To examine the cytotoxicity of syringic acid and cytoprotective effects against UVB, HaCaT cells were treated with syringic acid at concentrations of 1, 2, 5, 10, and 20 μM and incubated for 25 h before WST-1 assay. We found that 1 μM of syringic acid resulted in a 105% cell survival rate, 2 μM resulted in a 108% cell survival rate, 5 μM resulted in a 104% cell survival rate, 10 μM resulted in a 96% cell survival rate, and 20 μM resulted in an 88% cell survival rate (Fig. 1). To investigate the cytoprotective effects of syringic acid against cell damage caused by UVB, the HaCaT cells were treated with syringic acid at concentrations of 1, 2, 5, and 10 μM and irradiated with 15 mJ/cm2 UVB. Compared with the cell survival rate of 72% for the control group irradiated with UVB, that of cells treated with 1 μM of syringic acid increased to 78%, that of cells treated with 2 μM of syringic acid increased to 88%, that of the cells treated with 5 μM of syringic acid increased to 92%, and that of cells treated with 10 μM of syringic acid increased to 102% (Fig. 2).
In addition, we confirmed that ROS in HaCaT cells harmed by UVB were eliminated by syringic acid. After ROS produced by UVB were fluorescently stained with DCF-DA, flow cytometry (BD Biosciences, USA) was used to measure the change in values. It revealed that 15 mJ/cm2 UVB caused a 2.6-fold increase in ROS, but after treatment with syringic acid at concentrations of 2, 5, and 10 μM, the ROS levels in the UVB-exposed HaCaT cells decreased by 2.1-, 1.5-, and 1.2-fold, respectively. In particular, 10 μM of syringic acid was more effective in eliminating ROS than the same concentration of N-acetyl-l-cysteine (NAC; Calbiochem, USA), which acts as a ROS scavenger (Fig. 3). We used qRT-PCR to verify that SOD1 mRNA, which decreased in response to UVB, had an antioxidant effect of superoxide anion radical elimination due to UVB and found that exposure to 15-mJ/cm2 UVB caused the expression level of SOD1 to decrease 0.7-fold, but after treatment with 2, 5, and 10 μM of syringic acid, the SOD1 mRNA expression after exposure to 15 mJ/cm2 UVB increased 0.9-, 1.1-, and 1.2-fold, respectively (Fig. 4). We also used qRT-PCR to verify that GPX1 mRNA, which decreased in response to UVB, had an antioxidant effect of superoxide anion radical elimination caused by UVB and found that 15-mJ/cm2 UVB caused the expression level of GPX1 to decrease 0.2-fold, but after treatment with 2, 5, and 10 μM of syringic acid, the SOD1 mRNA expression after exposure to 15 mJ/cm2 UVB increased 0.8-, 1.4-, and 2.2-fold, respectively (Fig. 5). In this experiment, qRT-PCR was used to determine whether catalase (CAT) mRNA, which decreased in response to UVB, could be recovered to some extent by syringic acid and found that 15-mJ/cm2 UVB caused the expression level of CAT mRNA to decrease 0.2-fold, but after treatment with 2, 5, and 10 μM of syringic acid, CAT mRNA expression after exposure to 15 mJ/cm2 UVB increased 0.4-, 0.7-, and 0.9-fold, respectively (Fig. 6).
To examine the changes in MMP expression and MMP-1 production caused by syringic acid through the AP-1 mechanism, qRT-PCR was used to determine the degree of reduction in c-Jun and c-Fos mRNA expression due to syringic acid after their increase in response to UVB. Our results revealed that 15-mJ/cm2 UVB caused a 2.1-fold increase in the expression level of c-Jun mRNA, but after treatment with 2, 5, and 10 μM of syringic acid, c-Jun mRNA expression decreased 1.9-, 1.3-, and 1.1-fold, respectively. Exposure to 15-mJ/cm2 UVB caused a 1.2-fold increase in the expression level of c-Fos mRNA, but after treatment with 2, 5, and 10 μM of syringic acid, c-Fos mRNA expression after exposure to 15 mJ/cm2 UVB decreased 1.1-, 1.0-, and 0.9-fold, respectively (Fig. 7). In this experiment, AP-1 luciferase reporter vector (BPS Bioscience, CA, USA) with AP-1-responsive elements located in front of luciferase in the promoter region was used for transfection before luciferase gene expression was measured via luciferin luminescence measurement. The results confirmed the transcriptional activity of the transcription factor AP-1 promoter, which affects skin aging via inflammation, immune response, cell proliferation, and collagen degradation. The luminescence of luciferin increased twofold due to 15-mJ/cm2 UVB, but after syringic acid treatment at concentrations of 2, 5, and 10 μM, it decreased 1.5-, 1.2-, and 1.0-fold, respectively, after exposure to 15 mJ/cm2 UVB. We confirmed that the decrease in the luminescence of luciferin due to syringic acid was concentration-dependent. This finding confirmed that syringic acid inhibited the transcription activity of the transcription factor AP-1 promoter, which affects skin aging via skin inflammation, immune response, cell proliferation, and collagen degradation (Fig. 8).
Change in MMP mRNA expression
The effect of syringic acid on MMP-1, MMP-2, and MMP-9 expressions was confirmed by qRT-PCR. It demonstrated that after exposure to 15-mJ/cm2 UVB, the expression levels of MMP-1, MMP-2, and MMP-9 increased 2.5, 4.3, and 1.8-fold, respectively, but after treatment with 2, 5, and 10 μM of syringic acid, the MMP-1 mRNA expression after exposure to 15-mJ/cm2 UVB decreased 1.9-, 1.4-, and 1.06-fold, respectively. Because of syringic acid treatments at concentrations of 2, 5, and 10 μM, MMP-2 mRNA expression after exposure to 15-mJ/cm2 UVB decreased 3.4-, 2.1-, and 0.9-fold, respectively, in a concentration-dependent manner, and MMP-9 mRNA expression decreased to 1.0-, 0.9-, and 0.8-fold, respectively, in a concentration-dependent manner (Fig. 9). ELISA was used to determine the effects of syringic acid on the production of MMP-1, which is a collagen inhibitor that increases in response to UVB. It demonstrated that exposure to 15-mJ/cm2 UVB caused a 1- to 1.9-fold increase in MMP-1 production, but after treatment with 2, 5, and 10 μM of syringic acid, MMP-1 production after exposure to 15-mJ/cm2 UVB decreased 1.8-, 1.5-, and 1.3-fold, respectively (Fig. 10). Therefore, it seems that syringic acid has anti-aging effects in HaCaT cells.