Low level laser irradiation stimulates mitochondrial membrane potential and disperses subnuclear promyelocytic leukemia protein.
OBJECTIVE
Low level laser irradiation (LLLI) is used to promote wound healing. Molecularly it is known to stimulate mitochondrial membrane potential (MMP), cytokine secretion, and cell proliferation. This study was designed to determine the influence of LLLI on the kinetics of MMP stimulation and decay, specific cytokine gene expression, and subcellular localization of promyelocytic leukemia (PML) protein on HaCaT human keratinocytes.


METHODS
The cells were irradiated by a 780 nm titanium-sapphire (Ti-Sa) laser with 2 J/cm(2) energy density. MMP was monitored with Mitotracker, a mitochondrial voltage-sensitive fluorescent dye. Cytokine gene expression was carried out using semi-quantitative-reverse transcription polymerase chain reaction. Subcellular localization of PML protein, a cell-cycle checkpoint protein, was determined using immunofluorescent staining.


RESULTS
The fluorescence intensity of MMP was increased immediately after the end of LLLI by 148 +/- 6% over control (P<0.001). Subsequently it decayed, reaching 51 +/- 14% of the control level (P < 0.01) within 200 minutes. This decay was characterized by an exponential curve (R = 0.96) with a lifetime of 79 +/- 36 minutes (P < 0.05). Following irradiation, the expression of interleukin-1alpha, interleukin-6, and keratinocyte growth factor (KGF) genes were transiently upregulated; but the expression of the proinflammatory gene interleukin-1beta, was suppressed. The subnuclear distribution of PML was altered from discrete domains to its dispersed form within less than 1 hour after LLLI.


CONCLUSIONS
These changes reflect a biostimulative boost that causes a shift of the cell from a quiescent to an activated stage in the cell cycle heralding proliferation and suppression of inflammation. Further characterization of MMP kinetics may provide a quantitative basis for assessment of the effect of LLLI in the clinical setting.
Gavish
Lilach
L
Department of Molecular Virology, The Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, Israel. lilach_g@md.huji.ac.il
Asher
Yael
Y
Becker
Yechiel
Y
Kleinman
Yosef
Y
D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D015603:Keratinocytes; D028022:Low-Level Light Therapy; D008564:Membrane Potentials / Q000528:radiation effects*; D008928:Mitochondria / Q000528:radiation effects*; D009363:Neoplasm Proteins / Q000528:radiation effects*; D009687:Nuclear Proteins / Q000528:radiation effects*; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors / Q000528:radiation effects*; D025521:Tumor Suppressor Proteins
D016207:Cytokines; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C070576:PML protein, human