Abstract: Tian Liming, Peng Yuan, Hu Rongyi, Cheng Yang, Jiang Honghao, Chen Hongying, Tian Qingjun, Zhang Chong, Wang Ping Department of Dermatology, Wuhan No.1 Hospital, Hospital of Traditional Chinese and Western Medicine Affiliated to Hubei University of Chinese Medicine, Wuhan Hospital of Traditional Chinese and Western Medicine Affiliated to Huazhong University of Science and Technology, Wuhan 430022, China（Tian LM, Hu RY, Cheng Y, Jiang HH, Chen HY, Tian QJ）; College of Basic Medicine, Hubei University of Chinese Medicine, Wuhan 430065, China （Peng Y, Zhang C, Wang P） Corresponding author: Wang Ping, Email: pwang@aliyun.com 【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 typeⅡ （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 of the autophagy-related protein LC3, reverse transcription PCR （RT-PCR） to measure the mRNA of SMP30, and Western blot analysis to measure the protein 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 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 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-Ⅰratio （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 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 of SMP30 and LC3-Ⅱ in NHSFs, and accelerate the senescence of NHSFs.