The Effect Of XIAP-SHRNA On Primary Auditory Cortex Gaba Nerves Apoptosis Of Presbycusis Mouse

Background and Objective:Presbycusis is the cumulative effect of aging on hearing.Also knowm as presbycusis,itis defined as aprogressive bilateral symmetrical age-related sensorineural hearing loss.Thehearing loss is most marked at higher frequency.The etiopathogenisis is result of geneticfactor and environmental agent Jointly, recently, scholar pay attention to effect of gene ofhereditary deafness on presbycusis evelopment，and made a lot of achievements. Theresearch in environmental agent about pesbycusis, focus on noise, ototoxicity medicine,diabetes and angiocardiopathy.The research demonstrate apoptosis of auditio nerves was pathology alloeosis ofpresbycusis,and the clinical research demonstrate prebsycusis possibly was the relust of theperipheral auditory system and the center auditory system function reduces the decayjiontly, X-chromsome-linked inhibitor of apoptosis protein(XIAP),as animportant member of mammal Inhibtor of apoptosis protein(IAP),It can express in nervussensorialis and motor nervous system simultaneously.and can containment apoptosis ofnerve cell by inhibiting caspase protease cascade reaction.The mian inhibitory neuronGABAergic have a significance effective in architecture and signal coding of auditorycortex,This experiment interact the XIAP expression of neuron GABAergic auditory cortexby RNAi technique, to confirm XIAP have significance role in apoptosis ofGABAergic,and to discusses the relation between presbycusis and GABAergic, knewthoroughly the pathogenesis of presbycusis,and provides the new mentality for thepresbycusis treatment.Materials and methods:1.The effect of neuron GABAergic auditory cortex on presbycusisSeparately selects20young C57BL/6J mouse (2month old) and20aged C57BL/6J mouse(10month old).detection the ABR,then get auditory cortex,to test apoptosis ofGABAergic and expression of XIAP by immunofluorescence，and discuss difference ofyoung group and aged group.2.Construct XIAP-ShRNA plasmid and accreditConstruct XIAP-ShRNA plasmid A、B、C,and analysis effectiveness in cellular level,cultured cell and depuration,to accredit,test expression of XIAP after transfection into cell.3.Test the effective of XIAP-ShRNA plasmid on presbycusis in vivoImport XIAP-ShRNA plasmid into A1by using stereotaxic apparatus,andexamines ABR on pro-import,14days later,test the apoptosis of GABAergic by tuenl,testGABAergic and the expression of XIAP by IF，definite the degree of apoptosis and analysisdifference and significance.Results:1.In young group,the results of ABR examines1KHz,2KHz,4KHz,8KHz,16KHzhearing threshold respectively was23.50±3.15dB SPL,32.75±4.99dB SPL,40.50±5.36dB SPL,60.25±5.73dB SPL,66.00±6.19dB SPL，in aged group. the relsults was60.50±6.69dB SPL,62.25±4.02dB SPL,65.75±4.55dB SPL,81.50±4.77dB SPL,85.50±4.44dB SPL.two group of hearing threshold all have the obvious difference in variousfrequencies(P<0.01).2.In A1of young group the numbers of apoptosis of GABAergic was10.22±0.84,inaged group was19.17±4.06,there are signifficant difference between them(P<0.01).thenumber of XIAP of expression in GABAergic of young group was47.83±6.77,in agedgroup was13.27±6.94,there are signifficant difference between them(P<0.01).3.To construct a RNAi sequence targeting XIAP gene as XIAP-ShRNA-A,XIAP-ShRNA-B,XIAP-ShRNA-C.and transfect into cortex cellula nervosa of primaryculture,then RT-PCR, Western-Blot was performed to determine the expression level ofXIAP.and flow cytometry was performed to determine the apopotsis of nerve cell.theXIAP-ShRNA-C was the best.4.Get XIAP-ShRNA-C into A1as experiment group,PBS as controlgroup,2week later, the ABR test have been taken on all groups with1KHz,2KHz,4KHz,8KHz,16KHz hearing threshold respectively and thehearing threshold was determined respectively,The results of young experiment group was39.25±5.07dB SPL,44.50±6.87dB SPL,52.25±5.80dB SPL,70.00±6.52dB SPL,79.50±5.68dB SPL， the results of youngcontrol group was27.25±4.87dB SPL,38.75±6.68dB SPL,48.25±5.07dBSPL,64.00±5.37dB SPL,70.50±6.30dB SPL,in aged group the results ofexperiment group was74.25±4.81dB SPL,75.75±6.75dB SPL,78.75±7.89dB SPL,85.50±4.44dB SPL,87.75±2.94dB SPL,the result of aged controlgroup was68.50±5.02dB SPL,70.75±6.37dB SPL,73.75±6.10dB SPL,81.75±3.96dB SPL,84.50±4.15dB SPL,two group of hearing threshold all havethe obvious difference in various frequencies(P<0.05),1month later, theresults of ABR examines1KHz,2KHz,4KHz,8KHz,16KHz hearing thresholdrespectively was44.00±6.04dB SPL,49.50±5.45dB SPL,56.00±4.06dBSPL,75.00±5.92dB SPL,82.00±5.57dB SPL，in young experiment group. theresults of young control group was34.50±5.89dB SPL,44.50±4.71dBSPL,52.00±4.30dB SPL,71.25±4.14dB SPL,72.25.±8.31dB SPL,in agedgroup the results of experiment group was77.25±4.60dB SPL,80.00±3.87dB SPL,83.75±3.83dB SPL,88.25±2.86dB SPL,88.50±2.29dB SPL,theresult of control group was71.50±5.26dB SPL,76.75±3.63dB SPL,81.50±5.94dB SPL,84.75±5.36dB SPL,87.00±4.30dB SPL,except for16KHz, twogroup of hearing threshold have the obvious difference in variousfrequencies(P<0.05).5.Two weeks later,the number of apoptosis of GABAergic in young experiment groupwas18.21±3.90by TUNEL,the number of young control group was12.22±0.63, twogroup of hearing threshold all have the obvious difference(P<0.05).the number of agedexperiment group was7.18±2.10,the number of aged control group was17.42±3.39,thenumber of GABAergic with XIAP expression in young experiment group was35.29±9.83,the number in young control group was52.47±4.58,the number of aged expriementgroup was8.45±6.62,the number of aged control group was16.77±8.35,and aftertransfection,the XIAP expression was decreased by23.61%in young expriement grouprespectively compared with control group, the number was20.73%decreased by in agedgroup(P<0.05). Conclusion：1.The hearing of C57BL/6J mouse decrease when aging,simulataneously the numer ofthe GABAergic neuron apoptosis in the A1of the C57BL/6J mouse increase obviousely,the GABAergic neuron apoptosis in A1possibly is an important attribute.2.The level of XIAP expression in C57BL/6J mouse A1have the obvious differencein various age groups, moreover，the XIAP expression in GABAergic neuron of C57BL/6JA1have the obvious difference in various age,the degression of XIAP expression possiblyis an important factor for apoptosis of GABAergic neuron apoptosis in A1.3.The XIAP expression of cortical neuron of C57BL/6J mouse in primary culture wasdegression by RNAi,and then, there can induce theapoptosis of cortical neuron.4.In vivo experiment，the XIAP expression in cortical neuron of C57BL/6J mouse wasdegression by RNAi,and then, there can increase the apoptosis of GABAergic neuron in A1,to lead to the hearing degression,XIAP possbily in the presbycusis occurrence,thedeveloping process the vital role,they participated in the regulative process of theGABAergic neuron apoptosis in the A1of C57BL/J mouse.