Mechanism Of HMGB1/FoxO1 In Atopic Dermatitis

Background:Atopic dermatitis（AD）is a chronic,relapsing inflammatory skin disease characterized by skin barrier dysfunction,inflammation,and chronic pruritus,and its incidence is increasing worldwide.The etiological factors of atopic dermatitis involve environmental factors,genetic susceptibility,skin barrier disruption,microbiome alterations,immune dysregulation with Th2 cell activation and Ig E-mediated hypersensitivity.High mobility group box 1（HMGB1）is a highly conserved and ubiquitously expressed nuclear protein that can be involved in the pathology of various chronic inflammatory and autoimmune diseases as a key damage-associated molecular pattern after release.HMGB1 exerts its biological effects mainly by binding to receptors and can mediate activation of the MAPK pathway through the RAGE receptor.FoxO1 is an important transcription factor in cells,and studies have shown that activation of the MAPK pathway is closely related to phosphorylation and nuclear translocation of FoxO1 and is involved in the regulation of barrier function and inflammatory response.However,whether HMGB1 can regulate barrier function and inflammatory response in atopic dermatitis by regulating nuclear translocation of FoxO1 remains to be investigated.Objective:To identify the role of HMGB1 in atopic dermatitis,explore the regulatory mechanism of HMGB1/FoxO1 on skin barrier and inflammatory response in atopic dermatitis,and provide new targets for the treatment of atopic dermatitis.Methods:1.BALB/c mice were randomly divided into 3 groups,6 mice in each group.In addition to the normal control group,2,4-dinitrochlorobenzene（DNCB）was applied to the model group and the model+inhibitor group,and glycyrrhizin,an HMGB1inhibitor,was intraperitoneally injected into the inhibitor group.Western blot was used to detect the expression levels of HMGB1;thick gauge was used to detect ear and skin thickness;HE and toluidine blue staining were used to observe histopathological changes;flow cytometry was used to detect the expression levels of TNF-α,IL-4,and IL-13 in single cell suspensions of skin tissues as well as in spleen and lymph nodes;ELISA and q-PCR were used to detect the levels of inflammatory factors and chemokines in tissues;Western blot was used to detect the expression of inflammatory factors,RAGE receptors,tight junction protein,FoxO1 nuclear translocation,FoxO1phosphorylation,and MAPK pathway phosphorylation in tissues;immunohistochemistry was used to detect the expression of tight junction proteins in skin and ear tissues.2.Si RNA was constructed,TNF-αwas used to stimulate cells,immunofluorescence and ELISA were used to detect the expression levels of intracellular and extracellular HMGB1,ELISA and q-PCR were used to detect the expression levels of cellular inflammatory factors and chemokines;Western blot and immunofluorescence were used to detect the expression levels of tight junction proteins;transmembrane resistance（TER）and FITC-dextran permeability of cell monolayer epithelial cells were detected;and Western blot was used to detect the phosphorylation expression levels of RAGE,FoxO1 nuclear translocation,and MAPKs pathway.Cells were stimulated with human recombinant HMGB1,cell viability was measured by MTT assay,TER and FITC-dextran permeability were used to detect the effect of different concentrations on cell permeability;Western blot was used to detect the expression levels of cellular inflammatory factors and tight junction protein;cells were pretreated with RAGE receptor inhibitor TFA,FoxO1 inhibitor AS1842856,JNK inhibitor SP600125,ERK inhibitor SCH772984,and p38 MAPK inhibitor SB203580,respectively,and Western blot was used to detect the phosphorylation expression of cellular inflammatory factors,tight junction protein,and MAPKs pathways,TER and FITC-dextran permeability were detected,and q-PCR was used to detect the expression levels of inflammatory factors and chemokines.3.Hmgb1^f/fgene and K14-Hmgb1-Cre gene were identified by PCR,and HMGB1 knockout was verified by HE and immunohistochemistry.Staining was used to observe histopathological changes;flow cytometry was used to detect the expression levels of inflammatory cells and inflammatory factors in single cell suspensions of skin tissues,ELISA and q-PCR were used to detect the levels of inflammatory factors and chemokines in tissues;Western blot was used to detect the expression of inflammatory factors,RAGE receptor,tight junction protein,FoxO1 nuclear translocation,FoxO1phosphorylation and MAPKs pathway phosphorylation in tissues;immunohistochemistry was used to detect the expression of tight junction protein in skin and ear tissuesResults:1.Part 1:In DNCB model group,HMGB1 expression was significantly increased in ear tissue and skin tissue,ear and skin epidermis were thickened and there was significant inflammatory cell and mast cell infiltration,mast cell degranulation was significant,spleen index increased lymph node weight,eosinophil and neutrophil ratio in skin was significantly increased,inflammatory factor TNF-α,IL-4,IL-13 expression was increased in spleen and lymph nodes,tissue IL-1β,IFN-γ,TSLP,IL-33,IL-23,IL-17,IL-31,IL-5,CXCL11,CXCL17 expression was increased by ELISA,IL-17A,CXCR2,CXCR4,CXCL10,CCL3,CCL5 expression was increased in tissues by q-PCR,FoxO1 expression was decreased in cytoplasm of increased nuclear expression in tissues by Western blot,P-FoxO1 and MAPKs pathway phosphorylation expression was increased;intraperitoneal injection of HMGB1 inhibited,reducing skin and ear thickness,reducing inflammatory cell infiltration and mast cell degranulation,reducing spleen index and lymph node weight.Reduce the proportion of eosinophils and neutrophils,reduce the expression of the above inflammatory factors and chemokines,inhibit FoxO1 nuclear expression and phosphorylation,and reduce MAPKs pathway phosphorylation.2.Part 2:After transfection with si HMGB1,HMGB1 expression in cells and culture medium was significantly decreased;cytokine and inflammatory chemokines CXCR2,CXCR4,CXCL1,CXCL10,CCL3,CCL5 expression was significantly decreased,tight junction protein FLG,occludin,ZO1 and CLDN1 expression was significantly increased,transmembrane resistance value was increased,FITC-dextran permeability was decreased;recombinant HMGB1 stimulation of HaCaT cells significantly increased epithelial-derived cytokine and inflammatory cytokine expression levels at100 ng/ml,decreased tight junction protein expression,and had no significant effect on occludin expression at 300 ng/ml;TER expression was most significantly decreased at24 h,TER was significantly decreased at 6 h,FITC-dextran permeability was significantly increased at 12 h;RAGE inhibitor TFA pretreatment cells,FoxO1 nuclear expression was significantly decreased,FoxO1 phosphorylation expression was significantly increased in the cytoplasm;MAPK phosphorylation was decreased.Pre-treatment of cells with FoxO1 inhibitor AS1842856 significantly increased tight junction expression;increased TER,decreased FITC-dextran permeability,and decreased inflammatory factors and chemokine expression levels;compared with HMGB1 group,pretreatment of cells with ERK inhibitor SCH772984and p38 MAPK inhibitor SB203580 significantly inhibited FoxO1 phosphorylation and inhibited FoxO1 nuclear translocation;while JNK inhibitor SP600125 inhibited FoxO1 nuclear translocation but had no significant effect on its phosphorylation;SP600125,SCH772984,and SB203580 significantly increased TER and decreased FITC-dextran permeability,all of which significantly decreased inflammatory factors and chemokine expression levels.3.Part 3:Compared with AD-Hmgb1^f/f,AD-K14-Hmgb1 c KO mice had reduced skin damage,dander and inflammatory cell infiltration,reduced ear thickness,but no significant effect on skin thickness,reduced spleen index,reduced lymph node weight,significantly reduced expression of inflammatory factors in spleen and lymph nodes,decreased proportion of eosinophils and neutrophils in skin,lower expression of chemokines and inflammatory factors in skin and ear tissues,and more expression of barrier proteins FLG,occludin,ZO1 and CLDN1 in skin and ear tissues;lower nuclear expression of FoxO1,higher expression in cytoplasm,decreased phosphorylation expression,but no significant change in total FoxO1 expression content;as well as lower expression of RAGE and lower phosphorylation expression of JNK,ERK,p38MAPK.Conclusion:1.HMGB1 expression is increased in atopic dermatitis and has pro-inflammatory and skin barrier disrupting effects;2.Changes in FoxO1 subcellular localization affect the tight junctions of skin epidermal cells and participate in the regulation of skin barrier function.3.HMGB1 is involved in the inflammatory process of atopic dermatitis by regulating skin barrier function through the RAGE/MAPKs/FoxO1 pathway.4.Conditional knockout of HMGB 1 inhibited barrier disruption and inflammation in atopic dermatitis.