Protective Effects of Xanthorrhizol-Rich Extracts Against PM-Induced Skin Damage in Human Keratinocytes and 3D-Reconstructed Skin Models

Abstract

Background: Particulate matter (PM) is a major environmental pollutant that induces oxidative stress, inflammation, and extracellular matrix (ECM) degradation, leading to skin damage and accelerated aging. Xanthorrhizol (XAN), a bioactive compound derived from Curcuma xanthorrhiza Roxb., exhibits anti-inflammatory and antioxidative properties, making it a promising candidate for protecting against PM-induced skin damage. This study investigated the protective effects of XAN and C. xanthorrhiza supercritical extract (CXSE) on PM-exposed skin cells. Methods: A 3D-reconstructed skin model and HaCaT human keratinocytes were exposed to PM (100 µg/mL) with or without CXSE or XAN. Histological analysis, enzyme-linked immunosorbent assay (ELISA), Western blot, reverse transcription-polymerase chain reaction (RT-PCR), and reporter gene assays were performed to assess the ECM integrity, inflammatory cytokine production, aryl hydrocarbon receptor (AhR) activation, and oxidative stress responses. Results: PM exposure activates AhR and mitogen-activated protein kinases (MAPK) signaling, increases reactive oxygen species (ROS) levels, and upregulates matrix metalloproteinases (MMPs) and inflammatory cytokines. CXSE and XAN suppresses AhR-mediated transcriptional activity and downregulates the expression of AhR target genes. Additionally, CXSE and XAN reduces ROS production by upregulating antioxidant enzyme-related genes. Conclusions: CXSE and XAN protect against PM-induced skin damage by inhibiting oxidative stress, inflammation, and ECM degradation, highlighting their potential as natural anti-pollution skincare ingredients.

Keywords: Curcuma xanthorrhiza supercritical extract (CXSE); aryl hydrocarbon receptor (AhR); particulate matter (PM); skin damage; xanthorrhizol (XAN).

Figure 1

Effects of CXSE and XAN on the histological changes and pro-inflammatory cytokine production in a PM-stimulated, 3D-reconstructed human skin model. (A) The chemical structure of XAN. (B) Schematic overview of the experimental design. The 3D-reconstructed human skin tissues were treated with CXSE or XAN following PM exposure. (C,D) Histological changes in the epidermis after treatment with CXSE (C) and XAN (D) in the presence of 100 µg/mL of PM were assessed by H&E staining (magnification, ×100; scale bar, 20 μm). (E,F) Production of IL-6 and IL-8 following treatment with CXSE (E) or XAN (F) after PM stimulation, as measured by ELISA. All treatment with CXSE and XAN were performed after exposure to 100 µg/mL of PM for 72 h. Data are presented as the mean ± SD (% of untreated control) from three individual experiments. Statistical analysis was performed using one-way ANOVA followed by Duncan’s test. ^# p < 0.5 and ^## p < 0.01 vs. untreated control, and * p < 0.05 vs. PM-treated control. CXSE and XAN were used at concentrations of 0.5 and 1 µg/mL or µM, respectively. Symbols “-” in the figure indicate no treatment, and symbols “+” in the figure indicate treatment with PM.

Figure 2

Inhibitory effects of CXSE and XAN on the expression of AhR and cytochrome P450 enzymes and the production of ROS in HaCaT cells. (A) Protein expression of AhR and phosphorylated p38 in cells treated with PM, benzo[a]pyrene (BaP), and resveratrol (Res). (B,C) Effect of CXSE (B) or XAN (C) on AhR protein expression following PM stimulation. (D,E) Relative mRNA expression of CYP1A1 and CYP1B1 in PM-exposed cells treated with CXSE (D) or XAN (E), as measured by RT-PCR. (F,G) The intracellular ROS levels and mRNA expression of antioxidant enzymes (CAT, SOD, and GPx) in PM-stimulated cells treated with CXSE (F) or XAN (G). All data are expressed as the mean ± SD (% of untreated control) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Duncan’s multiple range test. ^# p < 0.5 and ^## p < 0.01 vs. untreated control, and * p < 0.05 and ** p < 0.01 vs. PM-treated control. CXSE and XAN were used at 0.5 or 1 μg/mL or μM, respectively. Symbols “-” in the figure indicate no treatment, and symbols “+” in the figure indicate treatment with PM.

Figure 3

The inhibitory effects of CXSE and XAN on the MAPK pathway and MMP expression in HaCaT cells. (A,B) Phosphorylation levels of MAPK proteins (p-ERK, p-JNK, and p-p38) in PM-stimulated cells treated with CXSE (A) or XAN (B), as assessed by Western blotting. (C,D) The relative mRNA expression of MMP-1, MMP-2, and MMP-13 in PM-treated cells following treatment with CXSE (C) or XAN (D), as measured by RT-PCR. (E,F) Protein expression of MMP-1 in CXSE-treated (E) or XAN-treated (F) cells after PM stimulation. Data are expressed as the mean ± SD (% control) from three individual experiments. Group differences were assessed using one-way analysis of variance (ANOVA) followed by Duncan’s test. ^# p < 0.5 and ^## p < 0.01 vs. untreated control, and * p < 0.05 and ** p < 0.01 vs. PM-treated control. All data are expressed as the mean ± SD (% of untreated control) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Duncan’s multiple range test. ^## p < 0.01 vs. untreated control, and * p < 0.05 and ** p < 0.01 vs. PM-treated control. CXSE and XAN were used at 0.5 or 1 μg/mL or μM, respectively. Symbols “-” in the figure indicate no treatment, and symbols “+” in the figure indicate treatment with PM.

Figure 4

Inhibitory effects of CXSE and XAN on PM-induced inflammation in HaCaT cells. HaCaT cells were exposed to 100 µg/mL of PM for 24 h and subsequently treated with CXSE or XAN. (A) Protein expression of NF-κB and COX-2 was evaluated by Western blotting using α-tubulin as a loading control. (B,C) mRNA expression level of IL-6 and IL-8 were determined by RT-PCR after treatment with CXSE (B) or XAN (C). (D,E) IL-6 and IL-8 concentration was determined on the PM-treated cells following treatment with CXSE (D) or XAN (E). All data are expressed as the mean ± SD (% of untreated control) from three independent experiments. Statistical significance was determined using one-way ANOVA followed by Duncan’s multiple range test. ^# p < 0.5 and ^## p < 0.01 vs. untreated control, and * p < 0.05 and ** p < 0.01 vs. PM-treated control. CXSE and XAN were used at 0.5 or 1 μg/mL or μM, respectively. Symbols “-” in the figure indicate no treatment, and symbols “+” in the figure indicate treatment with PM.