The Role Of Inflammatory Cytokine TNF-αregulating Connexin 43 In UVA-induced Photoaging Of Fibroblasts

ObjectiveUltraviolet radiation（UVR）could cause skin photoaging by inducing inflammatory response.The regulatory and inflammatory factors of skin immune system（SIS）play important roles in maintaining homeostasis and mediating photoaging in skin.However,the detailed mechanism is unclear.This study is based on the role and the mechanism of ultraviolet A（UVA）radiation-mediated photoaging of fibroblasts in skin,so as to provide experimental basis for the prevention and treatment of UVA-mediated photoaging of skin.As an important cell in the dermis of skin,fibroblasts play an important role in maintaining the normal physiological function of skin tissue.Studies have shown that connexin 43（Cx43）,a connexin protein expressed by fibroblasts in skin tissue,plays a key role in mediating intercellular communication and maintaining the normal physiological function of skin tissue.It could also be regulated by many types of inflammatory factors.UVA radiation could induce HDFs activation.On the other hand,UVA radiation could also induce inflammatory response in local skin tissues and cause skin photoaging.Moreover,the photoaging is closely related to a variety of skin diseases.However,the detailed mechanisms underlying in which UVA radiation induces skin photoaging through inflammatory responses remain unclear.In the early study,for the first time,we found that Cx43 in HDFs,as an important factor,could regulate matrix metalloproteinases（MMPs）,an important marker of skin photoaging.Based on the above results,we hypothesized that UVA may induce the local immune response of skin tissue and release inflammatory cytokines,and then affect the MMPs level of skin by affecting the expression and function of Cx43,ultimately resulting in skin photoaging.A variety of cellular and molecular biological methods were used in this study to explore the potential mechanism of skin photoaging induced by UVA-caused inflammation,which may provide a new theoretical basis for the development of anti-skin aging and skin tumor drugs.MethodsCCK-8 assay was used for screening the optimum conditions of UVA irradiation for HDFs.The levels of MMP-1,MMP-9 and TNF-αin supernatant were detected by ELISA,Cx43 m RNA and protein expressions were detected by q RT-PCR and Western Blot.HDFs was stimulated with recombinant human TNF-α,and the expressions of Cx43,MMP-1 and MMP-9 in cells were detected by Western Blot.HDFs were divided into blank group,negative control group,si RNA-1 group and si RNA-2 group.si RNAs for each group were transiently transfected into HDFs,respectively,and the levels of MMP-1 and MMP-9 in supernatant were detected.Cells were divided into control group,UVA group,UVA+TNF receptor 1 neutralizing antibody group and TNF receptor 1neutralizing antibody group.The expression of Cx43 m RNA and the levels of MMP-1and MMP-9 in the supernatant of the cells were detected by q RT-PCR and ELISA,respectively.Results1.5 J/cm~2 UVA irradiation increased the level of MMP-1 and MMP-9 in HDFs in a time-dependent manner.2.UVA irradiation significantly elevated the production of TNF-αand reduce the expression of Cx43 in HDFs.3.Gene silence of Cx43 significantly promoted HDFs to produce more MMP-1 and MMP-9.4.Inflammatory cytokine TNF-αsignificantly inhibited the expression of Cx43 and increased the levels of MMP-1 and MMP-9 in HDFs.5.Blocking TNFR1 of HDFs with an anti-TNFR1 antibody significantly attenuated the changes on the levels of Cx43 m RNA expression and MMP-1 level which were affected by UVA irradiation.Conclusion1.UVA may induce photoaging of HDFs by promoting the production of TNF-αand reducing Cx43 expression,thereby promoting the secretion of MMP-1 and MMP-9.2.Blocking TNFR1 on HDFs surface can significantly improve the UVA-induced inhibition of Cx43 and elevation of MMP-1 release.