The Observation About The Effort Of Nrf2-HDAC2 Axis In Sudden Hearing Loss Induced By Oxidative Stress

Objective At present,Sudden sensorineural loss is a high otology diseases.Its pathogenesis is unknown clearly.There have some reports argue that oxidative stress may take part in the pathogenesis of Sudden sensorineural loss which by producing amounts of reactive oxygen species destroy the cells of the cochlear and spiral ganglia and cause hearing impairment.Nrf2 can translate from cytoplasmic to nuclear to active a series of Ⅱ detoxication enzymes and Antioxygen factor,under the ROS invade.As we know,Glucocorticoid is a useful drug but ineffective in parts of patients who’s hearing have no improve after treatment,we called this glucocorticoid resistance which may have related with HDAC2 as our former research.To this project we study:1.Collected SSNHL patients fasting plasma before and after the treatment,detect the antioxidant index,judge the oxidative stress involved in the pathogenesis of SSNHL or not.2,gathering PBMC fromSSNHL patients’s blood.detected the Nrf2/HDAC2 level,compare Nrf2 in SSNHL patient and the relationship between HDAC2 and GC.3,on the basis of develop auditory cells(HEI-OC1),construct oxidative stress model,the research on interference Nrf2 HEI-OC1 cells under the stimulus accept OS,HDAC2 expression changes,study the correlation of Nrf2 and HDAC2.To SSNHL pathogenesis to find the reason and to further find SSNHL patients with GC resistance cause of HDAC2 expression of decline,targeted therapy,and improve ear hearing.Methods Clinical trials:Patients with SSNHL have treatment in Nanjing Gulou hospital otolaryngology department,collected blood in patients on an empty stomach before and after treatment,and collected blood in the healthy volunteers on an empty stomach as control group.Ultraclean PBMC and plasma extraction in patients after centrifugal and then make the following test:1,to detect SSNHL patients plasma antioxidant indicators before and after treatment:SOD content,to determine whether oxidative stress take part in the pathogenesis of SSNHL.2,detect the expression detectionof Nrf2/HDAC2 level:collected blood in patients on an empty stomach before and after treatment,Extraction of PBMC of total RNA and the nucleus protein,to detect patients with PBMC of Nrf2 mRNA expression level in the real-time PCR assay,ELISA and Peggy Sue Microprotein detection technology.3,Divided the patients into the GC and GC resistance group according to if the frequency of PTA improvement>15 db,compared the Nrf2/HDAC2 level expression in different groups.Cell experiment:1,Using different concentrations of H2O2 to deal with HEI-OC1 cells,CCK 8 experimental detection cell activity,groping the optimum condition of setting up HEI-OC1 in vitro cell oxidative stress model.2,In HEI-OC1 cell transfection Nrf2 interference fragment Si-RNA interference or Nrf2 expression plasmid expressing,after the success of the confirm transfection,again through the PCR/Western blot detection Nrf2,HDAC2 processing protein assays in oxidative stress model by HEI-OC1 cells between different groups(Nrf2-RNA interference group,Nrf2 plasmid expression group,normal oxidative stress stimulation group).3,Discuss the change of protein levels,the comparative analysis between the correlation between Nrf2 and HDAC2.Results Clinical trials:1,Before treatment of SSNHL patients plasma SOD content GC effective group(0.431 ±0.026)and GC resistance group(0.441±0.033)was significantly lower than the control group(0.497±0.049).2,SSNHL patients HDAC2 mRNA expression despite GC effective group(0.439±0.009)or GC resistance group(0.432±0.010)is higher than the normal control group(0.406±0.019),the GC effective group after treatment have improved,while GC resistance group after treatment have no obvious change.3,SSNHL patients HDAC2 protein expression despite GC effective group(0.442±0.045)or GC resistance group(0.463±0.042)is lower than the normal control group(0.578±0.023),the GC effective group after treatment have improved,while GC resistance group after treatment have no obvious change.4,SSNHL patients Nrf2 mRNA expression despite GC effective group(0.510±0.049)or GC resistance group(0.53±0.065)is lower than the normal control group(0.67±0.1119),the GC effective group after treatment have improved,while GC resistance group after treatment have no obvious change.5,Nrf2 protein expression is improved in GC effective group after treatment while GC resistance group have no change Cell experiment:1,H2O2 treatment can concentration dependence of HEI-OC1 induced cell death,and 1.0 tendency for 24 h/L H2O2 treatment is established in vitro HEI-OC1 cell the best condition of HEI-OC1 cell oxidative stress model.2,HEI-OC1 cells under the condition of same oxidative stress,HDAC2 protein expression in the Nrf2 Si-RNA interference group is lower than normal oxidative stress stimulation,and HDAC2 protein expression in Nrf2 plasmid expression group was higher than normal oxidative stress stimulation,Nrf2 and HDAC2 correlation between each other.3,CCK-8 experimental results confirmed that using the Si-Nrf2 small RNA interference expression can lead to HEI-OC1 cell oxidative stress under cell activity decreased.Conclusions 1,Oxidative stress may be involved in the pathogenesis of SSNHL and the decline o.f Nrf2 may also have effort to SSNHL.2,which show again about the former research:HDAC2 may effert the GC sensitivity in SSNHL patients.3,there have some relationship between Nrf2 and HDAC2 that can effect GC sensitivity.4,Through the study of HEI-OC1 cells,we confirmed that Nrf2 can effect HDAC2 in inner environment,demonstrate Nrf2-HDAC2 axis in inner environment.