The Effect Of Ipl Exposure On Cultured Human Skin Fibroblast

Skin aging includes chronological aging and photoaging which wasinduced mainly through UV irradiation, especially UVA. The treatment forphotoaging went through ablative and non-ablative method. Since themore side effect of the former, the implication of it gradually declined. IPLtechnique is a new non-ablative method for treating photoaged skin inrecent years. Clinical studies have certified the effectives of IPLimproving many symptoms of photoaging skin. As a result, it has gotgeneral use. Although there were some reports about IPL mechanism,there were a few controversial results. Moreover, most of the previousstudies were performed in normal tissue and cultured cells. There is greatdifference between normal cell and senescence cell. In the aspect ofcytobiology, there is no report about the effect of IPL irradiation ontelomere length so far.In the present study, we evaluated the effects of single IPL treatmenton cell morphology, cell viability and expression of MMP-1; multiple IPL treatment on the cell senescenc markers and multiple IPL treatment onPUVA-induced senescence fibroblast by use of cultured cells and directlycompared with UVA irradiation effects aiming at a better understanding ofthe underlying molecular mechanism concerning photoaging and therejuvenation effects of IPL.Part 1: Cells were divided into seven groups: one group withoutirradiation as a control, three groups receiving IPL treatment with 5, 15and 20 J per cm~2, and the other three groups receiving UVA irradiationwith 9, 11 and 13 J per cm~2. 24 hours after UVA and IPL treatment, cellmorphology were determined using a phase contrast microscope, cellviability was monitored using MTT and the level of MMP-1 was measuredby ELISA. Our results revealed that fibroblast cells showed cell shrinkageand their shape were getting round upon increasing dose of UVAirradiation while the cell morphology of fibroblast has no obvious changesupon IPL treatment. MTT analysis showed a dose-dependent decrease incell viability after UVA irradiation was observed and the cell viability isabout 80% at 9 J per cm~2 of UVA irradiation. In contrast, an increase incell viability in a dose-independent manner was observed upon IPLtreatment and 15 J per cm~2 of IPL treatment, which is the therapeuticaldose in clinical IPL rejuvenation, gave the highest cell viability. The levelof MMP-1 increased upon UVA irradiation within 24 hours and theMMP-1 level reached the highest upon UVA irradiation at 9 J/cm~2.MMP-1 expression decreased upon IPL treatment, but only with IPL doseof 15J/cm~2 showed statistical significance of differences. Based on theabove results, Cells were further divided into four groups: that is controlgroup, UVA9 J per cm~2 irradiation group, IPL15J per cm~2 and UVA9+ IPL15J per cm~2 (fibroblast cells were first treated with UVA irradiation at9J/cm~2, following irradiated by IPL at 15 J/cm~2 ) . At 24 hours afterirradiation, the MMP-1 protein level was determined by ELISA.Interestingly, the increased MMP-1 expression by UVA irradiation wasdrived down after IPL exposure to the level of normal control within 24h.Part 2: Cells were divided into three groups: one group withoutirradiation as a control, one group receiving IPL treatment with 15 J percm~2, and the last group receiving UVA irradiation with 9 J per cm~2. IPLand UVA irradiation were performed once a day during five days. On thesixth day, the cells were collected. Senescence-associatedβ-galactosidasestaining, cell cycle, reactive oxygen species and telomere length weredetermined. Our results showed that five consecutive days of IPLirradiation had no effect on the activity of SA-β-gal and telomere lengthand decreased the G1% of cell cycle and the level of ROS in comparisonwith the control group(p<0.05). On the contrary, five consecutive days ofUVA irradiation increased the activity of SA-β-gal and the level of ROS,shortened the length of telomere and no obvious change in the G1% of cellcycle in comparison with the control group.Part 3: Cells were divided into three groups: one group withoutirradiation as a control, one group receiving PUVA treatment, and the lastgroup receiving PUVA+IPL irradiation(IPL irradiation were performedwith 15 J per cm~2 once a day during five days). Our results showed that incomparison with the control cells, cells irradiated with PUVA and PUVA +IPL showed a general elevation in SA-β-gal activity (p<0.05). The numberof SA-β-gal-positive fibroblasts in the PUVA + IPL group was clearlylower than that in the PUVA group (p<0.05). Cell viability showed a time-dependent decrease in both the PUVA and PUVA + IPL groups, incomparison with the viability in the control group, and there was nodifference in cell viability between the PUVA and PUVA + IPL groups.PUVA treatment shortened telomere length and increased the level of ROS,in comparison with the corresponding findings for the control cells(p<0.05), while irradiation with IPL after PUVA exposure preventedtelomere shortening and decreased the ROS level, in comparison withPUVA treatment only (p<0.05).Conclusions:(1) The regulations of cell morphology, cell viability and MMP-1expression differ in single exposure to UVA and IPL in cultured skinfibroblast. The data of combined treatment of UVA and IPL suggestedthat IPL may attenuate the UVA-induced up-regulation of MMP-1therefore decreasing the protein destruction in extracellular matrix.(2) Multiple UVA irradiations induce cell senescence including increasingstaining of senescence-associatedβ-galactosidase (SA-β-gal),telomere shortening and induction of reactive oxygen species (ROS).On the contrary, multiple IPL treatments could not induce cellsenescence.(3) PUVA treatment induced cell senescence in cultured fibroblasts, whileIPL exposure protected cells against PUVA-induced aging byreducing ROS and preventing telomere shortening.