The Protective Effect Of Stilbene Glucoside Against UVB-induced Photoaging On Human Skin Fibroblasts

ObjectiveEstablish vitro photoaging models by using cultured human skin fibroblasts (HSF), repeated exposures to ultraviolet B (UVB) irradiation at a subcytotoxic level. And to clarify 2,3,5,4’-tetrahydroxystilbene-2-O-beta-D-glucoside (TSG) weather protect HSF from stress-induced premature senescence (SIPS) induced by UVB.Methods1. Establish stress-induced premature senescence model of human skin fibroblasts induced by UVBHuman skin fibroblasts (HSF) were repeatedly exposed to UVB at a subcytotoxic level. Cell morphology was observed under inverted microscope, cell viability was calculated by trypan blue staining, the senescence-associated bgalactosidase (Sa-β-gal) expression was detected by histochemistry staining. Matrix metalloproteinases-1(MMP-1)in cultured supernatant was detected by enzyme-linked immunosorbent assay (ELISA), the content of malondialdehyde(MDA)and the activity of superoxidedismutase (SOD) in Cell lysates were detected by TBA and WST-1 method. To determine the best UVB irradiation dose for constrcuting stress-induced premature senescence model of human skin fibroblasts.2. Effects of TSG on human skin fibroblasts of SIPSThe HSF were divided into six groups, i.e. blank control group receiving no treatment, UVB-SIPS group receiving UVB irradiation only, three combination group receiving UVB irradiation and post-irradiation treatment with TSG at 0.02、0.1 and 0.5mmol/L respectively, and TSG group treated with TSG 0.5mmol/L only. Irradiation was performed twice a day for three consecutive days, and TSG treatment was given immediately after each irradiation.cell counting kit-8(CCK-8)was used to evaluate the proliferative activity of cells, and β-galactosidase (SA-P-Gal)staining to estimate the degree of premature senescence in cells, ELISA to quantify the secretion level of MMP-1 in cultured supernatant, Intracellular MDA and SOD activity were detected by Enzymatic chemical method.One-way analysis of variance was used for statistical analysis.Results1. As the cumulative dose of UVB reached 60mJ/cm2, the secretion level of MMP-1 in cultured supernatant increased dramatically and strong positive result of SA-β-Gal cytochemical staining was observed.suggested an aging state of HSF. Under inverted microscope, cells become flat, wide, transparent cytoplasm, nucleus particles increased; intracellular SOD activity detected by enzymatic chemical method decreased while intracellular MDA increased significantly. A series of characteristics of senescence were markedly expressed in HSF after UVB irradiation.2. The proliferative activity(expressed as the absorbance value at 450 nm)of HSF was 0.3177±0.0175、0.3352±0.0289、0.3546±0.0221 and 0.2325±0.0296 respectively in the UVB+0.02mmol/L TSG group, UVB+0.1mmol/L TSG group, UVB+0.5mmol/L TSG group respectively (F=89.12, P<0.05), with the percentage of SA-β-Gal-positive cells being (85.33±3.82)%、(57.50±6.27)%、(47.00±4.82)%、(90.33±3.55)% respectively. (F=297.50, P<0.05). Compared with the UVB-SIPS group, the three combination groups showed significantly decreased of intracellular MDA while increased of intracellular SOD. Compared with the UVB-SIPS group,the three combination groups showed significantly decreased secretion level of MMP-1. In these combination groups,the changes in the above parameters were dependent on the concentration of TSG to a degree.Conclusion1. SIPS of HSF is induced by a cumulative dose of 60mJ/cm2 with repetitive subcytotoxic exposure of UVB, it can be applied as an in vitro model in photoaging and photo-biological research.2. The stilbene glucoside promotes the growth of UVB-irradiated HSF and can effectively inhibit the activation of SA-β-Gal, reduce the content of MMP-1 in cultured supernatant, increase SOD activity while against UVB-induced lipid peroxidation. These results suggest that TSG antagonizes photoaging of HSF induced by UVB to some extent.