Protective Effect Of Polydatin Against Damage Of UVB Irradiated Skin Cells And Its Molecular Mechanism

Objective:Polydatin as Polygonum cuspidatum rhizome extract, has antitussive, antiasthmatic, antibacterial, scavenging free radicals, and lipids, cholesterol; against Staphylococcus aureus, the card he cocci, alpha or beta chain Bacteria, Escherichia coli, which are inhibited. The research of protective effect of ultraviolet damage to skin have not been reported yet. The subject of the proposed establishment of ultraviolet B irradiation HACAT damage model to investigate the inhibition of HACAT damage and protection mechanisms.Methods:1 Cell culture, UVB irradiation and Determination of cell viability2 Measurement of SOD, GSH-px and MDA release3 Determination of Tyrosinase activity and melanochrome secretion4 Detection of intracellular ROS and Confocal microscopy5 Measurement of MMP-1,p53, IL-6, IL-18, caspase-3, bax expressionResults:1 Inhibitory effects of Polydatin on viability of UVB exposed skin cellsIn the experiment,two cells lines were chosen to determine the anti-oxidant activity of Polydatin. The immortalized human keratinocyte cell line HaCaT was purchased from KeyGEN Biotechnology. Melanoma cell were obtained from the China Union Medical University of Blood Institute. All cells were cultured in Dulbecco's modified Eagle's media containing 10% FBS, 2mmol/L glutamine,100U?ml penicillin and 100μg?ml streptomycin at 37℃humidified atmosphere of 5% CO2 in air.Polydatin has excellent performance in protecting from oxidative damage to HaCaT cells by MTT assay. When the UVB irradiation is at 30mj/cm2, the cell viability was reduced 30%. The results showed that Polydatinhas excellent anti-oxidative activity. The viability was dose-dependently enhanced and obtained the inhibition in UVB-induced skin cells.2 Measurement of SOD, GSH-px and MDA releaseThe supernatant and intracellular SOD, GSH–px and MDA level was measured by assy kit (Nanjing Jiancheng Bioengineering Institute). The result showed that Polydatin can increase the SOD, GSH–px and decrease the MDA. It indicated that Polydatin elevated the anti-oxidant ability when the UVB irradiation at 30mj/cm2.3 Measurement of the Tyrosinase activity and melanochrome secretionThe result shows the inhibitory effects of Polydatin on Tyrosinase activity and melanochrome secretion in UVB-induced Melanoma cells. It suggested that Polydatin has posses the function of whitening.4 Detection of intracellular ROS and Confocal microscopyWe selected A375cells which seeded in DMEM in exponential phase of growth, add Polydatin, then keeping on cultivating the A375 cells for 24h, then detected by 405nm. Each experiment was repeated at least three times.To avoid direct photooxidation of the fluorescent dye, cells were first treated with UVB irradiation and then loaded with the dye. In brief, HaCaT cells were irradiated with UVB (30mJ/cm2) in the presence or absence of Polydatin and then loaded with DCFH-DA (5μM). The intensity of DCF fluorescence was measured using a spectrofluorophotometer. Each experiment was repeated at least three times.The intensity of DCF fluorescence was measured using a spectrofluorophotometer. The result shows that Polydatin can reduce the intensity of DCF fluorescence in UVB-induced human dermal fibroblasts.The figure shows that the intensity of DCF fluorescence in UVB-exposed HaCaT cell was reduced. The inhibitory was enhanced with dose-dependently.5 The expression of P53 mRNA, caspase-3 mRNA, Bax mRNA, p21 mRNA, Bcl-2 mRNA and mdr-1 mRNA Expression of P53 mRNA, caspase-3 mRNA, Bax mRNA, IL-6 mRNA,IL-18 mRNA, MMP-1 mRNA in UVB-induced HaCaT was measured by RT-PCR. The result showed that P53 mRNA, caspase-3 mRNA, Bax mRNA, p21 mRNA, IL-6 mRNA, IL-18 mRNA, MMP-1 mRNA levels decreased.Conclusions:The report is the first to provide evidence that Polydatin can clean the free radicals derived from UVB-irradiated HaCaT and increase the antioxidant enzyme. The antioxidant enzyme plays an important role in the anti-oxidant damage. The cell viability is obviously augmentation. The result demonstrated that P53 mRNA, caspase-3 mRNA, Bax mRNA, IL-6 mRNA, IL-18 mRNA, MMP-1 mRNA levels decreased compare with control group. Polydatin prevented collagen degradation by blocking MMPs production in UVB-induced firblasts. Polydatin attenuated the UVB-induced toxicity of HaCaT. Further studies needed to clarify the molecular mechanism involved in effects on UVB-exposed cells. These results indicated that Polydatin possess excellent antioxidant. The dates provide strong evidence for the value of Polydatin as an antioxidant and anti-inflammatory agent.