噪声引起小型猪耳蜗炎症复合体的激活及蛋白质组学研究
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摘要
目的通过研究炎症复合体及相关通路在猪耳蜗中的激活,探讨噪声介导炎症复合体激活的关键分子机制,为噪声性耳聋的预防和治疗提供新靶点。方法采用小型猪为研究对象,建立噪声性耳聋模型,测试噪声暴露前后动物的ABR阈值,应用蛋白质组学iTRAQ、生物信息学、western blot、荧光实时定量PCR等技术,研究噪声刺激引起耳蜗炎症复合体的激活以及作用机制。结果正常小型猪ABR阈值为35.4±2.6 dB SPL,噪声后一天ABR阈值平均提高到72.1±4.1dB SPL,在4k Hz处听力损失最严重,高频听力损失较低频严重;噪声后七天平均ABR阈值恢复至52.8±4.7dB SPL,4k Hz以上听力恢复较低频稍差。iTRAQ实验共鉴定到蛋白质2158种,噪声暴露后较正常组具有显著差异表达的蛋白质共227个,富集在免疫过程的差异表达蛋白包括:ASC, caspase-1, IL-1 beta,CD59等。富集的KEGG pathway包括:阿尔兹海默病信号通路,帕金森病信号通路,MAPK信号通路,,氧化磷酸化通路等。结论噪声暴露后可能激活耳蜗内NLRP3受体介导的炎症复合体,通过caspase-1活化IL-1β、IL-18,并间接促进TNF-α等炎症因子上调,加剧耳蜗内炎症反应,导致耳蜗内重要结构的损伤,这一机制可能是噪声引起听力损失的重要原因。
Objective To study key molecular mechanisms of noise-induced inflammasome activation and related pathways in the cochlea for the goal of blocking inflammatory response at the source and providing a new target for the prevention and treatment of noise-induced hearing loss(NIHL). Methods Miniature pigs were used to establish a model of NIHL. Proteomics iTRAQ, bioinformatics, western blot and real-time quantitative PCR were used to study activation and mechanisms of cochlear inflammasome caused by acoustic injury. Results ABR(clicks) tests before noise exposure showed a normal threshold at 35.4±2.6 dB SPL. Average hearing threshold increased by 38.4±3.4 dB at one day after noise exposure, showing worst threshold at 4 kHz and worse hearing loss at high frequencies. ABR threshold elevation at seven days after noise exposure decreased to 20.1±4.9 dB, although recovery above 4 kHz was slightly less than lower frequencies. A total of 2158 proteins were identified by iTRAQ test. After noise exposure, 227 proteins showed significantly different expression levels compared with the normal control. The differentially expressed proteins enriched in the immune/inflammatory process including: ASC, caspase-1, IL-1 beta, CD59 and so on. Enriched KEGG pathways including: Alzheimer's disease signaling pathway, MAPK signaling pathway, oxidative phosphorylation pathway, Parkinson's disease signaling pathway and Huntington's disease signaling pathway. Conclusion Noise exposure may activate the NLRP3-inflammasome in the cochlea, which can activate IL-1β and IL-18 by caspase-1 and indirectly promote up-regulation of inflammatory factors such as TNF-α, hence aggravating inflammatory reaction in the cochlea,leading to damage of important inner ear structures as possibly an important cause of NIHL.
Objective To study key molecular mechanisms of noise-induced inflammasome activation and related pathways in the cochlea for the goal of blocking inflammatory response at the source and providing a new target for the prevention and treatment of noise-induced hearing loss(NIHL). Methods Miniature pigs were used to establish a model of NIHL. Proteomics iTRAQ, bioinformatics, western blot and real-time quantitative PCR were used to study activation and mechanisms of cochlear inflammasome caused by acoustic injury. Results ABR(clicks) tests before noise exposure showed a normal threshold at 35.4±2.6 dB SPL. Average hearing threshold increased by 38.4±3.4 dB at one day after noise exposure, showing worst threshold at 4 kHz and worse hearing loss at high frequencies. ABR threshold elevation at seven days after noise exposure decreased to 20.1±4.9 dB, although recovery above 4 kHz was slightly less than lower frequencies. A total of 2158 proteins were identified by iTRAQ test. After noise exposure, 227 proteins showed significantly different expression levels compared with the normal control. The differentially expressed proteins enriched in the immune/inflammatory process including: ASC, caspase-1, IL-1 beta, CD59 and so on. Enriched KEGG pathways including: Alzheimer's disease signaling pathway, MAPK signaling pathway, oxidative phosphorylation pathway, Parkinson's disease signaling pathway and Huntington's disease signaling pathway. Conclusion Noise exposure may activate the NLRP3-inflammasome in the cochlea, which can activate IL-1β and IL-18 by caspase-1 and indirectly promote up-regulation of inflammatory factors such as TNF-α, hence aggravating inflammatory reaction in the cochlea,leading to damage of important inner ear structures as possibly an important cause of NIHL.
引文
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12 Rybak LP,Ravi R,and Somani SM.Mechanism of Protection by Diethyldithiocarbamate against Cisplatin Ototoxicity:Antioxidant System[J].Fundam Appl Toxicol.1995,26(2):293-300.
13 Van Campen LE,Murphy WJ,Franks JR,et al.Oxidative DNADamage is Associated with Intense Noise Exposure in the Rat[J].Hear Res.2002,164(1-2):29-38.
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19 Han WJ,Shi X,and Nuttall AL.AIF and EndoG Translocation in Noise Exposure Induced Hair Cell Death[J].Hear Res.2006,211(1-2):85-95.
20韩维举,史晓瑞,Alfred Nuttall.噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移[J].中华耳鼻咽喉头颈外科杂志.2007,42(7):515-519.Han WJ,Shi XR,Nuttall AL.Camlmse 3 Activation and Apoptosis Inducing Factor Translocafion in Noise Exposure Induced Outer Hair Cells Apoptasis[J].Chin J Otorhinolaryngol Head Neck Surg.2007,42(7):515-519.
21 Yang S,Cai Q,Vethanayagam RR,et al.Immune Defense is the Primary Function Associated with the Differentially Expressed Genes in the Cochlea Following Acoustic Trauma[J].Hear Res.2016,333:283-294.
22胡博华.噪声性耳聋:基础研究进展和展望[J].中华耳科学杂志.2016,14(6):693-700.Hu BH.Noise-induced Hearing Loss:a Review of Recent Advances[J].Chinese Journal of Otology.2016,14(6):693-700.
23 Zhou R,Yazdi AS,Menu P,et al.A Role for Mitochondria in NL-RP3 Inflammasome Activation[J].Nature.2011,469(7329):221-5.
24 Bauernfeind FG,Horvath G,Stutz A,et al.Cutting Edge:NF-kappaB Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression[J].J Immunol.2009,183(2):787-91.
25 Vethanayagam RR,Yang W,Dong Y,et al.Toll-like Receptor 4Modulates the Cochlear Immune Response to Acoustic Injury[J].Cell Death Dis.2016,7(6):e2245.
26 Cai Q,Vethanayagam RR,Yang S,et al.Molecular Profile of Cochlear Immunity in the Resident Cells of the Organ of Corti[J].JNeuroinflammation.2014,11:173.
27 Lee CY,Landreth GE.The Role of Microglia in Amyloid Clearance from the AD Brain[J].J Neural Transm(Vienna).2010,117(8):949-60.
28 Kuemmerle-Deschner JB,Koitschev A,Tyrrell PN,et al.Early Detection of Sensorineural Hearing Loss in Muckle-Wells-syndrome[J].Pediatr Rheumatol Online J.2015,13(1):43.
29 Chakraborty S,Kaushik DK,Gupta M,et al.Inflammasome Signaling at the Heart of Central Nervous System Pathology[J].J Neurosci Res.2010,88(8):1615-31.
30 Cedikova M,Pitule P,Kripnerova M,et al.Multiple Roles of Mitochondria in Aging Processes[J].Physiol Res.2016,65(Supplementum 5):S519-531.
31 de Vries HE,Blom-Roosemalen MC,van Oosten M,et al.The Influence of Cytokines on the Integrity of the Blood-brain Barrier in Vitro[J].J Neuroimmunol.1996,64(1):37-43.
32 Shi X,Qiu S,Zhuang W,et al.NLRP3-inflammasomes are Triggered by Age-related Hearing Loss in the Inner Ear of Mice[J].Am J Transl Res.2017,9(12):5611-5618.
2 Sandberg A,L.G.,K?llstr?m BN,et al.Tumor Proteomics by Multivariate Analysis on Individual Pathway Data for Characterization of Vulvar Cancer Phenotypes[J].Mol Cell Proteomics.2012,11(7):M112.016998.
3 Latosinska A,V.K.,Makridakis M,et al.Comparative Analysis of Label-Free and 8-Plex iTRAQ Approach for Quantitative Tissue Proteomic Analysis[J].PLoS One.2015,10(9):e0137048.
4 Cai XZ,Z.W.,Xiang Y.iTRAQ-Based Quantitative Proteomic Analysis of Nasopharyngeal Carcinoma[J].J Cell Biochem.2015,116(7):1431-41.
5杨仕明.小型猪动物模型在耳科学领域的应用[J].中华耳科学杂志.2016.14(1):1-5.Yang SM.The Miniature Pig as an Animal Model in Otological Research[J].Chinese Journal of Otology.2016.14(1):1-5.
6 Zhong LL,Zhang Y,Liang XJ,et al.Inner Ear Structure of Miniature Pigs Measured by Multi-planar Reconstruction Techniques[J].Am J Transl Res.2018,10(3):709-717.
7陈志婷,王方园,冀飞,等.小型猪听性脑干反应分析及脉冲噪声暴露后的变化[J].中华耳科学杂志.2016,14(6):735-739.Chen ZT,Wang FY,Ji F,et al.Characteristics of Auditory Brainstem Responses in Miniature Pigs and Changes after Impulse Noise Exposure[J].Chinese Journal of Otology.2016,14(6):735-739.
8李强.四种食物对二点委夜蛾中肠消化酶活性的影响与中肠蛋白质组学研究[D].山东农业大学.2017.Li Q.Effects of Four Foods on Digestive Enzyme Activity in the Midgut of Spodoptera exigua and Midgut Proteomics[D].Shandong Agricultural University.2017.
9侯赟,郭维维,杨仕明,等.小型猪内耳发育形态学观察[J].中华耳科学杂志.2012,10(4):485-488.Hou Y,Guo WW,Yang SM,et al.Developmental of Inner Ear in Miniature pigs[J].Chinese Journal of Otology.2012,10(4):485-488.
10 Kopke RD,Weisskopf PA,Boone JL,et al.Reduction of Noise-induced Hearing Loss Using L-NAC and Salicylate in the Chinchilla.Hear Res.2000,149(1-2):138-46.
11韩维举,陈星睿.噪声暴露引起耳蜗损伤机制的研究[J].中华耳科学杂志.2013,11(3):357-362.Han WJ,Chen XR.Mechanisms of Noise Induced Cochlea Injury[J].Chinese Journal of Otology.2013,11(3):357-362.
12 Rybak LP,Ravi R,and Somani SM.Mechanism of Protection by Diethyldithiocarbamate against Cisplatin Ototoxicity:Antioxidant System[J].Fundam Appl Toxicol.1995,26(2):293-300.
13 Van Campen LE,Murphy WJ,Franks JR,et al.Oxidative DNADamage is Associated with Intense Noise Exposure in the Rat[J].Hear Res.2002,164(1-2):29-38.
14 Fetoni AR,Troiani D,Eramo SL,et al.Efficacy of Different Routes of Administration for Coenzyme Q10 Formulation in Noise-Induced Hearing Loss:Systemic Versus Transtympanic Modality[J].Acta Otolaryngol.2012,132(4):391-9.
15 Sha SH,Taylor R,Forge A,et al.Differential Vulnerability of Basal and Apical Hair Cells is Based on Intrinsic Susceptibility to Free Radicals[J].Hear Res.2001,155(1-2):1-8.
16 Choi CH,Du X,Floyd RA,et al.Therapeutic Effects of Orally Administrated Antioxidant Drugs on Acute Noise-induced Hearing Loss[J].Free Radic Res.2014,48(3):264-72.
17 Choi CH,Du X,Floyd RA,et al.NAC for Noise:from the Bench Top to the Clinic[J].Hear Res.2007,226(1-2):114-25.
18 Yang WP,Henderson D,Hu BH,et al.Quantitative Analysis of Apoptotic and Necrotic Outer Hair Cells after Exposure to Different Levels of Continuous Noise[J].Hear Res.2004,196(1-2):69-76.
19 Han WJ,Shi X,and Nuttall AL.AIF and EndoG Translocation in Noise Exposure Induced Hair Cell Death[J].Hear Res.2006,211(1-2):85-95.
20韩维举,史晓瑞,Alfred Nuttall.噪声刺激致豚鼠外毛细胞凋亡时半胱氨酸天冬氨酸蛋白酶3激活和凋亡诱导因子转移[J].中华耳鼻咽喉头颈外科杂志.2007,42(7):515-519.Han WJ,Shi XR,Nuttall AL.Camlmse 3 Activation and Apoptosis Inducing Factor Translocafion in Noise Exposure Induced Outer Hair Cells Apoptasis[J].Chin J Otorhinolaryngol Head Neck Surg.2007,42(7):515-519.
21 Yang S,Cai Q,Vethanayagam RR,et al.Immune Defense is the Primary Function Associated with the Differentially Expressed Genes in the Cochlea Following Acoustic Trauma[J].Hear Res.2016,333:283-294.
22胡博华.噪声性耳聋:基础研究进展和展望[J].中华耳科学杂志.2016,14(6):693-700.Hu BH.Noise-induced Hearing Loss:a Review of Recent Advances[J].Chinese Journal of Otology.2016,14(6):693-700.
23 Zhou R,Yazdi AS,Menu P,et al.A Role for Mitochondria in NL-RP3 Inflammasome Activation[J].Nature.2011,469(7329):221-5.
24 Bauernfeind FG,Horvath G,Stutz A,et al.Cutting Edge:NF-kappaB Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression[J].J Immunol.2009,183(2):787-91.
25 Vethanayagam RR,Yang W,Dong Y,et al.Toll-like Receptor 4Modulates the Cochlear Immune Response to Acoustic Injury[J].Cell Death Dis.2016,7(6):e2245.
26 Cai Q,Vethanayagam RR,Yang S,et al.Molecular Profile of Cochlear Immunity in the Resident Cells of the Organ of Corti[J].JNeuroinflammation.2014,11:173.
27 Lee CY,Landreth GE.The Role of Microglia in Amyloid Clearance from the AD Brain[J].J Neural Transm(Vienna).2010,117(8):949-60.
28 Kuemmerle-Deschner JB,Koitschev A,Tyrrell PN,et al.Early Detection of Sensorineural Hearing Loss in Muckle-Wells-syndrome[J].Pediatr Rheumatol Online J.2015,13(1):43.
29 Chakraborty S,Kaushik DK,Gupta M,et al.Inflammasome Signaling at the Heart of Central Nervous System Pathology[J].J Neurosci Res.2010,88(8):1615-31.
30 Cedikova M,Pitule P,Kripnerova M,et al.Multiple Roles of Mitochondria in Aging Processes[J].Physiol Res.2016,65(Supplementum 5):S519-531.
31 de Vries HE,Blom-Roosemalen MC,van Oosten M,et al.The Influence of Cytokines on the Integrity of the Blood-brain Barrier in Vitro[J].J Neuroimmunol.1996,64(1):37-43.
32 Shi X,Qiu S,Zhuang W,et al.NLRP3-inflammasomes are Triggered by Age-related Hearing Loss in the Inner Ear of Mice[J].Am J Transl Res.2017,9(12):5611-5618.