Study On The Role And Mechanism Of Fluorofenidone In UVA Induced Photoaging

OBJECTIVEThe aim of this study was to investigate the protection effect of Fluorofenidon(AKF-PD) on UVA induced human skin fibroblasts (HSF) senescence and mice skin photoaging,and preliminarily discussed the underlying mechanism of this action.METHODS1. We use MTT assay to evaluate the effect of UVA-induced proliferative inhibition influenced by AKF-PD in HSF.2. Select different concentration of AKF-FD(0,50,100,150μM) treat HSF after UVA (10J/cm2*3d).After24hours of the last irradiation,We use β-galactosidase staining Kit detect the activity of aging-related β-galactosidase,western blot detect the expression of sirtl,phosphorylated mTOR and aging related p16.What’s more, use qPCR dectect the level of sirtl mRNA.3. After each irradition,we pretreated with mTOR inhibitor rapamycin(RP)(50nM),and added AKF-PD one hour later.After24hours from the last irradiation, dectect the expression level of sirt1mRNA with RT-PCR and the protein expression with western blot. 4. Animal experiment:Select Fvb female mice born8weeks and with removal of back skin’s hair.Then measure the minimum erythermal dose(MED).With the mice irradiated three times a week and the irradiation doses increasing once every two weeks, irradiation doses from1MED up to15J/cm2starting remain unchanged until12weeks later. No process with the control group of mice,while the experimental group and the vehicle gel group of mice are swabbed the local skin of mice with2%AKF-PD gel or vehicle every day.After12weeks,we analyzed the wrinkle formation of mice dorse skin,and taken skin tissue for HE staining,observe the changes of epidermal thickness.RESULTS1. AKF-PD dose-dependently reversed the proliferative inhibition of HSF, β-galactosidase staining rate (p<0.05)and the increase of P16expression,which are induced by UVA.2. AKF-PD can inhibit the decline of sirt1expression and increase of phosphorylated mTOR level in HSF,which induced by HSF. This trend is also dose-independent(p<0.05).3. RP,pretreating HSF after UVA irradiation, can promote the transcription and protein level of sirtl more obviously than AKF-PD’s role in HSF irradiated by UVA(p<0.05).4. The dorsal skin of mice irradiated by UVA forms more obvious wrinkles and meanwhile the skin thickness increase.If mice are put on 2%AKF-PD gel,however,the photoaging phenotype will be significantly improved(p<0.05).CONCLUSION1. AKF-PD can increase the level of sirtl by inhibiting mTOR, thereby delaying the formation of senescence in human dermal fibroblasts.2.Mice skin photoaging Phenotype can be significantly improved by2%AKF-PD gel.