Mechanism And Intervention Of Ferroptosis In Auditory Central Degeneration

PartⅠ Establishment of age-related hearing loss rat modelObjective: To establish an age-related hearing loss rat modelMethods: A total of 150 1-month-old male Sprague-Dawley(SD)rats were randomly divided into two groups after one month of adaptive feeding: a control group and an experimental group.The experimental group was a simulated aging group.Rats in the simulated ageing group were injected subcutaneously with D-galactose(D-gal,600 mg / kg/ day)for 8 consecutive weeks.Meanwhile,rats in the control group were also injected with an equal amount of 0.9% NS.Each group of rats was divided into three subgroups: the4-month-old group(0 months after treatment),the 10-month-old group(6 months after treatment),and the 16-month-old group(12 months after treatment).Auditory brainstem response(ABR)was used to estimate the hearing level of rats.Transmission electron microscope(TEM)was used to observe neurodegeneration in the auditory cortex.TUNEL staining was used to evaluate apoptosis in the auditory cortex.q RT-PCR was used to detect mitochondrial DNA common deletions(CDs).Result: In the control groups,the hearing threshold of all frequencies increased significantly at 16 months of age.In the simulated aging groups,the hearing thresholds of high-frequencies,including 24 k Hz and 32 k Hz,increased at 10 months old,and at 16 months of age,the hearing thresholds of all frequencies increased significantly.In addition,the hearing threshold of high-frequencies(24k Hz and 32 k Hz)were significantly higher than that in the control group at 16 months old.With aging,there showed some ultrastructural changes of neurodegeneration,inclluding vacuolated mitochondria,abundant lipofuscin and dropsical even disrupted myelin.The simulated aging groups begun to show these changes earlier.TUNEL-positive cells in the auditory cortex of the two groups of rats increased with age,and the TUNEL-positive cells in the 16-month-old simulated aging group increased significantly compared with the age-matched control group.CD levels of the auditory cortex in the simulated aging groups were significantly higher than that in the age-matched control groups,and the differences showed an increasing trend with aging.Conclusions: With aging,the hearing thresholds of rats increased,and the high-frequency hearing thresholds in the simulated aging group increased earlier,accompanied by the increase of CD in the simulated aging group compared to the age-matched control group,which are consistent with the characteristics of age-related hearing loss.In addition,the ultrastructure of auditory cortex showed a degenerative change with aging,meanwhile apoptotic cells gradually increased.It can be proved that we successfully applied D-gal to build a model of age-related hearing loss.PartⅡ Changes in ferroptosis in rat auditory cortex with agingObjective: Age-related hearing loss is a growing public health problem,but its underlying molecular mechanisms have not been fully elucidated.Ferroptosis is a new discovered regulated cell death caused by lipid peroxidation,which is caused by iron and reactive oxygen species(ROS),and is involved in many pathological processes.But whether ferroptosis is involved in the degradation of the auditory system is unclear.The purpose of this study was to observe changes in ferroptosis in auditory cortex degeneration and to study the pathogenesis of central presbycusis.Methods: The level of iron,malondialdehyde(MDA),superoxide dismutase(SOD),and glutathione(GSH)in auditory cortex were detected by colorimetry.The characteristic structural changes of ferroptosis in the auditory cortex were observed by transmission electron microscope(TEM).Western Blot was used to detect the levels of auditory cortical ferroptosis marker protein(ACSL-4),intracellular iron content marker protein(FPN),and iron regulation related proteins(IRP-2,Tf R-1).The protein levels of IRP-2 and Tf R-1 were also detected by immunofluorescence(IF).Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the transcription levels of IRP-2 and Tf R-1 in the auditory cortex.Results: The iron content in auditory cortex increased with aging,and this trend in the simulated aging groups was more obvious.At 10 and 16 months of age,the iron content of the simulated aging groups increased compared to that of the age-matched control groups.The level of MDA showed the same trend,but the levels of SOD and GSH were opposite.Ultrastructural changes related to ferroptosis were found in the 10-month-old simulated aging group and both groups at 16 months old.Compared to the age-matched control groups,the levels of FPN,IRP-2,and Tf R-1 in the simulated aging groups were increased.At 10 and 16 months of age,ACSL-4 levels in the simulated aging groups were increased compared with the age-matched control groups.IF results showed that the levels of IRP-2and Tf R-1 increased with age;and compared to the age-matched control groups,the levels of IRP-2 and Tf R-1 in the simulated aging groups increased.The transcription levels of IRP-2 and Tf R-1 in the simulated aging groups increased compared to the age-matched control groups.Conclusions: With aging,the levels of proteins associated with iron regulation changed,resulting in the deposition of iron in the auditory cortex.Unbalanced iron state caused an increase in ROS and a weakening of the antioxidant system,eventually leading to ferroptosis of cells.The occurrence of ferroptosis may be closely related to central presbycusis.Part Ⅲ The role of ferroptosis in neuron senility and the mechanism of alleviating agingObjective: To explore the role of ferroptosis in the process of neuron senility and the potential mechanism of alleviating agingMethods: D-gal was used to establish a simulated aging model of PC12 cells.Then,D-gal-induced simulated aging PC12 cells were treated with iron chelator DFO or knockdown of IRP-2.The concentrations of D-gal and DFO were screened by colorimetry.Intracellular iron,MDA,SOD,and GPx levels were detected by colorimetry.TEM was used to observe the ultrastructure of cells.Cell senescence was observed by senescence-associated β-galactosidase staining.Apoptosis levels were detected by flow cytometry.CD was detected by q RT-PCR.Western Blot was used to detect knockdown efficiency and protein levels of FPN,ACSL-4 and 4-HNE.Results: The simulated aging model of PC12 cell was established with 15 mg/ml D-gal.si RNA successfully reduced the expression of IRP-2 and Tf R-1.The optimal concentration of DFO was screened at 50 μM.Both DFO treatment and IRP-2 knockdown partly reversed the increase of intracellular iron and MDA levels and the decrease of SOD and GPx activities caused by D-gal.Both DFO treatment and IRP-2 knockdown partly reversed the up-regulation of FPN,ACSL-4 and 4-HNE levels caused by D-gal.Both DFO treatment and IRP-2 knockdown partly reversed the increase in CD level caused by D-gal.Both DFO treatment and IRP-2 knockdown partly reversed the ultrastructural degradation,apoptosis,and increase of β-galactosidase-positive cells caused by D-gal.Conclusions: Both DFO treatment and IRP-2 knockdown can reduce the deposition of iron in cells through different ways,so they can inhibit the occurrence of ferroptosis,and thereby delay cell senescence.