Study Of Protective Mechanism Of Antioxidant Against Noise-induced Hair Cells Loss And Regulation To Apoptotic Ralated Gene Expression

NIHL is one kind of occupational diseases, have high disease rate. For many years, researchers are constantly going in for the study of the mechanism and precaution of NIHL. The death of HC is the main cause of hearing damage. Because HC of mammal cannot regenerate, the precautions of the death of HC become the key point of NIHL study. Confirmed, free radical plays an important role in strong noise induced damage of inner ears, the damage can become the antioxidant's rivalry. Further, the mechanism of free radical in the pathopoiesis of NIHL and the antioxidant in the pathway of clearance in free radical and damage rivalry of inner ears, are significant to the precaution of NIHL.Recently, establishing a stable animal model of NIHL which adopting multiple antioxidants to interfere the hearing functional disturbance and issue damage induced by noise. According to the measurement of noise in the ahead and post auditory threshold value, measure the protection of antioxidants in the hearing function of the model. Based on the membrane film hematoxylin nucleus staining and PI dyeing, ensure the death mode of cochlea hair cell induced by strong noise and depressant effect of apoptotic of cochlea cell by antioxidants. Advanced experimental technique can investigate the variation of MDA contents and SOD activity of cochlea issue of rats. To move forward a single step from biochemical indicator can see the pathogenic mechanism of NIHL induced by free radical and the pathway of antioxidants take part in the clearance of free radical. Imitated to use RT-PCR and immunohistochemical method to analysis the expression of Caspase-3,Caspase-9,Bcl-2 and NF-kB in cochlea issue, from transcription and protein level to identify the protection effects of antioxidants to the death of cochlea cell induced by noise exposure and possible regulatory mechanism. Discuss the multiple antioxidants and association affection to coordinative protection to NIHL, for the clinical application of precaution of antioxidants and theoretical evidence and experimental foundation of early treatment of NIHL.MethodDivide normal auditory without otitis media and healthy rats to seven groups randomly, 54 rats for the purely exposed group, 28 rats for others. With intragastric administration of VE, VC and Sodium Chloride individually for 30 d, in and after the exposing period , keeping on intragastric administration for 7 d. GSH group and VE+VC+GSH group in the noise exposure. Before one exposing hour, during the exposure and after exposure for 5 h of intraperitoneal injection of reducibility glutathione, and the normal group give the routine meal. All the rats in the experiments are constantly exposed to 116dBSPL,4KHz narrow noise for 6 hours. Based on the measurement by ABR, assess the different time points of audibility threshold changes and cut down the head for cochlea after the last measurement. Adopt the biochemical enzyme assessing kit to assess the content of MDA and SOD activity changes in each group of cochlea issue of rats; adopt PI staining method and basilar membrane film to observe the condition of the death of cochlear cell of each group; adopt immunity classification and RT-PCR to assess the expression of Caspase-3,Caspase-9,Bcl-2 and NF-kB in the cochlear issue of each group. On statistics, adopt mean±standard deviation ( x±S), base on the Sigma stat to analyze the single factor variance analysis, P<0.05 makes sense.Results:ABR assessing results: both purely exposed group and multiple antioxidants interfered group have obviously auditory damage, appearance is audibility threshold above moved (P<0.05). After the noise exposure for 1h, 1d, 2d, 3d, 7d to assess the audibility threshold value, according to the random designed several samples'mean comparison analysis, all have highly significant distinctions (P<0.05), except noise exposure for 1 h (P>0.05). Especially VE+VC+GSH group, exposure in noise for 1h has highly no different (P>0.05), others are significantly different (P<0.01), similar to the use of antioxidants group, all have highly significant differences (P<0.05).Variation of MDA content and SOD activity: both purely exposed group and multiple antioxidants interfered group cochlea MDA significantly higher than the normal group (P<0.01), among these is the interfered group significantly lower than the purely exposed group (P<0.05). Both purely exposed group and multiple antioxidants interfered group'SOD significantly lower than the normal group (P<0.01), among these results, interfered group is higher than the purely exposed group (P<0.05), VE+VC+CSH group significantly higher than other interfered groups (P<0.05).Basilar membrane film hematoxylin nucleus staining: staining coloration results, no exposed group cochlea HC eumorphism, its 3 row of OHC and IHC are to line up in order, structure clear and no depletion. Both purely exposed group and multiple antioxidants interfered group show up cell boundary obscure or vanish, low-return damage obvious, especially among the 3 row OHC, the third row of OHC damaged mostly, vanished significantly, the second row and the first row have different extents of vanish, similar to the parts of IHC. To number the total length of OHC to the basilar membrane, we can see the interfered group OHC depletion rate is significantly lower than the purely exposed group (P<0.05), especially for the VE+VC+GSH group (p<0.01), compared to other purely used antioxidant group has significantly different (P<0.05).Basilar membrane film PI staining: staining coloration results, no exposed group cochlea HC nuclus morphous is normal, its 3 row OHC and IHC are to line up in order, structure clear. Purely exposed group after exposure of four hours that hair cell turned to be nuclear swelling,pyknotic nucleus deletion, visible parts of OHC nucleus karyorrhexis and apoptotic body appeared, the main pattern of damage of hair cells is deletion.Immunity classification assess Caspase-9,Caspase-3 and Bcl-2 protein expressed in the cochlea. Masccline expression are Buffy or brown drops, Caspase-9,Caspase-3 mostly at the cell nucleus, few at the intracytoplasm. Through the hematoxylin counterstained, normal group cochlea cell turned to be blue. Adopt image analysis and software analyze the expression of protein gray scale value. As a result, VC and GSH interfered group Caspase-9 and Caspase-3 protein expression level significantly lower than the purely exposed group, (P<0.01), especially multiple factor interfered group, compared to other groups of interfered with antioxidants has significantly differences. In the VE interfered group, the activation of Caspase-3 and Caspase-9 have no significant deviation. In all the interfered groups, Bcl-2 protein expression higher than the purely exposed group (P<0.01), but VE+VC+GSH group Bcl-2 expression level have significantly difference than the interfered with multiple antioxidants group.RT-PCR results show that Caspase-9,Caspase-3 and Bcl-2 mRNA in each interfered group expression level match the expression regularity of accessing Caspase-9,Caspase-3 and Bcl-2 mRNA in cochlea with immunity classification.The adoption of immunofluorescence technic in accessing of VE in interfered group of NF-kB expression, show that purely exposed noise can induce cochlear hair cell NF-kB increased expression, laser copolymerization scanning software analysis of expression strength of fluorescence, VE interfered group has significantly lower expression of protein than the noise exposed group(p<0.01).Conclusion:The study is based on the stable NIHL animal model and multiple antioxidant interfere pretreatment of noise exposed animal model, individually observe the protection of antioxidants after hearing impairment induced by noise exposure.1. Noise induces MDA content increase and SOD activity depress of cochlea issue. Complementary of VE,VC and GSH can regulate MDA content and SOD activity of cochlea induced by noise, further to protect breakdown of auditory.2. Noise exposures induce ABR threshold value increase, auditory descend, and OHC of cochlea apoptosis. Apoptosis mainly happens in the OHC,SG and SV of cochlea. Complementary of VE,VC and GSH can reduce OHC of cochlea apoptosis, ABR threshold value descend.3. Caspase-9,Caspase-3 and Bcl-2 thimbleful of expression in the cochlea. After noise exposed, Caspase-9,Caspase-3 and Bcl-2 expression related to the apoptosis of OHC. Complementary of VE,VC and GSH can make the Caspase-9 and Caspase-3 expression descend, Bcl-2 expression increase.4. Complementary of VE,VC and GSH association affection compared to single factor interfered noise, have significantly counteract affection, like the content of MDA decrease and SOD activity increase, suppress the hair cell apoptosis, decrease cochlea Caspase-9 and Caspase-3 expression decrease, strengthen the expression of Bcl-2, further in coordination with the protection of auditory.5. Exogenous supply of VE can depress cochlear hair cell apoptosis, increase activation of antioxidase, protect the function of hearing, but not to influence expression activity of caspase-3 and caspase-9. VE probably through upstream transcription regulation factors NF-kB activity, to abide by the independence of caspase cell apoptosis system signal transmit pathway, depress the death of cochlear hair cell induced by the noise.To bring new ideas:1. Confirm the antioxidants of VC and VE interfered pretreatment of auditory protection in NIHL.2. Confirm VC,VE and GSH in the association of association of protection of interfered pretreatment of NIHL3. Investigated Caspase-9,Caspase-3 and Bcl-2 expression regularity after noise exposure and antioxidants of VC,GSH,VC+VE+GSH to regulate the expression regularity of Caspase-9,Caspase-3 and Bcl-2.4. First to confirm increased expression of NF-kB protein after noise injury. these findings provide evidence that VE protection to hair cells may occur through other pathways independent of caspase expression. Inhibition of NF-kB activity may play an important role in hair cells survival after noise injury.This research provides that clinical application of antioxidant therapy and preservation of NIHL detailed experimental evidence.