Curcumin-photodynamic Therapy Inhibits Cutibacterium Acnes Biofilm-induced Inflammatory Response In Keratinocytes Through Regulating Circ＿105040/miR-146a

Background and objectivesAcne is characterized by chronic inflammation affecting up to 85-100%in adolescence.Cutibacterium acnes（C.acnes）causes the acne inflammatory response,which is crucial to the pathological alterations of acne,by changing the composition of sebum and inducing a number of immunological responses.Studies have found that the biofilm state of C.acnes has stronger pathogenicity and resistance to antimicrobials than suspended bacteria.Circular RNAs（circRNAs）are a new type of endogenous noncoding RNAs（ncRNAs）with covalently looped structures,which are widely expressed in mammals.Recent studies have found that circRNA can act as competing endogenous RNA（ceRNA）to regulate gene expression through sponge microRNA（miRNA）.In our early research,we have confirmed that miR-146a inhibits C.acnes biofilm induced inflammatory reactions in human keratinocytes（KC）.However,it is unclear whether circRNAs effects miR-146a in the development of acne.Curcumin is a polyphenolic curcuminoid that has been proven to have antioxidant,anti-inflammatory,and antibacterial activities.Studies have found that curcumin combined with blue light,curcumin photodynamic therapy（curcumin-PDT）has good antiinflammatory effects recently.There has been no research on the effectiveness of curcumin-PDT in reducing acne inflammation.The goal of the current study is to investigate the role of circ₀105040 in acne inflammation progression,and to explore whether curcumin could control the C acnes biofilm-induced inflammatory response in keratinocytes,either alone or in combination with blue light photodynamic therapy.Materials and methods1.C.acnes was cultured under anaerobic conditions at 37℃ to build a biofilm.2.C.acnes biofilm and keratinocytes were co-culturing to build an acne inflammation model.3.RNA fluorescence in situ hybridization（FISH）was used to detect the expression level of circ₁05040 in acne lesions and normal tissues.Cytoplasmic nuclear separation assay was utilized to confirm the existence and expression pattern of circ₁05040.Luciferase reporter assay,biotin-labeled miRNA pull-down assay and RNA immunoprecipitation（RIP）were assessed to demonstrated that circ₁05040 and miR-146a could be combined directly.4.Quantitative reverse transcription PCR（qPCR）,western blot（WB）,Enzyme-linked immunosorbent assay（ELISA）and immunofluorescence（IF）were assessed to clarify the function and mechanism of circ₁05040 in acne.5.Following C.acnes biofilm stimulation,human primary keratinocytes were treated with 20μM curcumin solution alone or 5μM curcumin with combined blue light irradiation.The inflammatory cytokine expression was detected by qPCR and ELISA.Then the expression levels of Toll-like receptor 2（TLR2）and its downstream proteins were determined by WB.The nuclear factor translocation of nuclear factor kappa-B（NF-kB）was detected by IF.6.After overexpressing circ₁05040 in keratinocyte,C.acnes biofilm was added to induce inflammatory response.Then they were treated with curcumin alone or curcumin combined with blue light irradiation.qPCR and ELISA were assessed to detect the expression of inflammatory cytokines.Results1.According to FISH of tissue sectioning,we identified that circ₁05040 was significantly down regulated in acne lesions.Mechanistic studies reveal that circ₁05040 acts as ceRNA can sponge miR-146a.Functional analysis showed that circ₁05040 promoted keratinocyte inflammation through the activation of NF-κB and MAPK pathways.2.Treatment with 20 μM curcumin alone or 5 μM curcumin combined with blue light reduced the inflammatory response to C.acnes biofilms in keratinocytes by blocking the TLR2/MAPK/NF-κB pathway.3.The expression of inflammatory cytokines was significantly increased after overexpressed circ₁05040 in keratinocyte.Treatment with 20μM curcumin alone or 5μM curcumin combined with blue light irradiation could reduce this response.Conclusions1.Circ₁05040 promoted keratinocyte inflammation via a ceRNA mechanism and activated NF-KB and MAPK pathways.2.Curcumin alone,in sufficient concentrations,or low-concentration curcumin with blue light had anti-inflammatory activity on keratinocytes stimulated by C.acnes biofilms through inhibition of MAPK and NF-κB signaling pathways by downregulating TLR2 expression.3.Curcumin alone or curcumin combined with blue light inhibits C.acnes biofilm-induced inflammatory response in keratinocytes through regulating circ₁05040/miR-146a.