Protective effect of WSY6, a caffeic acid derivative, on oxidative stress injury in melanocytes and its mechanism / 中华皮肤科杂志

Objective To evaluate the protective effect of WSY6 (a caffeic acid derivative) on hydrogen peroxide (H2O2)-induced oxidative stress injury in melanocytes,and to explore its potential molecular mechanism.Methods In vitro cultured human primary melanocytes were divided into 5 groups:control group receiving no treatment,H2O2 group treated with 1 mmol/L H2O2,6.25,12.5,25 μmol/L WSY6 groups pretreated with 6.25,12.5,25 μmol/L WSY6 respectively followed by 1-hour treatment with 1 mmol/L H2O2.After 24-hour treatment,MTS assay was performed to determine the survival rate of melanocytes,and the lactate dehydrogenase (LDH)kit was used to detect the LDH leakage level.Some melanocytes were divided into 2 groups:inhibitor group pretreated with the p38 inhibitor for 1 hour followed by 1-hour treatment with 1 mmol/L H2O2,and H2O2 group treated with 1 mmol/L H2O2 for 1 hour.After 24-hour treatment,the LDH kit was used to detect the LDH leakage level.Some other melanocytes were pretreated with 25 μmol/L WSY6 for 1,2,4 hours separately,followed by 1-hour treatment with H2O2.Then,flow cytometry was conducted to detect the level of intracellular reactive oxygen species (ROS).Some melanocytes were treated with 6.25,12.5,25 μmol/L WSY6 separately for 1 hour,followed by 1-hour treatment with H2O2.Then,Western bolt analysis was performed to determine the protein expression of cytochrome c (cyto-c),caspase-3,caspase-9,phosphorylated (p)-p38 MAPK,p-ERK and p-JNK.Results Compared with the control group,the H2O2 group showed significantly decreased survival rate of melanocytes (29.22％ ± 1.31％,P ＜ 0.05),but significantly increased intracellular LDH leakage level (47.19％ ± 4.85％,P ＜ 0.05),elevated intracellular ROS level (18.37 ± 1.59,P ＜ 0.05),and increased expression of caspase-3 and caspase-9.Compared with the H2O2 group,the 6.25,12.5,25 μmol/L WSY6 groups showed significantly increased cell survival rate (52.48％ ± 1.17％,60.21％ ± 0.25％,78.32％ ± 1.73％,P ＜ 0.05),but significantly decreased LDH leakage level (21.99％ ± 0.22％,15.38％ ± 0.45％,13.18％ ± 0.38％,P ＜ 0.05),and the intracellular ROS level was significantly decreased in the 25 μmol/L WSY6 group after 1,2,4 hours of treatment (7.59 ± 1.00,6.22 ± 0.52,5.1 ± 0.48,P ＜ 0.05).The LDH leakage level in melanocytes was significantly lower in the p38 inhibitor group than in the H2O2 group (P ＜ 0.05).Western blot analysis revealed that after the pretreatment with 6.25,12.5,25 μmol/L WSY6 separately,the WSY6 groups all showed obviously decreased expression of caspase-3,caspase-9 and p-p38 compared with the H2O2 group.However,there was no obvious difference in the expression of p-ERK and p-JNK between the WSY6 groups and the H2O2 group.Besides,the WSY6 groups showed decreased expression of p-p53 (a downstream product of p38 MAPK),which decreased along with the increase in the concentration of WSY6.Conclusion WSY6 shows a markedly protective effect on H2O2-induced oxidative stress injury in melanocytes,likely through the p38 MAPK pathway.

Expression of interleukin-13 and its receptors in mycosis fungoides / 中华皮肤科杂志

Objective To measure the expression of interleukin-13 (IL-13) and its receptors in mycosis fungoides (MF) lesions,and to investigate their clinical significance.Methods A total of 34 paraffin-embedded specimens of MF,which was confirmed by clinical and histopathological features,immunophenotyping and/or T-cell receptor gene rearrangements,were collected from Hangzhou Third People's Hospital between January 2010 and March 2016.According to the tumor-node-metastasis (TNM) staging system,5 patients were at stage I A,9 at stage Ⅰ B,17 at stage Ⅱ A,and 3 at stage Ⅱ B.Ten normal skin tissue specimens served as controls.Immunohistochemical study was conducted to measure the expression of IL-13,IL-13Rα1 and IL-13Rα2.Results IL-13,IL-13Rα1 and IL-13Rα2 were all expressed in atypical lymphoid cells and epidermotropic lymphoid cells in MF lesions at various stages.IL-13Rα2 was highly expressed in all the MF lesions.None of IL-13 and its receptors were expressed in normal skin tissues and lymphocytes.The expression rates of IL-13 and its receptors in MF lesions increased along with the progression of MF.Additionally,the expression rates of IL-13 (10.00％ ± 3.14％),IL-13Rα1 (21.43％ ± 6.88％) and IL-13Ro2 (31.14％ ± 6.38％) significantly decreased in MF lesions at stage Ⅰ compared with those at stage Ⅱ (27.50％ ± 11.00％,39.45％ ± 9.43％,44.40％ ± 11.15％,respectively,all P ＜ 0.05),but no significant differences were observed between stage Ⅰ A and Ⅰ B,or between stage Ⅱ A and Ⅱ B (P ＞ 0.05).Conclusion IL-13 and its receptors,especially IL-13Rα2,may be expected to serve as biomarkers for early diagnosis of MF and prediction of its biological behaviors.

Therapeutic effect of narrow band-utraviolet B combined with interferon-alpha-2b for the treatment of mycosis fungoides and its correlation with Treg/Th17 cells / 中华皮肤科杂志

Objective To evaluate the therapeutic effect of narrow band-ultraviolet B (NB-UVB) alone or in combination with interferon-alpha-2b (INF-alpha-2b) for mycosis fungoides (MF),and to assess the correlation between the therapeutic effect and peripheral blood regulatory T (Treg)/T helper type 17 (Th 17) cells.Methods Thirty-three patients with stage ⅠA to ⅡA MF were randomly divided into two groups:NB-UVB group (n =15) receiving NB-UVB radiation alone,combined group (n =18) treated with NB-UVB radiation and intramuscular injection of INF-alpha-2b.Ten healthy volunteers were selected as the control group.Peripheral blood samples were collected before and 9 months after the start of treatment.Flow cytometry was performed to determine the percentages of Treg cells and Th17 cells.Statistical analysis was carried out by using t test,one-way analysis of variance,and Fisher's exact test.Results The average treatment duration was 9 months among these patients.Therapeutic outcomes were significantly better in the combined group than in the NB-UVB group (P =0.023).Among the 15 patients in the NB-UVB group,6 achieved complete remission,3 partial remission,6 showed no response;of the 18 patients in the combined group,12 experienced complete remission,5 partial remission,and 1 showed no response.Before the treatment,the percentages of both Treg and Th17 cells in peripheral blood were significantly higher in the NB-UVB group and combined group than in the control group (both P ＜ 0.05),but similar between the NB-UVB group and combined group (both P ＞ 0.05).After the treatment,the percentages of both Treg and Th17 cells in the NB-UVB group and combined group significantly decreased compared with those before the treatment,but were still higher than those in the control group (both P ＜ 0.05).Additionally,the degree of decrease in the percentages of Treg and Th17 cells was significantly greater in the combined group than in the NB-UVB group (both P＜ 0.05).The seven patients with no response also showed a significant decrease in the percentage of Treg cells (P ＜ 0.05),but no obvious changes in that of Th 17 cells (P ＞ 0.05) after the treatment.Conclusions The therapeutic effect of NB-UVB radiation combined with intramuscular INF-alpha-2b is superior to that of NB-UVB radiation alone for MF,which may be associated with the degree of decrease in peripheral blood Treg and Th 17 cells.

Protective effect of acetylated epigallocatechin gallate on melanocytes from oxidative stress-induced damage / 中华皮肤科杂志

Objective To investigate the protective effect of acetylated epigallocatechin gallate (AcEGCG) against H2O2-induced oxidative damage to human epidermal melanocytes,and to explore its possible mechanism.Methods Human epidermal melanocytes were isolated and cultured in vitro.Some melanocytes were classified into a H2O2 group induced by H2O2 only,EGCG groups and AcEGCG groups induced by H2O2 after pretreatment with different concentrations of EGCG and AcEGCG,respectively.Three concentrations (10,20 and 40 μmol/L) of EGCG or AcEGCG were used to treat melanocytes for 1 hour in MTS assay and lactate dehydrogenase (LDH) leakage assay and for 2 hours in Western blot assay,while only one concentration (40 μmol/L) was used to treat melanocytes for 0.5,1,2 and 4 hours respectively in flow cytometry assay.Some melanocytes treated with only culture medium and 0.1％ dimethyl sulphoxide (DMSO) served as the control group.After additional culture,MTS assay was performed to determine cell survival rate,flow cytometry to detect the level of reactive oxygen species (ROS) in melanocytes,Western blot to measure the expressions of caspase-9 and caspase-3 proteins.Lactate dehydrogenase (LDH) kit was used to detect the leakage of LDH to culture medium.Statistical analysis was carried out by using one-way analysis of variance for comparisons of multiple group means followed by Student-Newman-Keuls-q (SNK-q) test for multiple comparisons.Results Compared with the control group,the H2O2 group showed significantly decreased cell survival rate (22.99％ ± 0.53％,P ＜ 0.01),but increased LDH leakage level (36.58％ ± 0.73％,P ＜ 0.01),intracellular ROS level (19.08 ± 0.57,P ＜ 0.01),as well as caspase-9 (2.65 ± 0.079,P ＜ 0.01) and caspase-3 (2.36 ± 0.057,P ＜ 0.01) expressions.In comparison with the H2O2 group,the cell survival rate was significantly higher in the 10-,20-and 40-μmol/L AcEGCG groups (79.50％ ± 3.62％,86.52％ ± 5.13％,97.81％ ± 5.21％,respectively,all P＜ 0.01) and EGCG groups (43.19％ ± 1.68％,63.34％ ± 3.60％,70.82％ ± 2.1％,respectively,all P ＜ 0.01).However,the 10-,20-and 40-μ mol/L AcEGCG groups and EGCG groups all showed a significant decrease in the expression levels of caspase-9 (AcEGCG groups:1.44 ± 0.067,1.26 ± 0.059 and 1.10 ± 0.072 respectively;EGCG groups:2.31 ± 0.085,2.13 ± 0.091 and 1.35 ± 0.064 respectively,all P ＜ 0.05) and caspase-3 (AcEGCG groups:1.70 ± 0.053,1.57 ± 0.057 and 1.24 t 0.068 respectively,all P＜ 0.05;EGCG groups:2.09 ± 0.076,1.98 ± 0.093 and 1.79 ± 0.056 respectively,all P ＜ 0.05) compared with the H2O2 group.Similarly,a significant reduction was observed in the leakage level of LDH in these AcEGCG and EGCG groups (all P ＜ 0.01) and in ROS levels in the 40-μmol/L AcEGCG and EGCG groups when compared with the H2O2 group.Conclusions AcEGCG has a stronger protective effect against H2O2-induced oxidative damage to human epidermal melanocytes compared with EGCG,which may be realized through clearance of free radicals,antioxidant effects,and decrease of caspase-9 and caspase-3 expressions.

Association analysis between T helper type 17 cells, regulatory T cells and mycosis fungoides / 中华皮肤科杂志

Objective To investigate the changes of T helper type 17 (Thl7) cells and regulatory T (Treg) cells in different stages of mycosis fungoides.Methods Flow cytometry was performed to determine the percentage of Treg and Th17 cells in peripheral blood from 28 patients with mycosis fungoides (MF),13 patients with large plaque parapsoriasis (PP),17 patients with lichen planus (LP) and 10 healthy human controls,and immunohistochemistry to detect the expressions of forkhead box protein 3 (FOXP3) and interleukin (IL)-17 in tissue specimens from 40 patients with MF,13 with PP,17 with LP and 10 healthy human controls.Statistical analysis was carried out by one-way analysis of variance and Pearson correlation analysis.Results As far as the percentage of Treg cells in peripheral blood was concerned,MF,PP and LP patients were significantly higher than the healthy controls (8.09％ ± 1.68％,6.53％ ± 1.67％ and 2.84 ％ ± 1.16％ vs.1.01％ ± 0.35,all P＜ 0.05),PP patients were higher than LP patients and healthy controls (both P ＜ 0.05),and LP patients were higher than healthy controls (P ＜ 0.05).The percentage of Th17 cells in peripheral blood was significantly increased in MF patients compared with PP patients,LP patients and healthy controls (3.22％ ± 0.82％ vs.2.46％ ± 0.79％,1.38％ ± 0.47％ and 0.59％ ± 0.30％,all P ＜ 0.05).Elevated expression rate of FOXP3 was observed in MF,PP and LP lesions as compared with normal skin (14.94％ ± 4.46％,11.95％ ± 4.72％,6.32％ ± 2.81％ vs.3.43％ ± 1.79％,all P ＜ 0.05),and in MF and PP lesions compared with LP lesions (both P ＜ 0.05),but no significant difference was observed between MF and PP lesions (P ＞ 0.05).There was a significant increase in the expression rate of IL-17 in MF lesions compared with PP lesions,LP lesions and normal skin (15.89％ ± 4.27％ vs.12.02％ ± 3.34％,4.84％ ± 1.93％ and 2.62％ ± 0.89％,all P ＜ 0.05).The Th17/Treg ratio in peripheral blood was significantly lower in MF and PP patients than in LP patients and healthy controls (0.41 ± 0.11 and 0.39 ± 0.12 vs.0.50 ± 0.06 and 0.57 ± 0.19,all P ＜ 0.05).A positive correlation was observed between the proportion of Thl7 cells and Treg cells (r =0.423,P ＜ 0.05) in patients with early-stage MF,but not in those with tumor-stage MF.The proportion of Th17 cells decreased,but that of Treg cells continuously increased in patients with tumor-stage MF.However,no significant difference was noted in the proportion of Thl7 cells or Treg cells among patients with different stages of MF.Conclusion The imbalance between Treg and Th17 cells may be involved in the occurrence and development of MF.

Roles of protein kinase B and mitogen-activated protein kinase pathways in the protection by nicotinic acid against ultraviolet B-induced damage in keratinocytes / 中华皮肤科杂志

Objective To investigate the intracellular signal transduction pathways involved in the protective effect of nicotinic acid against ultraviolet B(UVB)-induced damage in human skin keratinocytes.Methods Cultured human keratinocyte HaCaT cells were divided into several groups to be treated with nicotinic acid,UVB irradiation,LY294002(an inhibitor of Akt),U0126(an inhibitor of extracellular signal-regulated kinase(ERK)1/2),SB203580(an inhibitor of P38)alone or in combination for different durations.Then,Western blot was performed to quantify the phosphorylation levels of the protein kinase B(Akt)/MAPK pathwayassociated proteins including Akt,P38,JNK and ERK1/2,MTT assay to evaluate the activity of HaCaT cells,enzyme-linked immunosorbent assay to determine the levels of endothelin-1(ET-1)and basic fibroblast growth factor(bFGF)in the culture supernatant of HaCaT cells,and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL)to evaluate the apoptosis in HaCaT cells.Results As Western blot showed,phosphorylated Akt,P38,JNK and ERK1/2 were markedly activated within 60 minutes after pretreatment with nicotinic acid and irradiation with UVB(all P ＜ 0.01),and the activation was more significant for phosphorylated Akt,P38,and ERK1/2 within 2 hours(all P ＜ 0.01).Nicotinic acid effectively suppressed the UVB-induced cell death and apoptosis in HaCaT cells.The levels of supernatant ET-1 and bFGF were significantly decreased in HaCaT cells treated with the above 3 inhibitors followed by UVB irradiation than in those treated with the inhibitors alone(all P ＜ 0.05),and nicotinic acid pretreatment only reversed the decrease in supernatant bFGF in HaCaT cells treated with SB203580 followed by UVB irradiation.Conclusion The Akt signaling pathway may play a regulatory role in the protection by nicotinic acid against UVB-induced damage in HaCaT cells.

Preparation and identification of Nogo-66 polyclonal antibodies / 中华检验医学杂志

Objective To prepare polyclonal antibodies of anti Nogo-66, the extracellular region of one central nervous system neurite regeneration inhibitor Nogo, which could be used to further identification and functional study of Nogo molecule.Methods Preparing rabbit anti rat Nogo-66 polyclonal antibodies with a purified Nogo-66 fusion protein expressed in E.coli system. Studying its specificity by Western-blot and immuno-histochemical techniques and identifying its biological activity in PC12 cells.Results The high titer (1∶[KG-*2]10 000) anti rat Nogo-66 polyclonal antibodies were obtained.This antibody could specifically recognize the Nogo protein expressed in E.coli system.Immuno-histochemical staining indicated that the Nogo was widely expressed in rat spinal cord neurons and oligodendrocytes.It could effectively block the neurite extensioninhibition of Nogo protein in PC12.Conclusion Successful preparation of anti rat Nogo polyclonal antibodies provides a useful tool in identification or further functional study of Nogo molecule.