The Research Of Graphene's Protection For Auditory Cell Damage Caused By Oxidative Stress

Objective:To establish the HEI-OC1 cell oxidative stress model,which was suffered oxidative stress from H2O2 damage,and verify the protective effect of 2d-graphene on the hair cells after oxidative stress.It also explores the possible mechanisms of graphene to reduce the hair cell damage caused by oxidative stress.Methods:We treated 2D-G as an experimental group and TCPs as a control group.Firstly,treating the HEI-OC1 cells grown on the two materials with different concentrations of H2O2 of 500?mol/L?750?mol/L?1mmol/L?2mmol/L?5mmol/L.Detect the differences in cell survival rates between two groups by CCK 8 method after H2O2 treating for 4 hours and 12 hours.Meanwhile,observe the survival situation of two groups of cells by immunofluorescence method.Nextly,we use the two fluorescent probes of DCFH-DA and Mito-sox,which respectively detects the whole ROS level of the cell and the ROS level of mitochondrial,testing the changes of intracellular ROS levels by the flow analysis and immunofluorescence method between the two groups after oxidative stress.We then examined the differences in the levels of apoptosis in the two groups of cells after oxidative stress by TUNNEL method.As the generation of ROS in cells mainly comes from mitochondria,so we test the cells of two groups after oxidative stress by transmission electron microscope,to observe the changes of cell cytoplasm and mitochondrial morphological in the two groups.Finally,we took advantage of the Mito-tracker fluorescence probe,which responds to mitochondrial membrane potential,to detect the changes in the fluorescence intensity of two sets of cells by immunofluorescence.Results:1.After H2O2 treatment,the experimental group and the control group HEI-OC1 cell viability experiments suggest that:(1)After H2O2 treatment for 4h,under the concentration of 500?mol/L,750?mol/L,1mmol/L,2mmol/L,5mmol/L,the cell survival rates of the experimental group are higher than those in the control group,especially under the concentration of 500mol/L,750mol/L,1mmol/L,the cell survival rates between the two groups are statistically significant.(2)After H2O2 treatment for 12h,the cell survival rates of the experimental group in the above five H2O2 concentrations are higher than that in the control group,and the cell survival rates of the two groups under 500?mol/L and 750?mol/L H2O2 concentrations are statistically significant between the two groups.According to the above experimental results,we select 500?mol/L H2O2 concentration as the optimum concentration,and 4h as the suitable treatment time for subsequent experiments.2.After H2O2 treatment,detect the level of ROS in HEI-OC1 cells between the experimental group and the control group.Experimental results show that the overall level of ROS and the ROS in mitochondria of cells in the experimental group is lower than those in the control group,and there is statistically significant.3.After H2O2 treatment,the two groups' cell apoptosis level detection results showed that apoptosis rate of the experimental group is significantly lower than the control group,and the difference between the two groups is statistically significant.4.After H2O2 treatment,the structures in cytoplasm and mitochondrial morphology observation results of the two groups show that more regular mitochondrion and less lipofuscin can be observed in the experimental group,while,more swollen mitochondrion and more lipofuscin appeared in the experimental group.5.After H2O2 treatment,the function test of mitochondria of HEI-OC1 cells between the two groups show that compared with the control group,the mitochondrial metabolism level in experimental group decreased,but there is no statistically significant between the two group.Conclusions:Two dimension graphene can significantly reduce the levels of ROS and apoptosis in the cytoplasm and mitochondria of the cochlear hair cell line HEI-OC1 after oxidative stress,and protect the cochlear hair cell injury caused by oxidative stress.In addition,Two dimension graphene can also reduce the swelling and deformation of mitochondrion and decrease the generation of lipofuscin in HEI-OC1 cell.Thus,graphene may play a protective role in hair cells by inhibiting apoptosis and reducing the levels of oxidative stress in both cytoplasm and mitochondria.