Adenovirus-mediated XIAP Overexpression In Primary Auditory Cortex Prevents Hearing Loss Of Presbycusis

Background and Objective：Presbycusis is a kind of the common diseases in old people. The nunmer of patien isgrowing up as china entering into aging society. The diseas brings more and more trouble topatien.Being short of sensitive treatment, presbycusis is a natural process of development.Doctors usually suggest patients wearing hearing aids for daily life. Although hearing aidshelping for hearing of presbycusis patients, there are still plenty of shortcomings. As aresult, hearing aids can not deal with acoustic signals fluently insteading of AuditoryPathway.More an more researchers focus on presbycusis because of its universality and theindeterminacy on clinical treatment. The researchers found that presbycusis is related tooxygen free radicals of local tissue, metabolic imbalance, mitochondrial DNA mutation,glycogen aggregation, microcirculation disturbance and apoptosis. Further researchindicates that presbycusis is related to apoptosis of neurons in primary auditory cortex.There are two groups neurons in brain of mammals, one group is excitation neurons whichmainly include glutamate energic neurons, the other group is GABA-ergic neurons. Thenumber of glutamate energic neurons is more than GABA-ergic neurons.. We consider that,the mainly pathophysiology of presbycusis is apotosis of GABA-ergic neurons in primaryauditory cortex. There will be a molecule combine with target on apoptotic pathway tocontrol the apotosis of GABA-ergic neurons, which is the most important way to treatmentpresbycusis.X-linked inhibitor of apoptosis protein is the most important member in IAPs., whichcan inhibit apoptosis in the path way of caspase-3, NF-κB/bcl-2, TAB1-TAK1-JNK, MEK1and PI3K-Akt. The function of XIAP is established in many fields such as oncology andapoptosis-related disease. Unfortunately, there aren’t researches about XIAP in presbycusis. Because of XIAP has anti-apoptotic function in cells, and the GABA-ergic neuronsis apoptosis in primary auditory cortex of presbycusis. This experiment is supported by theNational Natural Science. Foundation, at first we constructed a recombinant adenovirusvector carrying XIAP to verify the relationship between XIAP and nerve cell activity invitro; And then, built a animal models which is over expression of XIAP in the primaryauditory cortex to observe the change of high frequency in auditory threshold, to definitethe relationship between the apoptosis related proteins and GABA-ergic neurons, to explorethe molecular mechanisms in presbycusis. This part of experiment provides basic researchfor the clinical treatment of presbycusis.Methods1． Transfection of primary culture cortical neurons with recombinantadenovirus carrying X-linked inhibitor of apoptosis protein gene in vitro.1) Developing a method of primary culture cortical neurons of C57BL/6J mouse andestablishing experiment cell model of the neurons. Immuno-staining of β-Tublin is appliedto assess the culture purity.2) Transfection of primary culture cortical neurons with recombinant adenoviruscarrying X-linked inhibitor of apoptosis protein gene in vitro.3) The function of Ad-XIAP is tested by reverse transcription polymerase chainreaction (RT-PCR) and Westernblot.4) The test of MTT explore the important role of XIAP in cortical neurons.2． In primary auditory cortex, XIAP over-expression protects thehigh-frequency hearing loss in presbycusis by inhibiting the apoptosis of GABA-ergicneurons.1) In primary auditory cortex, over-expression of XIAP protects the high-frequencyhearing loss in presbycusis.①Built a animal models which is over expression of XIAP in the primary auditorycortex by sterotaxis injection.②The expressions XIAP is tested by Westernblot.③Observe the function of Ad-XIAP protecting high-frequency hearing in presbycusis indifferent time.2) Over-expression of XIAP protects the high-frequency hearing loss in primary auditory cortex by inhibiting apoptosis of GABA-ergic neurons.①XIAP over-expression inhibits expression of caspase-3in immunohistochemistytest.②A part of GABA-ergic neurons are apoptosus in primary auditory cortex inpresbycusis by TUNEL test.③XIAP over-expression protects GABA-ergic neurons in TUNEL andimmunohistochemisty test.Results1、cortical neurons are gained by the method of primary culture which are more than90%.2、Ad-XIAP transfect nerve cells smoothly in which XIAP is over expression.3、The expression of XIAP is the greatest after Ad-XIAP transfecting primary culturecortical neurons in72hours.4、Built a animal models which is over expression of XIAP in the primary auditorycortex by sterotaxis injection successfully.5、The expression of XIAP is the greatest after Ad-XIAP transfecting primary auditorycortex in2weeks. At the same time, the high-frequency hearing demonstrates a significantimprovement (p<0.05).6、On one hand, the number of GABA-ergic neurons in primary auditory cortex of oldgroup is less then young group(p<0.05), on the other hand, the number of apoptosisGABA-ergic neurons in old group is more then young group(p<0.05)7、After transfecting primary auditory cortex2wssks, the expression of caspase-3andGABA-ergic neurons apoptosis decreased(p<0.05).Conclusion1、Constructing a recombinant adenoviral vector carried XIAP which with has a highexpression of XIAP and relieves the apoptosis of nerve cells.2、The high expression of XIAP in the primary auditory cortex neurons can effectivelyprotect the high-frequency hearing of presbycusis.3、The over expression of XIAP in primary auditory cortex inhibit the expression ofcaspase-3and the apoptosis of GABA-ergic neurons and enhance the expression of bcl-2.4、The above literatures suggest that XIAP inhibit the apoptosis of GABA-ergic neurons in primary auditory cortex by the path way of caspase-3, which may be a newtherapeutic method for presbycusis.