Mechanism Of 5-aminolevulinic Acid Photodynamic Therapy Inhibit Keratinocyte Differentiation And Proliferation

Backgroud:The pathogenesis of acne is generally considered abnormal keratosis follicular ducts,sebaceous gland hyperactivity,propionibacterium and inflammation reactions.In recent years,a growing number of studies confirm that 5-aminolevulinic acid photodynamic therapy(ALA-PDT)is effective in treating acne.Although the efficacy of the treatment of acne has been recognized,but its mechanism of action is still not entirely clear,it may also act on the keratinocytes differentiation and proliferation.Objective:Based on the classic animal acne model of rabbit ear,we demonstrate the inhibition of ALA-PDT for follicular duct hyperkeratosis in vitro.To build the hyperkeratosis and proliferation model,HaCaT was induced by the fibroblast growth factor-10(FGF-10).To investigate the proliferation level of HaCaT and the K1,K6 K16,PKC,IL-1α expression after ALA-PDT.In this study,we detected that ALA-PDT is involved in the inhibition of differentiation and proliferation of keratinocytes through PKC pathway.Methods:1.Experiments in vivo1.1 Female rabitts were divided into following four groups.Five rabitts in the first group did not receive any treatment and served as a control.Five rabitts in the second group received a topical treatment of oleic acid on their ear skin.Five rabitts in the third group received ALA-PDT treatment.The rabitts in the fourth group received ALA-PDT followed by oleic acid.1.2 To investigate the size of comedo,dermascopy was performed.1.3 After treatment with polyester wax,the skin samples were sliced into 6-mm thicknesses.The sliced sections were treated with haematoxylin and eosin(H&E).1.4 To investigate the levels of proteins(K1,K6,K16),Immunohistochemical analysis was performed.2.Experiments in vivo2.1 HaCaT were obtained from circumcisions in accordance with the ethical committee approval process of Jiangsu Provincial People’s Hospital,Nanjing,Jiangsu,China.Specimens were sterilized in 70%ethanol,minced,and incubated in Dulbecco’s modified Eagle medium(DMEM)supplemented with 10%fetal bovine serum and 1%penicillin-streptomycin in an atmosphere of 5%CO2 at37℃.2.22.2.1 HaCaT were divided into following four groups:control,FGF-10,ALA-PDT,FGF-10+ALA-PDT.2.2.2 HaCaT were divided into following five groups:control,FGF-10,FGF-10+ALA-PDT,FGF-10+ALA-PDT+PKCa,FGF-10+ALA-PDT+PKCi.2.3 Cell proliferation was assayed using a CCK-8 Kit.2.4 To investigate the level of differential and proliferation-related proteins(K1,K6and K16 proteins),Western Blotting and immunofluorescences analysis was performed.2.5 To investigate the levels of IL-1α expression,RT-PCR and ELISA analysis was performed.2.6 To investigate the levels of PKC pathway-related protein(PKC,K1,K6 and K16),Western Blotting and immunofluorescences analysis was performed.Results:1.Experiments in vivo1.1 ALA-PDT suppresses OA-induced comedo enlargement in rabbit ear Because comedo enlargement is a major biomarker of acne,we evaluated the effect of ALA-PDT on OA-induced comedo enlargement.In a quantitative analysis,dermascopy demonstrated that OA induced increase in comedo(p<0.05 vs.the control group;n=5).ALA-PDT decreased the amount of OA-induced comedo enlargement respectively(p<0.05 vs.the OA group;n=5)1.2 ALA-PDT suppresses OA-induced hair follicle duct epithelial hyperkeratosis in rabbit ear Hematoxylin and eosin staining demonstrated that OA induced aggravate in hair follicle duct epithelial hyperkeratosis.ALA-PDT alleviate the OA-induced hair follicle duct epithelial hyperkeratosis respectively.1.3 ALA-PDT prevented OA-induced the expression of K1,K6 and K16 in rabbit ear Immunohistochemistry revealed that OA induced amplification of K1,K6 and K16 expression throughout the skin,especially in epidermis.OA+ALA-PDT-treated skin decrease the K1,K6 and K16 staining in skin,compared with OA-pretreated skin.3.Experiments in vivo3.1 ALA-PDT protects HaCaT against FGF-10 induced cell proliferation Compared with the control group,the cell viability of the FGF-10 group was significantly increased(P<0.05).FGF-10+ALA-PDT-treated HaCaT decreased the K1,K6 and K16 expressions,compared with FGF-10-pretreated skin.3.2 ALA-PDT decreased the level of K1,K16,and K16 proteins in the FGF-10 induced HaCaTThe levels of K1,K16,and K16 proteins in the FGF-10 group showed increase respectively,compared to the control group.The levels of K1,K16,and K16 proteins in the FGF-10 groups treated with ALA-PDT decreased significantly,compared to those of the FGF-10 group.3.3 ALA-PDT decreased the level of IL-1α expressions in the FGF-10 induced HaCaTThe levels of IL-1α expression in the FGF-10 group showed increase respectively,compared to the control group.The levels of IL-1α expression in the FGF-10 groups treated with ALA-PDT decreased significantly,compared to those of the FGF-10 group.3.4 ALA-PDT decreased the level of PKC pathway in the FGF-10 induced HaCaTCompared with the FGF+ALA-PDT group,the cell viability of the FGF+ALA-PDT+PKCa group was significantly increased(P<0.05).FGF+ALA-PDT+PKCi-treated HaCaT decreased the K1,K6 and K16 expressions,compared with FGF-10-pretreated skin.The levels of K1,K16,K16 and PKC proteins in the FGF+ALA-PDT+PKCa group showed increase respectively,compared to the FGF+ALA-PDT group.The levels of K1,K16,K16 and PKC proteins in the FGF-10 groups treated with ALA-PDT decreased significantly,compared to those of the FGF+ALA-PDT group.The levels of IL-1α expression in the FGF+ALA-PDT+PKCa group showed increase respectively,compared to the FGF+ALA-PDT group.The levels of IL-1α expression in the FGF+ALA-PDT+PKCi groups decreased significantly,compared to those of the FGF+ALA-PDT group.Conclusion:In summary,We observed that ALA-PDT significantly antagonizes differentiation and proliferation in vivo and in vitro,indicating the potential of ALA-PDT application for anti-acne treatment.