Study Of Different Concentrations Of Hydrogen Peroxide On Aging In Rat Skin Fibroblasts

Fibroblasts are the main components of dermis.Recent years studies have been shown that excessive ROS can induce the oxidative aging of fibroblasts and decrease the secretion of extracellular matrix,but other research suggests that ROS at low level are conducive to cell signal transduction and cell proliferation.As a strong oxidizing,H₂O₂ makes a wide range of applications in the study of cell oxidative stress,senescence and proliferation.However,there are few studies on the time point of aging of skin fibroblasts induced by different concentrations of H₂O₂ and on the expression rule of cell aging and proliferation related factors in the process of aging.Fibroblasts were extracted from the tail top of suckling SD rats by isolating and culturing tissue masses and passed to the third generation.The third generations skin fibroblasts were continuously stimulated with different concentrations of H₂O₂ which diluted with cell culturing medium.According to the concentration of H₂O₂ screened by the results of Senescence-associatedβ-galactosidase（SA-β-gal）and Cell Counting Kit-8（CCK-8）,fibroblasts were divided into four groups:0μM,50μM,100μM and 500μM.Four concentrations of H₂O₂ were used to stimulate fibroblasts for 12 hours and the treatment solution was changed every four hours,the corresponding indexes were detected at the time point of 0h,4h,8h and 12h.Reactive oxygen species（ROS）,catalase（CAT）and superoxide dismutase（SOD）levels were detected by kits to indicate the situation of oxidation and antioxidation.The expression of p16,p21,p53,transforming growth factor-β1（TGFβ1）,CCAAT enhancer binding protein（C/EBPβ）andγH2A.X were identified by Western blot.The cell proliferation and cell cycle were detected by cell count and flow cytometry.The levels of senescence-associated secretory phenotype（SASP）Interleukin-1α（IL-1α）,Interleukin-6（IL-6）and Interleukin-8（IL-8）were detected by enzyme linked immunosorbent assay（ELISA）.The expression of DNA damage indicator proteinγH2A.X were identified by immunofluorescence.Morphological observation of fibroblasts and Vimentin immunofluorescence proved that rat primary generation skin fibroblasts were isolated and culture successfully.The indexes of 0μM remained basically stable in all time periods with good morphology and did not expressγH2A.X all the time.Compared with the group of 0μM,the level of ROS,the activity of CAT and SOD increased continuously in 12 hours,the expression of p16,p21 and p53 were basically stable at 0h-8h but increased at 12h,the expression TGFβ1 and C/EBPβcontinued to increase,the proportion of fibroblasts in G2+S increased and the cells number increased significantly,the secretion of IL-1α,IL-6 and IL-8 stable at 0h-8h but increased at 12h,the expression ofγH2A.X began at 12h in the group of 50μM.The level of ROS,the activity of CAT and SOD increased at 0h-4h but decreased steady after 4h,the expression of p16,p21 and p53 increased continuously,the expression of TGFβ1increased at 0h-8h and then decreased after 8h,but C/EBPβincreased rapidly and then stabilized at a high level,the ratio of fibroblasts in G2+S first increased and then decreased while the number of cells first increased and then remaining unchanged,the secretion of IL-1α,IL-6 and IL-8 were climbing all the time and significantly higher than the group of 50μM at 12h,the expression ofγH2A.X began at 8h with nuclear enlargement in the group of 100μM.The level of ROS,the activity of CAT and SOD all increased significantly at 4h but decreased between 4h and 12h,the expression of p16,p21 and p53 increased continuously and higher than other three groups significantly,the expression of TGFβ1 and C/EBPβdecreased all the time,the cell cycle was blocked at G1 phase and it was difficult to detect the normal cell cycle in 8h and 12h,meanwhile,the number of cell decreased significantly,the secretion of IL-1α,IL-6 and IL-8 were rapidly increased at 0h-4h but decreased at 4h-8h,the expression ofγH2A.X began at 4h and increased continuously,nuclear enlargement happened at 4h and nuclear pyknosis happened at 8h to 12h in the group of 500μM.The results showed that the effect of H₂O₂ on fibroblasts is in dose and time-dependent manner.Higher concentration and longer treatment can lead fibroblasts aging or even death easily.On the contrary,short time and low concentration can promote cell proliferation.