Studies Of Expression And Regulation Of APE/Ref-1 In Rat Spiral Ganglion Cells And Its Potential Role After Oxidative Stress

Background: Damage of auditory neurons is one of the major causes of sensorineural deafess. Oxidative stress has been identified as a basic mechanism involved in spiral ganglion cells death caused by drug ototoxicity,noise exposure, Age-related causes. The multifunction DNA repair/redox enzyme APE/Ref-1 is involved in the repair of DNA damage as well as in the transcriptional regulation of genes. Indeed, APE/Ref-1 protects cells from the cytotoxicity of oxidizing agents and is able to interfere with gene expression by regulating the activity of several transcription factors .Despite abundance of APE/Ref-1 in neurons and correlations between alterations in APE/Ref-1 levels and various neuropathologies ,few ,if any, studys have addressed the question of expression of APE/Ref-1 in rat spiral ganglion cells and whether APE/Ref-1 is involved in preventing spiral ganglion cell after Oxidative stress.To address this question and begin to directly elucidate the role of APE/Ref-1 in auditory neurons afer Oxidative stress. First, we examined expression of APE/Ref-1 in cochlear of rat.We isolated and cultured spiral ganglion cells of the rat cochlea in vitro. Furthermore, We examined the effects of altering APE/Ref-1 expression in primary cultures of spiral ganglion cells on oxidative damage induced by exposure to H2O2.To alter the activity of APE/Ref-1 in spiral ganglion cells,we developed a method to improve APE/Ref-1 protein levels using recombinant adenovirus and to reduce APE/Ref-1 protein levels using silencing RNA (isRNA) methodology.Objective: This study was aimed to address the question of expression of APE/Ref-1 in rat spiral ganglion cell and whether APE/Ref-1 is involved in preventing spiral ganglion cells oxidative damage after Oxidative stress.Methods:1.The expression of APE/Ref-1 in rat cochlear was determined by Immunohistochemistry.2. Spiral ganglion cells of the rat cochlea was isolated and cultured in vitro. And Spiral ganglion cells were grown in culture for 48 hours and then treated with H2O2 (0,10,25,50,100,300μmol/L)for 1h,and then changed back into normal medium .The cell viability was determined by MTT and the apoptosis of Spiral ganglion cells was determined by terminal-deoxynucleotidyl transferase mediated nick end labeling(TUNEL).3. Directed cloning and homologous recombination were employed to generate adenovirus containing APE/Ref-1. Primary cultured Spiral ganglion cells were infected with the adenovirus containing APE/Ref-1. Following up expression of GFP, we detected the infective efficiency of virus response to different MOI(multiplicity of infection), and the positive rate of GFP expression at 24h, 48h and 72h after adenovirus infection, we ascertained the suitable infection time and MOI.4. RT-PCR and Western Blot were used to detect the level of APE/Ref-1 mRNA and protein in the infected cells to ensure APE/Ref-1 overexpression as a result of adenovirus infection. then combining with viability of Spiral ganglion cells cultured in oxidative stress environment was evaluated by MTT and the apoptosis of Spiral ganglion cells was determined by TUNEL.5.The silencing RNA expression vector PGenesil1-APE/Ref1 was constructed, then PGenesil1-APE/Ref-1 was transfected into Spiral ganglion cells by lipofectamineTM 2000.6.RT-PCR and Western Blot were used to detect the level of APE/Ref-1 mRNA and protein in the infected cells to ensure reduction of APE/Ref-1 expression as a result of a PGenesil1-APE/Ref1 infection. then combining with viability of Spiral ganglion cells cultured in oxidative stress environment was evaluated by MTT and the apoptosis of Spiral ganglion cells was determined by TUNEL.Results:1.The abundance of APE/Ref1-like-immunoreactivity was detected in a variety of tissues of rat cochlear, including Spiral ganglion cells , stria vascularis, spiral ligament and Corti organ.2.The Spiral ganglion cells was successfully cultured in vitro. When Spiral ganglion cells were grown in culture for 24h, the typic bipolar neurons and occasionally tri-pole neurons could be observed. At the end of the 8 days ,the cells began to die. The Spiral ganglion cells were identified using specific rabbit anti-NSE antibody according to red fluorescence.3.MTT showed that a statistically significant (P<0.01)reduction in cell viability in cultures treated with different concentration H2O2 from 50 to 300μmol/L. (H2O2:50μmol/L,OD=0.1917±0.0217; H2O2 : 100μmol/L,OD=0.1542±0.0165, H2O2 :300μmol/L,OD=0.1322±0.0151,P<0.01).inversely,the apoptosis of cells was significantly improved (H2O2:50μmol/L, 14.41±1.68%; H2O2:100μmol/L, 25.52±1.71%1; H2O2:300μmol/L,OD=43.51±2.13%,P<0.01).4.Via directed cloning and homologous recombination, infection-competent adenovirus was generated in the AD293 package cell line. Following repeatedly infecting, high titer adenovirus particles yielded.5. After Ad-APE/Ref-1,Ad-GFP infection, the positive rate of GFP expression in the host Spiral ganglion cells was growing up in MOI-dependent and time-dependent manner, When MOI increased up to 150 or above, the percent of cardiomyocytes expressing GFP exceeded 90%; For each strata of MOI, there was no more increase in the positive rate of GFP expression as the expression time exceeded 48h, suggesting that this time point seemed be suitable for consequent procedures.6. Findings with RT-PCR and Western Blot showed that infection of adenovirus resulted in APE/Ref-1 overexpression in the Spiral ganglion cells.7.Overexpression of APE/Ref-1 significantly improved cell viability in cultures treated with different concentration H2O2 from 50 to 300μmol/L. Inversely, the apoptosis of cells was significantly inhibited .8.Based on the rat APE/Ref-1 cDNA sequence ,Synthesized 64nt oligonucleotides containing a unique 21nt sequence derived from the target transcript were cloned into the vector pGenesil-1 .Silencing RNA (isRNA) PGenesil1-APE/Ref-1 was constructed.9.PGenesil1-APE/Ref-1 was transfected into Spiral ganglion cells using lipofectamineTM 2000,after 72h, Expression of APE/Ref-1 mRNA and protein was reduced,about 40～50%.10. After the expression of APE/Ref-1 was selectively reduced ,The cell viability was significantly inhibited (P<0.05),compared with control group. Inversely, the apoptosis of cells was significantly improved.Conclusion:1.APE/Ref-1 was expression in rat cochlear, including Spiral ganglion cells,stria vascularis, spiral ligament and Corti organ.2. The Spiral ganglion cells was successfully cultured in vitro. The spiral ganglion cells were induced to be Oxidative damaged by H2O2.3.Recombinant adenovirus expression Ad-APE/Ref1 was successly constructed.4.Overexpression of APE/Ref-1 could protect Spiral ganglion cells from oxidative damage.5.Short hairpin RNA (shRNA) PGenesil1-APE/Ref-1 was successlly constructed.6.The cell viability was significantly inhibited in oxidative environment flowing expression of APE/Ref-1 reduction. It shows that APE/Ref-1 is necessary for optimal levels of SGCs survival.7. APE/Ref-1 could protect Spiral ganglion cells from oxidative injury that may be involved in inhibition apoptosis of cells.