Noise Induced Microvascular Injury And Proteomics Changes Of Stria Vascularis

BackgroundNoise-induced hearing loss(NIHL)is a common sensorineural deafness.In addition to the mechanical damage,there are metabolic damage and other damage including a serious of biological,molecular changes.The stria vascularis located in lateral wall of the cochlea,and the blood-labyrinth barrier(BLB)in the stria vascularis is a specially differentiated capillary network that control exchanges between blood and intrastitial space in the cochlea,playing an essential role for the maintenance of endocochlear potential(EP).Dysfunction of stria vacularis,including blood-labyrinth barrier lead to inbanlance of endoclchlear microenvironment,is considered to be one of the etiologies of NIHL.The BLB is disrupted in the early stage of noise exposure,and damage and decrease of tight junction and adherens junction proein between ensothelial cells increase permeability.Vascular endothelial growth factor(VEGF)is a potent vascular permeability factor.VEGF in the stria vascularis is up-regulated after noise exposure.In addition to acting as cytokines,VEGF also play important role in regulating cell junction protein.Studies have shown that VEGF/VEGFR2 signaling can increase vascular permeability by activating tyrosine kinase,and knockdown of Src kinase can block the increase of permeability.Src kinase plays a crucial role in the regulation of VEGF on VE-cadherin.The isobaric tags for relative and absolute quantitation(iTRAQ)is an agent developed by ABI in the United States in 2004 to label proteins and peptides in vitro.Combined with liquid chromatography and mass spectrometry,it can label almost all peptides in protein samples,and has been widely used in protein identification and quantification of various samples such as human tissues,cells and body fluids.iTRAQ technology has been successfully applied to the study of microorganisms,animals and plants,brain nerve tissue and plasma.It can discover disease-specific proteins in non-physiological conditions,which is helpful for elucidating the pathogenesis of diseases and the prevention,diagnosis,treatment and prognosis monitoring of diseases.Parallel reaction monitoring(PRM)is a technique for targeting proteomics.It 1s applied to the detection of target proteins.It is mainly used for large-scale verification of differential proteins.It has the advantages of high throughput,specificity and high sensitivity.On the basis of constrcuction of a noise-induced deafness model.Immunohistochemistry,immunoblotting and laser confocal microscopy were used to observe the changes of VEGF,Src expression and microvascular permeability in stria vascularis after noise exposure.The VEGFR2 inhibitor SU5416 and Src inhibitor PPI were injected intraperitoneally to compare the changes of VE-cadherin expression after noise exposure,and to observe the changes of microvascular permeability.We found that the expression of Src in vascular endothelial cells and VEGF in marginal cells increased after niose exposure,while the expression of VE-cadherin decreased and the permeability of microvessels increased.The expression of Src in SU5416 group after application of SU5416 was decreased than that of NE group.The expression of VE-cadherin in PP1 group was significantly higher than that in NE group after Src inhibitor PP1,and the vascular microvascular permeability was lower than that of NE group.To study the proteomics change of stria vascularis after noise exposure.The iTRAQ technique was used to scree the differential protein of stria vascularis after niose exposure.GO analysis and KEGG analysis of differential proteins were performed using bioinformatics analysis.A total of 2852 proteins were identified by iTRAQ technology,and 171 differential proteins were expressed after noise exposure,of which 56 were up-regulated and 115 were down-regulated.Among the up-regulated proteins,proteins with higher expression levels include keratin type Ⅱ cytoskeleton 8(Krt8),vimentin(Vim),keratin type Ⅰ cytoskeleton 18(Krt18),and keratin type Ⅱ cytoskeleton 7(Krt7).Proteins with higher expression in down-regulated proteins include Kcnj10 protein(Kcnj10),gap junction protein beta-2(Gjb2),neuronal adhesion molecule 1(Ncaml),glutamyl ammonia synthase(Glul),gap junction protein beta-6(Gjb6)and apolipoprotein D(Apod).Proteins with a differential fold greater than 1.5 fold include Kcnj protein(Kcnj10),ubiquitin carboxy terminal hydrolase 14(Usp14),neuronal adhesion molecule 1(Ncaml),and gap junction protein beta-6(Gjb6).Proteins associated with n transport include Kcnj10 protein(Kcnj10),inward rectifier potassium channel 13(Kcnj13)and solute carrier family 12(Slc12a9).GO analysis found that up-regulated proteins are mainly involved in metabolic reactions such as immune response and oxidative phosphorylation,and down-regulated proteins are mainly about ion transport-related proteins.KEGG analysis of significant protein-rich pathways including Regulation of lipolysis in adipocytes,prion disease,MAPK signaling pathway,EPstein-Barr virus infection and nitrogen metabolism signaling pathway.PRM technology was used to verify the differential proteins screened in the second part.Among the nine proteins whose PRM verification 1s consistent with the iTRAQ quantification results,the up-regulated proteins include histidine-rich glycoprotein(Hrg),cAMP-dependent protein kinase catalytic subunit(Prkaea),keratin,type Ⅰ cytoskelcton 18(Krt18),Tetraspanin(Ca63)and cell adhesion molecule 4(Cadm4).Down-regulated proteins include apolipoprotein D(Apod),inositol proton transporter(Slc2a13),Kcnj10 protein(Kcnj10),and Myolb protein(Myo1b).GO analysis of its biological processes mainly involves cell adhesion,tight junctions,apoptosis,protein phosphorylation,oxidative stress,inflammatory factor response and K+ transport.After the noise exposure,the metabolic reaction such as immune reaction and oxidative phosphorylation is increased,and the down-regulation of ion transport-related proteins and potassium ion channels plays an important role in the regulation of potassium ion and microcirculation balance.Noise-mediated oxidation,catalytic activity,and stress response are the main causes of stria vascularis damage.The VEGF-VEGFR2 signaling pathway,VEGF/VEGFRZ activates Src kinase to regulate the expression of VE-eadherin,is an important mechanism for the changes of microvascular permeability in the stria vascularis.The focal adhesion and the MAPK signaling pathway also play an important role in the damage of stria vascularis after noise exposure.