| Objective: People are prone to Trichophyton rubrum,about 90%pathogenic infection of dermatophytoses was caused by Trichophyton rubrum.Study showed that dermatophytoses caused by Trichophyton rubrum mostly happened in summer which had no obvious inflammatory with longer duration but seriously affected the quality of life.Vitamin D was a kind of steroid derivatives which had an important influence on the body’s immune system,vitamin D has attracted more people’s attention than before.Vitamin D combinated with the vitamin D receptor(VDR)to play physiological role.VDR belonges to the steroid/thyroid hormone receptor superfamily members and was a kind of nuclear transcription factor relyed on ligands.VDR was an important component of the keratinocytes cell which had normal function.The skin was an important source of vitamin D in the human body.Vitamin D can be conversed from 7-dehydrocholesterol stored in the skin by sunlight.The activity form of vitamin D was 1,25(OH)2D3,which serum contains more during the summer.Vitamin D storage had a rising trend in summer which lead to a decline in the inflammatory factor and immune tolerance to pathogenic microorganisms.IL-12 was a heterodimeric proinflammatory cytokine,which could induce IFN-γ production and promoted the differentiation of Th1 cells that played the powerful proinflammatory role.IL-10 could suppress the viability and cytokines production of T cells,monocytes and macrophages.Thus,IL-10 played an important role in the process of limitation of host immune reaction to pathogens.It helped the host to clear the pathogens and protected from the immune injury.In this study,we discussed the relation of vitamin D receptor and IL-10,IL-12 in keratinocytes interacted with Trichophyton rubrum invitro.We wanted to investigate the immune tolerance and longer duration of Trichophyton rubrum mediated whether through VDR signaling pathways.Methods:1 Culture of HaCaT cellsHaCaT cells were provided by Shanghai Sai Li Biological Technology Co.Ltd.Certificate number: MXC138.HaCaT cells were cultured in 90% DMEM and 10% fetal bovine serum medium contains 1% Penicillin-Streptomycin Solution.HaCaT cells were dissociated into single cell suspension to climb on the slide after 5 days culture.Cells were fixed by acetone and identified by HE staining and immunohistochemical SP method,which was used to detect the expression of keratin 10 in the HaCaT cells.2 Experimental strainsStrains of Trichophyton rubrum saved from the Second Hospital of Hebei Medical University.3 Activation and preparation of suspensionThe type strain of Trichophyton rubrum rewarmed and activated on Potato Dextrose Agar(PDA)for three times.Sterile water with 0.1% Tween 80 was added to the colony and beat blended through a straw.Suspension was grindered in milling bottle and counted using a hemcytometer and finally adjusted to 4-6 × 105 CFU / m L.4 The experimental groups and treatmentCells were divided into two groups:the control group and the experimental group.HaCaT cells were collected after 7-day culture.To wash the cells with PBS for three times,then 0.25% trypsin was added to digest the cells.After that to terminate the digestion with the culture medium already prepared.Cells were centrifuged and the concentration of the suspension was adjusted to 1-2×106/m L,then inoculated these cells in the 24-well culture plate.1 m L Trichophyton rubrum suspension was added for each well in experimental group.1 m L sterile water supplemented with 0.1% Tween 80 was added for each well in the control group.The cells supernatant of theexperimental group and control group were collected at 2,4,8 and 16 hours respectively.3 parallel holes were seted for each well.Supernatant were collected and stored at-20℃.5 The levels of IL-10,IL-12 detected by ELISAThe concentrations of IL-10 and IL-12 in supernatants in two groups were detected according to kit instructions.6 The expression level of VDR detected by Western blot assayPBS was used to wash the cells in the 24-well culture plate,then scrape off the cells from the bottom of the 24-well culture plate with a cell scraper,all the cells were collected in the EP tubes.Suspension was centrifuged,and then to wash these cells with PBS for twice.Lyse these cells with RIPA buffer.Total protein extracted from all samples were run on SDS-Tris-HCl Ready Gels,and then transferred onto PVDF membrane.Milk blocking solution was used to block PVDF membranes,and subsequently incubated with primary antibodies VDR,1:1000;β-actin,1:1000 then corresponding second antibodies(1:3000).Finally,double color infrared laser scanner scanned membranes.The results were integrated by Image J.7 Statistical analysesUsing SPSS 13.0 to calculate the statistics.Results are expressed as the mean ± standard deviation.Using One-Way ANOVA to tested the statistical significances between different groups,to test differences within groups with SNK-q method.Correlation between VDR,IL-10 and IL-12 were analyzed by Pearson correlation method.For all test results,P value <0.05 was considered statistically significant.Results:1 The morphology of HaCaT cellsUnder inverted microscope,most cells were adhesion,strong refraction and had 2 to 3 pseudopodia.The cell membrane was complete.Cultured for 5days later,the cells were polygonal and confluent.The appearance of cells was cobblestone arrangement.HE staining showed that the cytoplasm of the HaCaT cells were stained shallow pink and rich,the nuclear was big andstained blue.Immunohistochemical stain showed that the positive reaction of keratin 10 was found in the cell membrane and cytoplasm.2 The results of the expression level of IL-10 in the supernatants by ELISAAt 2h,4h,8h,and 16 h,the expression level of IL-10 in the experimental group were 1427.71±15.952,1562.09±12.24,1826.57±16.12,and 2363.807±53.465pg/m L,respectively.As the extension of the time,the expression trend was increasing.While the expression level of IL-10 in control group were1100.580±2.460,1160.8±7.044,1285.070±5.185 and 1400.96±11.657pg/m L,respectively.As the extension of the time,the expression trend was increasing.The expression level of IL-10 was significantly higher in the experimental group.F=6.591,P=0.042(P<0.05),the difference was statistically significant.3 The results of the expression level of IL-12 in the supernatants by ELISAAt 2h,4h,8h,and 16 h,the expression level of IL-12 in the experimental group were 296.850±0.6,285.430±2.030,275.190±6.030,249.180±3.720pg/m L,respectively.As the extension of the time,the expression trend was decreasing.The expression level of IL-12 in control group were 294.85±4.601,317.360±7.026,340.123±18.905 and 385.05±6.593pg/m L,respectively.As the extension of the time,the expression trend was increasing.The expression level of IL-12 was significantly lower in the experimental group,F=7.012,P=0.038(P<0.05),the difference was statistically significant.4 The results of the VDR expression level detected by Western blot assayAt 2h,4h,8h,and 16 h,the expression level of VDR in the experimental group were 0.3940±0.0110,0.4680±0.0190,0.458±0.0210 and 0.6160±0.1560,respectively.The expression level in control group was 0.27±0.014,0.294±0.015,0.301±0.002 and 0.413±0.01,respectively.With the prolonging of time,expression level of VDR in both experimental and control group increased.But the expression level of VDR was significantly higher in the experimental group,F=8.404,P=0.027(P<0.05),the difference was statistically significant.5 The correlation of the expression level between VDR and IL-10,IL-12 was analyzedPearson correlation analysis showed a positive correlation between the expression levels of VDR and IL-10,r = 0.955 P = 0.045(P <0.05).While a negative correlation was shown between the expression level VDR and IL-12,r=-0.968,P=0.032(P<0.05).Conclusions:1 Trichophyton rubrum interacted with human keratinocytes in vitro,the expression level of IL-10 increased with the prolonging of time,but the expression level of IL-12 decreased with the prolonging of time.2 Trichophyton rubrum interacted with human keratinocytes in vitro,the expression level of VDR gradually increased time-dependently.3 Trichophyton rubrum interacted with human keratinocytes in vitro,there was positive correlation with the expression level of VDR and IL-10 and negative correlation with the expression level of VDR and IL-12.In conclusion,Trichophyton rubrum interacted with human keratinocytes in vitro,with time increasing,the expression trend of VDR and IL-10 was increasing,the expression trend of IL-12 was decreasing.We presumed that human keratinocytes through VDR signaling pathway secreted IL-10 and inhibited the secretion of IL-12 was one of the reasons of the immune tolerance and longer duration of Trichophyton rubrum mediated. |
