【作者】 苗龙；

【导师】 浦跃朴；

【作者基本信息】 东南大学 ， 劳动卫生与环境卫生学， 2023， 博士

【摘要】 研究背景与目的噪声性听力损失(Noise-induced hearing loss,NIHL)是人类第二大最常见的感音神经性听力障碍。在中国,NIHL已成为第二大有害性职业病,约占每年职业病增加量的六分之一,严重影响着作业工人的身心健康。NIHL是一种复杂性的疾病,其确切发病机制尚不完全清楚。越来越多的研究证据表明蛋白质和代谢物的异常表达与疾病的发生发展密切相关。蛋白质组学和代谢组学在描述疾病发生过程中蛋白质和内源性代谢物总体变化特征、寻找生物标志物和阐明发病机制方面显示出强大的潜力。NIHL的发生具有显著的个体遗传易感性差异,在稳定噪声作业环境下,遗传性因素对NIHL发生的贡献率超过50%。近年来,针对NIHL易感性研究主要以单一基因水平研究为主,已发现氧化应激基因(GSTM1、PON2、SOD1、SOD2等)、钾离子循环基因(KCNQ1、KCNQ4、GJB1等)、热休克蛋白基因(HSP70)、钙黏蛋白基因(CDH23、PCDH15)、头状转录因子(GRHL2)等基因的某些单核苷酸多态性(Single nucleotide polymorphism,SNP)位点与NIHL易感性相关,可能是NIHL遗传易感生物标志物。目前NIHL易感性研究仅解释了少量的NIHL发病风险,因此还需进一步发掘和探索新的NIHL致病基因和遗传变异。基于以上背景,本课题旨在采用横断面调查法分析江苏省内重点职业病监测企业噪声作业工人NIHL现状及其影响因素。利用蛋白质组学和代谢组学技术分析NIHL工人和听力正常工人血浆样本中蛋白质和内源性代谢物表达谱差异,筛选与NIHL发生发展相关的差异表达蛋白和内源性代谢物及异常信号通路。基于NIHL小鼠模型,利用蛋白质组学和代谢组学技术分析NIHL小鼠和听力正常小鼠耳蜗组织中蛋白质和代谢物表达谱差异,进一步发掘NIHL关键差异表达蛋白和代谢物及异常信号通路。筛选NIHL工人血浆和NIHL小鼠耳蜗组织中相同的差异表达蛋白,探讨其在NIHL中的功能作用及潜在分子调控机制。此外,筛选NIHL关键差异表达蛋白或异常信号通路中关键调控基因功能性SNPs位点,探讨其与NIHL易感性间的关系及其潜在的分子作用机制。研究方法1.收集2015-2020年间江苏省内重点职业病监测企业77990名在岗噪声作业工人职业健康体检资料,分析噪声作业工人NIHL患病率。按性别、年龄、接噪工龄、噪声暴露水平、吸烟和饮酒分为不同亚组,分析各组间噪声作业工人NIHL患病率,并应用多因素Logistic回归分析评估各变量与NIHL发病风险间的关联强度。2.利用TMT标记的定量蛋白质组学技术对3名NIHL工人和3名听力正常工人血浆样本进行蛋白表达谱差异分析,利用非靶向HPLC/Q-TOF-MS代谢组学技术对62名NIHL工人和62名听力正常工人血浆样本进行代谢表达谱差异分析,筛选NIHL的差异表达蛋白和内源性代谢物。应用Western blot法对候选差异表达蛋白POU4F3、ATG5和COLEC11进行验证。采用RT-q PCR检测工人外周血中PIK3CA、AKT2、Beclin1和ATG5基因表达水平,并应用Elisa法检测工人血浆中SOD、GSH-Px、IL-6和TNF-α的水平。3.选用6-8周龄雄性C57BL/6品系小鼠作为实验研究对象,将其分为NIHL组和对照组。通过给予NIHL组小鼠120 d B SPL宽带白噪声4 h暴露,对照组小鼠不给予噪声暴露构建NIHL小鼠模型。对小鼠进行听性脑干反应(Auditory brainstem response,ABR)检测,提取耳蜗组织进行耳蜗基底膜铺片和免疫荧光染色分析。利用TMT标记的定量蛋白质组学技术对6只NIHL小鼠和6只听力正常小鼠的耳蜗组织进行蛋白质表达谱差异分析,利用非靶向HPLC/Q-TOF-MS代谢组学技术对5只NIHL小鼠和5只听力正常小鼠的耳蜗组织进行代谢表达谱差异分析,筛选NIHL的差异表达蛋白和代谢物及信号通路。采用Western blot对候选差异表达蛋白ITGA1、KNG1、CFI、FGF1、AKT2、ATG5和POU4F3进行验证。采用Elisa检测小鼠耳蜗组织中SOD、MDA、IL-6和TNF-α的水平。应用Western blot、冰冻切片和免疫荧光染色技术对小鼠耳蜗组织中SOD1和SOD2蛋白表达及定位进行分析。4.应用不同浓度(0、0.25、0.5、1、10μg/m L)的LPS给予小鼠耳蜗毛细胞HEI-OC1不同时间(24、48、72 h)处理构建HEI-OC1细胞炎症模型。采用CCK-8法检测细胞增殖活性,采用流式细胞术和免疫荧光染色技术检测细胞凋亡和活性氧(Reactive oxygen species,ROS)水平,并应用Elisa检测细胞培养上清液中IL-6和TNF-α的水平。采用Western blot检测LPS处理后细胞内POU4F3、ATG5、LC3B、p62、Bax及Bcl-2蛋白表达变化。此外,应用Western blot检测自噬激活剂Rapamycin(RAP)和自噬抑制剂3-MA处理后细胞内LC3B、p62、Bax和Bcl-2蛋白表达变化。构建POU4F3和ATG5敲降的HEI-OC1细胞株,采用流式细胞术、免疫荧光染色、Western blot技术检测细胞凋亡和ROS水平变化,以及LC3B、p62、Bax和Bcl-2蛋白表达变化。5.收集江苏省内重点职业病监测企业噪声作业工人职业健康体检资料,将其分为NIHL组和听力正常对照组。基于NCBI db SNP,1000Genomes和Hap Map数据库,按照最小等位基因频率(Minimum allele frequency,MAF)>0.01和连锁不平衡r~2>0.8筛选自噬相关基因PIK3CA、PIK3R1、PIK3R3、AKT1、ATG4、ATG5、ATG7功能性SNPs位点,并采用多重PCR结合二代测序技术对其进行基因分型。利用生物信息学数据库(TFtarget、Func Pred)、定点突变、质粒构建、RT-q PCR、双荧光素酶报告基因、RNA干扰、细胞转染和Western blot实验探讨PIK3R3 rs7536272、ATG5 rs510432、转录因子SP1以及C/EBPβ参与NIHL发生过程的潜在分子调控机制。研究结果1.噪声作业工人的NIHL现状调查及影响因素分析77990名噪声作业工人的平均噪声暴露水平为87.79±7.24 d B(A),26650人被诊断为NIHL,NIHL总体患病率为34.17%。男性、年龄>35岁、接噪工龄>5年、噪声暴露水平>85 d B(A)、吸烟和饮酒工人NIHL患病率显著升高(P<0.001)。Logistic回归分析显示,男性、年龄>35岁、接噪工龄>5年、噪声暴露水平>85 d B(A)、吸烟和饮酒与NIHL发病风险相关,是影响NIHL的危险因素。2.NIHL工人的血浆蛋白质组学和代谢组学研究2.1基于TMT标记定量技术的NIHL工人的血浆蛋白质组学研究按照差异倍数(Fold change,FC)>1.2且P<0.05,NIHL组和对照组间共筛选出22个差异表达蛋白。KEGG通路富集分析显示,差异表达蛋白显著富集于免疫炎症、自噬、蛋白合成与加工、糖代谢相关信号通路中。Western blot结果显示,NIHL工人血浆中ATG5和POU4F3蛋白表达较对照组显著降低(P<0.05),COLEC11蛋白表达较对照组显著升高(P<0.05)。2.2基于HPLC/Q-TOF-MS技术的NIHL工人的血浆代谢组学研究根据变量重要性投影(Variable important for the projection,VIP)>1且P<0.05,NIHL组和对照组间共鉴定到20种差异代谢物。KEGG通路富集分析显示,差异代谢物显著富集于磷脂代谢、氨基酸代谢、自噬相关代谢通路中。RT-q PCR结果显示,相比于对照组,NIHL组工人外周血中PIK3CA、AKT2、Beclin1和ATG5基因m RNA表达显著降低(P<0.05)。Elisa结果显示,NIHL组工人血浆中SOD和GSH-Px水平较对照组显著降低(P<0.01),IL-6和TNF-α水平较对照组显著升高(P<0.001)。3.基于NIHL模型的小鼠耳蜗组织的蛋白质组学和代谢组学研究3.1 NIHL小鼠模型的构建与对照组相比,NIHL组小鼠在4、8、12、16、24和32 k Hz测试频率下的ABR阈值显著升高(P<0.001)。NIHL组小鼠耳蜗中段和底段外毛细胞(Outer hair cells,OHCs)计数较对照组显著减少(P<0.01)。3.2基于TMT标记定量技术的NIHL小鼠耳蜗组织的蛋白质组学研究根据FC>1.2且P<0.05,两组间共筛选到221种差异表达蛋白,表达上调蛋白110个,表达下调蛋白111个。KEGG通路富集分析显示,差异表达蛋白显著富集于免疫炎症、蛋白合成与加工、细胞增殖、分化和凋亡、自噬、糖代谢相关信号通路。Western blot结果显示,NIHL小鼠耳蜗组织中ITGA1、KNG1和CFI蛋白表达较对照组显著升高(P<0.05),FGF1、AKT2、ATG5和POU4F3蛋白表达较对照组显著降低(P<0.05)。3.3基于HPLC/Q-TOF-MS技术的NIHL小鼠耳蜗组织的代谢组学研究基于VIP>1且P<0.05,两组间共鉴定出86种差异代谢物,48种表达上调差异代谢物和38种表达下调差异代谢物。KEGG通路富集分析显示,差异代谢物显著富集于糖代谢、氨基酸代谢、磷脂代谢、自噬相关代谢通路中。免疫荧光染色和Western blot分析结果显示,SOD1和SOD2特异性表达于小鼠耳蜗组织螺旋神经节中,且SOD1和SOD2在NIHL组小鼠耳蜗组织中的表达较对照组显著降低(P(27)0.05)。Elisa结果显示,NIHL组小鼠耳蜗组织中SOD活性较对照组显著降低(P<0.01),MDA、TNF-α和IL-6水平较对照组显著升高(P<0.05)。4.POU4F3和ATG5在HEI-OC1细胞炎症中的分子机制研究4.1 LPS诱导HEI-OC1细胞炎症损伤模型的构建CCK-8结果显示,与对照组(0μg/m L)相比,1μg/m L LPS处理48 h组HEI-OC1细胞增殖活性显著降低(P<0.001),且随着LPS浓度和时间的增加而加剧。Elisa结果显示,1μg/m L LPS处理48 h后细胞培养上清液中TNF-α和IL-6水平较对照组显著升高(P<0.01)。流式细胞凋亡和ROS检测结果显示,1μg/m L LPS处理48 h组细胞凋亡和ROS水平较对照组明显升高(P<0.01)。4.2 POU4F3和ATG5在HEI-OC1细胞炎症损伤中的分子调控机制研究Western blot结果显示,POU4F3蛋白表达随着LPS浓度的增加显著降低(P<0.001)。ATG5和LC3-II蛋白表达随着LPS浓度的增加先升高后降低,在1μg/m L LPS作用下表达水平最高(P<0.001)。相较于对照组,Bax和Cleaved Caspase-9蛋白在1μg/m L和10μg/m L LPS作用下表达显著升高(P<0.001),Bcl-2蛋白表达显著降低(P<0.001)。LPS+3-MA组LC3-II和Bax蛋白表达较LPS组显著降低(P<0.001),p62和Bcl-2表达较LPS组显著增加(P<0.001)。与LPS组相比,LPS+RAP组LC3-II和Bax蛋白表达显著增加(P<0.001),p62和Bcl-2表达显著降低(P<0.001)。LPS+si-POU4F3组LC3-II和Bax蛋白表达较LPS组显著升高(P<0.001),p62和Bcl-2表达较LPS组显著降低(P<0.01)。流式凋亡和ROS结果显示,与LPS组相比,LPS+si-POU4F3组细胞凋亡和ROS水平均显著升高(P<0.01)。相较于LPS组,LPS+si-ATG5组LC3-II、ATG5和Bax蛋白表达显著降低(P<0.001),p62和Bcl-2表达显著增加(P<0.001)。流式凋亡和ROS结果显示,LPS+si-ATG5组细胞凋亡和ROS水平较LPS组显著降低(P<0.01)。5.PIK3R3和ATG5基因多态性与NIHL易感性的关系及其机制研究5.1 PIK3R3和ATG5基因多态性与NIHL易感性的关系纳入688名NIHL工人作为NIHL组和667名听力正常工人作为对照组,年龄、性别、接噪工龄、噪声暴露水平、吸烟和饮酒情况在两组间分布差异均无统计学意义(P>0.05)。NIHL组双耳高频平均听阈(Binaural high frequency threshold on average,BHFTA)显著高于对照组(P<0.001)。PIK3R3 rs7536272和ATG5 rs510432与NIHL显著相关(P<0.05),rs7536272 G等位基因显著增加NIHL的发病风险(adjusted OR=1.22,95%CI=1.03-1.45),rs510432 T等位基因显著降低NIHL的发病风险(adjusted OR=0.77,95%CI=0.66-0.90)。5.2 PIK3R3和ATG5基因多态性影响NIHL易感性的机制研究RT-qPCR结果显示,携带rs7536272 GG基因型的个体外周血中PIK3R3基因m RNA表达水平显著低于携带AA/AG基因型的个体(P<0.01),携带rs510432 CT/TT基因型的个体外周血中ATG5基因m RNA表达水平显著高于携带CC基因型的个体(P<0.01)。双荧光素酶报告基因结果显示,rs7536272野生型A等位基因和SP1过表达质粒共转染组HEI-OC1细胞荧光素酶活性较对照组和突变型G等位基因和SP1过表达质粒共转染组显著增加(P<0.001)。rs510432突变型T等位基因和C/EBPβ阴性对照质粒共转染组细胞荧光素酶活性较野生型C等位基因和C/EBPβ阴性对照质粒共转染组明显增加(P<0.001);突变型T等位基因和C/EBPβ过表达质粒共转染组细胞荧光素酶活性较突变型T等位基因和C/EBPβ阴性对照质粒共转染组显著降低(P<0.001)。Western blot分析结果显示,与阴性对照组相比,si-SP1转染组LC3-II、Caspase-3和Bax蛋白表达显著升高(P<0.01),p-PI3K/PI3K、p-AKT/AKT和Bcl-2蛋白表达显著降低(P<0.01)。si-C/EBPβ转染组ATG5、LC3-II、Bcl-2蛋白表达较阴性对照组显著增加(P<0.05),Caspase-3和Bax蛋白表达较阴性对照组显著降低(P<0.05)。研究结论1.噪声作业工人NIHL患病率较高,男性、年龄、接噪工龄、噪声暴露水平、吸烟和饮酒与NIHL发病风险密切相关,是影响NIHL的危险因素。2.ATG5和POU4F3蛋白的异常表达参与NIHL发生发展过程,可能是NIHL潜在生物标志物。3.免疫炎症、氧化应激和自噬在NIHL中扮演关键角色,可能是NIHL重要的病理生理机制。4.自噬在LPS诱导的HEI-OC1细胞炎症过程中起促细胞凋亡的作用,LPS可诱导HEI-OC1细胞自噬性死亡。5.POU4F3和ATG5在LPS诱导的HEI-OC1细胞损伤过程中可通过调控自噬活性影响细胞凋亡进而影响细胞对炎症的敏感性。6.PIK3R3 rs7536272和ATG5 rs510432与NIHL易感性相关,可能是NIHL潜在易感生物标志物;转录因子SP1和C/EBPβ可通过调控细胞自噬和凋亡参与NIHL发生过程,可能是NIHL潜在治疗靶点。更多还原

【Abstract】 Background and objectivesNoise-induced hearing loss(NIHL)is the second most common sensorineural hearing disorder in humans.NIHL has become the second most harmful occupational disease in China,accounting for about one-sixth of the annual increase in occupational diseases,seriously influencing the physical and mental health of workers.NIHL is a complex disease,and its exact pathogenic mechanisms are not fully understood.More and more evidence show that the abnormal expression of proteins and metabolites is closely associated with the occurrence and development of diseases.Proteomics and metabolomics have great potential in describing the overall change characteristics of proteins and endogenous metabolites in the process of diseases occurrence,clarifying the pathogenesis,and searching for biomarkers.There are obvious individual susceptibility differences in the occurrence of NIHL,the contribution rate of genetic factors to the occurrence of NIHL exceeds 50%under the stable noise working environment.Nowadays,although some progress has been made in the research of susceptibility to NIHL,however,most of the single nucleotide polymorphisms(SNPs)studied were not functional,which might not be the genetic variation directly involving in the regulation of the susceptibility to NIHL,therefore,it is difficult to reveal the real cause of NIHL susceptibility and cannot be used as the genetic markers for the diagnosis of NIHL susceptibility.Based on the background mentioned above,this study aims to analyze the present situation and influencing factors of NIHL of workers who exposed to noise in the key occupational disease monitoring enterprises in Jiangsu using cross-sectional survey.Proteomics and metabolomics techniques were used to analyze the differences of expression profiles of protein and endogenous metabolite in the plasma samples of NIHL workers and normal hearing workers,and screen differentially expressed proteins,endogenous metabolites,and abnormal signal pathways related to the occurrence and development of NIHL.Based on the NIHL mouse model,proteomics and metabolomics techniques were used to analyze the differences of protein and metabolite expression profiles in the cochlear tissues of NIHL mice and normal hearing mice,and further explore the key differentially expressed proteins,metabolites,and abnormal signal pathways of NIHL.To screen the same differentially expressed proteins in the plasmas of NIHL workers and the cochlear tissues of NIHL mice and to explore its functional roles and potential molecular regulatory mechanisms in NIHL.Moreover,to screen the functional SNPs of key differentially expressed proteins or key regulatory genes in abnormal signal pathways of NIHL and to explore its relationships with susceptibility to NIHL and potential molecular mechanisms.Methods1.The occupational health examination data of 77990 noise-exposed workers from the key occupational disease monitoring enterprises in Jiangsu during 2015-2020 were collected to analyze the prevalence of NIHL among noise-exposed workers.The gender,age,noise exposure time,noise exposure level,smoking and alcohol consumption were divided into different subgroups and the prevalence of NIHL of noise-exposed workers was analyzed,and multivariate logistic regression analysis was performed to evaluate the strength of the association between each variable and the risk of NIHL.2.TMT-labeled quantitative proteomics was conducted to analyze the differences of protein expression profiles in plasma samples from 3 NIHL workers and 3 normal hearing workers,and non-targeted HPLC/Q-TOF-MS metabolomics was performed to analyze the differences of metabolic expression profiles in plasma samples of 62 NIHL workers and 62 normal hearing workers,and to screen differentially expressed proteins,and endogenous metabolites of NIHL.The candidate differentially expressed proteins POU4F3,ATG5 and COLEC11 were verified by Western blot.RT-q PCR was used to detect the expression levels of PIK3CA,AKT2,Beclin1,ATG5 genes in the peripheral blood of workers,and Elisa was performed to detect the levels of SOD,GSH-Px,IL-6 and TNF-αin the plasma of workers.3.Male C57BL/6 mice aged 6-8 weeks were selected as experimental objects and divided into NIHL group and control group.The mice in NIHL group were exposed to 120 d B SPL broadband white noise for 4 h,while the control mice were not exposed to noise to construct the mouse model of NIHL.Auditory brainstem response(ABR)was carried out in mice,the cochlear tissues of mice were extracted for cochlear basement membrane preparation and immunofluorescence staining analysis.Based on the NIHL mouse model,TMT-labeled quantitative proteomics was used to analyze the differences of protein expression profiles in cochlear tissues of 6 NIHL mice and 6 normal hearing mice,and non-targeted HPLC/Q-TOF-MS metabolomics was performed to analyze the differences of metabolic expression profiles in cochlear tissues of 5 NIHL mice and 5 normal hearing mice,to screen the differentially expressed proteins,metabolites,and signal pathways of NIHL.Western blot was used to verify the candidate differentially expressed proteins ITGA1,KNG1,CFI,FGF1,AKT2,ATG5 and POU4F3.The levels of SOD,MDA,IL-6 and TNF-αin cochlear tissues of mice were measured by Elisa.Western blot,frozen section and immunofluorescence staining were used to analyze the expression and localization of SOD1 and SOD2 proteins in cochlear tissues of mice.4.Mouse cochlear hair cell lines HEI-OC1 were treated using LPS at different concentrations(0、0.25、0.5、1、10μg/m L)at different times(24,48,72 h)to construct the inflammatory model of HEI-OC1 cells.Cell proliferation activity was determined with CCK-8,cell apoptosis and reactive oxygen species(ROS)level were detected by flow cytometry and immunofluorescence staining,and the levels of IL-6 and TNF-αin the supernatant of cell culture were detected by Elisa.Western blot was used to detect the expression change of POU4F3,ATG5,LC3B,p62,Bax,and Bcl-2 proteins in LPS treated cells.Western blot was also choosed to determine the expression levels of LC3B,p62,Bax and Bcl-2 proteins in the cells treated with autophagy activator Rapamycin(RAP)and autophagy inhibitor 3-MA.The HEI-OC1 cell lines with POU4F3 and ATG5 knockdown were constructed,the apoptosis and ROS level,and the expression levels of LC3B,p62,Bax,and Bcl-2 were detected by flow cytometry,immunofluorescence staining and Western blot.5.The occupational health examination data of workers exposed to noise in key occupational disease monitoring enterprises in Jiangsu were collected and divided into NIHL group and normal hearing control group.Based on NCBI db SNP,1000Genomes and Hap Map databases,functional SNPs of autophagy related genes PIK3CA,PIK3R1,PIK3R3,AKT1,ATG4,ATG5 and ATG7 were screened based on the minimum allele frequency(MAF)>0.01 and the linkage disequilibrium r~2>0.8,and the multiplex PCR combined with second-generation sequencing was then performed for genotyping.The bioinformatics database(TFtarget,Func Pred),site mutation,plasmid construction,RNA interference,cell transfection,dual-luciferase reporter assays,RT-q PCR,and Western blot were conducted to explore the potential molecular regulatory mechanism of PIK3R3 SNP rs7536272,ATG5 SNP rs510432,transcription factor SP1 and C/EBPβinvolved in the process NIHL.Results1.Survey on NIHL status of noise-exposed workers and influencing factors analysisThe average noise exposure level of 77990 noise-exposed workers was 87.79±7.24 d B(A),26650 workers were diagnosed with NIHL,the prevalence of NIHL was 34.17%.The prevalence of NIHL was significantly increased in male,aged>35 years,noise exposure time>5 years,noise exposure level>85 d B,smoking and drinking workers(P<0.001).Logistic regression analysis showed that male,age>35 years,noise exposure time>5 years,noise exposure level>85 d B(A),smoking and drinking status were associated with the risk of NIHL and were risk factors affecting NIHL.2.Plasma proteomics and metabolomics of NIHL workers2.1 Plasma proteomics of NIHL workers based on TMT-labeling quantitative techniqueAccording to fold change(FC)>1.2 and P<0.05,a total of 22 differentially expressed proteins were screened between the NIHL group and control group.KEGG pathway enrichment analysis results showed that the differentially expressed proteins were significantly enriched in signaling pathways related to the immune inflammation,autophagy,protein synthesis and processing,and glucose metabolism.Western blot results demonstrated that the expression of ATG5 and POU4F3proteins in the plasma of NIHL workers was significantly decreased in relation to the controls(P<0.05),the expression of COLEC11 increased remarkablely in relation to the control group(P<0.05).2.2 Plasma metabolomic of NIHL workers based on HPLC/Q-TOF-MS techniqueAccording to variable important for the projection(VIP)>1 and P<0.05,a total of 20 different metabolites were identified between the NIHL group and control group.KEGG pathway enrichment analysis demonstrated that differential metabolites were significantly enriched in the metabolic pathways related to phospholipid metabolism,amino acid metabolism,and autophagy.The results of RT-q PCR showed that the m RNA expression of PIK3CA,AKT2,Beclin1 and ATG5 genes in peripheral blood of NIHL workers was obviously decreased compared with the controls(P<0.05).The results of Elisa revealed that the SOD and GSH-Px levels in plasma samples of NIHL workers were significantly lower than those of control group(P<0.01),while IL-6 and TNF-αlevels were obviously higher than that of control group(P<0.001).3.Proteomics and metabolomics of mice cochlea tissues based on NIHL model3.1 Construction of NIHL mouse modelIn relation to the control group,the ABR thresholds of mice in NIHL group were significantly increased at test frequencies of 4,8,12,16,24 and 32 k Hz(P<0.001).The number of outer hair cells(OHCs)in the middle and basal segments of the cochlea in NIHL group mice was significantly lower than that in the control group(P<0.01).3.2 Proteomic study of NIHL mice cochlea tissue based on TMT labeling quantitative techniqueAccording to FC>1.2 and P<0.05,totally,221 differentially expressed proteins were identified between the two groups,with 110 up-regulated proteins and 111 down-regulated proteins.KEGG pathway enrichment analysis revealed that differentially expressed proteins were significantly enriched in signaling pathways related to immune inflammation,protein synthesis and processing,cell proliferation,differentiation and apoptosis,autophagy,and glucose metabolism.Western blot results showed that the protein expression of ITGA1,KNG1 and CFI in the cochlear tissue of NIHL mice was significantly increased compared to the control group(P<0.05),while the protein expression of FGF1,AKT2,ATG5 and POU4F3 was significantly decreased compared to the control group(P<0.05).3.3 Metabolomic study of NIHL mice cochlea based on HPLC/Q-TOF-MS techniqueBased on VIP>1 and P<0.05,a total of 86 differential metabolites were identified between the two groups,of which 48 metabolites were up regulated and 38 metabolites were down regulated.KEGG pathway enrichment analysis results demonstrated that the identified differential metabolites were prominently participanted in the metabolic signaling pathway related to glucose metabolism,amino acid metabolism,phospholipid metabolism,and autophagy.Results of immunofluorescence staining,and Western blot showed that SOD1 and SOD2 were specifically expressed in the cochlear spiral ganglion of mice,and the expression of SOD1 and SOD2 in the cochlea of NIHL mice was significantly decreased than that of control group(P<0.05).Elisa results showed that the SOD activity in the cochlea of NIHL mice was significantly lower than control group(P<0.01),MDA,TNF-αand IL-6 levles were notably higher than ontrol group(P<0.05).4.Study on the molecular mechanism of POU4F3 and ATG5 in the inflammation of HEI-OC1 cells4.1 Construction of LPS-induced inflammatory injury model of HEI-OC1 cellsCCK-8 results demonstrated that in relation to the control group(0μg/m L),the proliferation activity of HEI-OC1 cells was significantly decreased after 48 h treatment with 1μg/m L LPS(P<0.001)and aggravated with the increase of LPS concentration and time.The results of Elisa revealed that the TNF-αand IL-6 levels in the supernatant of cell culture treated with 1μg/m L LPS for 48 h were significantly higher tcompared to the control group(P<0.01).Flow cytometry and ROS detection results showed that the cell apoptosis and ROS levels in the group treated with 1μg/m L LPS for 48 h were significantly higher than those in the control group(P<0.01).4.2 Molecular regulation mechanisms of POU4F3 and ATG5 in the inflammatory injury of HEI-OC1cellWestern blot results show that,POU4F3 protein expression significantly decreased with the increase of LPS concentration(P<0.001).The expression of ATG5 and LC3-II proteins firstly increased and then decreased with the increase of LPS concentration,and the expression level reached the highest under the treatment of 1μg/m L LPS(P<0.001).Compared to the control group,the expression of Bax and Cleaved Caspase-9 proteins was obviously increased after treatment with 1μg/m L and 10μg/m L LPS(P<0.001),Bcl-2 protein expression was significantly reduced(P<0.001).LC3-II and Bax protein expressions in LPS+3-MA group were significantly decreased compared with the LPS group(P<0.001),while p62 and Bcl-2 protein expression were significantly increased compared with the LPS group(P<0.001).Compared to the LPS group,LC3-II and Bax proteins expressions in LPS+RAP group were dramatically increased(P<0.001),the expression of p62 and Bcl-2 proteins was significantly decreased(P<0.001).The expression levels of LC3-II and Bax proteins in the LPS+si-POU4F3 group were dramatically increased compared to the LPS group(P<0.001),the expression of p62 and Bcl-2 was significantly decreased conpared to the LPS group(P<0.01).The results of flow cytometry and ROS revealed that compared to the LPS group,apoptosis and ROS levels were significantly increased in the LPS+si-POU4F3 group(P<0.01).Compared to the LPS group,the expression of LC3-II,ATG5 and Bax proteins in LPS+si-ATG5 group were significantly decreased(P<0.001),the expression of p62 and Bcl-2 was significantly increased(P<0.001).Flow cytometry and ROS results showed that the levels of apoptosis and ROS in the LPS+si-ATG5 group were clearly decreased when compared to the LPS group(P<0.01).5.Study on the relationship between PIK3R3 and ATG5 gene polymorphisms and NIHL susceptibility and its mechanisms5.1 Relationship of PIK3R3 and ATG5 gene polymorphisms and NIHL susceptibilityA total of 688 NIHL workers were recruited as the NIHL group and 667 normal hearing workers were recruited as control group,there were no significant differences in the distribution of age,gender, noise exposure time,noise exposure level,smoking and drinking status between the the two groups(P>0.05).The binaural high frequency threshold on average(BHFTA)in NIHL group was significantly higher than that in control group(P<0.001).PIK3R3 rs7536272 and ATG5 rs510432were significantly associated with NIHL(P<0.05),the rs7536272 G allele significantly increased the risk of NIHL(adjusted OR=1.22,95%CI=1.03-1.45),the rs510432 T allele significantly decreased the risk of NIHL(adjusted OR=0.77,95%CI=0.66-0.90)。5.2 Study on the mechanism of PIK3R3 and ATG5 gene polymorphism affecting NIHL susceptibilityRT-q PCR results showed that the m RNA expression level of PIK3R3 gene in the peripheral blood of individuals with rs7536272 GG genotype was significantly lower than that of individuals carrying AA/AG genotype(P<0.01),the m RNA expression level of ATG5 gene in peripheral blood of individuals with rs510432 CT/TT genotype was significantly higher than that of individuals carrying CC genotype(P<0.01).Dual-luciferase reporter gene results showed that the luciferase activity of HEI-OC1 cells co-transfected rs7536272 wild A allele and SP1 overexpression plasmid was significantly increased compared with the control group and mutant G allele and SP1overexpression plasmid co-transfected group(P<0.001).The luciferase activity of rs510432 mutant T allele transfected with C/EBP-βnegative control plasmid was significantly increased compared to the wild C allele transfected with C/EBP-βnegative control plasmid(P<0.001);the activity of luciferase in mutant T allele and C/EBPβoverexpression plasmid co-transfected group was significantly decreased compared with mutant T allele and C/EBPβnegative control plasmid co-transfected group(P<0.001).Western blot assay demonstrated that compared to the negative control group,the expression of LC3-II,Caspase-3,and Bax protein in the si-SP1 transfection group was significantly increased(P<0.01),while the expression of p-PI3K/PI3K,p-AKT/AKT,and Bcl-2proteins was significantly decreased(P<0.01).The expression of ATG5,LC3-II and Bcl-2 protein in si-C/EBPβtransfection group was significantly increased compared to the negative control group(P<0.05),the expression of Caspase-3 and Bax protein was significantly decreased in relation to negative control group(P<0.05).Conclusion1.The prevalence of NIHL in the noise-exposed workers was higher,male,age,noise exposure time,noise exposure level,smoking and drinking were closely related to the risk of NIHL and were risk factors influencing NIHL.2.The abnormal expression of ATG5 and POU4F3 was involved in the occurrence and development of NIHL and may be potential biomarkers of NIHL.3.Immune inflammation,oxidative stress and autophagy played key roles in NIHL and may be important pathophysiological mechanisms of NIHL.4.Autophagy could promote apoptosis in LPS-induced inflammation of HEI-OC1 cells,and LPS could induce autophagic death of HEI-OC1 cells.5.POU4F3 and ATG5 could influence apoptosis by regulating autophagy activity in the process of LPS-induced HEI-OC1 cell injury,thus affecting the sensitivity of cells to inflammation.6.PIK3R3 rs7536272 and ATG5 rs510432 were associated with susceptibility to NIHL and may be potential susceptible biomarkers of NIHL;Transcription factors SP1 and C/EBPβwere involved in the NIHL by regulating autophagy and apoptosis pathways,which may be potential therapeutic targets for NIHL.更多还原