title: ALA-PDT elicits oxidative damage and apoptosis in UVB-induced premature senescence of human skin fibroblasts.
abstract: BACKGROUND
  5-Aminolevulinic acid photodynamic therapy (ALA-PDT) has been used for the treatment of skin photoaging. It can significantly improve the appearance of fine lines, dotted pigmentation, and roughness of photoaged skin. However, the mechanisms by which ALA-PDT yields rejuvenating effects on photoaged skin have not been well elucidated. Thus, in this study we explored the effects of ALA-PDT in photoaged fibroblasts.
  
  
  METHODS
  We established a stress-induced premature senescence (SIPS) model by repeated exposures of human dermal fibroblasts (HDFs) to ultraviolet B (UVB) irradiation. Cells were irradiated by red light laser at 635nm wavelength (50mW/cm(2)). Intracellular protoporphyrin IX (PpIX) was detected by confocal microscopy. Intracellular reactive oxygen species (ROS) level and mitochondrial membrane potential (MMP) change were detected by fluorescence microscopy and flow cytometry. Morphological changes were observed by optical microscopy. Proliferative activity was measured by a cell counting kit-8 (CCK-8). Cell apoptosis was detected by fluorescence microscopy using Hoechst staining and flow cytometry using annexin V/propidium Iodide double staining.
  
  
  RESULTS
  Intracellular PpIX fluorescence in UVB-induced premature senescent HDFs (UVB-SIPS-HDFs) reached the highest intensity after incubation with 1.00mmol/L ALA for 6h (P<0.05). Compared with control group, intracellular ROS level, MMP, and apoptotic rate were increased (P<0.05) and proliferative activity was decreased (P<0.05) in UVB-SIPS-HDFs treated with ALA-PDT, which were positively correlated to ALA incubation time and red light laser dose.
  
  
  CONCLUSIONS
  Our study demonstrated that ALA-PDT elicits oxidative damage and apoptosis in photoaged fibroblasts in vitro, which may be the basis for the rejuvenating effects on photoaged skin.
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China. Electronic address: bingrong.2002@163.com.
authors.forename: Bing-Rong
authors.initials: BR
authors.lastname: Zhou
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
authors.forename: Li-Chao
authors.initials: LC
authors.lastname: Zhang
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
authors.forename: Felicia
authors.initials: F
authors.lastname: Permatasari
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China.
authors.forename: Yang
authors.initials: Y
authors.lastname: Xu
authors.affiliation: Department of Dermatology, The First Affiliated Hospital of Nanjing Medical University, 300 Guangzhou Road, Nanjing 210029, China. Electronic address: daniluo2013@njmu.edu.cn.
authors.forename: Dan
authors.initials: D
authors.lastname: Luo
mesh_terms: D000622:Aminolevulinic Acid / Q000494:pharmacology / Q000627:therapeutic use; D017209:Apoptosis; D016922:Cellular Senescence / Q000528:radiation effects; D005347:Fibroblasts / Q000187:drug effects / Q000528:radiation effects*; D005434:Flow Cytometry; D006801:Humans; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010778:Photochemotherapy; D017382:Reactive Oxygen Species; D012867:Skin / Q000528:radiation effects*; D014466:Ultraviolet Rays
chemical_list: D017382:Reactive Oxygen Species; D000622:Aminolevulinic Acid
keywords: ALA; Cell apoptosis; Human dermal fibroblasts; Oxidative stress; Photodynamic therapy; Premature senescence