The Effect Of Bacteria Superantigen SEB On Glucocorticoid Receptor In Human Keratinocyte And Associated Mechanisms

Background:Inflammatory skin disease is the most common type of disease in the department of dermatology which includes atopic dermatitis,contact dermatitis,eczema,psoriasis and many other skin diseases.For clinical treatment of this type of disease,the topical glucocorticoid with its good anti-inflammatory,anti-allergic,immunosuppression and so on,has long been one of the main choices of the clinician.Although topical glucocorticoid achieves a certain effect in the treatment of inflammatory skin,with the extension of treatment time its effect will be gradually weakened.This kind of phenomenon is called topical glucocorticoid resistance.Bacterial colonization is one of the important features of inflammatory skin diseases,the study confirmed that the staphylococcal enterotoxin B(SEB)secreted by staphylococcus aureus may play an impotent role in the process of topical glucocorticoid resistance.To investigate the role and associated mechanisms of SEB in the topical glucocorticoid resistance has an important significance for regulating the clinical use of glucocorticoid topical formulations,reducing the dose of glucocorticoid as well as improving the treatment effect of inflammatory skin diseases.Objective:To investigate the effect of bacteria superantigen SEB on glucocorticoid receptor in human keratinocyte and associated mechanisms.Methods:Human immortalized keratinocyte cell line(HaCaT cell)was used as research model.HaCa T cells were treated with SEB for 24 h at different concentrations,and then the mRNA and protein levels of glucocorticoid receptor(GR)were measured by RT-PCR and western blot.After pretreated with SEB,HaCaT cells were stimulated with dexamethasone for various durations to observe the intracellular localization of GRα.HaCaT cells were treated with SEB for various durations,then the activation of MAPK was measured by western blot.HaCaT cells were stimulated with SEB and MAPK inhibitor,western blot and immunofluorescence were used to detect the expression and intracellular localization of GR.Results:1.The effects on the mRNA and protein expressions of GR with bacteria superantigen SEB in HaCaT cells:(1)After treated with SEB,the mRNA expression of GRα in HaCaT cells was not significantly different from the control group(p>0.05);(2)After treated with SEB,the mRNA expression of GRβ were increased in a dose-dependent manner,reaching a maximum at 100 ng/ml in HaCaT cells;(3)After treated with SEB,the protein expression of GRα in HaCaT cells was not significantly different from the control group(p>0.05);(4)After treated with SEB,the protein expression of GRβ were increased in a dose-dependent manner,reaching a maximum at 100 ng/ml in HaCaT cells;2.The effects on nuclear translocation of GRα with bacteria superantigen SEB in HaCaT cells:(1)10-6 mol/L dexamethasone could induce the translocation of GRα from cytoplasm to nucleus;(2)HaCaT cells were treated with 100 ng/ml SEB for 1 h and subsequently treated with 10-6 mol/L dexamethasone for 8 h.The GRα in SEB group and control group mainly distributed in the cytoplasm,and the GRα in dexamethasone group translocated to nucleus;while the GRα in dexamethasone+SEB group was mainly localized in cytoplasm.3.The effects on GR with bacteria superantigen SEB in HaCaT cells by activating MAPK signaling:(1)After treated with SEB,p38 signaling pathway was not activated in HaCaT cells;(2)After treated with SEB,JNK signaling pathway was not activated in HaCaT cells;(3)After treated with SEB,ERK1/2 signaling pathway was activated in HaCaT cells;(4)After treated with ERK1/2 inhibitor U0126 and SEB,the protein expression of GRβ in HaCaT cells was not significantly different from the control group(p>0.05);(5)HaCaT cells were treated with 100 ng/ml SEB and 20 μmol/L U0126 for 1 h and subsequently treated with10-6 mol/L dexamethasone for 8 h.The GRα in dexamethasone group and dexamethasone+SEB group translocated to nucleus.Conclusions:1.SEB increased the mRNA and protein levels of GRβ in a dose-dependent manner,reaching amaximum at 100 ng/ml in HaCaT cells.Neither the mRNA nor protein level of GRα was affected by SEB.2.Dexamethasone could induce the translocation of GRα from cytoplasm to nucleus.However,the translocation of GRα from cytoplasm to nucleus induced by dexamethasone was partly inhibited by SEB.3.SEB could activate the ERK1/2 signaling pathway in HaCaT cells.The up-regulation of GRβ and the inhibition of GRα nuclear translocation induced by SEB were suppressed by the inhibition of ERK1/2 signaling pathway.