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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 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.
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Objective To analyse the possible risk factors associated with the progression of vitiligo.Methods A questionnaire survey was carried out to collect the clinical data on 1088 patients with vitiligo.The relationship between possible inducements to the progression of vitiligo and lesion area was statistically analyzed in patients with the same clinical course of vitiligo. Paired t test was performed to compare the mean area index of involvement between patients with inducements and those without Results A significant difference was observed in the mean area index of involvement between patients with isomorphic response and those without (t = 6.770, P ＜ 0.01 ) as well as between patients negatively affected by psychiatric factors and those unaffected (t = 6.704, P ＜ 0.01 ), but not between patients with family history and those without (t = 1.499,P ＞ 0.05). Conclusion A rapid progression of vitiligo is more likely to be observed in patients negatively affected by psychiatric factors and patients with isomorphic response.
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Objective To investigate the role of activation of Akt/mTOR pathway in denfense against UVB-induced apoptosis in cultured human skin keratinocyte cell line HaCaT. Methods HaCaT cells were irradiated with UVB at different doses for various durations. Western blotting was performed to detect dynamic changes of Akt/mTOR pathway-related signaling molecule, such as phosphorylated-epidermal growth factor receptor (EGFR), -Akt, -4EBP1, etc; apoptosis was estimated by staining with DNA dye Hoechst 33342. To evaluate the role of signaling molecules in defense against UVB-induced apoptosis, HaCaT cells were pretreated before irradiation with EGFR inhibitor (PD153035), PI3K inhibitor (LY294002), mTOR inhibitor (rapamycin) followed by the detection of expressions of signaling molecule and apoptosis. Results UVB could activate Akt/mTOR pathway in a dose- (5 ～ 30 mJ/cm2) and time- (5 ～ 30 min) dependent manner. PD153035,LY 294002 and rapamycin could inhibit UVB-induced activation of the Akt/mTOR pathway. The apoptosis rate in HaCaT cells was upregulated by pretreatment with rapamycin and LY294002. Conclusion The activation of Akt/mTOR signaling pathway could inhibit the UVB-induced apoptosis in cultured HaCaT cells.
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Objective To investigate the relationship between apoptosis in epidermal keratinocytes and efficacy of epidermal grafting. Methods Epidermal specimens were obtained from donor sites and depigmented area of 44 patients with vitiligo receiving epidermal grafting. The apoptosis in keratinocytes was determined by terminal deoxynucleotidyl transferase end-labeling (TUNEL) assay, and the expressions of caspase 3, 8, 9 as well as bcl-2 and P53 were evaluated by immunohistochemistry. Results As TUNEL assay showed, the number of apoptotic keratinocytes in epidermis from depigmented area differed significantly from that from donor sites.The expressions of caspase 3, 8 and 9 were mainly located in the membrane and cytoplasm of keratinocytes,and positive keratinocytes were predominately distributed in the middle and lower layer of the epidermis. Of the 44 patients, 19, 15 and 16 were positive for the expressions of caspase 3, 8 and 9 in the depigmented epidermis,respectively, and 9, 5 and 4 for those in the donor epidermis, respectively. P53 was expressed in neither donor epidermis nor depigmented epidermis, while Bcl-2 was weakly positive in donor epidermis and negative in depigmented epidermis. The number of apoptotic keratinocytes was higher in donor epidermis from patients failing to respond to the transplantation than in that from patients successfully treated by transplantation (15.83 ± 2.69 vs.9.24 ± 1.80, t = 10.96, P < 0.01 ). Conclusions There is an obvious apoptosis in keratinocytes from depigmented epidermis of patients with vitiligo, together with an increase in the expression of caspase 3, 8 and 9. The apoptosis in keratinocytes may be related to the efficacy of epidermal transplantation.
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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.
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Objective To investigate quercetin's protective effect against oxidative stress in and impact on the biological activity of mouse B10BR melanocytes. Methods B10BR cells were cultured and treated with different concentrations of quercetin followed by additional culture. Then, cell viability was measured by using MTT assay, hydrogen peroxide-induced cell apoptosis by flow cytometry, and cell morphological changes by microscopy. The tyrosinase activity in and melanin synthesis by B10BR cells were measured by dopa oxidation assay and sodium hydroxide (NaOH)-lysis method, respectively. Results After treatment with quercetin of 33.33 μmol/L for 24 hours, the survival rate of B10BR cells reached (94.22 ± 3.36)%, tyrosinase activity (107.15 ± 10.96)%, and melanin content (111.85 ± 9.49)%. A significant difference was observed in tyrosinase activity and melanin content between hydrogen peroxide-induced and 33.33 μmol/L quercetin-treated B10BR cells and those only induced by hydrogen peroxide (both P < 0.01). Flow cytometry revealed that quercetin inhibited hydrogen peroxide-induced apoptosis in melanocytes. Conclusion The protective effect of quercetin against hydrogen peroxide-induced apoptosis in melanocytes may provide a new idea for the treatment of vitiligo.
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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.
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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.
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Objective To investigate the relationship between the efficacy of autologous epidermal grafting and the levels of epidermal cytokines in vitiligo. Methods A total of 57 patients with stable vitiligo receiving autologous epidermal grafting were included in this study. Before grafting, 17 patients were irradiated with narrow-band UVB on vitiliginous sites. Suction blister fluid was collected from the recipient site (vitiligous lesions) and donor site (normal skin) in these patients (including the 17 patients irradiated with NB-UVB). ELISA was used to detect the levels of endothelin-1 (ET-1 ) and stem cell factors (SCF) in suction blister fluid. Clinical efficacy was evaluated through a 3-month follow-up. Resttlts Among these 57 patients, 45 successfully responded to autologous epidermal grafting. In these 45 patients, the levels of ET-1 and SCF in vitiligous lesions were 728.97±286.12 ng/L and 329.97±114.13 ng/L respectively, significantly higher than those in nomal skin (503.16±251.44 ng/L, 224.73±107.91 ng/L, t = 5.443, 5.897, respectively, both P < 0.05 ). In those who responded poorly, significant difference was also observed in the level of SCF between the normal skin and vitiligous lesions (309.00±163.89 ng/L vs 204.22±83.25 ng/L, t = 3.03, P < 0.05), but not in the level of ET-1. Increased level of ET-1 was observed in both vitiligous lesions and normal skin of patients who responded well compared to those who responded poorly, while no difference was noticed in the level of SCF between these two groups of patients. The level of ET-1 was statistically higher in vitiligous lesions in patients exposed to NB-UVB than in those without exposure (t = 1.44, P > 0.05). In those patients who responded successfully, the level of ET-1 was lower in the 15 patients exposed to NB-UVB compared to the other 30 patients without exposure (548.48±230.22 ng/L vs 794.60±278.72 ng/L, P<0.05); no significant difference in the level of SCF was noted. Conclusions ET-1 and SCF may both play important roles in the repigmentation of vitiligo, with ET-1 exerting a more important role.
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Objective To establish a fluorescent quantitative polymerase chain reaction method for quantifying the ICP4 gene expression of herpes simplex virus type 2(HSV2).Methods According to the HSV2 ICP4 gene sequence,we designed and synthesized PCR primer.The purified PCR product was sequenced after connecting with pMD-18 T plasmid.According to the sequence assay results,the primer and probe of fluorescent quantitative PCR was designed and synthesized.Standard recombinant plasmid extracted from the positive bacteriumclone was used as standard substance.The plasmid as standard substance was diluted for 10 times,then PCR reaction proceeded.The sensitivity and dependability of the real-time fluorescent quantitative PCR were analyzed.Results The sequence result indicated that there was non-sense mutation of A-G and T-C.And the detection sensitivity was 101 copy.The Ct value were 14.275土0.137,17.988?0.162,22.081土0.259,25.957土0.345,29.565?0.203,33.269土0.287,37.737?0.698,respectively with 107~ 101 copies/?l.The coefficient of variability were 0.965%,0.902%,1.174%,1.329%,0.686%,0.862% and1.851%,respectively.There was a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen.The coefficient of regression was 0.998.Conclusion The method of quantification of ICP4 gene of HSV2 with real-time fluorescent quantitative PCR is successfully established,and the method has good sensitivity and dependability,which can be used to quantitative detecting HSV2 ICP4.