregulated the protein expression levels of intercellular adhesion molecule-1. Compounds 2, 6, 12, 4, and 14 inhibited nuclear factor-kappa B p65 translocation to the nucleus; however, compound 13 showed no significant effects. In addition, extracellular signal regulatory kinase-1/2 phosphorylation was only inhibited by compound 14, whereas p38 phosphorylation was inhibited by compounds 13 and 4. Taken together, the compounds from C. tricuspidata showed potential to be further developed as therapeutic agents to suppress inflammation in skin cells.

Keywords: Cudrania tricuspidata; HaCaT; ICAM-1; NF-κB; inflammation.

Figure 1

Structure of compounds 1–16.

Figure 2

Effects of the 70% EtOH extract from Cudrania tricuspidata on the viability of HaCaT cells (A) and, IL-6 (B) and IL-8 (C) production in HaCaT cells stimulated with TNF-α + IFN-γ. (A): The cells were incubated with the indicated concentrations for 24 h; (B): cells were pre-treated with the 70% EtOH extract for 3 h and stimulated with TNF-α + IFN-γ for 24 h. Each value represents the mean ± SD. ** p < 0.01 and *** p <0.001 as compared with TNF-α + IFN-γ only.

Figure 3

Effects of the six compounds from C. tricuspidata on RANTES (A) and TARC (B) production in TNF-α + IFN-γ-treated HaCaT cells. HaCaT cells were incubated with TNF-α + IFN-γ (20 ng/mL) in the presence or absence of the six compounds at the indicated concentrations. After 24 h, the chemokine secretion levels were analyzed using enzyme-linked immunosorbent assay (ELISA). Data are presented as the mean ± standard deviation. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with TNF-α + IFN-γ only. TI: TNF-α + IFN-γ.

Figure 4

Protein expression levels of COX-2 (A,B) and ICAM-1 (C,D) in TNF-α + IFN-γ-treated HaCaT cells. (A,B) Cells were pretreated with the six compounds from C. tricuspidata at the indicated concentrations for 3 h and stimulated with TNF-α + IFN-γ (20 ng/mL) for 24 h. (C,D) Cells were pretreated with the six compounds from C. tricuspidata at the indicated concentrations for 3 h, and then stimulated with TNF-α + IFN-γ (20 ng/mL) for 6 h. Western blotting analysis was performed as described in the Materials and Methods section. Representative stains from four independent experiments are presented. The immunoblot was quantified using ImageJ software. The band intensity was normalized to that of actin. * p < 0.05, *** p < 0.001 compared with TNF-α + IFN-γ only. COX-2, cyclooxygenase-2; ICAM-1, intercellular adhesion molecule-1.

Figure 5

Effects of the six compounds from C. tricuspidata on the degradation of IκB-α (A,B), the phosphorylation of IκBα (C,D), and the translocation of NF-κB p65 to the nucleus (E,F) in HaCaT cells. Cells were pretreated with the six compounds from C. tricuspidata at the indicated concentrations for 3 h and stimulated with TNF-α + IFN-γ (20 ng/mL) for 10 min. IκBα, p-IκBα, and NF-κB p65 were analyzed using Western blotting, as described in the Materials and Methods section. Representative blots from four independent experiments are presented. The immunoblots were quantified using ImageJ software. The band intensity was normalized to that of actin or anti-proliferating cell nuclear antigen (PCNA). * p < 0.05, ** p < 0.01, *** p < 0.001 compared with TNF-α + IFN-γ only.

Figure 6

Effect of the six compounds from C. tricuspidata on JNK-1/2, ERK-1/2, and p38 phosphorylation in HaCaT cells. Cells were pretreated with the six compounds at the indicated concentrations for 3 h, and then stimulated with TNF-α + IFN-γ (20 ng/mL) for 2 h (A,B), 15 min (C,D), or 6 h (E,F). Cell extracts were analyzed using Western blotting and antibodies specific for phosphorylated JNK1/2 (p-JNK1/2), p-ERK1/2, or p-p38. The membrane was stripped and reprobed to determine the total abundance of each MAPK, as a control measure. Representative blots from four independent experiments are presented. Immunoblots were quantified using ImageJ software. The band intensity was quantified and normalized to each total protein concentration. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with TNF-α + IFN-γ only. JNK, c-Jun N-terminal kinase; ERK, extracellular signal regulatory kinase; MAPK, mitogen-activated protein kinase.