Study On The Regulatory Mechanism Of TNF-α In Age-related Hearing Loss

Objective: Age-related hearing loss(ARHL),also referred to as presbycusis,is one of the most common in the elderly ARHL sensory disease.Presbycusis is a progressive,irreversible,and symmetrical bilateral neuro-sensory hearing loss.The main pathological features of ARHL are progressive hair cells loss and degeneration of spiral ganglion neurons(SGNs).Inflammation is an important factor in the study of age-related chronic diseases,and its effect on hearing loss has also attracted extensive attention in the field of audiology and auditory neuroscience.Inflammation is involved in the pathogenesis of ARHL,in which tumor necrosis factor-α(TNF-α)plays an important role in the progression of ARHL,but its specific molecular mechanism remains unclear.We choose the DBA /2J mice as ARHL animal model,this project intends to investigate the role of TNF-α in the hair cells loss.Methods: Eighty DBA/2J mice aged from 2 weeks to 8 weeks were used as experimental subjects.(1)About 8 mice were randomly selected from(2w 、4w 、6w 、8w)DBA/2J mice of different age groups for audiological examination,in which the change of hearing threshold was detected by brainstem evoked potential(ABR),and the change of hair cells was detected by distortion product otoacoustic emission(DPOAE);(2)The cochlear tissues of DBA/2J mice of different age groups were collected.Histological analyses of hair cells,spiral ganglion neurons and stria vascularis were observed by H&E staining(hematoxylin and eosin),the inflammatory cell infiltration was also detected by H&E staining;(3)The cochlea of DBA/2J mice at the age of 2w 、4w 、6w and 8w were taken respectively,the inner ear tissues were ground,and after extracting its total RNA,it is retrotranscribed into c DNA.m RNA expression levels of TNF-α,NF-κB,c-FLIP,Caspase-8,Caspase-3 and Caspase-6 were detected by q PCR,and protein expression levels of NF-κB and Caspase-3 were detected by Western blotting;(4)The localization expression of Caspase-3 and NF-κB in inner ear tissue of 4w was detected by immunohistochemistry;(5)We add different concentrations of recombinant TNF-α into HEI-OCI inner ear cells.m RNA expression levels of NF-κB,Caspase-3 and Caspase-8 in different groups were detected by q PCR,and cell proliferation was detected by CCK-8;(6)HEI-OC1 inner ear cell model was transfected with TNF-α si RNA.After confirming the successful transfection,m RNA expression levels of NF-κB and Caspase-3 in different groups were detected by q PCR.Results: 1 ABR demonstrate that the hearing threshold of DBA/2J mice increased gradually from 2 weeks to 8 weeks under different frequency stimulation,and DPOAE demonstrate that the vibration amplitude of hair cells decreased with age.The hearing loss of DBA/2J mice was gradually aggravated,which could be used as the animal model of presbycusis.2 H&E staining demonstrated that IHC and OHC is relatively complete in the 2 weeks,outer hair cell(OHC)is missing,and inner hair cells(IHC)still exist at 6 weeks,the urvival of SGNs gradually decrease over weeks,the widths of SV is gradually narrowed.3 Cochlear inflammatory cells in the cochlea wall increase gradually,mainly for the macrophage infiltration and their activation was obvious at 8 weeks.4.The expression of TNF-α in the cochlea of DBA/ 2 J is gradually increased with the increase of week age.Caspase-8,Caspase-3 and Caspase-6 was the highest at 2w and then decreased,while the expression of NF-κ B and c-FLIP was the highest at 4 w and then decreased.5.WB showed that the expression of NF-κ B protein was the highest at 4 w,and then decreased,while the expression of Caspase-3 protein gradually increased.6.Immunohistochemistry demonstrate that Caspase-3 and NF-κB were mainly located on hair cells and SGNs,Few are located on SV.7 Low concentration of TNF-α could increase NF-κB,while Caspase-3 and Caspase-8 had little change.High concentration of TNF-α could increase NF-κB,Caspase-3 and Caspase-8 in HEI-OC1 cells.CCk-8 showed that high concentration of TNF-α induced decreased proliferation and increased apoptosis of HEI-OC1 cells.8.The m RNA expression levels of NF-κB,caspase-3 and caspase-8 in the group transfected with TNF-α si RNA were significantly lower than those in the control group.Conclusion: TNF-α increased gradually with the age of DBA/2J mice.Low concentration of TNF-α can activate NF-κB transcriptional response to mediate hair cell survival,while high concentration of TNF-α can activate Caspase-3 cascade to mediate hair cell apoptosis.TNF-α mediated inflammatory signaling pathway plays an important role in the pathogenesis of ARHL..