Objective To evaluate the effect of human dermal mesenchymal stem cells (DMSCs) on the expression and secretion of interleukin (IL)-13 by perilesional CD8+ T lymphocytes from patients with vitiligo.Methods Tissue specimens were obtained from the perilesional region of six patients with active vitiligo,and CD8+ T lymphocytes were isolated from both the tissue specimens and peripheral blood of these patients.DMSCs and melanocytes were obtained from the foreskin tissue of healthy males.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to estimate the effect of different concentrations of recombinant IL-13 on the proliferation of melanocytes,reverse transcripition-PCR and Western blotting to detect the mRNA and protein expressions of IL-13 in perilesional and peripheral blood CD8+ T lymphocytes respectively,real-time quantitative reverse transcription (RT)-PCR and enzyme-linked immunosorbent assay (ELISA) to detect the IL-13 mRNA expression in,and IL-13 protein expression in the culture supematant of,CD8+ T lymphocytes before and after coculture with DMSCs,respectively.Statistical analysis was done by t test.Results No obvious changes were observed in the proliferation of melanocytes treated with different concentrations (10,50,100,250,500 μg/L) of recombinant IL-13 for various durations (24,48,72 and 96 hours)compared with untreated melanocytes (all P ＞ 0.05).Both perilesional and peripheral blood CD8+ T lymphocytes expressed IL-13,and the expression was stronger in perilesional than in peripheral blood CD8+ T lymphocytes.A significant decrease was noted in IL-13 mRNA expression (0.100 0 ± 0.002 4 vs.0.383 2 ± 0.018 7,P ＜ 0.05) and protein level in the culture supernatant ((1 509.62 ± 48.44) ng/L vs.(5 507.98 ± 34.11) ng/L,P ＜ 0.05) of CD8+ T lymphocytes cocultured with DMSCs compared with monocultured CD8+ T lymphocytes.Conclusions There is a strong expression of IL-13 by perilesional CD8+ T lymphocytes in patients with vitiligo,which may be inhibited by DMSCs and serve as a target for the treatment of vitiligo.

Objective To study the effects of Fructus ligustri lucidi and its monomers, tyrosol and oleanotic acid, on the migration of mouse melanoblast cell line (NCCmelb4M5). Methods Cultured NCCmelb4M5 cells were treated with Fructus ligustri lucidi (0.0625, 0.125, 0.25, 0.5, 2 mg/mL), tyrosol (0.02, 0.04, 0.08, 0.16, 0.8 mg/mL) and oleanolic acid (0.0625, 0.125, 0.25, 0.5, 2.5 mg/mL), respectively,for 48 hours followed by the detection of cell proliferation with MTT assay. The working concentration of the three drugs was determined according to the results of MTT assay. Scratch and transwell assays were performed to observe the effect of Fructus ligustri lucidi and its monomers at working concentration on the migration of NCCmelb4M5 cells. Results Based on the results of MTT assay, the working concentration of Fructus ligustri lucidi, tyrosol and oleanolic acid was determined at 0.125 mg/mL, 0.08 mg/mL and 0.0625 mg/mL respectively, and at these concentrations, these drugs exhibited a cytotoxity lower than that of absolute alcohol with no obvious stimulation of cell proliferation. Scratch and transwell assay revealed a promoting effect of both Fructus ligustri lucidi and tyrosol on melanoblast migration (P<0.05), while oleanolic acid had little effect on melanoblast migration. Conclusions The extract of Fructus ligustri lucidi has a significant stimulatory effect on the migration of mouse melanoblasts, and tyrosol may be an active component of Fructus ligustri lucidi associated with confirmative effect on migration of mouse melanoblasts.

ObjectiveTo assess the relationship of interleukin-17(IL-17) and transforming growth factor-β(TGF-β) with the development of vitiligo.MethodsEnzyme-linked immunosorbent assay (ELISA) was carried out to measure the levels of IL-17 and TGF-β in sera from 120 patients with vitiligo and 60 healthy controls.The correlations of serum IL-17 and TGF-β levels with patients' gender,stage and duration of disease,involved body area and presence of family history were assessed.ResultsThe level of serum IL-17 was significantly higher in patients with active vitiligo than in the controls and patients with stable vitiligo (both P ＜0.05).With the rise in involved body area,the level of serum IL-17 gradually increased (x2 =12.656,P ＜0.05).The level of TGF-β in patients with active vitiligo was a little higher than that in the controls and patients with stable vitiligo,with no significant difference between these groups(both P ＞ 0.05).Conclusions The levels of serum IL-17 and TGF-β are somewhat correlated with the activity of vitiligo,and both of them may play a certain role in the pathogenesis of vitiligo.

Objective To evaluate the protective effects of tea polyphenols against the destruction of melanocytes by CD8+ T cells from vitiligo patients.Methods Skin tissue was resected from the margin of vitiligo lesions followed by the isolation and culture of CD8+ T lymphocytes,and from the normal skin of vitiligo patients followed by the isolation and culture of melanocytes.Flow cytometry was carried out to evaluate the purity of CD8+ T cells.The melanocytes were cocultured with the CD8 + T cells at different ratios followed by the evaluation of killing effect of CD8+ T cells.Various concentrations (200 and 400 μg/ml) of tea polyphenols were added into the co-culture system of CD8+ T cells and melanocytes at a ratio of 5 ∶ 1 followed by an additional culture of 48 hours.Then,flow cytometry was performed to detect the apoptosis in melanocytes in the coculture system.Results CD8+ T lymphocytes were successfully obtained from the marginal area of vitiligo lesions with a purity of more than 90％,which highly expressed the antigens CD137 and CD69.The coculture with CD8+ T cells markedly accelerated the apoptosis in melanocytes,while the accelerative effect was inhibited by tea polyphenols of 200 and 400 μg/ml.Conclusions The CD8+ T cells infiltrating the edge of vitiligo lesions display a potential destructive effect on autologous melanocytes from vitiligo patients,and tea polyphenols have a protective effect against the destruction of melanocytes by CD8+ T cells.

Objective To evaluate the effect of injuries on monobenzone-induced vitiligo-like depigmentation in mice.Methods Forty C57BL/6 mice were randomly and equally divided into four groups:negative control group topically treated with vaseline cream,model group induced by topical monobenzone (40％) cream,acupuncture group receiving acupuncture treatment (15 times) once every three days,and acupuncture combined with monobenzone group receiving both monobenzone induction and acupuncture treatment.The treatment lasted 50 days and mice were sacrificed 15 days after the end of treatment.Hair decolorization was observed with naked eyes,and skin decolorization with reflectance confocal microscopy (RCM) on a daily basis.Tissue specimens were obtained from depigmented skin at monobenzone-uninduced sites,and subjected to hematoxylin and eosin (HE) staining for the cvaluation of lymphocytic infiltration as well as immunofluorescence staining for the detection of CD8+ T cell expression.Statistical analysis was done by t test.Results Varying degrees of depigmentation were observed in both monobenzone-induced and-uninduced sites in both the model group and acupuncture combined with monobenzone group,and the latter group showed earlier,larger and more stable depigmentation than the former group.At 15 days after the end of treatment,the decolorization area index in the model group and acupuncture combined with monobenzone group was 3.45 ± 0.17 and 3.90 ± 0.25 at monobenzone-induced sites respectively(t =7.433,P ＜ 0.05),1.90-± 0.12 and 2.85 ± 0.27 at monobenzone-uninduced sites respectively (t =7.529,P ＜ 0.05).Significant differences were observed in the fluorescence intensity of CD8 + T cells at monobenzone-uninduced depigmented sites between the model group and acupuncture combined with monobenzone group (175.528 ± 10.711 vs.645.928 ± 12.652,t =8.105,P ＜ 0.05),and there was a more evident infiltrate with lymphocytes and CD8+T cells in the monobenzone-uninduced depigmented sites in the acupuncture combined with monobenzone group.Conclusion Local destruction of skin barrier may promote monobenzone-induced vitiligo-like decolorization in mice.

Objective To investigate the level of NF-E2 related factor 2 (Nrf 2) in vitiligo lesions.Methods Tissue samples were obtained by press suction blisters at lesional and donor sites of 12 patients with vitiligo who were managed with epidermal transplantation. Four lesional samples from the patients were subjected to primary culture and the level of Nrf 2 was detected by AEC immunohistochemistry after 48hours of culture. Western blotting was utilized to further detect the level of cytoplasmic and nuclear Nrf 2 in tissue samples from the other 8 patients with vitiligo. Results Immunohistochemistry revealed that Nrf 2 was predominantly expressed in cytoplasm, rather than nuclei, of keratinocytes in vitiligo lesions compared with the normal skin of patients. The level of nuclear Nrf 2 was significantly lower in lesions than that in normal skin (0.10 ± 0.03 vs 0.26 ± 0.03, P ＜ 0.01) of the patients. In contrast, there was no significant dif- ference in the level of cytoplasmic Nrf2 between lesional and normal skin (0.61 ± 0.03 vs 0.60 ～ 0.02, P ＞0.05) of patients. Conclusion These results reveal an abnormality of nuclear translocation of Nrf 2 in vitili-go lesions.

Objective To express and purify the epitope peptide of human melanin-concentrating hormone receptor 1, and to evaluate its performance in the detection of autoantibodies in vitiligo patients. Methods The target gene encoding the epitope peptide of human melanin-concentrating hormone receptor 1 was synthesized, cloned to prokaryotic expression vector pGEX-4T-2 which was then transferred to E. coli BL21. The protein expression was induced by isopropy-β-D-thiogalactoside (IPTG) and identified with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Blocking ELISA was carried out with membrane proteins extracted from melanocytes as the blocking antigen. The antigenicity of the peptide was detected in sera from 100 patients with progressive vitiligo and 30 healthy human controls. Results The recombinant expression vector was successfully constructed, and the target protein was successfully expressed in E.coli, which was evidenced by SDS-PAGE and Western blot. With the glutathione S-transferase (GST) purification kit, the purity of the recombinant protein reached 100% when the sampling weight was less than 0.625 μg.The binding of the target protein with serum IgG antibodies from vitiligo patients could be blocked by natural membrane antigen of melanocytes. Of the 100 sera from patients with progressive vitiligo, 36 were reactive with the target protein. Conclusions The epitope peptide of human melanin-concentrating hormone receptor 1 has been successfully expressed and purified. The purified protein can bind with serum IgG antibodies from vitiligo patients, and may be applied to the detection of autoantibodies against human melanin-concentrating hormone receptor 1.

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.

Objective To investigate the effect of nuclear translocation of E2p45 related factor 2 (Nrf2)on the biological activity of melanocytes.Methods Plasmid vectors containing wild-type nrf2 gene (pcDNA-nrf2) and nls-deleted nrf2 gene (pcDNA-nrf2△nls) were constructed.B10BR normal murine melanocytes were classified into three groups,i.e.,untransfected group,wild-type nrf2 group transfected with pcDNA-nrf2,and mutated nrf2 group transfected with pcDNA-nrf2△nls.Each of the above groups were further divided into three subgroups:control subgroup receiving no treatment,hydrogen peroxide (H2O2) subgroup treated with H2O2 of 200 μmol/L for 24 hours,and combined subgroup pretreated with tert-butyl hydroquinone (TBHQ) followed by treatment with H2O2 of 200 μmol/L for 24 hours.Subsequently,methyl thiazolyl tetrazolium (MTT) assay was performed to evaluate the proliferative activity of cells,dopa oxidation assay to determine tyrosinase activity,Transwell assay to estimate cell migration ability,Western blot to quantify the expressions of Nrf2 and his tag fusion protein.Results TBHQ significantly enhanced the nuclear expression of Nrf2 in B10BR cells transfected with pcDNA-nrf2 or pcDNA-nrf2△nls (both P ＜ 0.01).No significant difference was observed in tyrosinase activity between untreated wild-type nrf2 group,mutated nrf2 group,and untransfected group (P ＞ 0.05).There was a statistical decrease in tyrosinase activity in the two H2O2-treated transfected groups compared with the untreated transfected groups (both P ＜ 0.05),and the decrease was reversed by TBHQ pretreatment in the wildtype nrf2 group (P ＜ 0.05),but not in the mutated nrf2 group (P ＞ 0.05).Further more,the proliferative activity of B10BR cells experienced no obvious changes in the wild-type nrf2 group (P ＞ 0.05),but was significantly reduced in the untransfected group (P ＜ 0.05) and mutated nrf2 group (P ＜ 0.01) after the H2O2 treatment compared with the corresponding untreated groups.TBHQ could protect the pcDNA-nrf2-transfected B10BR cells,but not pcDNA-nrf2△nls-transfected B10BR cells,from H2O2-induced oxidative damage.Transwell assay showed no significant difference in migration ability among these nine groups (P ＞ 0.05).Conclusions Abnormal nuclear translocation of Nrf2 could affect antioxidant activity of,proliferative activity of and tyrosinase activity in melanocytes.TBHQ may enhance the tyrosinase activity in,proliferative activity and antioxidant activity of melanocytes via activating the nuclear expression of wild type Nrf2.

Objective:To investigate the efficacy of systemic glucocorticoid treatment and its related factors in progressive vitiligo patients with vitiligo disease activity (VIDA) scores ≥ 2 points.Methods:A total of 272 progressive vitiligo patients with VIDA scores ≥ 2 points and skin lesion area < 1% of body surface area, who received no systemic glucocorticoid treatment, were collected from Department of Dermatology, the Third People′s Hospital of Hangzhou from June 2018 to June 2019. The area and type of skin lesions, VIDA scores, predisposing factors and special clinical markers (trichrome vitiligo, confetti-like depigmentation, Koebner phenomenon and inflammatory vitiligo) were analyzed. These patients were randomly divided into 3 groups by a random number table: topical glucocorticoid group (62 cases) , oral prednisone + topical glucocorticoid group (76 cases) and compound betamethasone injection + topical glucocorticoid group (134 cases) , and the latter two groups were also called as the systemic and topical glucocorticoid group. The patients in the topical glucocorticoid group were treated with halometasone cream or 0.05% clobetasol propionate cream once a day; during the oral prednisone treatment, the dose was adjusted once every 7 days, and gradually reduced from 30 mg/d to 20, 15, 10 and 5 mg/d, and the treatment lasted 35 days; during the treatment with compound betamethasone injection, intramuscular injection was performed once every 20 days at a dose of 1 ml for 2 sessions. The stable disease rate (defined as the proportion of patients experiencing no progression during the study among the analyzed patients) was calculated in these groups after 3 months of treatment, and changes in vitiligo types were evaluated after 1 year of follow-up. Statistical analysis was carried out by using Kruskal-Wallis H test, χ2 test and Fisher′s exact test. Results:After 3-month treatment, there was a significant difference in the expansion rate of skin lesion area among the 3 groups ( H = 12.468, P < 0.001) , and the expansion rate of skin lesion area was significantly lower in the oral prednisone + topical glucocorticoid group and compound betamethasone injection + topical glucocorticoid group than in the topical glucocorticoid group ( P < 0.001, = 0.005, respectively, α = 0.016 7) ; among the patients with slowly progressive vitiligo (VIDA scores = 2 or 3 points) , the stable disease rate was significantly higher in the systemic and topical glucocorticoid group than in the topical glucocorticoid group ( χ2 = 23.973, 11.877, respectively, both P < 0.001) ; the stable disease rate also significantly differed among the patients with different VIDA scores (VIDA scores = 2, 3 or 4 points) in the systemic and topical glucocorticoid group ( χ2 = 17.122, P < 0.001) . After 3-month treatment, the patients with predisposing factors or special clinical markers showed significantly decreased stable disease rate (47.3% [35/74], 41.2% [47/114], respectively) compared with those without predisposing factors or special clinical markers (70.6% [96/136], 87.5% [84/96]; χ2 = 11.098, 47.548, respectively, both P < 0.001) . After 1 year of follow-up, the proportion of patients with localized vitiligo converted into non-localized vitiligo was significantly higher in the topical glucocorticoid group (41.9%, 26/62) than in the systemic and topical glucocorticoid group (21.9%, 46/210; χ2 = 10.328, P = 0.006) , and higher in the group with predisposing factors or special clinical markers than in that without predisposing factors or special clinical markers respectively (both P < 0.01) . Conclusions:Early systemic glucocorticoid treatment should be performed in the progressive vitiligo patients with high VIDA scores, predisposing factors and special clinical markers.