基于蛋白质组学的噪声性隐性听力损失机制研究

Mechanism of noise induced hidden hearing loss based on proteomics

目的通过蛋白质组学对噪声性隐性听力损失机制进行初步探索。方法于2022年10月,按照单纯随机抽样的方法将64只SPF级雄性C57BL/6J小鼠分为对照组和噪声暴露组,每组32只。噪声暴露组在声压级100 dB、2000~16000 Hz宽频噪声下暴露2 h,构建小鼠隐性听力损失模型。采用听性脑干反应(ABR)测试小鼠噪声暴露后第7天的听力阈值变化情况,免疫荧光观察基底膜毛细胞损伤情况,4D-Label-free定量蛋白组学鉴定并分析各组小鼠内耳差异表达蛋白质,并用蛋白印迹(Western blotting)法进行验证,采用方差分析和t检验对结果进行统计分析。结果噪声暴露后第7天,短声(click)、8000 Hz声刺激时两组小鼠听力阈值差异均无统计学意义(P>0.05),16000 Hz声刺激时噪声暴露组小鼠听力阈值明显高于对照组(P<0.05);共聚焦免疫荧光显示噪声暴露组小鼠耳蜗组织基底膜毛细胞排列整齐,但中回和底回突触前膜C末端结合蛋白2抗体(CtBP2)相对表达均明显少于对照组(P<0.05)。GO富集分析显示,差异表达蛋白功能主要集中在膜电位调节、配体门控通道活性、配体门控离子通道活性等。KEGG通路富集分析发现,差异表达蛋白明显富集的通路包括磷脂酰肌醇3激酶-蛋白激酶B(PI3K-Akt)信号通路、NOD样受体信号通路、钙信号通路等。Western blotting结果显示,噪声暴露组小鼠肌醇1,4,5-三磷酸受体3型(Itpr3)表达量升高,溶脂载体家族38成员2(2Slc38a2)表达量降低(P<0.05)。结论在构建的小鼠噪声性隐性听力损失模型中,通过蛋白质组学分析、筛选并验证差异表达蛋白Itpr3和Slc38a2,初步证实以Itpr3和Slc38a2为代表的谷氨酸能突触相关通路可能参与了隐性听力损失的发生。

ObjectiveTo explore the mechanism of noise-induced hidden hearing loss by proteomics.MethodsIn October 2022,64 SPF male C57BL/6J mice were divided into control group and noise exposure group with 32 mice in each group according to random sampling method.The noise exposure group was exposed to 100 dB sound pressure level,2000-16000 Hz broadband noise for 2 h,and the mouse hidden hearing loss model was established.Auditory brainstem response(ABR)was used to test the change of hearing threshold of mice on the 7th day after noise exposure,the damage of basal membrane hair cells was observed by immunofluorescence,and the differentially expressed proteins in the inner ear of mice in each group were identified and analyzed by 4D-Label-free quantitative proteomics,and verified by Western blotting.The results were statistically analyzed by ANOVA and t test.ResultsOn the 7th day after noise exposure,there was no significant difference in hearing threshold between the control group and the noise exposure group at click and 8000 Hz acoustic stimulation(P>0.05).The hearing threshold in the noise exposure group was significantly higher than that in the control group under 16000 Hz acoustic stimulation(P<0.05).Confocal immunofluorescence showed that the basal membrane hair cells of cochlear tissue in noise exposure group were arranged neatly,but the relative expression of C-terminal binding protein 2 antibody of presynaptic membrane in middle gyrus and basal gyrus was significantly lower than that in control group(P<0.05).GO enrichment analysis showed that the functions of differentially expressed proteins were mainly concentrated in membrane potential regulation,ligand-gated channel activity,and ligand-gated ion channel activity.KEGG pathway enrichment analysis showed that differentially expressed proteins were significantly enriched in phosphatidylinositol 3 kinase-protein kinase B(PI3K-Akt)signaling pathway,NOD-like receptor signaling pathway,calcium signaling pathway,etc.Western blotting showed that the expression of inositol 1,4,5-trisphosphate receptor 3(Itpr3)was increased and the expression of solute carrier family 38 member 2(Slc38a2)was decreased in the noise exposure group(P<0.05).ConclusionThrough proteomic analysis,screening and verification of the differential expression proteins Itpr3 and Slc38a2 in the constructed mouse noise-induced hidden hearing loss model,the glutaminergic synaptic related pathways represented by Itpr3 and Slc38a2 may be involved in the occurrence of hidden hearing loss.