中华皮肤科杂志 ›› 2015, Vol. 48 ›› Issue (12): 849-852.

• 论著 • 上一篇    下一篇

绿原酸抗人皮肤成纤维细胞衰老的研究

陈婷1,1,2,江智茂3,于波4,马刚5   

  1. 1. 北京大学深圳医院皮肤科；深圳市妇幼保健院皮肤科
    2. 深圳市妇幼保健院皮肤科
    3. 北京大学深圳医院中心实验室
    4. 深圳市北京大学深圳医院皮肤科
    5. 北京大学深圳医院皮肤科
  • 收稿日期:2015-09-07 修回日期:2015-08-05 出版日期:2015-12-15 发布日期:2015-12-01
  • 通讯作者: 马刚 E-mail:szmagang@medmail.com.cn

Inhibitory effect of chlorogenic acid on senescence of human skin fibroblasts

  • Received:2015-09-07 Revised:2015-08-05 Online:2015-12-15 Published:2015-12-01

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摘要：

目的 探讨绿原酸在乙二醛诱导的人皮肤成纤维细胞衰老中的保护作用。 方法 使用1 mmol/L乙二醛处理体外培养的人皮肤成纤维细胞，构建细胞衰老模型。用5、10、20、40、80 μmol/L绿原酸和乙二醛共同处理人皮肤成纤维细胞，MTT法检测细胞增殖活性，筛选出绿原酸的有效浓度。1 mmol/L乙二醛分别与有效浓度的绿原酸（10、20、40 μmol/L）共同作用于成纤维细胞，细胞衰老β半乳糖苷酶（SA-β-gal）染色法及实时荧光定量PCR法检测衰老细胞比例和衰老基因p16INK4a mRNA的表达情况。统计分析采用单因素方差分析和LSD法。 结果 与空白对照组人皮肤成纤维细胞增殖（100% ± 6.90%）相比，乙二醛可明显抑制细胞增殖（55.65% ± 2.00%），两组差异有统计学意义（P < 0.01）。与乙二醛组细胞增殖相比，除乙二醛 + 5 μmol/L绿原酸组（55.36% ± 2.58%）外，乙二醛 + 10、20、40、80 μmol/L绿原酸组对细胞增殖均有不同程度地保护作用（细胞增殖率分别为60.75% ± 1.32%、67.65% ± 1.90%、75.71% ± 3.25%、75.69% ± 2.38%），呈剂量依赖性，40 μmol/L绿原酸达高峰，80 μmol/L绿原酸与40 μmol/L组相比，细胞活力的差异无统计学意义（P > 0.05），因此筛选出绿原酸的有效浓度为10 ~ 40 μmol/L。与空白对照组衰老细胞比例（13.00% ± 2.22%）比较，加入乙二醛后，衰老细胞明显增多（35.65% ± 2.24%），差异有统计学意义（P < 0.01）。与乙二醛组细胞SA-β-gal染色阳性率相比，分别加入10、20、40 μmol/L绿原酸与乙二醛共培养后，细胞SA-β-gal染色阳性率呈剂量依赖性减少（31.50% ± 2.13%、22.31% ± 3.11%、19.32% ± 3.01%），差异均有统计学意义（P < 0.05）。与空白对照组衰老基因p16INK4a mRNA的表达比较，乙二醛组明显增高（2-ΔΔCt：1.00 ± 0.06比0.26 ± 0.05，P < 0.01），分别加入10、20、40 μmol/L绿原酸与乙二醛共培养后表达下降（0.88 ± 0.08、0.73 ± 0.06、0.68 ± 0.04，P < 0.05）。 结论 绿原酸在乙二醛致人皮肤成纤维细胞衰老中具有潜在的保护作用。

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.