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.

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.

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

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.