Malonic Acid Isolated from Pinus densiflora Inhibits UVB-Induced Oxidative Stress and Inflammation in HaCaT Keratinocytes

Abstract

Skin aging is caused by exposure to various external factors. Ultraviolet B (UVB) irradiation induces oxidative stress, photoaging, and inflammation in skin cells. Pinus densiflora Sieb. et Zucc. (red pine) has various antimicrobial and antioxidant activities. However, the anti-inflammatory effects of red pine on skin have rarely been reported. The protective effects of malonic acid (MA) isolated from Pinus densiflora were investigated against UVB-induced damage in an immortalized human keratinocyte cell line (HaCaT). MA increased levels of the antioxidant enzymes superoxide dismutase 1 (SOD-1) and heme oxygenase 1 (HO-1) via activation of nuclear factor-erythroid 2-related factor-2 (Nrf2), resulting in a reduction in UVB-induced reactive oxygen species (ROS) levels. Additionally, the inhibition of ROS increased HaCaT cell survival rate. Thus, MA downregulated the expression of ROS-induced nuclear factor-κB, as well as inflammation-related cytokines (interleukin-6, cyclooxygenase-2, and tumor necrosis factor-α). Furthermore, MA significantly suppressed the mitogen-activated protein kinase/activator protein 1 signaling pathway and reduced the expression of matrix metalloproteinases (MMPs; MMP-1, MMP-3, and MMP-9). In contrast, MA treatment increased the expression of collagen synthesis regulatory genes (COL1A1 and COL3A1) via regulation of Smad2/3 signal induction through transforming growth factor-β. In conclusion, MA protected against UVB-induced photoaging via suppression of skin inflammation and induction of collagen biosynthesis.

Keywords: Pinus densiflora; UVB; inflammation; malonic acid; oxidative stress.

Figure 1

Isolation and purification of malonic acid (MA) from P. densiflora. (A) The P. densiflora n-BuOH layer has been separated and identified by nuclear magnetic resonance (NMR) to be MA. (B) 1H-NMR (300 MHz, D₂O, δH) 3.45 (2H, s, H-2); 13C-NMR (75 MHz, D₂O, δc) 167.5 (C-1,3), 62.2 (C-2). One methylene proton signal δH 3.45 (2H, s, H-2), two carboxyl δc 167.5 (C-1,3), and one methylene carbon signal δc 62.2 (C-2) in 1H- and 13C-NMR spectra suggest that it is MA. (C) Chemical structure of MA.

Figure 2

Effects of malonic acid (MA) on reactive oxygen species (ROS) levels and viability of ultraviolet (UV) B-irradiated HaCaT cells. HaCaT cells have been pre-treated with 2 or 4 μM MA and 4 μM epigallocatechin-3-gallate (EGCG) for 4 h and then exposed to UV-B irradiation (15 mJ/cm²). After UVB irradiation, cells have been incubated for 1, 6, 12, and 24 h. (A,B) Protective effects of MA against UVB-induced cell death, as measured by WST-1 assay and crystal violet staining. Scale bars, 100 µm. (C) Intracellular ROS levels induced by UVB-irradiation are measured using the 2′, 7′-dichlorodihydrofluorescein diacetate (DCF-DA) method. The data were analyzed in triplicate and displayed as the mean ± the standard error of the mean (SEM). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Figure 3

Effect of malonic acid (MA) on antioxidant enzymes, nuclear factor (NF)-κB activation, and inflammatory mediators in ultraviolet (UV) B-irradiated HaCaT cells. (A-F) HaCaT cells have been treated with MA for 4 h and then exposed to UVB irradiation (15 mJ/cm2). After an overnight incubation following UV exposure, the antioxidant enzymes (heme oxygenase 1 (HO-1), superoxide dismutase 1 (SOD-1), and nuclear factor-erythroid 2-related factor-2 (Nrf2)) are evaluated by (A) real-time polymerase chain reaction (PCR) and (B) western blot analysis. NF-κB, p50, and p65 are evaluated by (C) real-time PCR and (D) western blot analysis. The inflammatory mediators (tumor necrosis factor (TNF)-α, cyclooxygenase-2 (COX-2), and interleukin-6 (IL-6)) are evaluated by (E) real-time PCR and (F) western blot analysis. +, - means UVB irradiation or non-irradiation and treatment or non-treatment of MA and EGCG. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as an internal control for western blot analysis. Epigallocatechin-3-gallate (EGCG) is used as a positive control. The data were analyzed in triplicate and displayed as the mean ± the standard error of the mean (SEM). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Figure 4

Effect of malonic acid (MA) on ultraviolet (UV) B-induced mitogen-activated protein kinase/activator protein 1 (MAPK/AP-1) signaling and matrix metalloproteinase (MMP) mRNA expression in UVB-irradiated HaCaT cells. (A–E) HaCaT cells have been treated with MA for 4 h and then exposed to UVB irradiation (15 mJ/cm²). After an overnight incubation following UV exposure, phosphorylation of (A) MAPK signaling (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38) and (B) AP-1 complex (c-Jun and c-Fos) are assessed using western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as an internal control for western blot analysis. Epigallocatechin-3-gallate (EGCG) is used as a positive control. (C–E) UVB-induced MMP expression in HaCaT cells. The expression of mmp-1 (C), mmp-3 (D), and mmp-9 (E) mRNA, as measured using real-time polymerase chain reaction (PCR). +, - means UVB irradiation or non-irradiation and treatment or non-treatment of MA and EGCG. The data were analyzed in triplicate and displayed as the mean ± the standard error of the mean (SEM). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Figure 5

Effect of malonic acid (MA) on the expression of transforming growth factor (TGF)-β/Smad signaling components, collagen synthesis factor, and type I procollagen mRNA in ultraviolet (UV) B-irradiated HaCaT cells. (A-D) HaCaT cells have been treated with MA for 4 h and then exposed to UVB irradiation (15 mJ/cm²). After an overnight incubation following UV exposure, (A) Smad3 mRNA expression is evaluated by real-time polymerase chain reaction (PCR), and (B) the protein level of phosphorylated Smad2/3 is measured by western blot analysis. (C) col1a1 and col3a1 mRNA expression are evaluated by real-time PCR, and (D) the protein levels of Col1a1 and Col3a1 are measured using western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is used as an internal control for western blot analysis. Epigallocatechin-3-gallate (EGCG) is used as a positive control. +, - means UVB irradiation or non-irradiation and treatment or non-treatment of MA and EGCG. The data were analyzed in triplicate and displayed as the mean ± the standard error of the mean (SEM). (*) p < 0.05, (**) p < 0.01, (***) p < 0.001, and (****) p < 0.0001.

Figure 6

A schematic model of malonic acid (MA) inhibiting ultraviolet (UV) B-induced reactive oxygen species (ROS) activity. MA inhibits UVB-induced ROS in skin cells and suppresses mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB signaling pathways through the reduced activity of the MAPK/activator protein 1 (AP-1) complex, matrix metalloproteinase (MMP) expression, and NF-κB activity. In addition, the transforming growth factor (TGF)-β/Smad signaling pathway is increased to promote collagen production. These results suggest MA inhibits skin inflammation and photoaging. (Blue arrow: ROS induced signaling without MA treatment, Red arrow: ROS signaling with MA treatment).