Protective Effect Of Schizandrin B Against Oxidative Damage Of UVB Irradiated Skin Cells And Its Molecular Mechanism

Objective: Schizandrin B extract from schisandra chineisis (turcz.) baill show excellent activity in cleaning free radicals, anti-lipid overoxidation, and elevating antioxidant enzyme, and the ability of anti-oxidant more better Vitamin E, which were reported in hepatic cells. And on this property, the molecular biological technology methods were applied to test and analysis the antioxidant ability of Schizandrin B. The photoprotection effect of Schizandrin B on skin cells against oxidative damage of UVB irradiated skin cells and its molecular mechanism is not defined. This paper has studied the ability of anti-oxidant, photoprotection on skin cells and its molecular mechanism.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 p53, p21, pcna, cox-2, IL-10, IL-6, IL-18, tnf-a, mmp-1, caspase-3, bax expression6 Western-blotResults:1 Inhibitory effects of Schizandrin B on viability of UVB exposed skin cellsIn the experiment,three cells lines were chosen to determine the anti-oxidant activity of Schizandrin B. The immortalized human keratinocyte cell line HaCaT was purchased from KeyGEN Biotechnology. Human dermal fibroblasts and 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.Schizandrin B has excellent performance in protecting from oxidative damage to HaCaT cells and FB cells by MTT assay. When the UVB irradiation is at 30mj/cm2, the cell viability was reduced 30%, but Schizandrin B was added to UVB-exposed keratinocytes and fibroblastscan, the viability was increased 15%. The results showed that Schizandrin B has 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 Schizandrin B can increase the SOD, GSH-px and decrease the MDA. It indicated that Schizandrin B 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 Schizandrin B on Tyrosinase activity and melanochrome secretion in UVB-induced human dermal fibroblasts. It suggested that Schizandrin B 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 Schizandrin B, 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 Schizandrin B 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 Schizandrin B 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 mRNAExpression of P53 mRNA, caspase-3 mRNA, Bax mRNA, p21 mRNA, IL-1 mRNA, IL-6 mRNA, IL-10 mRNA,IL-18 mRNA,tnf-a mRNA,cox-2 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-1 mRNA, IL-6 mRNA, IL-10 mRNA,IL-18 mRNA,tnf-a mRNA,cox-2 mRNA , mmp-1 mRNA levels decreased.6 Immunohistochemical staining and Western-blotThe experiments showed Schizandrin B was associated with the decreased of cox-2 and the increased of typeⅠp rocollagen. Conclusions:The report is the first to provide evidence that Schizandrin B 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, p21 mRNA, IL-1 mRNA, IL-6 mRNA, IL-10 mRNA,IL-18 mRNA,tnf-a mRNA,cox-2 mRNA , mmp-1 mRNA levels decreased compare with control group. Schizandrin B prevented collagen degradation by blocking MMPs production in UVB-induced firblasts. It can modulate the cox-2 secretion. Schizandrin B attenuated the UVB-induced toxicity of HaCaT and Human dermal fibroblasts. Further studies needed to clarify the molecular mechanism involved in effects on UVB-exposed cells. These results indicated that Schizandrin B possess excellent antioxidant. The dates provide strong evidence for the value of Schizandrin B as an antioxidant and anti-inflammatory agent.