Study The Mechanism Of Catechin Dimers On Inhibiting PC-12 Cells Injury Induced By Oxidative Stress

Backgroud:It is well-known that food-derived antioxidants, which are considered to be harmless sources of natural antioxidants possess strong protective effects against a variety of chronic health problems such as ageing, cardiovascular diseases and cancer.In particular, great interest has arisen in the possibility that naturally occurring antioxidants from edible materials may slow ageing and reduce the risk of age-related degenerative diseases. Numerous antioxidants such as phenolic compounds（e.g.,polyphenols, flavonoids and tannins）, carotenoids, organic acid and polysaccharides discovered from various natural sources have already been applied in foods and medicines. Catechin dimers（CD） extracted from Cortinarius purpurascens Fr.（Family:Cortinariaceae）, which have prominent anti-oxidation effect can play an important role against oxidative stress in PC-12 cells. Not only have CD potential advantages in preventing oxidative stress damage, but also opening a new application in the treatment of age-related degenerative diseases.Objective:To probe the protective effect and molecular mechanism of cortinarius purpurascens extract catechins dimers to oxidized stress damage of PC-12 cells induced by H₂O₂. To determine the influence of catechins dimers to the changes of ROS in neurogenic PC-12 cells.To explore the role of ROS signaling in the apoptosis process of PC-12 cells induced by H₂O₂, and to research the effect of Mitochondrial ROS in the cell apoptosis process of PC-12 cells induced by H₂O₂.And further possible mechanistic insight on the cell protection of catechins dimers.Methods:1. PC-12 cells in a 96 well plate were treated with different concentrations of H₂O₂（50, 100, 150, 200, 250, 300μmol/L）, respectively for 2h,4h,6 h, 12 h, 24 h and 48 h. Then incubated with MTT 10μL/ well cell for 3-4 h, removal of culture medium and added 150μL /well of DMSO to dissolve. The application of Microplate Reader was used to detect the cell viability;2. PC-12 cells were treated with H₂O₂（150μmol/L） for 2h, then treated with Vitamin C（150 μmol/L）or CD（4 μg/m L）for 12 h, The application of MTT assay was used to detect the cell viability;3. PC-12 cells were treated with H₂O₂（150μmol/L） for 2h, then treated with Vitamin C（150 μmol/L）or CD（4 μg/m L）for 12 h, and incubated with the DCFH-DA. The distribution of ROS in the PC-12 cells were observed by inverted fluorescence microscope, The application of FCM（ flow cytometry） was used to detect the variation degree of ROS in the PC-12 cells;4. PC-12 cells were treated with H₂O₂（150μmol/L） for 2h, then treated with Vitamin C（150 μmol/L）or CD（4 μg/m L）for 12 h, and incubated with Hoechst 33342 fluorescent dye to stain nucleus. Changes of cell nucleus in different drug treatment groups were observed by inverted fluorescence microscope;5. PC-12 cells were treated with H₂O₂（150μmol/L） for 2h, then treated with Vitamin C（150 μmol/L）or CD（4 μg/m L）for 12 h, and incubated with the JC-1. The cell membrane potential changes were observed by inverted fluorescence microscope.The application of FCM（ flow cytometry） was used was used to detect the change of cell membrane potential;6. PC-12 cells were treated with H₂O₂（150μmol/L） for 2h, then treated with Vitamin C（150 μmol/L）or CD（4 μg/m L）for 12 h. Protein expression of caspase-9, Bax,Bcl-2, cleaved caspase-3 and PARP-1 in PC-12 cells was detected by Western Blot assay.Results:1. Established PC-12 cells oxidative stress damage model induced by H₂O₂successfully;2. H₂O₂ inhibited the activity of PC-12 cells and induced cell apoptosis. Treatment combined with vitamin C or CD reduce the toxicity effect to PC-12 cells caused by H₂O₂;3. H₂O₂ increased the concentration of ROS in cytoplasm and mitochondrial of the PC-12 cells with dose-dependently and time-dependently. Treatment combined with vitamin C or CD decrease the concentration of ROS in cytoplasm and mitochondrial of the PC-12 cells;4. CD can enhance the vitality of PC-12 cells which was induced by H₂O₂, reduced the cell death caused by H₂O₂, decreased the increasing intracellular ROS induced by H₂O₂,eased the apoptosis caused by mitochondrial dysfunction;5. H₂O₂ reduced mitochondrial membrane potential and its stability of the PC-12 cells to induce the cell apoptosis, treatment combined with vitamin C or catechins dimers increased the mitochondrial membrane potential of the PC-12 cells,decreased its apoptosis;6.H₂O₂ raised the cracked PARP-1, cracked caspase 3, caspase-9 and the expression of Bax, treatment combined with vitamin C or CD decreased the cracked PARP-1,cracked caspase 3, caspase-9 and and the expression of Bax levels induced by H₂O₂;7. H₂O₂ reduced the expression of Bcl-2, treatment combined with vitamin C or CD enhanced the expression of Bcl-2 levels and the viability of PC-12 cells, decreased its apoptosis.Conclusions:H₂O₂ induced the mitochondrial dysfunction of PC-12 cells, increased the level of ROS in cell cytoplasm and mitochondria, ROS signals participated in the regulation of mitochondrial pathways of apoptosis of the PC-12 cells induced by H₂O₂.Treatment with CD can significantly reduced the levels of ROS in PC-12 cells,stabled cell mitochondrial membrane potential, enhanced the ability of cells against oxidative stress and reduced the PC-12 cell apoptosis.