| Atopic dermatitis(AD)is a refractory inflammatory skin disease,and characterized by chronic inflammation,impairment of the cutaneous-epidermal barrier and hypersensitivity to environmental allergens induced by immunoglobulin E(IgE)and the production of type 2 cytokines including IL-4,IL-5,IL-9 and IL-13.Due to the increasing incidence of AD year by year,it has become one of the major global health problems.As a chronic disease which cannot be cured clinically,AD has affected the quality of people’s life in a serious way.Medical treatments like corticosteroids are the representative therapeutics to tackle AD,which are potent to control the symptoms but not efficient to reduce the relapse of the disease.Therefore,it is urgent for us to further explore the pathogenesis of AD and seek efficient preventive approaches.Traditional Chinese medicine(TCM)possesses unique prevention advantages in AD recurrence.We found out that cimifiugin,one of the main components of Saposhnikovia divaricate,was an effective ingredient again st AD recurrence.Taking the Chinese medicine theory,previous research reports and data acquired by our group into consideration,the pivotal role of tight junction proteins(TJs)in epithelial cells(EC)in the pathogenesis of AD is highlighted.However,the upstream regulatory mechanism of TJs is not cl ear.As a small non-coding RNA,in our previous study,miR-155-5p was found to be highly expressed in the process of AD recurrence,especially in the remission phase when the inflammation has regressed.Although miR-155-5p has been reported to increase significantly in patients with AD,its role in AD pathogenesis has not been elucidated.Whether miR-155-5p could affect epithelial susceptibility,promote the formation of allergic microenvironment and ultimately lead to the occurrence and development of AD by regulating epithelial TJs needs to be investigated.This paper includes the following contents:miR-155-5p inhibitor was utilized on the FITC-induced AD mice model to investigate the inhibitory effect of miR-155-5p,as well as the effect on mice ear swelling,inflammatory cell infiltration and Th2 cytokines.The miRanda and TargetScan databases were used to predict potential targets of miR-155-5p,and the predicted target genes were analyzed in our previous mRNA array.Protein kinase inhibitor α(Pkiα)shows the highest scores in the databases.A luciferase reporter assay was used to confirmed that Pkia mRNA was a specific target of miR-155-5p.The expression of miR-155-5p and PKIa were investigated in vivo and in vitro.The mice of AD and at the initial stage of AD were treated with miR-155-5p inhibitors,and the expressins of PKIa,TJs,TSLP and IL-33 were detected.HaCaT cells were treated with miR-155-5p inhibitors or TNF-α,and the effects of miR-155-5p on PKIα were detected by western blot and immunofluorescence.HaCaT cells were transfected with miR-155-5p mimic to investigate the effects of miR-155-5p on PKIα,TJs,TSLP and IL-33.Futhermore,PKIα siRNA and mimic were transfected in vitro to investigate the effects of PKIa on TJs,TSLP and IL-33.HaCaT cells were stimulated with TNF-α,and PKA was detected by western blot.HaCaT cells were treated with FSK and H89,and TJs were detected by western blot and immunofluorescence.After being stimulated with TNF-α,the mRNA levels of CLDNs(1,2,3,4,5,7,8,18),Occludin and Zo-1 in HaCaT cells were detected by PCR.HaCaT cells were transfected with PKIα siRNA,and the effect of PKIα on TJs mRNA was detected by PCR.The relationship between PKIα and transcription factors was predicted and analyzed.PKIα was silenced or overexpressed in HaCaT cells,and the changes of FoxO1 was observed by immunofluorescence subsequently.HaCaT cells were co-transfected of siRNAs of PKIa and FoxO1,and the effect of PKIα on CLDN-1 through regulation of FoxO1 was investigated.The co-localization of PKIα and FoxO1 in HaCaT cells were investigated.In vivo,FITC-induced AD mice were treated with cimifugin,and the efficiency of cimifugin on ear thickness and histopathological changes were examined.The expression of Thl/Th2 cytokines,TJs,TSLP/IL-33 and miR-155-5p,PKIα were detected,and ILC2 was analyzed by flow cytometry.The effects of cimifugin on miR-155-5p and PKIα were also detected by PCR in vitro.After being stimulated by TNF-α,HaCaT cells were transfected with CLDN-1 siRNA,and the production of TSLP were detected by ELISA.In order to observe the role of miR-J155-5p in the regulation of AD by cimifugin,HaCaT cells were treated with miR-155-5-p mimic and/or cimifugin,the expression of CLDN-1 and TSLP were detected by western blot or ELISA.Results:1.miR-155-5p is required for allergic inflammation in mice AD.Silencing miR-155-5p attenuated the thickening of the epidermis in AD and reduced the infiltration of inflanmmatory cells and the secretion of Th2 cytokines.Pkia was identified as a direct target of miR,155-5p.2.miR-155-5p regulates the expression of TJs and TSLP/IL-33 by targeting Pkia.miR-155-5p increased predominantly in epithelium and its upregulation correlated with PKIa downregulation in the AD model and HaCaT cells.When epithelial cells were transfected with an miR-155-5p inhibitor,the expression of PKIa,Occludin,and CLDN16 increased and that of TSLP decreased significantly,whereas the overexpression of miR-155-5p resulted in the opposite changes.The increased expression of PKIa and TJ proteins,with reduced TSLP and IL-33,was also detected in miR-155-5p-blocked mice,in both the initial and elicitation stages of AD.The expression of TJ proteins(CLDN-1、Occludin、Zo-1、CLDND1及CLDN16)also decreased when cells were transfected with PKIa siRNA.TJ proteins increased,and TSLP and IL-33 decreased significantly after the overexpression of PKIa.3.PKIα impacts TJs expression by inhibited the accumulation of FoxO1 in nucleus.After TNF-a stimulation,the change of PKA was negligible.Activation or inhibition of PKA activity had no significant effect on TJs.The gene expressions of TJs were down-regulated after the stimulation of TNF-α.Silencing PKIα significantly inhibited the mRNA levels of CLDN-1 and Occludin.It was predicted that there was a physical binding relationship between PKIca and TJs transcription factor FoxO1.Moreover,silencing of PKIa could promote the nucleation of FoxO1 in vitro.Overexpression the PKIa inhibited the accumulation of FoxO1 in the nucleus.HaCaT cells were co-transfected with PKIa and FoxO1 siRNA.FoxO1 could interfere with the regulation of CLDN-1 by PKIa,indicating that the regulation of TJs by PKIa requires the participation of FoxO1.The co-localization of PKIa and FoxO1 was found by immunofluorescence assay,indicating that PKIa may directly act on FoxO1 and thus regulate TJs.4.Cimifugin suppresses allergic inflammation by restoring the expression of TJs via regulating miR-155-5p.Cimifugin(50 mg/kg)significantly inhibited Th2 inflammation and the level of miR-155-5p in AD mice,restored the expression of PKIa and TJs,and reduced the production of TSLP,IL-33 and ILC2.Cimifugin(1λ,M)inhibited the expression of miR-155-5p in HaCaT cells induced by TNF-α and restored the expression of PKIa.The effects of cimifugin on TJs and TSLP depend on miR-155-5p.Conclusions:1.Cimifugin alleviated AD by inhibiting the abnormal expression of miR-155-5p,which subsequently restored epithelial barrier function,reduced epithelial susceptibility,and decreased Th2 inflammatory cytokines.2.Our data firstly confirmed that miR-155-5p was critical for allergic inflammation in a mouse model of AD by directly regulating PKIa and epithelial TJ expression.These findings suggested that targeting miR-155-5p might be novel and potent therapeutic strategy against allergic disorders. |
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