Effect of hydrogen peroxide on senescence marker protein-30 and autophagy-related protein LC3-Ⅱ in human skin fibroblasts
10.3760/cma.j.issn.0412-4030.2017.12.009
- VernacularTitle:H2O2对人皮肤成纤维细胞衰老标记蛋白30及自噬相关蛋白LC3Ⅱ表达的影响
- Author:
Liming TIAN
1
;
Yuan PENG
;
Rongyi HU
;
Yang CHENG
;
Honghao JIANG
;
Hongying CHEN
;
Qingjun TIAN
;
Chong ZHANG
;
Ping WANG
Author Information
1. 湖北中医药大学附属中西医结合医院华中科技大学附属武汉中西医结合医院武汉市第一医院皮肤科
- Keywords:
Skin aging;
Hydrogen peroxide;
Fibroblasts;
Autophagy;
SMP30;
LC3-Ⅱ
- From:
Chinese Journal of Dermatology
2017;50(12):899-903
- CountryChina
- Language:Chinese
-
Abstract:
Objective To evaluate the effect of hydrogen peroxide (H2O2) on a senescence marker protein-30 (SMP30) and an autophagy-related protein microtubule-associated protein 1 light chain 3 type Ⅱ (LC3-Ⅱ) in normal human skin fibroblasts (NHSFs).Methods NHSFs were isolated from the foreskin of children,and subjected to culture in vitro.The second-to fourth-passage NHSFs were treated with 150 μmol/L H2O2 for 2 hours to establish a model for cellular senescence,while un-treated NHSFs served as control group.Senescence-associated β-galactosidase (SA-β-gal) staining was performed to determine the percentage of senescent cells,indirect immunofluorescence assay to determine the expression of the autophagy-related protein LC3,reverse transcription PCR (RT-PCR) to measure the mRNA expression of SMP30,and Western blot analysis to measure the protein expression of SMP30 and LC3.Results The percentage of senescent (SA-β-gal-positive) cells was significantly higher in the H2O2 group than in the control group (41.70％ ± 2.95％ vs.3.03％ ± 0.25％,t =22.59,P ＜ 0.05).Indirect immunofluorescence assay showed that the percentage of LC3-positive cells was significantly lower in the H2O2 group than in the control group (12.60％ ± 1.57％ vs.23.67％ ± 3.04％,t =5.61,P ＜ 0.05).As Western blot analysis showed,no significant difference in the expression of LC3-Ⅰ (LC3-Ⅰ/glyceraldehyde-3-phosphate dehydrogenase [GAPDH] ratio) was observed between the H2O2 group and control group (0.40 ± 0.02 vs.0.41 ± 0.04,P ＞ 0.05),while the H2O2 group showed significantly lower expression of LC3-Ⅱ (LC3-Ⅱ/GAPDH ratio:0.20 ± 0.02 vs.0.80 ± 0.03,t =29.69,P ＜ 0.05) and lower LC3-Ⅱ/LC3-Ⅰ ratio (0.51 ± 0.03 vs.1.98 ± 0.23,t =10.967,P ＜ 0.05) compared with the control group.Moreover,the mRNA and protein expression of SMP30 (SMP30/GAPDH ratio) was significantly lower in the H2O2 group than in the control group (mRNA:0.16 ± 0.01 vs.0.35 ± 0.01;protein:0.27 ± 0.02 vs.0.63 ± 0.02,both P ＜ 0.05).Conclusion H2O2 can decrease the expression of SMP30 and LC3-Ⅱ in NHSFs,and accelerate the senescence of NHSFs.
