| Psoriasis is a chronic recurrent systemic inflammatory skin disease characterized by scaling,thickening,and erythema commonly seen in dermatology.Psoriasis is characterized by a chronic interaction between proliferating keratinocytes and infiltrating reactive immune cells.Activation of multiple signaling pathways are involved in hyperproliferation of keratinocytes,and activation of the nuclear factor-κB（NF-κB）signaling pathway plays a key role in abnormal activation of keratinocytes.Clinical therapeutic drugs for psoriasis can be divided into five categories:oral medications,topical medications,physiotherapy.These therapeutic drugs are often accompanied by adverse drug reaction,thus their clinical applications are limited.Therefore,it is important to develop drugs for the clinical treatment of psoriasis that are economical and have few side effects.GCK is a degradation product of ginseng in the intestinal tract and the main form of ginsenoside absorption in vivo.Our previous study found that GCK had anti-inflammatory effects on animal models of autoimmune arthritis.It was found that GCK could improve skin damage and alleviate keratinocyte proliferation in a psoriasis model induced by IMQ in BALB/c mice,but the mechanism is still unclear.Glucocorticoid receptor（GR）-mediated anti-inflammatory signaling is an important signaling pathway for GC to exert anti-inflammatory effects in vivo.GR activated by GC enters the nucleus as a homodimer and binds to the glucocorticoid receptor element（GRE）on the corresponding target gene to upregulate the expression of anti-inflammatory target genes.It can also enter the nucleus as a GR monomer and bind to NF-κB to inhibit the transcription of NF-κB target genes,and exert anti-inflammatory effects.It was found that the anti-inflammatory effect of GCK was associated with GR and could exert anti-inflammatory effects by competitively binding GR.It has been shown that GR is located in the nucleus of normal human epidermal and cultured keratinocytes but in the cytoplasm of psoriatic epidermal and cultured keratinocytes.It is suggested that abnormal GR signaling is associated with abnormal activation of psoriatic keratinocytes.Whether the therapeutic effect of GCK on psoriasis is related to the activation of GR in keratinocytes and the regulation of keratinocyte function deserves further investigation.This study investigated the therapeutic effects of GCK on psoriasis by establishing an IMQ-induced psoriasis model in mice.Normal human keratinocytes（NHEKs）were used to investigate whether GCK inhibited the hyperactivation of keratinocytes by affecting the nuclear translocation of GR and inhibiting the activation of NF-κB,and alleviated psoriasis-like inflammation in vitro.ObjectiveTo investigate whether GCK exerts a therapeutic effect on psoriasis by affecting GR nuclear translocation,inhibiting NF-κB activation,and suppressing keratinocyte hyperactivation.MethodsThe IMQ-induced psoriasis model was established,and the mice were randomly divided into following groups:model group,GCK(14 mg·kg-1,28 mg·kg-1,56 mg·kg-1,112 mg·kg-1)group,methotrexate(MTX,2 mg·kg-1)group,and dexamethasone(DEX,5 mg·kg-1)group.The psoriasis lesion area and severity index（PASI）scores were used to evaluate the severity of the lesions.HE staining was used to observe the pathological changes of the skin.Western blot was used to detect the expression of keratin（K）6,16,17,1,10,Involucrin,Loricrin,NF-κB pathway-related proteins（IκBα,p-IκBα,p65,p-p65）.The detection of proliferating cell nuclear antigen（PCNA）,K17 and p-p65 in mouse skin tissue were done by immunohistochemistry.The protein of proliferation marker Ki-67 in mouse skin tissues were detected by immunofluorescence.IL-6,IL-8,TNF-α,ICAM-1 mRNA in mouse skin and keratinocytes were detected by q PCR.Enzyme-linked immunosorbent assay（ELISA）were adopted to detect the expression of IL-23 and IL-17 in mouse skin and serum,and IL-6,IL-8,TNF-αand ICAM-1 in keratinocytes.In vitro,NHEKs cells were transfected with siRNA GR.Immunofluorescence,western blot were used to detect the expression of GR in NHEKs cells.High internal content was used to detect proliferation of NHEKs cells.Western blot was used to detect the expression of K6,K16,K17,K1,K10,Involucrin,Loricrin,NF-κB pathway related proteins in NHEKs cells.ELISA was used to detect the leves of IL-6,IL-8,TNF-α,ICAM-1 in NHEKs cells in the supernatant of NHEKs cells.Results1.Therapeutic effect of GCK on IMQ-induced psoriasis in miceThe skin of mice in the normal group were smooth and flat,while the severity of skin lesions were increased with increasing days of modeling in IMQ-induced mice.The severity of mice skin lesions was improved in the GCK(28 mg·kg-1,56 mg·kg-1,112 mg·kg-1),MTX(2 mg·kg-1),and DEX(5 mg·kg-1)groups compared with the mice in the IMQ group.The PASI scores of mice skin in the normal group were 0 consecutive seven days,and the PASI scores of IMQ-induced mice were consistently elevated.The PASI scores of mice were increasing in GCK(14 mg·kg-1,28 mg·kg-1,56 mg·kg-1,112mg·kg-1),MTX(2 mg·kg-1),and DEX(5 mg·kg-1)groups from the first day to the third day,but the PASI scores showed a decreasing trend compared with the model group from the fourth day to the seventh day.The hyperkeratinization of the mouse stratum corneum,thinning or even loss of the granular layer,hypertrophy of the spinous layer,prolongation of the dermatome,and inflammatory cell infiltration appeared in IMQ-induced mice.GCK(28 mg·kg-1,56mg·kg-1,112 mg·kg-1),MTX(2 mg·kg-1),and DEX(5 mg·kg-1)inhibited IMQ-induced skin lessions in mice.GCK(112 mg·kg-1),MTX(2 mg·kg-1),and DEX(5 mg·kg-1)significantly reduced IMQ-induced mRNA expression of TNF-α,IL-6,CXCL8,and ICAM-1 in mice skin,and decreased Barker score and mouse epidermal thickness.The levels of IL-23,IL-17 in skin and serum,and the expression of p-IκBα,p-p65 in skin were reduced in GCK(112 mg·kg-1),MTX(2 mg·kg-1),and DEX(5 mg·kg-1)groups.GCK(28 mg·kg-1,56 mg·kg-1,112 mg·kg-1),MTX(2 mg·kg-1)and DEX(5mg·kg-1)could reduce the splenic index,but there was no significant difference in the thymic index among the groups.2.GCK inhibited abnormal activation of NHEKs cellsNHEKs cells were activated by stimulating with the combination of TNF-α,VEGF165 and IFN-γ.DEX（1μM）,GCK（0.1μM,1μM,10μM）reduced NHEKs cell viability.DEX（1μM）,GCK（1μM,10μM）inhibited NHEKs cell proliferation and reduced TNF-α,IL-6,CXCL8,ICAM-1 mRNA levels in NHEKs cells.DEX（1μM）and GCK（10μM）decreased expression of K6,K16,K17,increased expression of cell early differentiation marker protein K1,K10,increased expression of cell late differentiation marker protein Loricrin,and decreased expression of Involucrin.In addition,DEX（1μM）and GCK（10μM）decreased the expression and nucleation of p-IκBαand p-p65,promoted GR nuclear translocation and expression level of TNF-α,IL-6,CXCL8,ICAM-1 in the supernatant of NHEKs cells.3.Role of GR in the inhibition of NHEKs cell activation and NF-κB activation by GCKNHEKs were transfected with siRNA GR,and then stimulated with TNF-α,VEGF165 and IFN-γ.Stimulation of TNF-α,VEGF165 and IFN-γenhanced the proliferation of NHEKs cells,significantly increased the expression of cell proliferation marker proteins K6,K16 and K17,decreased the expression of early cell differentiation marker proteins K1 and K10,decreased the expression of late cell differentiation marker protein Loricrin,and increased Involucrin expression.The expression of p-IκBαand p-p65 were increased,and protein expression levels of TNF-α,IL-6,CXCL8 and ICAM-1 were increased in stimulated NHEKs cells.GCK（0.1μM,1μM,10μM）had no effect on the proliferation of NHEKs cells after transfecting siRNA GR,as well as the expression of K6,K16,K17,K1,K10,Loricrin,Involucrin,p-IκBα,p-p65,TNF-α,IL-6,CXCL8,and ICAM-1.Conclusion1.GCK had a therapeutic effect on IMQ-induced psoriasis in mice.2.GCK alleviated psoriasis-like dermatitis by inhibiting excessive activation of NHEKs cell.3.Inhibitory effect of GCK on keratinocytes hyperactivation was associated with promoting GR nuclear translocation and inhibition of NF-κB. |
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