D-galactose Induces Oxidative Stress, Mitochondrial Damage And Apoptosis In The Inner Ear Of Rats And The Effect Of A Long-term High-fat Diet On The Inner Ear Of D-galactose-induced Aging Rats

Part Ⅰ D-galactose induces oxidative stress, mitochondrial damage and apoptosis in the inner ear of ratsObjective:Oxidative damage to mitochondrial DNA (mtDNA) and apoptosis are heavily implicated in aging. We have previously established a mimetic rat model of aging in the inner ear using D-galactose (D-gal) and revealed that chronic injection of D-gal can increase oxidative stress and mtDNA common deletion (CD). However, there is no direct evidence of the increased accumulation of mtDNA CD caused by oxidative damage and the sources of reactive oxygen species (ROS) and the occurrence of apoptosis have not been investigated in the inner ear of rats in this model.Methods:Forty5-week old male Sprague-Dawley rats were randomly divided into two groups:①D-gal group rats were injected subcutaneously with500mg/kg of D-gal once a day for8weeks, and②the control group rats received the same volume of the vehicle (0.9%saline) for8weeks. After the experiment termination, the auditory function of rats in the two groups was evaluated by auditory brainstem response (ABR). The measurements of serum H2O2, total superoxide dismutase (T-SOD) activity and malondialdehyde (MDA) were performed by colorimetric method. The accumulation of mtDNA CD and the mRNA levels of NADPH oxidase3(NOX3) and P22phox in the inner ear of the two groups were detected by real-time PCR. The ultrastructure of mitochondria in the cells of inner ear of rats from the two groups was observed by (Transmission Electron Microscopy, TEM). The expression of8-hydroxy-2-deoxyguanosine (8-OHdG) and the protein levels of NOX3and P22phox in the inner ear of rats from the two groups were investigated by immunohistochemical analysis. The protein levels of cleaved caspase-3in the inner of rats from the two groups were evaluated by western blot. The apoptotic cells in the inner ear of rats from the two groups were determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling (TUNEL) assay.Results:The levels of serum H2O2and MDA in the D-gal group were significantly higher than that in the control group, but the levels of serum T-SOD in the D-gal group remarkably decreased compared with the control group (P<0.01). The accumulation of mtDNA CD was significantly increased by D-gal (P<0.01). Numerous mitochondria in the cells of inner ear of rats from the D-gal group were swollen with a reduced electron density in the matrix or serious degeneration. The levels of8-OHdG, NOX3, P22phox and cleaved caspase-3in the D-gal group were significantly higher than that in the control group (P<0.01). TUNEL-positive cells were found only in the cochleae of D-gal-treated rats. A small number of TUNEL-positive cells were limited to the stria vascularis (SV) of the basal turn of the cochleae. There was no significant difference in the ABR threshold between the D-gal group and the control group (P>0.05).Conclusion:These findings suggest that NOX3-associated oxidative stress may contribute to the accumulation of mtDNA mutations and activate a caspase-3-dependent apoptotic pathway in the inner ear during aging. Part II A long-term high-fat diet increases oxidative stress, mitochondrial damage and apoptosis in the inner ear of D-galactose-induced aging ratsObjective:To investigate the effect of a long-term high-fat diet (HFD) on the inner ear of Sprague-Dawley rats and on the D-galactose (D-gal)-induced aging process in the inner ear.Methods:One hundred and four male Sprague-Dawley rats (5weeks old) were randomly divided into four groups, a control group, a D-gal group, a HFD group and a D-gal+HFD group (n=26for each group). The rats in the control group and the D-gal group were fed a basic diet (BD,5%fat,20%casein,63%cornstarch,6%sucrose,1%sodium carboxymethylcellulose,3.5%mineral mix, and1.5%vitamin mix) for12months. The rats in the HFD group and the D-gal+HFD group were fed a high-fat diet (HFD,15%fat,20%casein,53%cornstarch,6%sucrose,1%sodium carboxymethylcellulose,3.5%mineral mix, and1.5%vitamin mix) for12months. In the first8weeks of the12month-diet regimen, the rats in the D-gal group and the D-gal+HFD group were injected subcutaneously with D-gal (500mg/kg/d), while the rats in the control group and the HFD group were injected subcutaneously with an equal volume of the vehicle (0.9%saline). At the end of the12-month experimental period and12hours after the last feeding, the rats were anaesthetised with ketamine (30mg/kg, i.p.) and chloropromazine (15mg/kg, i.m.), the auditory function of rats in the different groups was evaluated by auditory brainstem response (ABR). The rats were weighed. The measurements of serum triglycerides (TG), total cholesterol (TC), nonesterified fatty acids (NEFA) and H2O2were performed by colorimetric method. The accumulation of mtDNA CD and the mRNA levels of NADPH oxidase3(NOX3) and P22phox, uncoupling protein (UCP2) and uncoupling protein3(UCP3) in the inner ear of the different groups were detected by real-time PCR. The protein expression of NOX3and the protein levels of P22phox, UCP2, UCP3and cleaved caspase-3were investigated by immunohistochemical analysis and western blot, respectively. The ultrastructure of mitochondria in the strial marginal cells of inner ear of rats from the different groups was observed by (Transmission Electron Microscopy, TEM). The apoptotic cells in the inner ear of rats from the different groups were determined by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end-labelling (TUNEL) assay.Results:After12months, the ABR thresholds in the D-gal and D-gal+HFD groups were significantly higher at all four frequencies tested compared to the control group (P<0.05). The ABR thresholds of the HFD group were quantitatively higher than the control group at all four frequencies but were not statistically distinguishable from the control group except at32kHz (P<0.05). However, the ABR thresholds of the D-gal+HFD group were significantly higher at16and32kHz than the D-gal group (P<0.05). The body weights were significantly greater for the rats in the HFD and D-gal+HFD groups than for the rats fed a basic diet (P<0.01). The levels of serum TG, TC and NEFA in the HFD and D-gal+HFD groups were significantly higher than in the control group (P<0.01). Additionally, the levels of serum H2O2in the D-gal, HFD and D-gal+HFD groups were significantly higher than in the control group (P<0.01). The combined treatment of D-gal and HFD caused significant increases in the levels of serum H2O2as compared to that of the D-gal group (P<0.01). The levels of NOX3, P22phox, UCP2, UCP3and cleaved caspase-3were significantly higher in the inner ear of rats from the D-gal, HFD and D-gal+HFD groups that from the control group (P<0.01). The levels of NOX3, P22phox, UCP2, UCP3and cleaved caspase-3in the D-gal+HFD group were significantly increased compared to the D-gal and HFD groups (P<0.01). The accumulation of mtDNA CD in the inner ear were significantly increased in the D-gal, HFD and D-gal+HFD groups compared to that in the control group (P<0.05). TEM revealed that the stria vascularis (SV) of the rats in the control group were rich in mitochondria, and the shape and size of the mitochondria were normal, In contrast, numerous mitochondria from the rats in the D-gal, HFD and D-gal+HFD groups were swollen with a reduced electron density in the matrix. In the basal turn of the cochleae, few TUNEL-positive cells were observed in the S V of cochleae from the control group. In the D-gal and HFD groups, TUNEL-positive cells were observed in the SV. Moreover, histological sections from the rats in the D-gal+HFD group exhibited a greater number of TUNEL-positive cells in the SV and a small number of TUNEL-positive cells in the organ of Corti.Conclusion:Long-term HFD may induce oxidative stress, mitochondrial damage and apoptosis in the inner ear, and it provided evidence regarding the link between HFD and link between HFD and an increased risk of age-related hearing loss.