Investigation on the Intracellular Sognal Transduction Mechanism of Human Dermal Fibroblasts of Photorejuvenation in Photodynamic Therapy

Abstract

The clinical studies have reported over years that the photodynamic therapy (PDT) has effects on the rejuvenation of photoaged skin. However, there have been no studies on the intracellular mechanism of photorejuvenation following PDT. The purpose of this study was to investigate the effects of PDT on the signal transduction mechanism in human dermal fibroblast and the role of reactive oxygen species (ROS) in vitro. Normal human fibroblasts were treated with various sublethal and relatively low doses of 5-aminolevulinic acid (ALA) and red light. The inhibition of fibroblast proliferation was not observed at the ALA concentration of 0.1mM, ALA incubation time of 30 minutes, and 3 J/cm2 of red light energy. In this condition, I found that a small quantity of ROS that continues to be produced after 8 hours of PDT was processed with 2,7-dichlororofluorescein diacetate (DCFDA) and visualized with fluorescence microscopy and confocal microscopy. Moreover, extracellular-signal-regulated kinase (ERK) signal, among the mitogen-activated protein (MAP) kinases which is known to be associated with cell proliferation, differentiation and survival, also remained to be activated after 8 hours of PDT in the same PDT condition, like ROS, and this was confirmed through western blotting and immunohistochemical stains. The expression of transforming growth factor (TGF)-β mRNA by semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was increased at 6 hours of PDT compared to that in other treatments. Matrix metalloproteinase (MMP)-1 and MMP-3 mRNA by real-time PCR significantly increased in PDT group compared with control group showing a maximal increase 24 h following PDT. TGF-β mRNA increased in PDT group at 6, 12 h post-PDT. Collagen Iα1 mRNA levels also increased after PDT in a time-dependent manner. MMP-3 and collagen Iα1 protein levels increased after PDT at 24, 48 h compared with control group. Results of this study indicate that a small quantity of ROS after PDT in dermal fibroblasts may be a critical aspect of the prolonged activation of ERK signaling that may lead to fibroblast proliferation and activation. This study provides valuable evidence of the photorejuvenation effect of PDT in vivo.