Study On The Mechanism Of TNFAIP3 And Occludin Participating In UVB Mediated Inflammation And Skin Barrier Damage In Chronic Actinic Dermatitis

[Introduction]Chronic actinic dermatitis(CAD)is a common group of chronic photosensitive inflammatory disease mediated by immunity,which appear chronic dermatitis changes in exposed and non exposed areas.The disease mainly occurs in middle-aged and old men,especially in plateau areas with long sunshine and strong ultraviolet(UV).The pathogenesis of CAD is complex,which is mainly related to delayed type hypersensitivity induced by immune dysfunction,oxidative stress and abnormal apoptosis of keratinocyte(KC),but there is no conclusive evidence.For a long time,UV has been considered to be involved in many aspects of the pathogenesis of CAD.It has been reported that UVB radiation with a dose greater than 2 times the minimum erythema dose(MED)can cause significant inflammatory infiltration in CAD.It is also believed that UVB can induce the change of cytokines to produce immunosuppressive effect by acting on KC,Langerhans cell(LC),T cell and other immune cells,thus causing KC damage and inflammatory reaction.As an important part of skin immune cells,KC is closely related to the inflammation of CAD,but the mechanism of KC inflammation induced by UVB is still unclear.In recent years,increased transdermal water loss(TEWL)has been observed in CAD patients,confirming the presence of impaired skin permeability barrier in CAD.The stratum corneum(SC)and tight connection(TJ)are the main structures of the function of the epidermis permeability barrier,which play an important role in maintaining the normal function of the epidermis permeability barrier(EPB).In the process of irreversible differentiation of keratinocytes into terminal cells,Keratin expression was changed,K,and K14 were down-regulated,K1 and K10 were up-regulated.Filaggrin,Loricrin,Involucrin and other proteins are assembled to form the keratinized sheath(CE),which is then sealed in a "brick wall" structure and bound with lipids to form the structural basis of EPB.The synthesis of intercellular lipids is closely related to the lamellar bodies in the granular layer,under the action of Serine Palmitoyl Transferase(SPT),Fatty Acid synthesis Acid synthase(FASN),trihydroxytrimethylglutaryl-coa(HMGCOA)and so on,the lamellar bodies are rich in lipid synthesis and hydrolase,they can synthesize lipids.TJ is composed of different types of transmembrane proteins and cytoplasmic proteins.The major transmembrane proteins are Occludin,Claudins and JAM,and the major cytoplasmic proteins are the band protein ZO-1 and so on.It is reported that the damage of EPB may be related to the alteration of intercellular lipids,CE,tight junction associated protein and abnormal expression of Keratin.However,the specific molecular mechanism of EPB damage in CAD has not been reported,and the correlation between inflammatory reaction and skin barrier remains to be studied.Based on the results of high-throughput sequencing,we screened the mRNA related to related to inflammation and skin permeability barrier in CAD,in order to explore mechanism of target gene on inflammatory response and skin barrier damage in CAD.Our previous research analyzed the results of mRNA expression profiles of CAD tissues,mainly from the two aspects of influencing CAD inflammation and epidermal permeability barrier,and screened the mRNA related to the differential expression,among them,TNFAIP3 was the most differentially expressed mRNA associated with inflammation,and Occludin was the most differentially expressed mRNA associated with permeability barrier.TNF inducible protein 3(TNFAIP 3),also known as A20.As a regulatory factor,A20 can be used to control NF-?B signal transduction.When activated,NF-?B can induce its expression rapidly.A20 controls its expression through a negative feedback loop and produces inflammatory mediators.Occludin is an important part of TJ and is mainly influenced by protein kinase C(PKC).PKC is a phospholipid-dependent protein kinase that regulates cell metabolism by catalyzing the phosphorylation of various proteins.Previous studies have shown that the abnormal expression of Occludin leads to the increase of cell TEER and affects the function of cell paracellular osmotic barrier.Therefore,on the basis of the results of whole transcriptome sequencing in CAD,the target molecules A20 and Occludin,which were differentially expressed in CAD,were found from two aspects of CAD inflammation and impaired skin permeability barrier function.This paper focuses on studing the role of A20 and Occludin in the regulation of CAD inflammation and permeability barrier function,and discusses the specific mechanism of A20 and Occludin in UVB-mediated CAD inflammation and skin barrier function damage,it will be helpful to understand the pathogenesis of CAD.[Objective]:To elucidate the role of A20 in UVB mediated inflammatory response in the pathogenesis of CAD and the mechanism of Occludin induced skin barrier damage and inflammatory response.[Methods]:Part 1:RNA sequencing(RNA seq)was used to screen the differentially expressed mRNA in the lesions of CAD,and the most obvious mRNA related to inflammation was screened;Immunohistochemistry(IHC)was used to detect evaluate the expression of A20 in skin lesions of the exposed parts of CAD patients and the skin of the exposed parts of healthy controls skin tissue;qRT-PCR was used to detect the expression of proteins related to skin permeability barrier in CAD(CLDN1,CLDN5,CLDN7,ZO-1,filaggrin,loricrin,involucrin and HMGCR,FAS and SPT).HaCaT cells were irradiated by UVB(10-40mJ/cm2,0-72h).CCK-8 and flow cytometry were used to detect cell viability and apoptosis level.qRT-PCR and ELISA were used to detect the expression of inflammatory factors induced by UVB.Western blot was used to detect the effect of UVB on the expression of A20.Then,the strategy of lentivirus stable knockdown or over-expression of A20 was used to transfect HaCaT cells to study the function and mechanism of A20 participating in UVB mediated inflammatory response.The level of inflammatory cytokines was detected by ELISA after A20 knockdown or over expression.The effect of A20 on the transmembrane electrical resistance(TEER/TER)was measured by cell transmembrane resistance meter;Western blot was used to detect the expression of NF-?B and MAPK related proteins(including NF-?B p-p65,p-I?Ba,MAPK-JNK,ERK,P-38)and the proteins related to skin permeability barrier and lipid key synthetase after A20 knockdown or overexpression;Cell immunofluorescence was used to detect the entry of NF-?B p65 into the nucleus after A20 knockdown or overexpression;Western blot was used to detect the expression of NF-?B p65.Part 2:RNA sequencing(RNA seq)was used to screen the differentially expressed mRNA in the lesions of CAD,and the most obvious mRNA related to skin permeability barrier was screened;Immunohistochemistry(IHC)was used to detect evaluate the expression of Occludin in skin lesions of the exposed parts of CAD patients and the skin of the exposed parts of healthy controls skin tissue;Western blot was used to detect the effect of UVB on the expression of Occludin and PKC.Cell transmembrane resistance was used to measure the effect of UVB on TER.Then,the mechanism of Occludin induced skin barrier damage and inflammatory response was studied by the strategy of the synthetic siOcludin and Occludin over expression plasmids transfection into HaCaT cells.The level of inflammatory cytokines was detected by ELISA after Occludin knockdown or over expression.The effect of Occludin on the TER was measured by cell transmembrane resistance meter;EdU was used to detect the proliferation of cells after Occludin knockdown.After the HaCaT cells were treated with siOcludin and PKCi,the secretion of IL-6 and TER were detected by ELISA and cell transmembrane resistance.[Results]:Part 1:A20 was the most significant mRNA related to inflammation in CAD(P<0.05).The results of immunohistochemistry showed that the expression of A20 in the skin lesions of CAD was significantly lower than that of the normal facial control skin(P<0.05),and the expression trend was identical with that of RNA-seq.In vitro,compared with the control group,the expression of A20 in HaCaT cells after UVB irradiation were decreased significantly(P<0.05).After UVB irradiation,the release of IL-1?,IL-6 and TNF-? were increased(P<0.01,P<0.01,P<0.05),and the secretion of IL-10 was decreased(P<0.05);After knocking down A20,the secretion of IL-1?,IL-6 and TNF-? were decreased(P<0.05,P<0.05).The expression of NF-?B p-p65 and p-I?Ba were increased after A20 knockdown(P<0.05,P<0.05),and NF-?B p-p65 was increased in the nucleus.After NF-kappaB p65 inhibitor(BAY11-7082)and A20 upregulation,the inflammatory level of IL-1? in HaCaT cells was partially restored(P<0.01,P<0.05);After interference or over-expression of A20,the expression of Occludin,CLDN1,CLDN5,cldn7,ZO-1,filaggrin,loricrin,involucrin,HMGCR,FAS,SPT,JNK,ERK and P-38 had no significant change;After interference or over-expression of A20,the expression of TER in cells had also no significant change.Part 2:Occludin was the most significant mRNA related to skin barrier(P<0.05).The results of immunohistochemistry showed that the expression of Occludin in the skin lesions of CAD was significantly lower than that of the normal facial control skin(P<0.05),and the expression trend was identical with that of RNA-seq.In vitro,compared with the control group,the expression of Occludin in HaCaT cells after UVB irradiation were decreased significantly(P<0.01,),and the expression of PKC was increased(P<0.05).The level of IL-6 was increased after occludin knockdown(P<0.05),but there was no significant change in IL-1?,TNF-?,IL-10(P>0.05,P=0.73;P>0.05,P=0.92;P>0.05,P=1.13).After Occludin knockdown,TER was decreased;IL-6 level was increased;K1 expression was increased,K5 expression was decreased;HaCaT cell proliferation was promoted(P<0.01,P<0.05,P<0.01,P<0.05,P<0.05);PKCi could reverse Occludin expression down-regulated by UVB(P<0.05),PKCi could partially recover TER decreased by UVB(P<0.05),PKCi could also increase IL-6 secretion level induced by Occludin down-regulation(P<0.01).[Conclusion]:1.The expression of A20 was down-regulated in CAD;2.A20down-regulation contributes to the UVB-mediated anti-inflammatory role via negative regulating the NF-?B signal pathway,which increased the release of IL-1?,IL-6 and TNF-?,and decreased the secretion of IL-10;3.There was no significant correlation between A20 and skin permeability barrier.4.The expression of Occludin was down-regulated in CAD;5.siOccludin could decrease the level of IL-6 and promote the inflammatory response and proliferation of HaCaT cells6.UVB contributes to the permeability barrier damage,pro-inflammatory and pro-proliferated role via regulating PKC-dependent downregulation of Occludin pathway,which decrease its TER,damage the permeability barrier,increase the release of IL-6,and promote the inflammatory response.