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Objective To evaluate the effect of hydrogen peroxide (H2O2) on autophagy in melanocytes,and to explore its possible regulatory mechanisms.Methods Normal human melanocytes at exponential growth phase were divided into several groups:blank control group receiving no treatment,positive control group treated with 100 nmol/L sirolimus solution,and experiment groups treated with H2O2 solution at different volume fractions of 10-7-10-3 respectively.After 4-hour treatment,cell counting kit-8 (CCK-8) assay and flow cytometry were performed to evaluate the cellular proliferative activity and detect apoptosis of melanocytes respectively.Acridine orange staining was performed to detect autophagosome formation,transmission electron microscopy to observe ultrastructural changes of autophagosomes,and Western blot analysis to measure the expression of autophagy-specific protein Beclin 1 and microtubuleassociated protein 1 light chain 3B (LC3B).A total of 84 autophagy-related genes were analyzed by RT2 Profiler PCR Array,so as to screen differentially expressed autophagy-related genes.Results After the treatment with H2O2 at different volume fractions of 10-3,5 × 10-4,10-4,5 × 10-5,10-5,5 × 10-6 and 10-6,experiment groups showed significantly decreased cellular proliferative activity,but significantly increased apoptosis rate compared with the blank control group (F =286.95,301.23,respectively,both P ＜ 0.05).With the increase in volume fractions of H2O2,the cellular proliferative activity was significantly gradually decreased (P ＜ 0.05),while the apoptosis rate showed an opposite trend (P ＜ 0.05),except that the 5 ×10-6 H2O2 group showed no significant differences in the apoptosis rate compared with the 10-5 H2O2 group and 10-6 H2O2 group.Acridine orange staining and electron microscopy showed autophagosome formation in the 10-5 H2O2 group,10-6 H2O2 group and positive control group.Western blot analysis revealed that Beclin1 expression and LC3B-Ⅱ/LC3B-Ⅰ ratio were significantly higher in the 10-5 H2O2 group,10-6 H2O2 group and positive control group than in the blank control group (all P ＜ 0.05).RT2 Profiler PCR Array showed significant up-regulation of ATG12,ATG3,ULK1,PIK3CG,PTEN and PIK3C3 genes and significant downregulation of EIF2AK3 gene in the 10-5 H2O2 group,10-6 H2O2 group and positive control group compared with the blank control group.In the 10-5 H2O2 group and positive control group,the mTOR gene was significantly up-regulated,and the ULK2 gene was significantly down-regulated.The 10-6 H2O2 group showed no obvious changes in the expression of mTOR gene,but significant up-regulation of AMPK and JNK1 genes.Conclusion H2O2 at volume fractions of 10-5 and 10-6 can induce autophagy in melanocytes,likely by influencing the expression of some related signaling molecules.
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Objective To evaluate the efficacy of systemic glucocorticoids and intravenous immunoglobulin (IVIG)for the treatment of toxic epidermal necrolysis (TEN). Methods Clinical data on TEN inpatients treated with systemic glucocorticoids alone or in combination with IVIG were collected from the Department of Dermatology, First Affiliated Hospital of Nanjing Medical University from January 2006 to December 2012. Therapeutic outcomes were evaluated in these patients. Statistical analysis was carried out by using a multiple linear regression analysis, binary logistic regression analysis and Cox regression analysis with the SPSS 16.0 software. Results A total of 48 inpatients with TEN were included in this study. Multiple linear regression analysis showed that the maximum daily dose of glucocorticoids for disease control was decreased gradually over years (β=-0.461, P=0.004). However, binary logistic regression analysis revealed no obvious changes in the frequency of administration of IVIG over years. Cox regression analysis showed that both hospitalization duration (RR=0.351, 95.0%CI:0.150-0.825)and the time required for the control of skin lesions (RR=0.492, 95.0%CI:0.245-0.986)decreased with the increase in the frequency of IVIG administration. In addition, with the increase in the maximum daily dose of glucocorticoids for disease control, the time required for the control of skin lesions was also shortened (RR=0.997, 95.0%CI:0.994 -1.000), while no obvious changes were observed in hospitalization duration. Conclusions IVIG shows superiority in controlling lesions, reducing complications and improving the prognosis of TEN. Compared with systemic glucocorticoids, IVIG shows better therapeutic efficacy and less adverse effects, and may be preferentially selected.