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.