Abstract:

Chen Ting*, Jiang Zhimao, Yu Bo, Ma Gang. *Department of Dermatology, Shenzhen Maternity and Child Healthcare Hospital, Shenzhen 518000, China Corresponding author: Ma Gang, Email: szmagang@medmail.com.cn 【Abstract 】 Objective To evaluate the inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts （HSFs）. Methods Fibroblasts isolated from human foreskin were treated with 1 mmol/L glyoxal in vitro to develop a model for cellular senescence. In order to select effective concentrations of chlorogenic acid, some HSFs were treated with 1 mmol/L glyoxal alone or in combination with chlorogenic acid at different concentrations （5, 10, 20, 40, 80 μmol/L） for 3 days, with those receiving no treatment serving as the blank control group. Then, methyl thiazolyl tetrazolium （MTT） assay was performed to evaluate the proliferative activity of HSFs. Some HSFs were divided into 5 groups to be cultured alone （blank control group）, or treated with 1 mmol/L glyoxal （glyoxal group） or the combination of 1 mmol/L glyoxal and chlorogenic acid at effective concentrations of 10, 20 and 40 μmol/L （glyoxal + chlorogenic acid groups）. Senescence associated β-galactosidase （SA-β-gal） staining and real-time fluorescence-based quantitative PCR were conducted to determine the percentage of senescent cells and expression level of p16INK4a mRNA respectively. Statistical analysis was carried out by one-way analysis of variance followed by the least significant difference （LSD）-t test. Results Compared with the blank control group, the glyoxal group showed significantly decreased cellular proliferative activity of HSFs （55.65% ± 2.00% vs. 100% ± 6.90%, P < 0.01）, while chlorogenic acid increased the proliferative activity of HSFs in a dose-dependent manner, and the increase reached a peak at 40 μmol/L. Concretely speaking, the glyoxal + 10-, 20-, 40-, 80-μmol/L chlorogenic acid groups all significantly differed from the glyoxal group in cellular proliferative activity （60.75% ± 1.32%, 67.65% ± 1.90%, 75.71% ± 3.25% and 75.69% ± 2.38% vs. 55.65% ± 2.00%, all P < 0.05）, but no significant difference was observed between the glyoxal group and glyoxal + 5-μmol/L chlorogenic acid group or between the glyoxal + 40-μmol/L chlorogenic acid group and glyoxal + 80-μmol/L chlorogenic acid group （both P > 0.05）. Therefore, 10 - 40 μmol/L was selected as the effective concentrations of chlorogenic acid. The glyoxal group showed significant increases in the percentage of senescent （SA-β-gal-positive） cells （35.65% ± 2.24% vs. 13.00% ± 2.22%, P < 0.01） and expression level of p16INK4a mRNA （2-ΔΔCt: 1.00 ± 0.06 vs. 0.26 ± 0.05, P < 0.01） compared with the blank control group, while the glyoxal + 10-, 20-, 40-μmol/L chlorogenic acid groups showed significantly decreased percentage of senescent cells （31.50% ± 2.13%, 22.31% ± 3.11% and 19.32% ± 3.01% respectively） and expression level of p16INK4a mRNA （2-ΔΔCt: 0.88 ± 0.08, 0.73 ± 0.06 and 0.68 ± 0.04 respectively） compared with the glyoxal group （all P < 0.05）. Additionally, the percentage of senescent cells decreased with the increase in chlorogenic acid concentrations in the glyoxal + chlorogenic acid groups. Conclusion Chlorogenic acid can protect HSFs from glyoxal-induced senescence.