Effects Of Genistein On Puva-induced Photoaging In Human Derma Fibroblasts

Objective: To investigate the photoprotective effects of genistein on8-methoxypsoralen plus ultraviolet-A irradiation(PUVA)-induced cell agingin human dermal fibroblasts in vitro, and to explore the underlyingmechanisms.Methods: Primary human dermal fibroblasts(HDFs) were isolated andcultured by trypsin-collagenaseⅠ, and identified by vimentinimmunofluorescence staining. Cells were assigned into three groups: thecontrol group, the PUVA group and the genistein group. We established astress-induced premature senescence model by8-methoxypsoralen plusultraviolet-A irradiation(PUVA). Cells proliferation or viability and thecell growth curve were detected by MTT assay. The cell morphology wasobserved under the invert microscope or stained by Wright-Gimsa.Senescence cells were stained by senescence-associated-β-galactosidase(SA-β-Gal) kit. Flow cytometry was performed to analyze the cell cycleand intracellular reactive oxygen species(ROS) levels. Matrixmetalloproteinases (MMPs) and estrogen receptor β (ER-β) mRNAexpression were detected by real-time fluorescent quantitative polymerase chain reaction (PCR).Results: The cells were isolated and cultured successfully，and99%were dermal fibroblasts. In a suitable range of concentration, genisteinsignificantly promoted proliferation of fibroblasts with a dose-dependentmanner.24h after UVA irradiation, The percentage of postmitoticfibroblasts in the PUVA group accounted for (99.7±0.58)%, while (7.7±1.15)%in the control group,(57.7±3.51)%in the genistein group (P＜0.01). According to the growth curve, the cells in the control group was atypical type-S curve, while cells in the genistein group did not reach thestagnate phase, but in the PUVA group, cells were not proliferating at all,and displayed as a horizontal line. The expression of senescence-associatedβ galactosidase in the control group、the PUVA group and the genisteingroup were respectively (0.67±0.58)%、(96.67±1.53)%、(51.67±2.08)%,and the results are statistically significant(P＜0.01). Phase of cell cycleanalysis by flow cytometer (FCM) showed that cells in the PUVA groupwas stalled in S phase, while in the genistein group, cells get though to theG2/M phase. The fibroblasts in the PUVA group displayed an increasingexpression of MMPs mRNA and ROS，while genistein shows a protectionin varying degrees, and the datas were statistically significant(P＜0.05).The estrogen receptor β (ER-β) mRNA expression in the genistein groupwas much higher than the control group and the PUVA group(P＜0.01).Conclusions: The proper concentration of genistein could protect 8-methoxypsoralen plus ultraviolet-A irradiation(PUVA)-induced cell agingin HDFs in vitro in some extent. Genistein could protect8-methoxypsoralen plus ultraviolet-A irradiation(PUVA)-induced cell agingin human dermal fibroblasts in vitro, and may be related to restoring thecell growth inhibition, down-regulating expression of MMPs, suppressingoxidative stress, and up-regulating expression of ER-β.