| Atopic dermatitis(AD)is one of the common chronic skin diseases with inflammation,pruritus and high recurrence.Its incidence is increasing year by year around the world.Although the drugs used in the clinical treatment of AD such as glucocorticoid have significant effects,there exist more serious adverse reactions.Yu-ping-feng-san(YPFS),as an ancient classic prescription,has the remarkable curative effects of invigorating qi and consolidating superficies in the treatment of allergic diseases.Our previous studies found that YPFS and its effective components,such as formononetin(FMN),could effectively alleviate the symptoms of FITC-induced mice AD model.Its mechanism was related to the functional regulation of epithelial cells,which could significantly inhibit the secretion of the key allergenic promoter TSLP.This is consistent with the effects of "invigorating qi and consolidating superficies" of YPFS.However,the specific regulation mechanism is still unclear and needs further research.In the early stage,our team used gene chip technology to analyze the gene changes in the mice model of AD and we found that the expression of A20 and TSLP was significantly changed in the model group after the screening analysis.In addition,current studies also indicated that A20 could be involved in the production of allergic diseases as a key negative regulatory factor of NF-?B.So is A20 related to TSLP secreted by epithelial cells?Do the active components of YPFS such as FMN have regulatory effects on A20?In order to answer the above questions,this paper maiuly studied from the following aspects.1.Correlation and mechanism between A20 and TSLPWe used TNF-? and Poly(I:C)combined stimulation on HaCaT cells at different time points or overexpressing and silencing A20 in HaCaT cells in vitro to detect the effects of A20 on the expression of TSLP protein(by ELISA)and mRNA(by qPCR).The results were listed as follows.(1)The expression of A20 and TSLP proteins and their mRNAs showed opposite dynamic changes,that is,there was a negative correlation between both expression in HaCaT cells.(2)Overexpressing A20 could observably restrain the expression of TSLP protein and its mRNA whereas silencing A20 could markedly up-regulate their expression.(3)It could inhibit TSLP expression in a dose-dependent manner by overexpressing different concentrations of A20.The above results suggested that A20 negatively regulated the expression of TSLP.The whole length of A20 and its N-and C-terminal proteins were overexpressed in HaCaT cells to detect the influence of different structural regions of A20 on TSLP expression and the combination of TSLP and Ub by immunoprecipitation reaction.GeneCards,Alggen and ChiPBase were searched for the prediction of TSLP transcription factor.TNF-a and Poly(I:C)were combined to stimulate HaCaT cells or silencing A20 expression by siRNA to detect the expression of NF-kB p65,STAT1,STAT3 and STAT6 proteins(by Western Blotting).Then we observed the effects on TSLP protein expression via adding the inhibitor of each transcription factor separately.The results were as follows.(1)Overexpression of the full-length and C-terminal A20 proteins had a conspicuous inhibition on TSLP expression but its N-terminal had no effect on TSLP.(2)TSLP could not be modified by Ub.(3)Combined stimulation of TNF-a and Poly(I:C)could activate the expression of p-STAT1,p-STAT3 and p-STAT6 in HaCaT cells.(4)After silencing A20,p-STAT1 and p-STAT3 expression increased significantly but p-STAT6 had no obvious changes,which manifested that A20 had a negative regulation on the expression of p-STAT1 and p-STAT3.(5)PDTC(the inhibitor of NF-?B p65),Fludarabine(the inhibitor of STAT1)and Stattic(the inhibitor of STAT3)all prominently inhibited TSLP expression.These results testified that A20 negatively regulated TSLP through its C-terminal proteins(the active end of ubiquitination),which was involved in inhibiting the expression of p-NF-?B p65,p-STAT1 and p-STAT3.The protein synthesis inhibitor CHX was administered in HaCaT cells and A20 was overexpressed in vitro for the detection of STAT1 and STAT3 expression.Then we overexpressed A20 in vitro again and gave the proteasome inhibitor MG132 and the autophagosome inhibitor Chloroquine to detect STAT1 expression.Immunoprecipitation was used to test the combination between STAT1,STAT3 and Ub,K48 and K63 chains respectively.After overexpressing and silencing A20 in HaCaT cells,the effects of A20 on K48 and K63 chains were detected.The results showed that(1)The expression of STAT1 and STAT3 was significantly decreased after the administration of CHX,which indicated that STAT1 and STAT3 could be degraded.(2)Overexpression of A20 memorably inhibited the expression of p-STAT1 and p-STAT3.(3)Overexpressing A20 could significantly down-regulate STAT1 expression and MG 132 would antagonize this effect,while Chloroquine had no significant effect.It suggested that A20 could regulate STAT1 expression through the proteasome pathway.(4)Both STAT1 and STAT3 could be combined with Ub,K48 and K63 chains.(5)Overexpressing A20 had a significant increase on the expression of K48 chain while silencing A20 restrained significantly its expression.And K63 chain was not visibly affected,which all demonstated that A20 mainly affected the expression of K48 chain in HaCaT cells.The above results suggested that the negative regulation of A20 on TSLP might be related to promoting ubiquitination of K48-chain dependent STAT1 and STAT3.2.The effective components of YPFS affect TSLP expression by regulating A20 in HaCaT cellsWe used active components of YPFS including astragaloside IV,cimifugin,calycosin and FMN in HaCaT cells to investigate their effects on TSLP,A20 and its downstream signaling pathways.FMN was used in HaCaT cells in vitro or in the initial stage of the AD sensitization model in vivo to detect its effects on TSLP,A20 and its downstream signaling pathways.Then we silenced A20 in HaCaT cells and used FMN to determinate the effects on TSLP,A20 and its downstream signaling pathways after silencing A20.The results manifested that(1)the effective components of YPFS could up-regulate the expression of A20 to different degrees,suppress the activation of p-NF-KB p65,p-STAT1,p-STAT3 and p-STAT6,and down-regulate TSLP production.Thereinto,FMN had the strongest effect.(2)According to further studies in vivo and in vitro,,FMN significantly up-regulated the expression of A20 and its mRNA,inhibited p-STAT1 and p-STAT3 expression,and down-regulated TSLP protein and its mRNA.(3)After silencing A20 in HaCaT cells,the inhibitory effect of FMN on TSLP was antagonized.These suggested that FMN inhibited the expression of p-STAT1 and p-STAT3 via up-regulating A20,thereby inhibiting TSLP.3.Studies on the regulation mechanism of A20 by FMNDifferent estrogen receptor agonists or inhibitors were used in HaCaT cells to observe their effects on A20 expression.The molecular docking(DS 3.0 software)was applied to investigate the combination of FMN and ER-?,ER-?,GPER.Then GPER agonist G1 or inhibitor G15 was given in HaCaT cells,or GPER expression was interfered by siRNA to investigate the role of GPER in the regulation of A20 through FMN.The FITC-induced initial model of AD sensitization was established in vivo,and G1,G15 and FMN were given severally to investigate the effect of GPER in the process of FMN alleviating AD.The results were listed as follows.(1)On the one hand,GPER agonist G1 significantly up-regulated A20 expression while PPT(ER-a agonist)and DPN(ER-? agonist)showed no distinct effect.On the other hand,GPER inhibitor G15 signally inhibited the expression of A20 while ER-a inhibitor MPP and ER-? inhibitor PHTPP had no significant effect.(2)The binding ability of GPER to FMN was stronger than that to ER-a and ER-?.(3)After G15(GPER inhibitor)was given or silencing GPER,the up-regulation effect of FMN on A20 was antagonized whereas FMN with G1(GPER agonist)could synergistically up-regulate A20 expression.(4)In the initial stage of AD sensitization model,G1 and FMN could significantly up-regulate A20 and inhibit the production of TSLP,and G15 could antagonize the effect of FMN on A20 and TSLP.These all showed that FMN up-regulated A20 expression by activating GPER.Research conclusions:1.A20 negatively regulates TSLP through its C-terminal structure,which promotes the ubiquitination degradation by facilitating the formation of K48 chains on STAT1 and STAT3,thereby inhibiting TSLP expression.2.FMN significantly inhibits IgE in the initial stage of the FITC-induced AD sensitization and the expression of the key allergenic promoter TSLP to effectively relieve the symptoms of AD.The mechanism is to activate GPER and then to up-regulate A20 expression to affect the ubiquitination of STAT1 and STAT3,thereby inhibiting the production of TSLP. |
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