D-galactose Induced SGNs ROS Generation And Its Correlation With Ageing And DNA Methylation

Objective:To explore the mechanism of the damage of ROS generation to spiral ganglion neurons and it’s correlation with senescence and DNA methylation.Methods:Primary cultured SGNs isolated from postnatal2-3day SD rats were exposed to different concentration of D-galactose,D-glucose and mannitol. CCK8assay was used to evaluate the toxicity of different concentration of each reagent and ROS status was examined using flow cytometry. Beta-galactosidase staining was taken to estimate the senescence status of SGN. Using RT-PCR, we calculated the mtDNA4834deletion level of each group.The mRNA level of DNMT1,PGC-la,BDNF were evaluated using RT-PCR. Immunofluorescence was used to image the expression of NF200, NeuN, TUJ1,Dnmt1,TFAM,NRF1in SGN.Results:25mg/ml D-galactose had a siginificant toxic influence on SGNs and for D-glucose and mannitol the concentration were30mg/ml and35mg/ml, which shows that we can exclude the influence of osmotic presure on SGNs. Flow cytomentry showed a20%ROS elevation after5days’20mg/ml D-galactose stimulation. However, D-gal was not enough to induce accererated senescence of SGNs nor the elevation of mtDNA4834deletion. After30days in vitro, mtDNA4834deletion had no significant difference compared with9days in vitro.The mRNA level of DNMT1and PGC-la had no significant change after D-galactose stimulation nor30days in vitro. However, the mRNA level of BDNF increases significantly after exposure to D-galactose or30days in vitro.Conclusion: D-galactose can induce the ROS generation in spiral ganglion neurons, but not enough to induce senescence of SGN. Together with previous work on D-galactose induced mimic ageing model of SD rat and in vitro accelerated senescence model of marginal cell, we might think that D-galactose was more likely to induce a matebolic presbyacusis model. Further investigation should be made to find a suitable method to induce a senescence model of SGN.