Fisetin Inhibits UVA-Induced Expression of MMP-1 and MMP-3 through the NOX/ROS/MAPK Pathway in Human Dermal Fibroblasts and Human Epidermal Keratinocytes

Abstract

Fisetin is a flavonoid found in plants and has been reported to be effective in various human diseases. However, the effective mechanisms of ultraviolet-A (UVA)-mediated skin damage are not yet clear. In this study, we investigated the protective mechanisms of fisetin regarding UVA-induced human dermal fibroblasts (HDFs) and human epidermal keratinocytes (HEKs) damages. Fisetin showed a cytoprotective effect against UVA irradiation and suppressed matrix metalloproteinases (MMPs), MMP-1, and MMP-3 expression. In addition, fisetin was rescued, which decreased mRNA levels of pro-inflammatory cytokines, reactive oxygen species production, and the downregulation of MAPK/AP-1 related protein and NADPH oxidase (NOX) mRNA levels. Furthermore, UVA-induced MMP-1 and MMP-3 were effectively inhibited by siRNAs to NOX 1 to 5 in HDFs and HEKs. These results indicate that fisetin suppresses UVA-induced damage through the NOX/ROS/MAPK pathway in HDFs and HEKs.

Keywords: NADPH oxidase; fisetin; matrix metalloproteinase; reactive oxygen stress; ultraviolet A.

Figure 2

Fisetin inhibited the expression of pro-inflammatory cytokines in UVA-irradiated HDFs and HEKs. (A) HDFs were treated with fisetin (10 μM) and exposed to UVA (8 J/cm²). (B) HEKs were treated with fisetin (5 μM) and exposed to UVA (5 J/cm²). After 4 h, pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) mRNA levels were analyzed using real-time PCR. GAPDH was used as an internal control. (C–F) Inhibitory effects of fisetin on UVA-induced reactive oxygen species (ROS) production in HDFs (C,D) and HEKs (E,F). HDFs and HEKs were treated with fisetin, irradiated with UVA, and then incubated for 1 h. Cells were then washed with PBS and incubated with DCF-DA for 30 min at room temperature. Intracellular ROS production was measured using DCF-DA fluorescence using a FACS flow cytometer. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA.

Figure 1

Effects of fisetin on HDF and HEK viability. Cells were treated with fisetin at various concentrations, and cytotoxicity was evaluated after 24 h. (A,B) After pretreatment with fisetin, cells were irradiated with UVA and incubated for 24 h. Cell viability was analyzed using the MTT assay. Fisetin inhibited UVA-induced MMP-1 and MMP-3 expression in HDFs and HEKs. HDFs and HEKs were treated with fisetin, irradiated with UVA, and then incubated for 24 h. (C,E) MMP-1 and MMP-3 protein expression was analyzed using Western blotting; β-actin was used for loading control. (D,F) MMP-1 and MMP-3 mRNA levels were analyzed using real-time PCR; GAPDH was used for internal control. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA.

Figure 3

Fisetin suppressed UVA-induced MAPK activation in HDFs and HEKs. (A,B) Fisetin inhibits UVA-induced AP-1 activation in HDFs. (C,D) HDFs and HEKs were treated with fisetin and exposed to UVA. After 30 min, total ERK, p38, JNK and phospho-ERK, phospho-p38, and phospho-JNK levels were analyzed using Western blotting. β-actin was used for loading control. After 2 h, nuclear extracts were prepared. Western blotting was performed to determine the nuclear levels of the AP-1 (p-c-Jun, p-c-Fos) subunits. PCNA was used as an internal control for the nuclear fraction. (E,F) To assess phosphorylation levels, the density of each band was quantified using ImageJ software (ver. 1.52 for MS Windows), and the relative density ratio of each protein was calculated accordingly. To assess phosphorylation levels, the density of each band was quantified using the ImageJ software (ver. 1.52 for MS Windows), and the relative density ratio of each protein was calculated accordingly. (G) HDFs were treated with fisetin and irradiated with UVA, and the promoter activity of AP-1 was measured using the dual-luciferase reporter assay. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA. ND: not detected.

Figure 4

Fisetin inhibited the expression of NOX in UVA-irradiated HDFs and HEKs. (A) HDFs were treated with fisetin (10 μM) and exposed to UVA (8 J/cm²). (B) HEKs were treated with fisetin (5 μM) and exposed to UVA (5 J/cm²). NOX1–NOX5 mRNA levels were analyzed using real-time PCR. GAPDH was used as an internal control. The data are expressed as the means ± SE (n = 3). # p < 0.01 vs. untreated control, * p < 0.01 vs. UVA. Regulation of MMP-1 and MMP-3 expression by NOX. NOX1–NOX5 were knocked down in HDFs and HEKs using NOX siRNA. Then, the cells were irradiated with UVA and incubated for 24 h. (C–F) * p < 0.01 vs. negative siRNA UVA-treated group. The data are expressed as the means ± SE (n = 3).

Figure 5

Schematic diagram of protective mechanisms of the fisetin on UVA-induced HDF and HEK cell damages via MMP-1 and MMP-3 expression through the NOX/ROS/MAPK pathway.