Study On The Role And Mechanism Of ADMA In Cellular Senescence Of Human Skin Fibroblasts

ObjectiveStudy on the role and mechanism of ADMA in cellular senescence of human skin fibroblasts.Methods1. To measure the concentration of ADMA from cutaneous cell and tissue of natural aging and photoaging using HPLC.2. In order to ensure the best drug concentration, we use MTT assay to evaluate the effect of ADMA to proliferation of HSF (primary Human skin fibroblasts)3. We detect the activity of beta-galactosidase and concentration of ROS of primary HSF after treatment of different ADMA concentration (10,30,50μM) using Galactosidase, β-, Staining Kit and Reactive oxygen species assay kit, while detect expression of P16,MMP-1, phosphorylation-P38using western blot.4. We pretreated primary HSF with SB203580(inhibitor of P38)and antioxidant NAC(lOuM), and added ADMA(50uM) after one hour. Then we detect the beta-galactosidase, concentration of ROS, and expression of PI6, MMP-1, and phosphorylation-P38using methods mentioned in step3.Results1. The content of ADMA is markedly higher in the skin tissue and cells of old age group than in young age group (P<0.05). After UVA irradiation, The content of ADMA is significantly increased in HSF (P<0.05)。 2. ADMA could inhibit proliferation of HSF, and the inhibition effect is increasing with ADMA concentration raising. IC50=51.47±1.013uM.3. After treatment of different ADMA concentration (10,30,50μM),cell density of HSF decrease,beta-galactosidase dyeing rate,P16increase with ADMA concentration raising, and appearance of the cultured HSF become big and round comparing to control group (P<0.05)4. After treatment of different ADMA concentration (10,30,50μM), ROS concentration, expression of phosphorylation-P38, MMP-1is increasing with ADMA concentration rising (P<0.05)5. Pretreated HSF with P38inhibitor SB203580(lOuM) before added ADMA, the beta-galactosidase dyeing rate, expression of P16and MMP-1decreased.Pretreated HSF with NAC (10μM) before added ADMA, the beta-galactosidase dyeing rate, expression of PI6, ROS concentration,phosphorylation-P38, MMP-1decreased preparing to the group only treated with ADMA.Conclusion1The ADMA content increased significantly of Natural aging and photoaging in the skin tissue and fibroblasts. ADMA plays an important role in the process of skin aging.2ADMA could induce senescence of human skin fibroblasts, mediated by activating ROS and its downstream P38and MMP-1.