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Objective:To investigate the effect of human tumor suppressor folliculin (FLCN) on the expression of melanocyte chemokines (MC) mediated by immune factors in vitiligo.Methods:The MC of vitiligo patients that received autologous melanocyte transplantation in the Department of Dermatology, Hangzhou Third People′s Hospital from January to April 2019 were collected. The blister fluid of the white spot and the normal part was taken. Western blot was used to analyze the expression difference of MC and FLCN protein in normal, vitiligo patients and that induced by immune factors; FLCN shRNA lentivirus was constructed by shRNA and transfected into normal MC (FLCN shRNA MC) to interfere with the expression of silenced FLCN gene. The effect of immune factors on chemokines in FLCN shRNA MC was detected by ELISA.Results:The results of Western blot showed that FLCN protein was highly expressed in melanocytes of vitiligo patients, immune factors stimulated FLCN protein expression in normal melanocytes significantly increased ( t=1.27; P<0.001), chemokine CXCL10 and CCL20 also significantly increased ( t=104.53 and 60.21, respectively; P<0.001). The expression of FLCN in FLCN shRNA MC was significantly decreased ( F=1.95, P<0.001); and the high expression of CXCL10 and CCL20 induced by immune factors was significantly inhibited ( F=93.676 and 74.096, all P<0.001). Conclusions:Immune factors can stimulate the expression of CXCL10 and CCL20, which are closely related to vitiligo, while FLCN is a key protein involved in immune factors inducing melanocyte chemokine expression.
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Objective To assess the quality of life, prevalence of depression and their influencing factors in patients with alopecia, to investigate, and to provide evidences for relevant clinical therapeutic strategies to improve patients′ quality of life. Methods A questionnaire survey was carried out in 237 patients with androgenetic alopecia or alopecia areata, and their quality of life and depression were measured using dermatology life quality index(DLQI)and center for epidemiologic studies depression scale (CES-D), respectively. Factors influencing the quality of life and depression were analyzed by analysis of variance and logistic regression analysis. Results Among 237 patients with alopecia, 218 questionnaires were eligible with the mean score of DLQI being 9.1 ± 5.4. Alopecia had a moderate effect on the quality of life in general, and 38.07%of the patients were severely affected. The mean score of CES-D was 14.8 ± 9.9, and 37.61%of the patients showed depressive tendency. The DLQI score was positively correlated with CES-D score(r=0.29, P<0.01). One-way analysis of variance(ANOVA)showed that the DLQI score was not affected by age, gender, education level or the number of visits. Multivariate logistic regression analysis revealed that the risk factors for depressive tendency in patients with alopecia were the number of visits (OR = 1.81, 95% CI: 1.21- 2.69) and DLQI score (OR = 1.08, 95% CI: 1.03- 1.13). Conclusion Alopecia not only affects the quality of life, but also mental states of patients.
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To evaluate the efficacy of different courses of Narrowband Ultraviolet B phototherapy ( NB-UVB) in the treatment of vitiligo .50 vitiligo patients who had finished 3 courses of phototherapy and met the inclusion criteria were included in our study .Each course contained at least 30 times of phototherapy , and between two courses , there was a rest of 3~6 months .The repigmentation of each vitiligo lesion after every course of phototherapy was recorded .To the same lesion , the efficacy difference between the first course and the second course was not statisti -cally significant.But the efficacy of the first course was better than the third course (P<0.05),while the efficacy of the second course was also better than the third course ( P<0.05 ) .There was no significant difference among the three courses concerning the average single irradiation dose .In conclusion , when using NB-UVB in treating vitili-go, the efficacy of first course was equivalent to the second course , but it reduced in the third course .Whenever a plateform stage occurs , a rest of more than 3 months is long enough for the vitiligo lesion to recover initial light sen-sitivity.
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Objective To evaluate the therapeutic efficacy of autologous melanocyte transplantation for the treatment of vitiligo in patients with abnormal thyroid function.Methods A total of 60 patients with vitiligo were enrolled in this study,including 30 with abnormal thyroid function and 30 without.Epidermal sheets were obtained by suction blister biopsy from the normal skin of all the patients followed by melanocyte isolation and culture.After 2-5 passages of subculture,the melanocytes were transplanted onto vitiliginous lesions,which were abraded previously by ultra-pulsed CO2 laser,in the corresponding patients.All the patients were followed for 6-12 months.Results Of the 30 patients with abnormal thyroid function,7 patients achieved more than 90％ repigmentation,9 patients 50％-89％ repigmentation,53.3％ more than 50％ repigmentation,with the average repigmentation rate being 47％ within 6 months after the transplantation.Meanwhile,13 out of the 30 patients without abnormal thyroid function showed more than 90％ repigmentation,11 showed 50％-89％ repigmentation,with the average repigmentation rate being 75％.Both the cure rate and response rate were significantly higher in the patients without abnormal thyroid function than in those with (cure rate,43.3％ vs.23.3％,P＜ 0.05; response rate,80％ vs.53.3％,P＜ 0.05).Significant differences were also found in the response rate for lesions on the face or neck and for those sized more than 20 cm2 between the two groups of patients (both P ＜ 0.05).The lesions transplanted with epidermal melanocytes from the waist exhibited the lowest cure rate and response rate.Conclusion Clinical or subclinical thyroid dysfunction may have a negative impact on the efficacy of autologous melanocyte transplantation in vitiligo.
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A 3-year-old boy presented with a 3-month history of brown-yellow papules scattered on the trunk.The first skin biopsy showed a dermal infiltrate of many mononuclear histiocytes,eosinophilic granulocytes and a small number of lymphocytes.Immunohistochemical examination of the lesions demonstrated positive reactions with anti-CD68,-S100 and-CD1a (partial) antibodies.After the biopsy,the skin lesions gradually turned dark and partially regressed leaving hyperpigmentation,but new lesions continuously appeared.Four months later,a second biopsy was performed,and showed a dermal infiltrate of histiocytes with eosinophilic granulocytes and a few multinucleated giant cells.Immunohistochemistry showed that the histiocytes stained positive for CD68,but negative for S100 and CD1a.Based on the above findings,the patient was diagnosed with juvenile xanthogranuloma.
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Aim To study the effect of halometasone in combination with scutellaria baicalensis georgi on the vitiligo mice induced by monobenzone. Methods 40% monobenzone cream was applied to induce vitiligo in C57BL/6 mice. Through the halometasone, halo-metasone and scutellaria baicalensis georgi combined with 40% monobenzone cream, the influence of halo-metasone and scutellaria baicalensis georgi on mice de-colorizing was studied. Hair decolorizing was observed with the naked eye, the skin decolorizing was observed by reflectance confocal microscopy ( RCM ) , and CD8 +T cell infiltration was tested with immunofluores-cence detection. The serum levels of interleukin-6(IL-6 ) and tumor necrosis factor-α( TNF-α) were deter-mined by enzyme linked immunosorbent assay ( ELISA) . Results Mice in model group showed de-pigmentation at both the monobenzone application part and non-application part. The halometasone group did not show significant therapeutic efficacy. In halometa-sone and scutellaria baicalensis georgi treatment group, there was less decolorization, the occurrence ratio, the scores of occurring time and size were lower compared with model group. There were fewer infiltrated lympho-cytes and CD8 +T cells. Halometasone and scutellaria baicalensis georgi group also showed that the serum levels of IL-6,TNF-α decreased. Conclusion Halo-metasone and scutellaria baicalensis georgi have thera-peutic effect on vitiligo mice induced by monobenzone.
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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.
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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.
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Objective To establish a model for studying CD8+ cytotoxic T lymphocyte proliferation in vitro and to screen traditional Chinese drugs (TCDs) with immunosuppressive effects.Methods Spleen tissue was isolated from mice,and made into single cell suspensions followed by separation of CD8+ T lymphocytes with specific antibodies.Then,the CD8 + T lymphocytes were seeded into anti-CD3/CD28 antibody-coated 96-well plates and cocultured with the extracts of 23 TCDs (100 mg/L) separately for 96 hours.Those ceils cultured with and without the presence of anti-CD3/CD28 antibody alone served as the positive control and negative control respectively.The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt (MTS) assay was performed to evaluate the proliferation of cells and to select the top four TCDs with the strongest inhibitory effect.The relationship between the inhibitory effect and TCD concentrations was further assessed for the four selected TCDs.Enzyme-linked immunospot (ELISPOT) assay was carried out to estimate the influence of the four TCDs on the secretion of interferon (IFN)-γ by CD8+ T lymphocytes induced by anti-CD3/CD28 antibodies.Statistical analysis was done by nonparametric rank sum test.Results Of the 23 TCDs,14 significantly inhibited the proliferation of CD8+ T lymphocytes (all P ＜ 0.05),of which,Rhizoma Coptidis,Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae displayed the strongest inhibitory capacity with the 50％ inhibitory concentration being 25,35,50 and 60 mg/L respectively,and the 100％ inhibitory concentration being 200,100,200 and 200 mg/L respectively.The anti-CD3/CD28 antibody-induced secretion of IFN-γby CD8+ T lymphocytes was markedly suppressed by Radix Scutellariae,Radix Aucklandiae and Rhizoma Curcumae Longae at the concentration of 100 mg/L,but not by Rhizoma Coptidis at this concentration.Conclusions A model for studying the proliferation of CD8+ T lymphocytes is successfully developed in vitro,and four TCDs with strong inhibitory effects on the proliferation of CD8+ T lymphocytes have been screened out with this model.
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Objective To investigate the dermoscopic and confocal microscopic features of Riehl's melanosis,as well as their association with histopathological findings.Methods Ten patients with a previously established diagnosis of Riehl's melanosis were recruited.The lesions of the patients were observed using dermoscopy and in vivo confocal laser scanning microscopy (CLSM),followed by histopathologic analysis.Results On dermoscopy and CLSM,all the lesions showed the following features:pseudonetwork,liquefaction degeneration of the basal cell layer,and incontinence of pigment.Conclusion Both dermoscopy and in vivo CLSM can serve as noninvasive auxiliary diagnostic tools for Riehl's melanosis.
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Objective To analyze the clinical features and evolution ot segmental vitiligo in children.Methods A standardized questionnaire was used to clinically investigate segmental vitiligo in 170 children aged ＜ or =12 years and 174 adults with an age of onset ＞ 18 years.Data analysis was done by the software SPSS16.0.Results The female to male ratio was 108:62 in the 170 child patients,significantly different from that in the adult patients (x2 =14.44,P ＜ 0.05).Localized vitiligo occurred as the initial presentation in 82.9％ (141/170) ofthe children and 76.4％ (133/174) of the adults,and progressed into segmental vitiligo in half a year in 76.5％ of the 141 children and 63.9％ of the 133 adults.Vitiligo entered the quiescent stage after half-a-year progression in 71.6％ of the children and 67.3％ of the adults with localized vitiligo as the initial presentation,and after one-month progression in 62.1％ of the children and 41.5％ of the adults with segmental vitiligo as the first presentation (x2 =8.39,P ＜ 0.01).Head and face were affected at the onset of segmental vitiligo in 44.1％ of the childhood cases and 56.9％ of the adult cases.Single nerve segments were involved at the onset in 94.7％ of the child patients and 86.8％ of the adult patients (x2 =0.04,P ＞ 0.05).The proportions of patients with white hairs,autoimmune diseases,and family history were significantly lower in the child patients than in the adult patients (x2 =15.88,5.62,6.66 respectively,all P ＜ 0.05).Conclusions Childhood-onset segmental vitiligo shows a predilection for males,usually presents as localized vitiligo at the onset,and becomes quiescent after half-a-year progression in more than 70％ of patients.
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Objective To assess the microscopic features of lentigo by using in vivo confocal laser scanning microscopy (CLSM).Methods This study included 30 patients clinically diagnosed with solar lentigo and 10 patients with lentigo simplex.Lentigo lesions and perilesional normal skin were examined by in vivo CLSM.Tissue specimens were also obtained from lesional and perilesional normal skin and subjected to routine histopathologic examination.Results The CLSM features of lentigo were mainly observed at the dermo-epidermal junction.In all of these cases of lentigo,there was an increase in the length and number of rete ridges,with a marked hyperpigmentation of the basal layer.Several distinct microscopic patterns were observed,such as extended,irregularly shaped papillary dermis surrounded by highly refractile cells (presumed to be basal keratinocytes).Histopathologically,there was an infiltrate of a small quantity of melanophages and lymphocytes in superficial dermis.Conclusion As far as lentigo lesions are concerned,CLSM images are consistent with histopathological findings.
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Objective To characterize the progression of segmental vitiligo.Methods Clinical data were collected using questionnaires from 387 patients with segmental vitiligo at the Department of Dermatology,Third People's Hospital of Hangzhou,between October 2011 and October 2012.The progression of segmental vitiligo was analyzed.Results Among the 387 patients,329 initially sufferred from focal vitiligo that evolved into segmental vitiligo later,58 began with segmental vitiligo.Vitiligo progressed most rapidly in the initial three months,and tended to be stable after three years in 220 (66.9％) of the 329 patients experiencing the evolution from focal to segmental vitiligo.By contrast,vitiligo developed most rapidly in the first month,and tended to be quiescent after one year in 40 (69.0％) of the 58 patients presenting with segmental vitiligo only.The condition still remained active in 27.6％ (107/387) of these patients after several years of progression.Totally,333 (86.0％) patients showed the involvement of single ganglion at the onset of vitiligo,including 173 (52.0％) patients with the involvement of trigeminal ganglion.Autologous melanocyte transplantation was conducted for 62 patients whose condition had been quiescent for more than one year,and 56 (90.3％) patients achieved more than 80％ repigmentation.Conclusions Segmental vitiligo,which tends to remain quiescent after three-year progression,seems to have a more rapid onset than focal vitiligo.Autologous melanocyte transplantation appears to be an effective treatment for segmental vitiligo in stable stage.
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Objective To evaluate the effect of calcipotriol on the proliferation of and cytokine secretion by melanocytes and perilesional CD8+ cytotoxic T lymphocytes (CTLs) from patients with vitiligo.Methods Melanocytes isolated from abdominal skin and CD8+ CTLs from perilesional skin of patients with vitiligo were subjected to successive culture in vitro.After several passages,the melanocytes and CD8+ CTLs were cultured alone or in combination with or without the presence of various concentrations of calcipotriol for 24 to 48 hours.MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium,inner salt) method was used to evaluate the proliferative activity of cells,enzyme-linked immunosorbent assay to determine the levels of interleukin (IL)-6,tumor necrosis factor (TNF)-α and interferon (IFN)-γ in the culture supematant of cells,flow cytometry to detect cell apoptosis.Some co-cultured melanocytes and CTLs were treated with calcipotriol of 10-8 mol/L and anti-IL-6 antibody of various concentrations (0,1,2,2.5,5,10 mg/L) for two days followed by enumeration of cells.The concentrations of 108 and 10-9 mol/L (calcipotriol) were chosen for relevant tests.Results There was a marked apoptosis in MCs after coculture with CD8+ CTLs.The 24-hour treatment with calcipotriol of 104 and 10-9 mol/L had no obvious effect on the proliferation of melanocytes cultured alone (both P ＞ 0.05),but accelerated the proliferation of melanocytes cocultured with CTLs (both P ＜0.05) as well as that of CD8+ CTLs cultured alone or in combination with melanocytes (all P ＜0.05).A statistical decrease was observed in IL-6,TNF-α and IFN-γlevels in the supernatant of cocultured melanocytes and CTLs compared with those in the supernatant of melanocytes and CTLs cultured alone,and calcipotriol of 10-9 mol/L intensified the decrease in supernatant IL-6 level (t =2.89,P ＜0.05),but no statistical changes were noted for the level of TNF-α or IFN-γin the supernatant of the coculture system after treatment with calcipotriol of 104 or 104 mol/L compared with that before treatment (both P ＞ 0.05).In the coculture system pretreated with calcipotriol of 10-8 mol/L,the number of CD8+ CTLs significantly decreased (t =3.15,P ＜0.05),whereas that of melanocytes significantly increased (t =3.53,P ＜0.05) after the treatment with anti-IL-6 antibody of 5 mg/L.Conclusions Perilesional CD8+ CTLs have a specific cytotoxic effect on melanocytes,and calcipotriol may inhibit the cytotoxic effect of CD8+ CTLs by suppressing the secretion of IL-6,which may partly explain the therapeutic mechanism of calcipotriol for vitiligo.
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Objective To investigate the reflectance confocal microscopy (RCM) features of mycosis fungoides (MF).Methods Four male and two female patients with MF,who were aged 7-73 years with a clinical course of 1-5 years,were collected from June 2011 to May 2012 in the department of dermatology.Of the six patients,two were in patch stage,three in plaque stage,and one in tumor stage.All the cases were confirmed by histopathological,immunohistochemical and relevant examinations.For each patient,three lesions were selected and investigated using RCM,and one of the three lesions was subjected to histopathological examination.Results RCM features of MF included hyperkeratosis,disarray of honeycomb of stratum spinosum,reduced refractivity of dermal papillary rings,roundish or round scattered hyperrefractive cells in the epidermis,infiltration of a small quantity of inflammatory cells in the papillary dermis.Occasionally,some round to oval hyporefractive cells aggregated to form vesicle-like structures.These RCM features correlated well with histopathologic findings.Conclusion Combined with clinical manifestations,RCM may serve as a noninvasive auxiliary tool for the diagnosis of MF.
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Objective To detect the level of cytokines and their receptors secreted by peripheral blood CD8 + T lymphocytes from vitiligo patients,and to evaluate the influence of tea polyphenols on their secretion.Methods CD8+ T lymphocytes were isolated from the peripheral blood of 12 patients with progressive vitiligo,12 patients with stable vitiligo and 10 healthy controls,and cultured in vitro for 20 days.Then,some CD8+ T lymphocytes were treated with tea polyphenols of 100 mg/L for two days.Enzyme-linked immunosorbent assay (ELISA) was carried out to determine the levels of tumor necrosis factor (TNF)-α,interferon (IFN)-γ and interleukin-2 receptor (IL-2R) in the culture supernatants of CD8+ T lymphocytes before and after the treatment with tea polyphenols.Analysis of variance and t test were conducted to assess differences in these parameters among these groups and changes between pretreatment and posttreatment,respectively.Results There was a sequential decrease in the level of TNF-α ((191.302 ± 6.077) vs.(175.966 ± 2.467) vs.(173.664 ± 3.600) ng/L,F =4.784,P ＜ 0.05),but a sequential increase in that of IFN-γ ((280.182 ± 36.070) vs.(371.670 ± 24.352) vs.(447.147 ± 8.432) ng/L,F=9.036,P＜ 0.01) and IL-2R ((8.375 ± 0.161) vs.(8.845 ± 0.161) vs.(9.345 ±0.125) ng/L,F =9.639,P ＜ 0.01) in the culture supernatant of CD8+ T lymphocytes from patients with progressive vitiligo to patients with stable vitiligo and healthy controls.After two days of tea polyphenol treatment,a statistical decrease was observed in the level of TNF-α in the culture supernatant of CD8 + T lymphocytes from patients with progressive vitiligo ((164.797 ± 1.784) ng/L,P ＜ 0.01),patients with stable vitiligo ((166.150 ± 3.576) ng/L,P ＜ 0.05) and healthy controls ((155.028 ± 5.759) ng/L,P ＜ 0.05),but no statistical changes were noted for IFN-γor IL-2R (all P ＞ 0.05) despite a slight elevation in the level of IFN-γin the culture supernatant of CD8+ T lymphocytes from patients with stable vitiligo and in that of IL-2R from all the three groups as well as a mild reduction in that of IFN-γ from patients with progressive vitiligo and healthy controls.Conclusions The changes of cytokines and their receptors secreted by CD8 + T lymphocytes may be associated with the induction and progression of vitiligo.Tea polyphenols may treat vitiligo via suppressing the secretion of TNF-α by CD8+ T lymphocytes.
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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.
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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.
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Objective To assess the efficacy and safety of 308-nm excimer laser combined with topical tacrolimus 0.03％ cream for the treatment of childhood vitiligo on the face or neck.Methods Sixty-eight children aged ＜ or =14 years with vitiligo on the face or neck were enrolled in this study,and divided into two groups based on lesional sites,i.e.,skin group with lesions on the forehead,cheek,lower mandible,neck or in the periortic region and mucosa group with lesions in the perioral or periorbital region.All the patients received 308-nm excimer laser irradiation once or twice weekly and topical tacrolimus 0.03％ cream twice daily.Twenty sessions of irradiation served as a treatment course.Efficacy was evaluated at the end of the treatment course.The effects of erythema reaction and cumulative irradiation dose on efficacy were assessed.Results After 20 times of laser therapy,an excellent response was achieved in 78.4％ of the patients in the skin group,and 54.8％ in the mucosa group (P ＜ 0.05).During the treatment,the average frequency of erythema reaction lasting 48 hours or longer was significantly lower in the skin group than in the mucosa group (5.84 vs.9.12,P ＜ 0.01).After 10 and 20 times of laser therapy,the cumulative radiation dose was 4215 mJ/cm2 and 10 453 mJ/cm2 in the skin group,respectively,compared to 3364 mJ/cm2 and 7430 mJ/cm2 in the mucosa group,respectively (both P ＜ 0.01).Conclusions The 308-nm excimer laser combined with topical tacrolimus cream is effective and safe for the treatment of childhood vitiligo on the face or neck.Vitiligo on the forehead,cheek,lower mandible,neck and in the periortic region appears to respond better to this therapy with a weaker erythema reaction than that in the perioral or periorbital region.
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Objective To evaluate the effect of green tea polyphenol epigallocatechin-3-gallate (EGCG)and ultraviolet B (UVB) on the expression of aquaporin 3 and epidermal growth factor receptor (EGFR)/extracellular signal-regulated protein kinase (ERK) signaling pathway in keratinocytes.Methods Twenty healthy human subjects were enrolled in this study.Both legs of each subjects were separated into 4 areas to remain untreated (control area),be topically treated with 3％ and 1％ EGCG cream and the vehicle of EGCG cream respectively once a day for 2 weeks followed by the measurement of skin moisture content and transepidermal water loss (TEWL).Cultured keratinocytes were classified into various groups to be irradiated with different doses (10,20 and 30 mJ/cm2) of UVB,or be pretreated with different concentrations of EGCG (10-7,10-6,10-5 mol/L) or EGFR/ERK phosphorylation inhibitors for 1 hour followed by irradiation with UVB of 30 mJ/cm2.After various durations of additional culture,Western blot was conducted to quantify the expression of AQP3 and phosphorylated-EGFR (p-EGFR) and-ERK (p-ERK) of keratinocytes.Data were processed by SPSS 10.0 software,and statistical analysis was carried out by t test.Results Skin moisture content was significantly increased,while TEWL was decreased in healthy skin after treatment with 1％ and 3％ EGCG cream compared with vehicle-treated skin areas and untreated skin areas.Increased AQP3 expression was observed in keratinocytes pretreated with EGCG of 10-7,10-6,10-5 mol/L (172.36 ± 12.42,320.66 ± 15.51 and 368.10 ± 11.39 vs.100.00,t =12.16,26.75 and 38.62 respectively,all P ＜ 0.05) and in those pretreated with the EGFR inhibitor PD153035 of 1.0 μmol/L and ERK inhibitor U0126 of 10 μmol/L (413.85 ± 25.27 and 268.85 ± 16.33 vs.100.00,t =35.16,19.25 respectively,both P ＜ 0.05)compared with those irradiated with UVB of 30 mJ/cm2 alone.UVB irradiation stimulated the phosphorylation of EGFR/ERK in keratinocytes,and the stimulation was markedly inhibited by the pre-treatment with EGCG of 10-7,10-6 and 10-5 mol/L (all P ＜ 0.05).Conclusions EGCG can enhance skin barrier function.AQP3 expression is down-regulated by UVB irradiation in keratinoctyes,while EGCG can inhibit the downregulation likely by suppressing the UVB-induced activation of EGFR and ERK.