| Background and objective:Vitiligo is an acquired pigmentary disorder which the clinical efficacy is not satisfactory.Recent studies indicated that mitochondrial dysfunction and lower level of autophagy might contribute to weaker adhesion system of melanocytes and subsequent melanocytorrhagy.The enhancement of the level of mitophagy in melanocytes under oxidative stress would be helpful to mitochondrial turnover and to maintain cell homeostasis.Making an appropriate level adjustment to autophagy depends on the understanding of the molecular mechanism?s?underlying autophagy induced by oxidative stress.Hence,our study is aimed to explore the possible mechanisms of autophagy induced hydrogen peroxide at varying concentrations.Methods:Normal human melanocytes were treated with H2O2 at concentrations?v/v%?of 0,10-1%,5×10-2%,10-2%,5×10-3%,10-3%,5×10-4%,10-4%,5×10-5%and 10-5%.100nmol/L rapamycin was employed as a positive control of autophagy.After the treatment for 4 hours,the proliferative activity and apoptosis of melanocytes were assessed by CCK-8 and AnnexinV-FITC/PI flow cytometry respectively.The autophagy was observed through acridine orange staining,autophagic vacuoles under transmission electron microscopy and the expression of Beclin1 and LC3 detected by western blot.Analysis of 84 autophagy related genes was performed by RT2 Profiler PCR Array,and the differentially expressed genes in H2O2-treated melanocytes were identified from the normal melanocytes.Results:After the treatment with H2O2 at concentrations ranged from 10-1%to5×10-2%,the proliferative activity of melanocytes decreased significantly and the apoptosis rate reached up to 50%when compared with the normal control group.With the decrease of H2O2 concentration,the proliferative activity increased and the apoptosis rate decreased.There were no significant differences between 5×10-5%H2O2-treated and 10-5%H2O2-treated melanocytes.Therefore,we selected the concentrations of H2O2 at 10-2%,10-3%and 10-4%in the following autophagy related experiments.The results from AO staining and TEM indicated that autophagy occurred at low,basal levels in normal melanocytes.10-2%H2O2 did cause severe oxidative damages of mitochondria except autophagosomes.Obvious autophagy and the lighter degree of mitochondria injury were observed in melanocytes treated with10-3%H2O2,10-4%H2O2 and rapamycin.The level of Beclin1 was lower in 10-2%H2O2-treated melanocytes than that in normal melanocytes?P<0.05?,and there was no difference between the both groups in LC3-II/LC3-I ratio.The expressions of Beclin1 and LC3-II/LC3-I ratio in 10-3%H2O2,10-4%H2O2 and/or rapamycin were much higher than those in normal and/or 10-2%H2O2-treated melanocytes.There were 11 genes which were found to be differentially regulated with 10-3%H2O2,10-4%H2O2 and rapamycin treatment.There was up-regulation of ATG12,ATG3,ULK1,PIK3CG,PTEN,IGF1 and PIK3C3 genes and down-regulation of EIF2AK3,ATG5,ATG16L1 and ATG4A genes.The expression of MTOR and ULK2 genes in10-3%H2O2 and rapamycin treated melanocytes decreased and elevated respectively.In 10-4%H2O2-treated melanocytes,PRKAA1?AMPK?,MAPK8?JNK1?and MTOR genes was up-regulated but the fold change of MTOR<2 fold.Conclusions:Autophagy was successfully induced in melanocytes under the condition of oxidative stress mimicked by 10-3%H2O2 and 10-4%H2O2.According to the differentially expressed genes in H2O2-treated melanocytes identified from the normal melanocytes,we speculated that ATG12 conjugation to ATG3 facilitated autophagosome formation instead of the weakened conjugation system involved in ATG12 and ATG5.It might also play a key role in H2O2-induced autophagy through thiol modification of ATG4A which reduced form of ATG4A,or directly activation of PIK3C3.We also have found that there existed the differentially expressed genes between the 10-3%H2O2 and 10-4%H2O2 treated melanocytes,which suggested that different concentration of H2O2 induced autophagy in melanocytes underlying different molecular mechanisms.10-3%H2O2 induced autophagy could be mainly regulated by class I PI3K/AKT/mTOR dependent signaling pathway which was negatively regulated by PTEN.There were several signaling pathways involved in the control of autophagy induced by 10-4%H2O2 including PTEN independent mTOR signaling pathway,AMPK/ULK1 and AMPK/JNK1/BCL2/BECN1 signaling pathway.IGF1/AKT/mTOR and IGF1/AMPK/mTOR signaling pathway might exist in autophagy induced by H2O2,and mainly works in 10-3%H2O2-treated melanocytes.It was conjectured that IGF1 through a mechanism that depends on the Akt/mTOR or AMPK/mTOR pathway inhbited excessive autophagy and cell death caused by higher concentration of H2O2.It is worth noting that endoplasmic reticulum stress?ERS?could participate in regulation of autophagy induced by 10-3%H2O2 and 10-4%H2O2which might be mediated by EIF2AK3/eIF2?phosphorylation and subsequent up-regulation of ATG12. |
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