Effect of narrow-band ultraviolet B radiation on the autophagy of cultured human melanocytes in vitro / 中华皮肤科杂志

ABSTRACT

Objective To evaluate the effect of narrow-band ultraviolet (NB-UVB) radiation on the autophagy of cultured human melanocytes in vitro,and to explore possible mechanisms underlying the treatment of vitiligo by NB-UVB.Methods In vitro cultured human melanocytes were divided into 4 groups to be irradiated with NB-UVB at different irradiation doses of 0 (control group),50,100 and 200 mJ/cm2 (50-,100-and 200-mJ/cm2 NB-UVB groups) respectively.After 24-hour treatment,the cells were collected,and monodansylcadaverin (MDC) staining was conducted to detect changes of autophagosomes in melanocytes.Western blot analysis was performed to determine the protein expression of autophagy signals including phosphorylated AMP-activated protein kinase (p-AMPK),phosphorylated mammalian target of rapamycin (p-mTOR),microtubule-associated protein 1 light chain 3 Ⅱ/Ⅰ (LC3 Ⅱ/Ⅰ) and P62,and transmission electron microscopy to observe ultrastructural changes of autophagosomes and melanosomes in the melanocytes.Statistical analysis was done by using one-way analysis of variance (ANOVA) for the comparison of Western blot results,and by Kruskal-Wallis H test for the comparison of the number of melanosomes,autophagosomes and autolysosomes.Results MDC staining showed that the percentages of autophagosome-positive melanocytes were significantly higher in the 100-,200-mJ/cm2 NB-UVB groups (38.08％ ± 4.10％,40.23％ ± 1.45％,respectively) than in the control group (21.83％ ± 3.50％,both P ＜ 0.05) and 50 mJ/cm2 NB-UVB group (23.66％ ± 4.12％,both P ＜ 0.05).As Western blot analysis revealed,the 100-,200-mJ/cm2 NB-UVB groups showed significantly increased expression of p-AMPK and LC3 Ⅱ/Ⅰ,but significantly decreased expression of p-mTOR and P62 compared with the control group (all P ＜ 0.05).Transmission electron microscopy showed that the number of autophagosomes and autolysosomes was significantly higher in the 100-,200-mJ/cm2 NB-UVB groups (5.12 ± 1.13,5.25 ± 1.04) than in the control group (1.88 ± 1.18,both P ＜ 0.05).Meanwhile,the number of melanosomes was significantly higher in the 50-,100-and 200-mJ/cm2 NB-UVB groups (39.12 ± 9.42,57.38 ± 7.11,59.75 ± 15.15,all P ＜ 0.05) than in the control group (18.50 ± 4.18,all P ＜ 0.05).Conclusion NB-UVB radiation can not only promote the formation of melanosomes,but also activate the autophagy signal pathways in the melanocytes and promote the formation of autophagosomes and autolysosomes,which may be one of the mechanisms underlying the treatment of vitiligo by NB-UVB.