| [Background]The skin barrier can resist the external physical and chemical stimulation,and prevent both allergens and pathogens from invasing,and external harmful factors as well.Extracellular matrix(ECM)is an important structural component of the skin barrier,and its normal maintenance is indispensable to the skin barrier.The skin barrier reduces TEWL in a number of ways to complete the moisturizing function.Firstly,the intercellular membrane cross-linking within the stratum corneum forms an insoluble outer membrane called the cornified envelope(CE),the main component of which is ceramide(as well as cholesterol and sphingolipids,which are the main components of extracellular lipids),which is tightly lined between cells to restrict the flow of water across cells.Secondly,the evaporation of the skin mixed with the water secreted by the sweat glands to form a hydrolipidic membrane of epidermal sebum and lipids further reduces epidermal water evaporation.In addition,the skin barrier contains a variety of ingredients that are beneficial for moisturization,including natural moisturizing factors(NMFs),Aquaporins(AQPs),etc.Among them,amino acids,lactate,urea and other NMFs in ECM play a water retention role due to their strong water solubility.Caspase-14 is involved in the degradation of filaggrin(FLG)to produce some free amino acid NMFs.In addition,Aquaporin 3(AQP3)is an important barrier to TEWL reduction,which is both water permeable and aids glycerol transport.AQP3 is involved in lipid composition and metabolic processes by promoting glycerol transport and also contributes to skin barrier recovery and wound healing.The skin relies on its "brick wall structure" skin barrier to provide a mechanical barrier for the organism,shielding it from external damage and stimuli.The organism is in an external environment where it is constantly exposed to external physical and chemical stimuli for damage or pathogen invasion,requiring the skin barrier to have a continuous self-healing mechanism and frequently triggered immune response function.The extracellular matrix ECM is highly dynamic and is the result of continuous remodeling of the ECM by fibroblasts through the process of synthesis,degradation,recombination and chemical modification by secreting collagen precursors and matrix metalloproteinases(MMPs),which include both structural component rearrangements and inflammatory effects.Among them,MMP-9,also known as gelatinase B,is the most complex member of MMPs.It produces collagen peptides by degrading various ECM components such as type IV,V,VIII,and X collagen,and collagen peptides are involved in regulating cell proliferation,migration,apoptosis,and reduced angiogenesis through binding to integrins receptors.In addition,the ECM contains a variety of structural glycoproteins that act as ligands for cell surface receptors to participate in ECM-cell interactions and are also storage modes for some growth factors.These growth factors bind to the ECM and can be released upon protein hydrolysis.For example,Per promotes wound healing by promoting activation of fibroblast secretory stroma,contraction of local defective skin and hardening of local collagen fibers;on the other hand,Per is involved in inflammatory responses in skin barrier repair and remodeling by binding to αv integrin to activate and stimulate keratinogenic cells to secrete thymic stromal lymphopoietin(TSLP),which triggers the Th2 response.Moreover,during skin barrier repair,the organism regulates the secretion of hyaluronic acid(HA)by regulating Has2 and Has3 expression through EGF and TGF-β,and promotes the proliferation and migration of keratinocyte(KC)for barrier repair in skin trauma repair and inflammatory dermatoses,and HA is associated with epidermal proliferation,thickness and differentiation activities.The use of topical glucocorticoids is an important treatment for allergic skin diseases,but the long-term irregular use of glucocorticoids can cause a series of manifestations of skin barrier disruption,such as corticosteroid addictive dermatitis(CAD)and corticosteroid-induced rosacea-like dermatitis(CIRD),which are currently not described as independent diseases in foreign skin monographs,and the side effects of topical glucocorticoids are also attributed to CAD in more domestic literature.In this paper,CAD refers to long-term external hormone-induced dermatitis,which covers the local dermatitis caused by long-term,excessive,and non-indicative external use of glucocorticoid,including but not limited to topical corticosteroids withdrawal dermatitis.Previous studies have suggested that short-term topical use of glucocorticoid can delay the recovery of the skin permeability barrier,increase the incidence of abnormal exfoliation of the stratum corneum,and long-term topical use of glucocorticoid can inhibit the proliferation and differentiation of epidermis.Clinical findings,long-term topical use of glucocorticoids can appear skin thinning and elasticity reduction,capillary dilatation and purpura,accompanied by different degrees of swelling,itching and burning and other glucocorticoid withdrawal responses,resulting in CAD.The mechanism of CAD is not well understood,and the active anti-inflammatory effects of GCs are thought to be achieved through the direct interaction of glucocorticoid receptors(GR)with inflammatory transcription factors that interfere with transcriptional factors,i.e.transcriptional repression.On the other hand,hormones exert anti-inflammatory effects,i.e.,transcriptional activation,by directly binding to transcriptional regulatory regions of chromatin rapidly and efficiently increasing the expression of multiple genes.For example,induced leucine zipper(encoded by the TSC22D3 gene)inhibits nuclear factor-κB(NF-κB)and activator protein 1(AP-1);upregulates NF-κBa inhibitor(IκBa)and thus inhibits NF-κB.glucocorticoid-dependent transcriptional repression can coexist with transcriptional activation,of which transcriptional activation may be the largest and most effective mode of repression of inflammatory genes,but may also be the main cause of adverse effects of hormone therapy.It has been found that glucocorticoids affect skin barrier function by inhibiting fibroblast proliferation and reducing ECM protein synthesis,including inhibiting COL-1,COL-III expression and inhibiting IL-4/13-induced Per expression(does not affect TGF-β-induced Per production).In addition,glucocorticoid can inhibit the expression of MMP-1 and MMP-3,inhibit the expression of MMP-9 gene stimulated by IL-1 and affect the dynamic remodeling of extracellular Matrix.Eucalyptol,also known as 1,8-cineole(Cin),is the main active ingredient of Eucalyptus oil.In animal experiments,Cin has been found to have anti-inflammatory effects by inhibiting the MMP-9 and Nuclear Factor Kappa-B(NF-κB)signaling pathways in murine pneumonia.It has been reported that Cin can moisturize and anti-aging,promote drug skin penetration and enhance the anti-microbial activity of epidermis.In addition,Eucalyptus has been found to have anti-microbial,anti-inflammatory,anti-oxidation and moisturizing effects.Recently,with the discovery of its skin penetration and anti-bacterial activity,its application value in dermatology has been gradually developed.However,studies on the repair of skin barrier by Eucalyptus is limitted.In this study,Clobetasol propionate(CP)was used as an intervention control to investigate the m RNA transcription level,protein expression level and tissue immunostaining of the related proteins CAP-14,AQP3,MMP-9,PER,Has2 of Cin on ECM in CAD of SD rats.[Objective]:Taking SD rats as object,and contact dermatitis was induced by 2,4-Dinitrochlorobenzene(DNCB),and clobetasol propionate(CP)was used as an intervention factor to further induce CAD.q RT-PCR,Western blot and immunohistochemical techniques were employed to explore the m RNA transcription,and protein expression levels for CAP-14,FLG,AQP3,MMP-9,Per,HAS2,TNF-α,NF-κB p65,IκBa after topical Cin solution on SD rat skin to provide a theoretical basis for clarifying the effect of Cin on skin barrier protection.[Method]:1、Animal preparation: SD rats with weight > 300 g were kept in the required cleaner environment and given behavior training for 3 days to receive topical drug..2% pentobarbital sodium(30mg/kg in dose)anesthetized with local anesthesia and deburred(shaved)for skin preparation,selected 3cm × 3cm test area.Blank control matrix(alcohol glycerin applicator)configuration: 30 ml 75% alcohol,add glycerin to 100ml;Cin,CP topical configuration: the required volume of Cin,the required mass of CP dissolved in 30 ml 75% alcohol,add glycerin to 100 ml.2.Pre-experiment:(1)The concentration of topical Cin was determined to be 8% by immunohistochemical detection of proliferative cell nuclear antigen(PCNA).(2)The duration of inducing SD rat corticosteroid addictive dermatitis animal model by CP 0.05% was 20 days as determined by skin damage assessment.(3)A model of allergic contact dermatitis was established initial sensitization with 7% DNCB and a re-sensitization with 0.1% after 14 days.3.Study design: Two experiments were designed to be performed.Normal SD rat skin group(NS group)and allergic contact dermatitis group(ACD group),and they are divided into several subgroups respectively as follow:3.1 Subgroups for NS group.Group NS-A-Normal control group: alcoholic glycerin liniment(matrix),twice daily for 20 days.Group NS-B-CP group: topical application of 0.05% CP liniment twice daily for 20 days.Group NS-C-Cin group: topical application of 8% Cin liniment,twice daily for 20 days.Group NS-D-Cin Intervention Group: 0.05% CP twice daily for 20 days,Subsequently,8% Cin twice daily for 20 days.Group NS-E-Cin + CP Mixed Group: 8% Cin + 0.05% CP liniment,twice daily for 20 days.3.2 Subgroups for ACD group.ACD-A-ACD control group: Topical application with alcoholic glycerin liniment,twice daily for 20 days after ACD.ACD-B – ACD CP group: Topical application with 0.05% CP liniment,twice daily for 20 days after ACD.ACD-C-ACD Cin group: Topical application with 8% Cin liniment,twice daily for 20 days after ACD.ACD-E-ACD CP+Cin Mixed group: Topical 8% Cin + 0.05% CP liniment,twice daily for 20 days after ACD.4.Testing methods and indicators.4.1 Observational indicators4.1.1 Scored definition for SD rats scratching(times/20min): 0 points = no scratching;1 point = 1 to 2 times;2 points = 3 to 6 times;3 points = 7 to 10 times;4 points = 11 to 14 times,and 5 points = 15 or more times.Definition of scratching: One scratching behavior was considered as the front limb of SD rat scratching the neck back or bending down to chew the back or the hind limb scratching the side abdomen,or scratch the experimental area at the neck back.Scratches without pauses are counted as one time4.1.2 Quantification assesment of skin condition in SD rats;Erythema means 1 score,edema 2 scores,papular debridement 3 scores,and epidermal thinning with vasodilation 3 scores as well.and scratches or erosions 4 scores.、4.2 Criteria for SD rat CAD model:(1)In NS group: CAD-related manifestations such as erythema,epidermal thinning,vasodilatation(protrusion of capillaries),etc.during continuous topical application of 0.05% CP(2)In NS group: SD rats scratching after continuous topical application of 0.05% CP showed scratching behavior,skin dysphoria,hormonal withdrawal reaction with skin scratches,etc.(3)Hormonal withdrawal reaction in NS group was improved with CP again: after 15 days of continuous topical application of 0.05% CP,the SD rats had local erythema aggravated at the site of an original application for 3 days,and the hormonal withdrawal reaction was exacerbated,such as desquamation and scratching behavior,and the symptoms improved after 0.05% CP was applied again.(4)In the model intervention group: contact dermatitis in SD rats induced by DNCB,after continuous topical application of 0.05% CP,original dermatitis disappeared,but continued to scratch frequently after continuous topical application of 0.05% CP daily, accompanied by skin scratches,showed hormonal withdrawal reaction after stopping the drug,improved after topical application of 0.05% CP,and repeated symptoms after withdrawal..4.3 indicators for SD rat gene m RNA transcription and protein expression: The PNCA colorimetric method was used to detect the proliferative activity of KC nuclei for each Cin group.The m RNA or protein expression of AQP3,FLG,Caspase-14,Per,MMP-9,Has2,TNF-α,NF-κB p65,IκBa for each subgroup was detected by q RT-PCR,Western blot and immunohistochemical techniques respectively.[Results]1.Animal model for both allergic contact dermatitis and corticosteroid addictive dermatitis were made successful.After initial sensitization with DNCB 7% liniment,Allergic contact dermatitis animal model was induced in SD rats when re-sensitize given at two weeks later with DNCB 0.1% liniment.And corticosteroid addctive dermatitis was induced with the CP 0.05% liniment twice daily to the SD rat with allgeric contact dermatitis.The least time to induce was 20 days with topical 0.05% CP liniment twice daily.2.Effect of topical Cin liniment on the improvement of anti-inflammatory and itching effects and skin status scores in dermatitis in SD rats.Topical application of 8% Cin liniment had anti-inflammatory and antipruritic effects;alleviated corticosteroid dermatitis-related symptoms in SD rats;delayed the time of CP-induced pruritus exacerbation.In the follow-up treatment of SD contact dermatitis,the skin condition score at 15 d was Cin<CP=CP+Cin<basal(control group),and those at 20 d,Cin<basal<CP=CP+Cin,the anti-inflammatory effect of Cin was better than others.3.Effect on Caspase-14 m RNA transcription and protein expression in various experiments groups:When compared with the control group,in NS group,both Cin,CP,Cin+CP significantly up-regulate Casepase-14 m RNA transcription by 56.5%,25%,31.6% respectively,but CP down-regulats the Casepase-14 protein expression by 36.2%(both P<0.05).In ACD group,CP down-regulated Casepase-14 m RNA transcription by 66.2% and protein expression by 42.7%;Cin down-regulated Casepase-14 protein expression by 46.3%;and combined with CP and Cin down-regulated Casepase-14 m RNA transcription by 86.6% and protein expression by 56%(both P<0.05).4、Effect on FLG m RNA transcription in various experiments groups:When compared with control subgroup in NS group,Cin up-regulats FLG m RNA transcription by 39.3% in NS group.But no significant effect for CP on FLG m RNA transcription.in ACD group,Cin up-regulate FLG m RNA transcription by 10%,when compared to control(both P>0.05);but both CP and CP combinated with Cin down-regulates FLG m RNA by 85.2% and 89.7%(both P<0.05).5.Effect on AQP3 m RNA and protein expression in various experiments groups:Compared to control subgroup in NS group,CP and Cin significantly up-regulate AQP3 protein expression by 54.5%,62.8% respectively and by 95.4% in subgroup of CP combined with Cin(all P < 0.05).In ACD group,CP and Cin significant up-regulate AQP3 protein expression by 51.2%,71.2% rexpectively(both P<0.05)and by 71.2% in subgroup of CP combined with Cin though they all down-regulate AQP3 m RNA transcription.6、The effect of each experimental group on MMP-9 m RNA and protein:Compared to control subgroup in NS group,CP significantly up-regulate AQP3 m RNA transcription by 35.6%(all P<0.05).In subgroup of subgroup of first CP evoked followed by the Cin intervention(NS-D)and CP combined with Cin(NS-E),MMP-9 protein expression were significantly up-regulated by 220.6%,87.4% respectively(both P<0.05).In addition,CP and Cin also up-regulated protein expression by 34.8%,43.8% respectively,but the difference was not significant(both p>0.05).In ACD group,CP significant down-regulate MMP-9 m RNA transcription by 13.2%(p<0.05),the subgroup of CP combined with Cin down-regulated MMP-9 m RNA transcription by 86.8% and protein expression by 13.2%(all P<0.05).In addition,Cin also up-regulated protein expression by 16.0%,but the difference was not significant(p>0.05).7、The effect of each experimental group on Periostin m RNACompared to control subgroup in NS group,the subgroup of CP combined with Cin similarly up-regulated transcription by 35.4%(p<0.05).While CP,Cin,Cin after CP,and Cin+CP all up-regulated Per protein expression by 60.3%,28.4%,49.8% and 35.0%,respectively(all p< 0.05).In ACD group,CP,CP combined with Cin groups all down-regulated Per RNA transcription by 72.1%,44.6% respectively,but CP,Cin,and CP+Cin all up-regulated Per protein expression by 24.3%,234.1% and 140.8%,respectively.(all P<0.05).8、The effect of each experimental group on HA m RNA and HAS2 protein:Compared to control subgroup in NS group,Cin up-regulated HA m RNA transcription by 86.3% and protein expression by 60.9%,and both CP,CP combined with Cin groups down-regulated HAS2 protein expression by 37.3%,22.0% respectively(all P<0.05).In subgroup of subgroup of first CP evoked followed by the Cin intervention(NS-D),HAS2 protein expression was significantly up-regulated by 130.2%(p<0.05).Compared to control subgroup in ACD group,CP and CP combined with Cin both significantly down-regulated HA m RNA transcription by 72.7%,44.6% respectively,and also down-regulated HAS2 protein expression by 91.0%,88.6% respectively(both p<0.05),Similarly,Cin down-regulated HAS2 protein expression by 83.2%(p<0.05).9.Effect of each experimental group on TNF-α,NF-κB p65,IκBa-related NF-κB pathway:(1)Early topical CP may exert anti-inflammatory effects by down-regulating TNF-α and up-regulating IκBa,but long-term topical CP may activate the NF-κB pathway and thus mediate the persistence of inflammation.(2)Cin may exert anti-inflammatory effects by downregulating TNF-α transcript expression and upregulating IκBa simultaneously.(3)CP may inhibit or mitigate CP-induced activation of NF-κB signaling pathways.[Conclusion]1.Based on the normal skin of SD rats and DNCB-induced contact dermatitis in SD rats,Corticosteroid Addictive Dermatitis was induced in SD rats by continuous topical application of 0.05% CP for 20 days.2.Cin promotes the expression of Cap-14,HAS2/HA in normal skin and can be used as a potential humectant in the skin.In addition,Cin can affect the expression of three skin barrier-associated proteins such as Casepase-14,AQP3 and Periostin and inflammatory substances such as TNF-ɑ,NF-κB and IκB in the skin ECM with Corticosteroid Addictive Dermatitis in different skin states in SD rats,and has anti-inflammatory and antipruritic effects by reducing scratching and reducing skin symptoms.3.The possible mechanism of anti-inflammatory and antipruritic effects of Cin is to exert anti-inflammatory effects by down-regulating TNF-α expression and regulating NF-Κb/IκBa signaling pathway.The detailed mechanism of its barrier protection needs further in-depth study. |
PDF Full Text Request