5-aminolevulinic Acid-mediated Photodynamic Therapy(ALA-PDT)induces Oxidative Damage And Apoptosis In Ultraviolet B-induced Premature Senescent Human Skin Fibroblasts

BackgroundIn recent years,clinical proof shows that 5-aminolevulinic acid photodynamic therapy(ALA-PDT),has a rejuvenation effect,can be used for the treatment of skin photoaging.It can significantly improve the appearance of fine lines,dotted pigmentation and roughness of photoaged skin,causing dermal collagen deposition.When we use ALA photodynamic therapy on the treatment of skin photoaging,We usually locally packet photosensitizer ALA for a certain time,ALA is absorbed and converted into photosensitizer Protoporphyrin ?(Pp ?)in the organization,then the appropriate dose of red light(635 nm)is irradiated.Pp IX can absorb photon energy,converting from the ground state to excited,producing a large number of reactive oxygen species(ROS),which regulate cell lipid peroxidation response,inducing the cytotoxic effect directly or indirectly.It is generally believed that the mechanism of ALA-PDT treating skin photoaging may mainly through selective photo-thermal effect stimulating the production of new dermal collagen,or the local inflammation reaction,which induces skin wound repairing mechanism.However,there was no research reporting about the effects of ALA-PDT on photoaging fibroblasts at present.ObjectBy treating normal and(UVB Stress-induced premature senescence,UVB-SIPS)fibroblasts with ALA for different incubation times and red light exposure doses,we detected the intracellular ROS and mitochondrial membrane potential levels,proving that ALA-PDT could induce oxidative damages to human skin fibroblasts,and showd a concentration-response dependency relation ship with ALA incubation time and the density of red light.On the basis of the first part,we furtherobserved cell morphological changes,proliferation activity and apoptosis levels under different ALA incubation times and red light dose,preliminarily discussing the mechanisms of ALA-PDT treating skin photoaging,as well as providing experimental foundation and theoretical basis for the clinical parameter settings and the evaluation of effects and side effects of photorejunenation.Methods1.Cell separation and culture Human dermal fibroblasts was purchased from the First Affiliated Hospital of Nanjing Medical University.Cells were cultured with DMEM medium containing 10% fetal bovine serum.Take 5-10 substituting cells in logarithmic growth phase experiment.2.UVB-SIPS-HDFs model According to the experimental design time quantitative UVB radiation,selecting the appropriate concentration of mangiferin added to the culture to the point in time with a total dose of UVB 50 m J/cm~2(5 times 10 m J/cm~2�d UVB irradiation).3.Red light irradiation and dose calculation Cells were irradiated with 635 nm red light(50 m W/cm~2);light dose = power � time(s).4.Drug concentration and incubation time screening The laser confocal microscope detect Pp IX fluorescence intensity in cells.5.Intracellular ROS level and mitochondrial membrane potential change detection Fluorescence microscopy and flow cytometry detect intracellular ROS level and mitochondrial membrane potential change.6.Cell proliferation activity detection CCK-8 assay cell proliferation activity.7.Apoptosis detection Optical microscope observe cell morphology change,Hoechst staining and flow cytometry Annexin V/PI staining detect cell apoptosis.Result1.The Pp? fluorescence intensity reached the maximum after UVB-SIPS-HDFs incubated with 1.00mmol/L ALA for 6 hours Laser confocal microscope observed that: intracellular Pp fl? uorescence intensity reached the top after 1.00 mmol/L ALA incubated with cells for 6 hours(P <0.05).2.Changes in reactive oxygen species content and mitochondrial membrane potential of UVB-SIPS-HDFs after ALA-PDT Compared with the control group,intracellular ROS and MMP fluorescence intensity of ALA-PDT groups increased(P < 0.05),which were positively correlated with the light dose and ALA incubation time.Under the same condition,the intracellular ROS and MMP fluorescence intensity of normal HDFs were higher than UVB-SIPS,the difference was statistically significant(P < 0.05).3.Changes in proliferation activity and apoptosis rate of UVB-SIPS-HDFs after ALA-PDT Compared with the control group,the cell proliferation activity of ALA-PDT groups rose,with apoptosis rate declined,which were positively correlated with the light dose and ALA incubation time.Under the same condition,the cell proliferation activity declined and apoptosis rate rose dramatically in normal HDFs than in the UVB-SIPS groups,the difference was statistically significant(P < 0.05).ConclusionThis study preliminarily confirmed the oxidative damages of ALA-PDT on UVB-induced senescent hunman dermal fibroblasts.It can induce cell apoptosis and declined proliferation activity,which showed dose-concentration-dependence with ALA incubation time and red light dose.This may prompt the mechanism of ALA-PDT used for rejuvenation.