The Effect Of ZHX1 In D-galactose-induced Hair Cell Senescence Model

?Objective?To explore the expression and effect of ZHX1 in D-galactose induced hair cell senescence model,for purpose of the new ideas to study and deal with presbycusis.?Methods?(1)Different concentration gradients of D-gal were used to treat the hair cell strain,CCK-8 assay was used to estimate the cell viability,finally selected the concentration of15mg/ml to establish the Senescence model.(2)Depending on the treatment conditions,we had four grous:1)group C: Control group,cells grew in normal medium.2)group D: D-gal group,cells were treated with medium containing 15mg/ml D-gal for48 h.3)group NC:transfection negative control group,transfered the cells with negative control si RNA for 6h,and then cultured in medium containing 15mg/ml D-gal for 48 h.4)group Si:transfection ZHX1 Si RNA group,transfered the cells with ZHX1-si RNA for6 h,and then cultured in medium containing 15mg/ml D-gal for 48 h.(3)Observed the cells morphology by light microscope;Observed the ultrastructural changes of cells by light transmission electron microscopy;RT-PCR was used to estimate the common deletion;Flow cytometry was used to estimate the apoptosis rate and ROS content;The cells were treated with JC-1 probe and observed by confocal microscopy to detect the change of mitochondrial membrane potential;Microplate assay was used to estimate the content of GSH?MDA?ATP and the activity of SOD and CAT;RT-PCR was used to detect the m RNA expression of P16?P21?ZHX1?TWIST1?Bcl-2?NRF-1;Western blot was used to detect the protein expression of ZHX1?Bcl-2?Bax?caspase-3?NRF-1.?Results?(1)TEM to observe the ultrastructural changes of cells;we found that;in group C,the cellular outline was regular and the cell membrane kept intact,the cytoplasm was uniformly and dense,and there were abundant organelles,the structure of mitochondria and endoplasmic reticulum was intact and clear;whereas,in group D,the cellular outline was irregular,and there was a relatively obvious invagination in cell membrane,the integrity of the nuclear membrane was broken,we found the nuclear fragment,the cytoplasm was loose and tangled,the organelles was out of shape,the mitochondria was with the abnormal appearance,such as the the turgidity of mitochondria,the reduction of mitochondrial crest,and even the vacuolation.(2)The CD of group D was higher than group C;and the m RNA levels of P16 and p21 kept the same tendency of CD.(3)The ROS content of group D was higher than group C.(4)Annexin V-FITC assay to detect apoptosis,we found that there was a higher apoptosis rate,when group D compared with group C,and group D and group NC showed no difference,the apoptosis rate of group Si was the highest among all the treatment groups.(5)JC-1 probe to detect the mitochondrial membrane potential,by using confocal microscopy,we found that the ??m of group D and group NC was lower than group C,and group Si was at the lowest level.(6)The content of GSH?ATP and the activity of SOD and CAT was lower,the content of MDA was higer,when group D and group C compared with group C;Future more,the content of GSH?ATP and the activity of SOD and CAT of group Si was lower than group NC,and the content of MDA turned out the opposite.(7)RT-PCR showed that,the m RNA expression of ZHX1?TWIST1?Bcl-2and NRF-1 of group D and group NC was lower than group C,and group Si kept lower than group NC expect for TWIST1.(8)Western blot showed that,the protein expression of ZHX1?NRF-1 of group D and group NC was lower than group C,the Ratio of Bcl-2/Bax was lower,the expression of the active caspase-3 was higher;When group Si compared with group NC,the protein expression of ZHX1?NRF-1 and the Ratio of Bcl-2/Bax was lower,and the expression of the active caspase-3 was higher.?Conclusion?(1)In this study,we found that,treated the hair cell strain with A high concentration of D-gal for some time,would resulted in an increase of the ROS content and CD,also with the higher m RNA expression of P16 and P21.Combined with the fact that cells showed degenerative changes,we successfully establish the hair cell senescence model.(2)We used Si RNA to interfere the expression of ZHX1,and found that the ability of the hair cell to resist to apoptosis and oxidative stress was lower,indicated the possible role of the ZHX1 in control apoptosis and oxidative stress during cell senescence.(3)ZHX1 might take effect via regulating the express of Bcl-2 and NRF-1.