The Effects Of High Mobility Group Box-1 On Atopic Dermatitis

Objective:Atopic dermatitis(AD)is a genetically related,chronic,recurrent,and inflammatory skin disease.The incidence for children is as high as 10%-20%in developed countries,and it is on the rise all over the world.This disease usually arises in infancy,and can persist into adulthood.Patients often have severe itching,which affects the quality of life seriously and brings sustained financial burden to the family.The exact pathogenesis of AD remains unknown,but abnormal immunity with the release of inflammatory mediators plays an important role in the disease.At present,the oral administration of antihistamines and immunomodulators,as well as the use of topical glucocorticoids,calcineurin inhibitors,and humectants,is the preferred treatment for AD.However,the treatment of chronic recurrent or refractory AD still remains a problem.Therefore,the study of pathogenic factors of AD and exploring new therapeutic targets and measures to control the symptoms of dermatitis effectively are of important theoretical and practical significance for improving the prognosis and life quality of patients.High-mobility group box1(HMGB1)is a highly conserved nonhistone chromatin protein in nucleus.Its main function is to stabilize the structure of nucleosomes and regulate gene transcription.However,HMGB1 can be released passively when cells damage or necrosis,or secreted actively by monocytes,macrophages or neutrophils into the extracellular matrix,and then binds to a variety of cell surface receptors to involve in immune and inflammatory reaction as a cytokine.Receptor for advanced glycation end products(RAGE)is one of the main receptors of HMGB1.Activated RAGE signaling pathway leads to the activation of nuclear factor kappa B(NF-?B).This transcription factor promotes the production of various proinflammatory factors,including HMGB1 and RAGE themselves.This mode of feedback makes the HMGB1-RAGE signaling pathway tightly connected to the occurrence and sustainment of inflammation.Although the systemic use of corticosteroids is effective in treating allergic diseases,its efficacy in treating AD is limited due to its adverse effects.Some clinical studies showed that topical licorice extract improved dermatitis effectively in patients with AD,but the mechanisms are still unknown.Glycyrrhizin(GL)is a monomer triterpepene and the major bioactive ingredient of licorice root having many pharmacological and biological functions,such as protecting liver cells and exerting anti-inflammatory,anti-viral,immunoregulatory and anti-tumor effects.In recent years,the anti-inflammatory effect of GL as a natural inhibitor of HMGB1 has gained attention.GL can bind directly to HMGBl to inhibit its cytokine activity,which plays a therapeutic role in inflammatory diseases,like infection,burn,ischemic injury,pancreatitis and ulcerative colitis.Mast cells originate from bone marrow CD34~+precursor cells.After migrating to their location,mast cells differentiate and mature under the influence of different growth factors and microenvironment.Mast cells are widely distributed in different parts of the body that communicate with the outside world,such as skin,respiratory tract,digestive tract and so on.Therefore,mast cells and antigen-presenting cells interact first with allergens and invade pathogens,and are vital for innate and adaptive immunity.The cytoplasm of each mature mast cell contains plenty of particles which stores a variety of bioactive mediators.One of the important characteristics of mast cells is degranulation with external stimuli such as specific IgE,complement,virus,bacteria and other allergens to release prestored mediators such as histamine,heparin,chondroitin sulfate,cytokines,chymase and tryptase.Moreover,the activated mast cells synthesize new mediators such as prostaglandins,leukotrienes,cytokine and chemokines.These mediators are closely related to allergy and inflammation.The transient release of mast cell mediators can cause urticaria-like symptoms,such as pruritus,erythema,and edema.However,if this process persists,it leads to chronic itching and inflammatory dermatitis symptoms of eczema and atopic dermatitis,even allergic rhinitis and asthma several years later.Early studies have shown that mast cells in skin lesions of patients with AD were more than those in nonskin lesions,and the latter increased more significantly than those in healthy human skin.Therefore,it is necessary to investigate the components and activating factors of mast cells thoroughly for the treatment of AD.This study aimed to investigate the effects and associated mechanism of HMGB1pathway in AD-like symptoms and mast cell activation so as to provide the basis for finding a new target for therapy of AD.Methods:In this study,the AD-like symptoms animal model was established by the application of DNCB to the skin of BALB/c mouse.Glycyrrhizin treatment was performed.The effects of GL were compared with those of dexamethasone(Dex),an acknowledged potent medicine commonly used to treat allergic disorders.The model was evaluated according to dermatitis score,serum IgE level and pathological changes of skin lesions;the infiltration of mast cells detected by toluidine blue staining;the expression of HMGB1/RAGE in skin lesions was detected by tissue immunofluorescence and Western blot;the phosphorylation level of NF-?B was detected by Western blot,and the expression of tumor necrosis factor alpha(TNF-?)and interleukin-6(IL-6)were detected by Western blot and RT-PCR.Then,fetal skin-derived mast cells(FSMC)were separated from day-16 fetal skin of BABL/c mice,and cultured for 14 days with recombinant murine IL-3 and stem cell factor(SCF),and then enriched by density-gradient centrifugation with Percoll.Toluidine blue staining and flow cytometry were used for identification.A mouse mast cell line P815 cells were cultured as well.The expression of HMGB1/RAGE in mast cells was detected by cell immunofluorescence and Western blot.Then,the effects of HMGB1and GL on the proliferation and activation of P815 cells were detected,including degranulation,intracellular calcium concentration,CD117 expression,NF-?B signaling pathway activation and the expression of MC Tryptase,TNF-?and IL-6.The mice were intraperitoneally injected with HMGB1 to study the effect of HMGB1on mast cells accumulation.Results:1.The AD-like lesions induced by the topical application of DNCB resulted in erythema,edema and excoriation followed by erosion,scaling and dryness on the back of ears and dorsal skin with increased serum IgE level.HE and TB staining of dorsal skin tissue sections revealed the histopathological features of AD-like lesions.Hyperkeratosis,parakeratosis,acanthosis and spongiosis occurred in the epidermis with infiltration of inflammatory cells,especially mast cells,in the dermis of DNCB group.2.Immunofluorescence results showed the high expression of HMGB1 and RAGE in the DNCB group.Moreover,significant differences were found between the DNCB group and the others.The expression of HMGB1 was confined to cell nuclei in the control,but it was also detectable in the cytoplasm in the DNCB group.RAGE was expressed in the stratum corneum of epidermis,hair follicles and sebaceous glands in the control.In the DNCB group,RAGE was highly expressed in all epidermal layers and dermis.3.Western blot analysis showed the significant upregulation of HMGB1?RAGE?pNF-?B?TNF-?and IL-6 protein of the DNCB group.RT-PCR showed the higher relative m RNA expression TNF-?and IL-6 in AD mice.4.GL treatment significantly relieved the development of dermatitis;decreased the serum IgE level;ameliorated the infiltration of inflammatory cells and mast cells;downregulated the expression of HMGB1?RAGE?pNF-?B?TNF-?and IL-6.There was no significant difference compared with the Dex treatment group.5.The isolated murine fetal skin cells were cultured for 14 days and purified by density gradient centrifugation.Toluidine blue staining showed that most of the cells are mature mast cells with rich purple metachromatic granules in cytoplasm.6.The purity of FSMC was>97%based on the surface expression of CD117 and Fc?RI determined by flow cytometry.7.Double immunofluorescence results showed that HMGB1 was localized in both cell nuclei and cytoplasm in MC-trypatase-positive FSMC,mainly in the form of granules.RAGE was strongly positive in the cytoplasm with MC-trypatase.8.Western blot revealed the protein expression of HMGB1,RAGE,TNF-?and IL-6 in FSMC.9.As to P815 cells,immunofluorescence staining showed that HMGB1 also expressed in the nucleus and cytoplasm;the positive expression of RAGE was located in the cytoplasm and a small amount in the nucleus;MC Tryptase was positive in particles and cytoplasm,basically consistent with the expression patterns in FSMC.10.The results of CCK-8 showed that GL or recombinant murine HMGB1(rmHMGB1)had no significant influence on the proliferation of P815 cells.11.Stimulation of P815 cells with rmHMBG1 induced increased release of?-Hexosaminidase.12.Flow cytometry analysis showed that stimulation of P815 cells with rm HMBG1 induced increased calcium influx.13.Flow cytometry analysis showed that stimulation of P815 cells with rmHMBG1 induced increased surface expression of CD117.14.Western blot analysis showed that the rmHMGB1 activated PI3K/AKT and ERK1/2 signaling pathways,promoted the phosphorylation of AKT,ERK1/2 and NF-?B,induced the upregulationg of RAGE?MC Tryptase?TNF-?and IL-6 in P815 cells.15.RT-PCR showed that stimulation of P815 cells with rmHMBG1 induced increased relative mRNA expression of HMGB1 and RAGE.However,the effects of rmHMGB1 on P815 cells activation were inhibited by GL.16.The intraperitoneal injection of rmHMGB1 resulted in a significant increase of peritoneal mast cells in time-dependent manner.Conclusion:The HMGB1 signaling pathway was activated abnormally in AD;HMGB1 participated in the development of chronic allergic dermatitis partly by continuously stimulating mast cells.