Topical epigallocatechin-3-gallate in the treatment of vitiligo.

OBJECTIVE: To preliminarily assess the efficacy and safety of the topical application of Epigallocatechin-3-gallate (EGCG) in treating vitiligo, a 6-month clinical trial was carried out. METHOD: Patients were randomly given topical application of EGCG on the assigned lesions, with pimecrolimus being used as the control for twice a day over a 6-month treatment period. Responses to treatment were assessed based on the changes in VASI score for percentage reduction in body surface area and the PGA scores. RESULTS: According to our results, both drugs were discovered to be markedly effective on repigmentation. The VASI of lesion had diminished from 1.19 ± 0.42 to 0.63 ± 0.38, in the EGCG-treated lesions, while from 1.18 ± 0.43 to 0.61 ± 0.36 in the pimecrolimus-treated lesions, and there was no statistically significant difference in VASI score between the EGCG-treated lesions and pimecrolimus-treated lesions (P = 0.755). Meanwhile, the mean PGA score on the EGCG applied side was 4.39 ± 2.23, while that was 4.43 ± 2.02 on the pimecrolimus applied side (P = 0.886). Furthermore, difference in the improvement degree between pimecrolimus side and EGCG side was not statistically significant (P = 0.845). Notably, no serious side effects were observed throughout the study. CONCLUSION: Findings of the study indicate that topical EGCG can be effective on treating vitiligo.

Caffeic acid derivative WSY6 protects melanocytes from oxidative stress by reducing ROS production and MAPK activation.

Purpose: Vitiligo is a chronic depigmentation disease caused by a loss of functioning melanocytes and melanin from the epidermis. Oxidative stress-induced damage to melanocytes is key in the pathogenesis of vitiligo. WSY6 is a caffeic acid derivative synthesized from epigallocatechin-3-gallate (EGCG). This study is to investigate whether the new chemical WSY6 protected melanocytes from H2O2-induced cell damage and to elucidate the underlying molecular mechanism. Patients and methods: The present study compared the antioxidative potential of WSY6 with EGCG in hydrogen peroxide (H2O2)-treated PIG1 cells. Western blotting was used to study the protein expression of cyto-c, cleaved-caspase3, cleaved-caspase9, and the activation of MAPK family members, including p38, ERK1/2, JNK and their phosphorylation in melanocytes. ROS assay kit to detect intracellular reactive oxygen species production; CCK8 and lactate dehydrogenase leak assay to detect cytotoxicity. Results: EGCG and WSY6 ameliorated H2O2-induced oxidative stress damage in PIG1 cells in a does-dependent manner, while WSY6 was much more effective. WSY6 reduced cellular ROS production, cytochrome c release, downregulated caspase-3 and caspase-9 activation. MAPK pathway signaling including phosphorylated p38, ERK and JNK were observed under oxidative stress and can be much protected by pre-treatment of WSY6. Conclusion: These results indicated that WSY6 could be a more powerful antioxidant than EGCG and protect melanocytes against oxidative cytotoxicity.

Target area treatment ratio of varied lesions in the cultured pure melanocyte transplantation repigmentation of vitiligo: A retrospective study.

Autologous cultured pure melanocyte transplantation (CMT) can be utilized to treat stable vitiligo cases, but clinical data are insufficient to improve its efficacy. To evaluate the influence of various factors on the therapeutic effect of CMT, this single-center retrospective study enrolled stable vitiligo patients who underwent CMT between 2009 and 2020. Univariate and multivariable analysis were used to determine the factors affecting the outcome of repigmentation. The study included 491 patients with long-term follow-up data (6-120 months). It was found that 69.7% of patients achieved an excellent re-color effect and 18.4% achieved a good re-color effect. There were statistically significant differences in pigmentation between patients with stable disease course, vitiligo type, and lesion site. Overall, a significant positive correlation between the target area treatment ratio of varied lesions and the percentage of repigmentation was found. CMT is effective and well tolerated in the treatment of stable vitiligo. Various factors, especially the target area treatment ratio of varied lesions, should be carefully assessed before using CMT. As the target area treatment ratio of varied lesions could further improve the post-operative repigmentation other than type of vitiligo. This clinic trial was approved by Hangzhou Third People's Hospital (number 2023KA015, national clinical record number MR-33-23-034502).

Exploring the association of POSTN+ cancer-associated fibroblasts with triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with a poor prognosis. Cancer-associated fibroblasts (CAFs) play a critical role in regulating TNBC tumor development. This study aimed to identify and characterize a specific subtype of CAFs associated with TNBC. Initially, using high-throughput bulk transcriptomic data in two cohorts, we identified three CAF-related subtypes (CS1, CS2, CS3) in TNBC samples. These three CAFs subtypes were closely linked to the tumor microenvironment. The CS1 subtype exhibited a relatively immune-rich microenvironment and a favourable prognosis, whereas the CS3 subtype displayed an immune-deprived tumor microenvironment and an unfavourable prognosis. Through WGCNA analysis, POSTN was identified as a key biomarker for CAFs associated with TNBC. Then, POSTN+CAFs was identified and characterized. Both POSTN and POSTN+CAFs showed significant positive correlations with stromal molecules HGF and MET at both the transcriptional and protein levels. Specifically co-localized with CAFs in the tumor stromal area, POSTN, produced by POSTN+CAFs, could modulate the HGF-MET axis, serving as a bypass activation pathway to regulate tumor cell proliferation in response to EGFR inhibitor and MET inhibitor. This study underscores the significance of POSTN and POSTN+CAFs as crucial targets for the diagnosis and treatment of TNBC.

[Impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome].

OBJECTIVE: To determine the impact on tyrosinase expression and export from endoplasmic reticulum by inhibition of 26S proteasome. METHODS: Western blot was used to detect 26S proteasome from 8 vitiligo patients and 4 healthy controls. Melanocytes were incubated with proteasome inhibitor (lactacystin) and further detected as follows: cell survival by MTT assay, proteasome activity with fluorescence, ultrastructure observation with electron microscope, co-localization of tyrosinase and calreticulin (endoplasmic reticulum marker) by confocal laser scanning microscopy and 26S proteasome and tyrosinase with Western blot. RESULTS: The 26S proteasome expression level from lesions of vitiligo (1.05 ± 0.40) was significantly lower than the donor sites (1.82 ± 0.88) and the healthy controls (1.88 ± 0.16) (P < 0.05). But no significant difference existed between the latter two groups (P > 0.05). Compared to the untreated group, a 12-h incubation of 10 µmol/L lactacystin showed inhibitory effects on melanocytes (0.999 ± 0.110 vs 1.372 ± 0.127, P < 0.05) and significantly decreased proteasome activity (0.234 ± 0.019 vs 1, P < 0.01). Expansion rate of endoplasmic reticulum in the lactacystin group (1.91 ± 0.17) was significantly higher than that of the untreated cells (1.17 ± 0.11) (P < 0.05). More tyrosinase co-localized with calreticulin in endoplasmic reticulum in lactacystin-treated cells was observed than that of the untreated group. Compared with the untreated group, significantly decreased levels of tyrosinase (146 ± 10 vs 269 ± 8, P < 0.01) and tyrosinase activity (0.159 ± 0.017 vs 0.221 ± 0.019, P < 0.01) were shown in the lactacystin group (P < 0.05). CONCLUSIONS: Significantly decrease of 26S proteasome is found in lesions of vitiligo patients. Inhibition of 26S proteasome may lead to expansion of endoplasmic reticulum of melanocytes, impact export of tyrosinase from melanocyte endoplasmic reticulum and expression of tyrosinase.

Pathogenic Th2 Cytokine Profile Skewing by IFN-γ-Responding Vitiligo Fibroblasts via CCL2/CCL8.

PURPOSE: Vitiligo is a T cell-mediated skin depigmentation disease. Though treatments arresting disease progression and inducing repigmentation are available, the efficacy of these options is often limited and poorly sustained. How stromal signals contribute to the interferon-γ-dominant skin niches is unclear. This study aims to determine how fibroblasts participate in the IFN-γ-dominant vitiligo niche. PATIENTS AND METHODS: Mouse vitiligo models were established. Fibroblasts from control and vitiligo mice were extracted for RNA sequencing. In vitro IFN-γ stimulation was performed to verify the JAK-STAT pathway by qPCR and Western blot. T cell polarization with chemokines was measured by flow cytometry. Protein levels in tissues were also examined by IHC. RESULTS: The vitiligo mouse model recapitulates the human CD8-IFN-γ pathway. RNA sequencing revealed elevated chemokine CCL2 and CCL8 in vitiligo fibroblast, which may be regulated by the JAK-STAT signaling. Such phenomenon is verified by JAK inhibitor peficitinib in vitro. Moreover, CCL2 addition into the naïve T polarization system promoted type 2 cytokines secretion, which represents a hallmark of vitiligo lesions. CONCLUSION: Dermal fibroblasts, a principal constituent of skin structure, respond to IFN-γ by skewing T cells towards a type 2 cytokine profile via CCL2 and CCL8, which can be abrogated by JAK inhibitor peficitinib.

EGR1 Mediated Reduction of Fibroblast Secreted-TGF-ß1 Exacerbated CD8+ T Cell Inflammation and Migration in Vitiligo.

Vitiligo is a T cell-mediated depigment skin disease caused by the complex interplay between melanocyte dysfunction, environmental stimulation, and dysregulated immune signals. Transforming growth factor-ß1 (TGF-ß1), which typically derives from regulatory T cells, has long been identified at low levels in the peripheral system of vitiligo patients. Here, through RNA-sequencing and transcription factor enrichment, we revealed that in response to CD8+ T cell-secreted interferon-gamma (IFN-γ), stromal fibroblast downregulates early growth response 1 (EGR1) activity, leading to TGF-ß1 deficiency. The defective immune regulation loop further exacerbated local CD8+ T cell inflammation and promoted inflammatory cell migration in vitiligo. Thus, fibroblast-derived TGF-ß1 plays an important stromal signal in vitiligo pathogenesis.

Narrowband Ultraviolet B Combined with Topical Simvastatin Solution in the Treatment of Vitiligo: A Case Report.

Background: Phototherapy is one of the treatments for vitiligo. To be specific, the combination of narrowband ultraviolet B (NB-UVB) with topical preparations has currently become the most common therapeutic modality. Moreover, the research on new topical drug has been a hot issue in the field of vitiligo. Objective: At present, simvastatin has been considered as a potential therapeutic agent for the treatment of vitiligo. To the best of our knowledge, this is the first case report concerning the successful application of NB-UVB combined with topical simvastatin in the treatment of vitiligo. Methods: In this article, a clinical case report was presented, where the patient was not responsive to NB-UVB but was markedly responsive to the treatment of UVB combined with topical simvastatin. Results: A 34-year-old Chinese female patient with vitiligo was cured by NB-UVB combined with topical simvastatin solution. Conclusions: NB-UVB combined with topical simvastatin may be a potential treatment against vitiligo. This research was approved by the Medical Ethics Committee of Third People's Hospital of Hangzhou.

[Corrigendum] The role of VIT1/FBXO11 in the regulation of apoptosis and tyrosinase export from endoplasmic reticulum in cultured melanocytes.

Following the publication of the above article, an interested reader drew to the authors' attention that the Transwell cell migration assay data shown in Fig. 4A appeared to be partly overlapping with data presented for experiments performed under different experimental conditions in Figs. 4D and E. The authors independently examined the figure and realized that inadvertent errors had been made during the assembly of Fig. 4; furthermore, owing to the time that has elapsed since this paper was published, the authors no longer had access to the original data. Accordingly, to further verify the conclusions reported in the study, the authors repeated these experiments, and the results obtained were found to be consistent with the original findings. The new version of Fig. 4 is shown below. The authors are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 26: 57-65, 2010; DOI: 10.3892/ijmm_00000435].

CXCL9 as a key biomarker of vitiligo activity and prediction of the success of cultured melanocyte transplantation.

This study aimed to investigate the potential biomarkers of vitiligo by evaluating the disease activity and curative effect of autologous cultured pure melanocyte transplantation (CMT) on patients. Altogether, 36patients with stable vitiligo were treated with CMT. Blister fluid samples were collected from patients with stable vitiligo. Patients with active vitiligo were matched with healthy controls. The chemokine levels in the serum and blister fluid samples were measured using Luminex. The curative effect on patients with stable vitiligo was evaluated 6 months after treatment. Treatment responses were defined according to the extent of repigmentation as effective (if 50% or more repigmentation was achieved) or ineffective (if less than 50% or worse repigmentation was achieved). Patients received re-transplantation if the initial treatment was ineffective. The levels of C-X-C motif chemokine ligand (CXCL)9 and CXCL10 in blister fluid samples were significantly lower in stable patients than in active participants. Receiver operating characteristic analysis revealed that the levels of CXCL9 and CXCL10 were sensitive and specific in diagnosing active vitiligo. Further, 65.6% (21/32) of patients who received CMT had effective treatment responses. The high CXCL9 level in the blister fluid was a significant predictor of ineffective treatment responses. The treatment response was significantly enhanced after treatment. Four patients with ineffective treatment responses received anti-inflammatory treatment and re-transplantation. The CXCL9 and CXCL10 levels in the blister fluid were related to the presence of active vitiligo. Also, the CXCL9 level was a predictor of the effectiveness of CMT in treating vitiligo.

Inhibition of Fam114A1 protects melanocytes from apoptosis through higher RACK1 expression.

Fam114A1 is a gene closely related to the development of nerve cells, melanocytes, and nerve cells that originate from the neural crest of the embryonic ectoderm. Recent studies showed that Fam114A1 has a role in the occurrence of ankylosing myelitis spondylitis and autoimmune enteritis; still, its cellular function remains poorly understood. In this study, we investigated the effect of Fam114A1 on the biological activity of melanocytes. We found that the expression of Fam114A1 in vitiligo melanocytes (MCV-L, MCV-N, PI3V) was higher than that in normal melanocytes, and the biological function of melanocytes was significantly affected when the Fam114A1 gene was silenced. Inhibition of Fam114A1 increased proliferation, migration, and melanin synthesis proteins, decreased apoptosis, while its overexpression reversed this process. Mechanistically, we discovered that RACK1 is a target protein of Fam114A1 and that RACK1 can be negatively regulated by Fam114A1. Further study showed that Fam114A1 inhibition could not protect melanocytes from apoptosis once the expression of RACK1 protein was silenced. In summary, Fam114A1 is an effective regulatory protein for regulating the function of melanocytes. Inhibition Fam114A1 protects melanocytes from apoptosis through increasing RACK1.

[Expression of InnVit/FBXO11 in vitiligo and its role in tyrosinase export from endoplasmic reticulum].

OBJECTIVE: To investigate the roles of InnVit (FBX011) gene in melanocytes by detecting the expression of InnVit gene in vitiligo and analyzing the impact of InnVit gene on morphology of endoplasmic reticulum (ER) and the tyrosinase export from ER. METHODS: The lesion tissues and the donor tissues were collected from 10 vitiligo patients to examine the InnVit gene expression by immunohistochemistry. Synthesized specific siRNA and constructed plasmid P3XF-P120 were separately transfected into cells for the silence and over-expression of InnVit gene with lipofectamine(TM) 2000. The untreated cells were used as control. Morphology of ER of cells from the above three groups was observed under electron microscope. Co-localization of tyrosinase and calreticulin was identified by confocal laser scanning microscopy. InnVit, tyrosinase and calreticulin were examined by Western blot. RESULTS: In vitiligo patients, the expression of InnVit gene in the lesions was markedly lower than that in the donor tissues. The normal morphology of ER was found in the untreated and the plasmid groups whereas inflated ER was shown in siRNA group. And the relative inflation rate in siRNA group (1.97 +/- 0.48) was higher than that in the untreated group (1.28 +/- 0.09) and plasmid group (1.24 +/- 0.13) (both P = 0.001). In the untreated and the plasmid groups, tyrosinase was expressed beyond the scope marked by ER marker protein calreticulin partly, but co-localized with calreticulin in ER in the siRNA group. Western blot showed that, contrast to the untreated group (0.320 +/- 0.020), a lower expression level of InnVit in the siRNA group (0.030 +/- 0.004, P = 0.001) and a higher expression of InnVit in the plasmid group were shown (0.710 +/- 0.040, P = 0.001). No significant difference about the expression level of calreticulin was observed among the three groups (P > 0.05). As compared with the untreated group (0.350 +/- 0.030), a higher tyrosinase level in the siRNA group (1.040 +/- 0.060, P = 0.001) and in the plasmid group (0.720 +/- 0.030, P = 0.001) was found. And the former was higher than the latter (P = 0.001). CONCLUSION: A lower expression of InnVit is observed in the lesion tissues than in the donor tissues from vitiligo patients. The InnVit gene can have an impact on the morphology of ER and tyrosinase export from ER. And it may further affect the function of melanocytes.

The susceptibility to vitiligo is associated with NF-E2-related factor2 (Nrf2) gene polymorphisms: a study on Chinese Han population.

Vitiligo is an acquired pigmentary disorder and its pathogenesis remains unclear. Oxidative stress is considered to be the initial pathogenic event in the melanocyte destruction. NF-E2-related factor2 (Nrf2) is a transcription factor regulating the expression of detoxifying and antioxidant genes. To investigate the association of the Nrf2 gene promoter polymorphisms with vitiligo in Chinese Han population, the genotypes of -686A/G, -684G/A and -650C/A and the genotyping of variable number of tandem repeat were detected. The data were analysed by the chi-square test and the risk was evaluated by calculating OR and 95% CI. There was statistically significant difference in genotypic and allelic frequencies of -650C/A between the two groups (P < 0.05). A(-650) allele was significantly associated with the risk for vitiligo (OR = 1.724, chi(2) = 18.096). Polymorphism of the Nrf2 gene promoter at -650C/A was associated with the development of vitiligo and A(-650) allele may be one of the risk factors.

Melagenine modulates proliferation and differentiation of melanoblasts.

Melagenine, extracted from human placenta, has been shown to be effective in treating patients with vitiligo, yet the mechanisms of melagenine in inducing the repigmentation of vitiligo patients have not been fully investigated. Recent studies have suggested that melagenine stimulates melanocyte proliferation and melanogenesis. In this study, we utilized the NCCmelb4M5 melanoblast cell line to investigate the effects of melagenine on proliferation and differentiation of immature melanocytes or melanoblasts. NCCmelb4M5 cells were treated with different concentrations of melagenine (50-400 microg/ml), and MTT assay was performed to evaluate the effects of melagenine on proliferation of melanoblasts. RT-PCR and Western blotting were used to determine the expression of c-KIT and tyrosinase (TYR). Our results show that melagenine stimulates proliferation of NCCmelb4M5 cells in a dose-dependent manner with an optimal concentration of 100 microg/ml. Multipolar and highly branched dendritic network, as well as cluster-like growing cell assembly were visible in melagenine-treated NCCmelb4M5 cells. Melagenine induced expression of c-KIT, TYR and MITF. Our results provide insights into the molecular mechanism of the beneficial effect of melagenine in the treatment of vitiligo.

[Abnormal nuclear translocation of nuclear factor-E2 related factor 2 in the lesion of vitiligo].

OBJECTIVE: To investigate whether abnormal translocation of nuclear factor-E2 related factor 2 (Nrf2) exists in the lesion of vitiligo. METHODS: Skin specimens from 8 vitiligo patients and 3 healthy controls were collected, half of them underwent laser co-focal microscopy to detect the Nrf2 location and half of them underwent cell culture. Blister fluid was collected form the 8 vitiligo patients and skin donor sites to detect the levels of serum superoxide dismutase (SOD), catalase (CAT), and malonyldialdehyde (MDA) by using detection kit. Expression of Nrf2 in epidermal cell of the 8 vitiligo patients and primary epidermal cell of the 3 healthy controls was identified with cell immunofluorescence histochemistry method. The nuclear and cytoplasmic proteins of all above samples were isolated to be identified by Western blotting. RESULTS: The levels of SOD and CAT in the lesion tissue were significantly lower than those in the skin donor site. The levels of MDA in the lesion tissue were significantly higher than those in the skin donor sites (both P < 0.05). Immunofluorescence histochemistry, showed that Nrf2 was predominantly cytoplasmic in the epidermal cells in the lesion, while Nrf2 expression could be seen in both the cytoplasm and nucleus in the epidermal cells in the normal skin donor sites and skins of the healthy controls. Western blotting showed that the nuclear Nrf2 level in the vitiligo skin lesion was (0.11 +/- 0.03), significantly lower than that in the normal skin donor site (0.27 +/- 0.06) and in the skins of the healthy controls (0.32 +/- 0.02) (both P < 0.01). However, there was no significant difference in the Nrf2 level of in cytoplasm among the three types of tissues (0.63 +/- 0.04, 0.61 +/- 0.03, and 0.65 +/- 0.04, all P > 0.05). CONCLUSION: Nrf2 does not translocate from cytoplasm into the nucleus in the lesion of vitiligo patients.

[Association of single nucleotide polymorphisms of Nrf2 promoter region with susceptibility to vitiligo].

OBJECTIVE: To investigate the association of the single nucleotide polymorphisms (SNPs) in Nrf2 promoter region with the susceptibility to risk of vitiligo. METHODS: Samples of peripheral blood were collected from 300 vitiligo patients and 300 healthy persons. The genotypes of -686A/G, -684G/A, and -650C/A were detected by direct-sequencing. Genotyping of variable number of tandem repeat (VNTR) was performed by gene scan analysis with an ABI 310 Sequencer. Genetic and allelic frequencies were analyzed by Chi-square test and the risk was evaluated by calculating OR and 95% CI. RESULTS: There was statistical significant difference in genotypic and allelic frequencies of -650C/A between the vitiligo group and healthy control group (P < 0.05), and A -650 allele was associated with risk for vitiligo statistically significantly (OR = 1.724, 95% CI: 1.345-2.211, chi2 = 18.096, P < 0.01). Homozygote of A allele increased the risk for vitiligo obviously (OR = 2.902, 95% CI: 1.624-5.188, P < 0.01). No significant difference was found in other three polymorphisms between the two groups. CONCLUSION: polymorphism of Nrf2 promoter region -650C/A was associated with the development of vitiligo and A -650 allele may be one of risk factors for vitiligo.

Decreased SUMOylation of the retinoblastoma protein in keratinocytes during the pathogenesis of vitiligo.

The role of SUMOylation in the pathogenesis of vitiligo has not been reported previously. The present study aimed to reveal abnormalities in small ubiquitin­like modifier (SUMO) conjugation in keratinocytes from depigmented lesions of patients with vitiligo and confirm the role of SUMOylation in keratinocytes from patients with vitiligo. Skin samples used for immunohistochemistry were obtained by punch biopsy from the depigmented lesions of 6 patients. Blisters were produced by vacuum and the roofs were collected for keratinocyte culture. HaCaT cells were transduced with SUMO1 knockdown vectors. The protein expression of SUMO1, SUMO­specific peptidase 1 (SENP1), ubiquitin­conjugating enzyme E2 I (Ubc9), SUMO­activating enzyme subunit 1 (SAE1), cyclin­dependent kinase (CDK)2, CDK6, proliferating cell nuclear antigen (PCNA), retinoblastoma protein (Rb), phosphorylated Rb (pRb) and ß­actin was assessed by western blotting. The SUMOylation status of proteins was assessed by immunoprecipitation. Cell cycle analysis was performed by flow cytometry and cell proliferation rate was investigated using a Cell Counting Kit­8. The results demonstrated that the levels of SUMO1­conjugated proteins were decreased in vitiligo lesions and vitiligo keratinocytes compared with normal controls. The protein expression of Ubc9 was decreased and SENP1 was increased in vitiligo keratinocytes compared with normal keratinocytes, with no alterations in SAE1 expression. Following knockdown of SUMO1 in HaCaT cells, the proliferation of HaCaT cells was reduced and the cell cycle was arrested in G1 phase. Furthermore, the protein expression levels of PCNA, CDK2, CDK6 and pRb were reduced in SUMO1­knockdown HaCaT cells, and SUMOylated Rb was also decreased markedly in keratinocytes from lesions of patients with vitiligo compared with normal keratinocytes. In conclusion, vitiligo lesions in the present study exhibited dysregulated SUMOylation and deSUMOylation balance and dysregulation of cell cycle progression may be present in SUMO1 knockdown HaCaT cells. These results indicate that deSUMOylation of Rb of keratinocytes may serve an important role in vitiligo, providing a novel direction for the study into the mechanism of vitiligo.

[Expression and purification of epitope peptide of human tyrosinase and its antigenicity in vitiligo patients].

OBJECTIVE: To express the epitope peptide of human tyrosinase (TYR), and discuss the application of the peptide in detecting autoantibody of the vitiligo patients. METHODS: The epitope areas 240 - 255, 289 - 294, 295 - 300, 435 - 447, and 461 - 479 of human TYR were synthesized and connected to the vector pGEM-T. The target gene was cloned to the prokaryotic expression vector pGEX-4T-2, which was then transferred to Escherichia coli BL21 host cells. Isopropy-beta-D-thiogalactoside (IPTG) was used to induce the protein expression that was examined with SDS-PAGE and Western blotting. Indirect ELISA was conducted to detect the antigenicity of the peptide in 100 blood specimens of active vitiligo patients and 30 healthy controls. RESULTS: The recombinant expression vector was constructed successfully. The SDS-PAGE and Western blotting results showed expression of the recombinant protein in E. coli. The amount of the recombinant protein reached about 70% of the total mass of bacterial protein with PAGE analysis system. With the glutathione S-transferase (GST) purification kit, the purity of recombinant protein reached over 90%. Indirect ELISA showed that reaction with the target protein was negative in all the 30 healthy controls and was positive in 64 of the 100 active vitiligo patients. CONCLUSION: The epitope peptide of human TRY is expressed successfully, and it has antigenicity in the serum of vitiligo patients.

Quercetin attenuates the effects of H2O2 on endoplasmic reticulum morphology and tyrosinase export from the endoplasmic reticulum in melanocytes.

Swollen endoplasmic reticulum (ER) is commonly observed in the melanocytes of vitiligo patients; however, the cause and proteins involved in this remain to be elucidated. Oxidative stress has been reported to be involved in the pathogenesis of vitiligo and previous studies have demonstrated that hydrogen peroxide (H2O2) induced melanocyte apoptosis, whereas quercetin exhibited cytoprotective activities against the effects of H2O2. The aim of the present study was to further investigate the role of H2O2 in the ER of melanocytes as well as its role in the export of tyrosinase from ER; in addition, the present study aimed to determine the mechanism by which quercetin protects against the effects of H2O2. The results demonstrated that melanocyte cells treated with H2O2 presented with swollen ER; however, a normal ER configuration was observed in untreated cells as well as quercetin/H2O2­treated cells. Furthermore, H2O2 inhibited tyrosinase export from the ER and decreased expression levels of tyrosinase; however, quercetin was found to attenuate the effects induced by H2O2. In conclusion, the results of the present study confirmed the hypothesis that H2O2 induced ER dilation and hindered functional tyrosinase export from the ER of melanocytes. It was also found that quercetin significantly weakened these effects mediated by H2O2, therefore it may have the potential for use in the treatment of vitiligo.

Interferon-γ induces senescence in normal human melanocytes.

BACKGROUND: Interferon-γ (IFN-γ) plays an important role in the proceedings of vitiligo through recruiting lymphocytes to the lesional skin. However, the potential effects of IFN-γ on skin melanocytes and the subsequent contribution to the vitiligo pathogenesis are still unclear. OBJECTIVE: To investigate the effects of IFN-γ on viability and cellular functions of melanocytes. METHODS: Primary human melanocytes were treated with IFN-γ. Cell viability, apoptosis, cell cycle melanin content and intracellular reactive oxygen species (ROS) level were measured. mRNA expression was examined by real-time PCR. The release of interleukin 6 (IL-6) and heat shock protein 70 (HSP-70) was monitored by ELISA. ß-galactosidase staining was utilized to evaluate melanocyte senescence. RESULTS: Persistent IFN-γ treatment induced viability loss, apoptosis, cell cycle arrest and senescence in melanocytes. Melanocyte senescence was characterized as the changes in pigmentation and morphology, as well as the increase of ß-galactosidase activity. Increase of p21Cip1/Waf1 protein was evident in melanocytes after IFN-γ treatment. IFN-γ induction of senescence was attenuated by siRNAs against p21, Janus kinase 2 (JAK2) or signal transducer and activator of transcription 1 (STAT1), but not by JAK1 siRNA nor by p53 inhibitor pifithrin-α. IFN-γ treatment increased the accumulation of intracellular ROS in melanocytes, while ROS scavenger N-acetyl cysteine (NAC) effectively inhibited IFN-γ induced p21 expression and melanocyte senescence. IL-6 and HSP-70 release was significantly induced by IFN-γ treatment, which was largely inhibited by NAC. The increase of IL-6 and HSP-70 release could also be observed in senescent melanocytes. CONCLUSION: IFN-γ can induce senescence in melanocytes and consequently enhance their immuno-competency, leading to a vitiligo-prone milieu.