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Background: The Chinese ethnic medicine Jie-Du-Huo-Xue Decoction (JDHXD) is used to alleviate neuroinflammation in cerebral ischemia (CI). Our previous studies have confirmed that JDHXD can inhibit microglial pyroptosis in CI. However, the pharmacological mechanism of JDHXD in alleviating neuroinflammation and pyroptosis needs to be further elucidated. New research points out that there is an interaction between autophagy and inflammasome NLRP3, and autophagy can help clear NLRP3. The NLRP3 is a key initiator of pyroptosis and autophagy. The effect of JDHXD promoting autophagy to clear NLRP3 to inhibit pyroptosis on cerebral ischemia-reperfusion inflammatory injury is currently unknown. We speculate that JDHXD can inhibit pyroptosis in CI by promoting autophagy to clear NLRP3. Methods: Chemical characterization of JDHXD was performed using LC-MS. Model of middle cerebral artery occlusion/reperfusion (MCAO/R) was established in SD rats. Neurological deficits, neuron damage, and cerebral infarct volume were evaluated. Western Blot and immunofluorescence were used to detect neuronal pyroptosis and autophagy. Results: 30 possible substance metabolites in JDHXD medicated serum were analyzed by LC-MS (Composite Score > 0.98). Furthermore, JDHXD protects rat neurological function and cerebral infarct size after CI. JDHXD inhibited the expression of pyroptosis and autophagy after CI. Our western blot and immunofluorescence results showed that JDHXD treatment can reduce the expression of autophagy-related factors ULK1, beclin1, and LC3-â ¡. The expression of NLRP3 protein was lower in the JDHXD group than in the I/R group. Compared with the I/R group, the expressions of pyroptosis-related factors caspase-1 P 10, GSDMD-NT, IL-18, and IL-1ß decreased in the JDHXD group. Furthermore, we observed an unexpected result: immunofluorescence demonstrated that Gasdermin D (GSDMD) was significantly absent in the infarct core, and highly expressed in the peri-infarct and contralateral cerebral hemispheres. This finding challenges the prevailing view that GSDMD is elevated in the ischemic cerebral hemisphere. Conclusion: JDHXD inhibited pyroptosis and autophagy after MCAO/R. JDHXD suppressed pyroptosis and autophagy by inhibiting NLRP3, thereby alleviating CI. In addition, we present a different observation from previous studies that the expression of GSDMD in the infarct core was lower than that in the peri-infarct and contralateral non-ischemic hemispheres on day 3 of CI.
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Inner ear disorders are a cluster of diseases that cause hearing loss in more than 1.5 billion people worldwide. However, the presence of the blood-labyrinth barrier (BLB) on the surface of the inner ear capillaries greatly hinders the effectiveness of systemic drugs for prevention and intervention due to the low permeability, which restricts the entry of most drug compounds from the bloodstream into the inner ear tissue. Here, we report the finding of a novel receptor, low-density lipoprotein receptor-related protein 1 (LRP1), that is expressed on the BLB, as a potential target for shuttling therapeutics across this barrier. As a proof-of-concept, we developed an LRP1-binding peptide, IETP2, and covalently conjugated a series of model small-molecule compounds to it, including potential drugs and imaging agents. All compounds were successfully delivered into the inner ear and inner ear lymph, indicating that targeting the receptor LRP1 is a promising strategy to enhance the permeability of the BLB. The discovery of the receptor LRP1 will illuminate developing strategies for crossing the BLB and for improving systemic drug delivery for inner ear disorders.
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Orelha Interna , Perda Auditiva , Sistemas de Liberação de Medicamentos , Orelha Interna/irrigação sanguínea , Orelha Interna/metabolismo , Perda Auditiva/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Preparações Farmacêuticas/metabolismoRESUMO
BACKGROUND: IFNLR1 has been recently identified to be related to autosomal dominant nonsyndromic sensorineural hearing loss (ADNSHL). It is reported to be expressed in the inner ear of mice and the lateral line of zebrafish. However, it remains unclear how defects in this gene lead to hearing loss. OBJECTIVES: To elucidate the global gene expression changes in zebrafish when the expression of ifnlr1 is downregulated. METHODS: Transcriptome analysis was performed on ifnlr1 morpholino knockdown zebrafish and the control zebrafish using RNA-seq technology. RESULTS: The results show that 262 differentially expressed genes (DEGs) were up-regulated while 146 DEGs were down-regulated in the E4I4-Mo zebrafish larvae compared to the control-Mo. Six pathways were significantly enriched, including steroid biosynthesis pathway, adipocytokine signaling pathway, cytokine-cytokine receptor interaction pathway, p53 signaling pathway, AGE-RAGE signaling pathway in diabetic complications, and terpenoid backbone biosynthesis pathway. Among them, three pathways (steroid biosynthesis pathway, cytokine-cytokine receptor interaction pathway and p53 signaling pathway) are immune-associated. CONCLUSIONS: The transcriptome analysis results contribute to the groundwork for future research on the pathogenesis of IFNLR1-associated hearing loss.
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Transcriptoma , Peixe-Zebra , Animais , Citocinas , Perfilação da Expressão Gênica , Imunidade , Receptores de Citocinas/genética , Esteroides , Proteína Supressora de Tumor p53/genética , Peixe-Zebra/genéticaRESUMO
ATP6V1B2 encodes the V1B2 subunit in V-ATPase, a proton pump responsible for the acidification of lysosomes. Mutations in this gene cause DDOD syndrome, DOORS syndrome, and Zimmermann-Laband syndrome, which share overlapping feature of congenital sensorineural deafness, onychodystrophy, and different extents of intellectual disability without or with epilepsy. However, the underlying mechanisms remain unclear. To investigate the pathological role of mutant ATP6V1B2 in the auditory system, we evaluated auditory brainstem response, distortion product otoacoustic emissions, in a transgenic line of mice carrying c.1516 C > T (p.Arg506∗) in Atp6v1b2, Atp6v1b2 Arg506*/Arg506* . To explore the pathogenic mechanism of neurodegeneration in the auditory pathway, immunostaining, western blotting, and RNAscope analyses were performed in Atp6v1b2Arg506*/Arg506* mice. The Atp6v1b2Arg506*/Arg506* mice showed hidden hearing loss (HHL) at early stages and developed late-onset hearing loss. We observed increased transcription of Atp6v1b1 in hair cells of Atp6v1b2Arg506*/Arg506* mice and inferred that Atp6v1b1 compensated for the Atp6v1b2 dysfunction by increasing its own transcription level. Genetic compensation in hair cells explains the milder hearing impairment in Atp6v1b2Arg506*/Arg506* mice. Apoptosis activated by lysosomal dysfunction and the subsequent blockade of autophagic flux induced the degeneration of spiral ganglion neurons and further impaired the hearing. Intraperitoneal administration of the apoptosis inhibitor, BIP-V5, improved both phenotypical and pathological outcomes in two live mutant mice. Based on the pathogenesis underlying hearing loss in Atp6v1b2-related syndromes, systemic drug administration to inhibit apoptosis might be an option for restoring the function of spiral ganglion neurons and promoting hearing, which provides a direction for future treatment.
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Dominant deafness-onychodystrophy (DDOD) syndrome is a rare autosomal dominant disorder caused by mutations in ATP6V1B2 gene. We previously generated an induced pluripotent stem cell (iPSC) line (CPGHi002-A) from a DDOD patient with a heterozygous c.1516 C>T mutation in the ATP6V1B2 gene. Here we genetically corrected the c.1516 C>T mutation in the ATP6V1B2 gene using CRISPR/Cas9 technology to generate an isogenic control, CPGHi002-A-1. The characterization of CPGHi002-A-1 demonstrates normal karyotype, pluripotent state, and potential to differentiate in vitro towards endoderm, mesoderm, and ectoderm.
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Células-Tronco Pluripotentes Induzidas , ATPases Vacuolares Próton-Translocadoras , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Heterozigoto , Humanos , Mutação , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Inflammation is involved in noise-induced hearing loss (NIHL), but the mechanism is still unknown. The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, which triggers the inflammatory cascade, has been implicated in several inflammatory diseases in response to oxidative stress. However, whether the NLRP3 inflammasome is a key factor for permanent NIHL is still unknown. In this study, quantitative real-time polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assays (ELISAs) demonstrated that the expression levels of activated caspase-1, interleukin (IL)-1ß, IL-18, and NLRP3 were significantly increased in the cochleae of mice exposed to broadband noise (120 dB) for 4 h, compared with the control group. These results indicate that the activation of inflammasomes in the cochleae of mice during the pathological process of NIHL as well as NLRP3, a sensor protein of reactive oxygen species (ROS), may be key factors for inflammasome assembly and subsequent inflammation in cochleae. Moreover, many recent studies have revealed that NEK7 is an important component and regulator of NLRP3 inflammasomes by interacting with NLRP3 directly and that these interactions can be interrupted by oridonin. Here, we further determined that treatment with oridonin could indeed interrupt the interaction between NLRP3 and NEK7 as well as inhibit the downstream inflammasome activation in mouse cochleae after noise exposure. Furthermore, we tested anakinra, another inflammatory inhibitor, and it was shown to partially alleviate the degree of hearing impairment in some frequencies in an NIHL mouse model. These discoveries suggest that inhibiting NLRP3 inflammasomes and the downstream signaling pathway may provide a new strategy for the clinical treatment of NIHL.
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Anti-Inflamatórios/uso terapêutico , Diterpenos do Tipo Caurano/uso terapêutico , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Quinases Relacionadas a NIMA/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Animais , Anti-Inflamatórios/farmacologia , Cóclea/efeitos dos fármacos , Cóclea/imunologia , Citocinas/imunologia , Diterpenos do Tipo Caurano/farmacologia , Perda Auditiva Provocada por Ruído/imunologia , Inflamassomos/imunologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Camundongos Endogâmicos C57BL , Quinases Relacionadas a NIMA/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
Overproduction of reactive oxygen species (ROS) and inflammation are two key pathogeneses of noise-induced hearing loss (NIHL), which leads to outer hair cell (OHC) damage and hearing loss. In this work, we successfully developed ROS-responsive nanoparticles as berberine (BBR) carriers (PL-PPS/BBR) for OHC-targeted therapy of NIHL: Prestin-targeting peptide 2 (PrTP2)-modified nanoparticles (PL-PPS/BBR), which effectively accumulated in OHC areas, and poly(propylene sulfide)120 (PPS120), which scavenged ROS and converted to poly(propylene sulfoxide)120 in a ROS environment to disintegrate and provoke the rapid release of BBR with anti-inflammatory and antioxidant effects. In this study, satisfactory anti-inflammatory and antioxidant effects of PL-PPS/BBR were confirmed. Immunofluorescence and scanning electron microscopy (SEM) images showed that PL-PPS/BBR effectively accumulated in OHCs and protected the morphological integrity of OHCs. The auditory brainstem response (ABR) results demonstrated that PL-PPS/BBR significantly improved hearing in NIHL guinea pigs after noise exposure. This work suggested that PL-PPS/BBR may be a new potential treatment for noise-associated injury with clinical application.
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Anti-Inflamatórios não Esteroides/farmacologia , Antioxidantes/farmacologia , Berberina/farmacologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Perda Auditiva Provocada por Ruído/tratamento farmacológico , Espécies Reativas de Oxigênio/química , Animais , Anti-Inflamatórios não Esteroides/química , Antioxidantes/química , Berberina/química , Modelos Animais de Doenças , Portadores de Fármacos/química , Potenciais Evocados Auditivos do Tronco Encefálico/efeitos dos fármacos , Cobaias , Estrutura Molecular , Nanopartículas/química , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Propriedades de SuperfícieRESUMO
BACKGROUND: Germline variants in PTPN11 are the primary cause of Noonan syndrome with multiple lentigines (NSML) and Noonan syndrome (NS), which share common skin and facial symptoms, cardiac anomalies and retardation of growth. Hearing loss is considered an infrequent feature in patients with NSML/NS. However, in our cohort, we identified a group of patients with PTPN11 pathogenic variants that were primarily manifested in congenital sensorineural hearing loss (SNHL). This study evaluated the incidence of PTPN11-related NSML or NS in patients with congenital SNHL and explored the expression of PTPN11 and the underlying mechanisms in the auditory system. METHODS: A total of 1502 patients with congenital SNHL were enrolled. Detailed phenotype-genotype correlations were analysed in patients with PTPN11 variants. Immunolabelling of Ptpn11 was performed in P35 mice. Zebrafish with Ptpn11 knockdown/mutant overexpression were constructed to further explore mechanism underlying the phenotypes. RESULTS: Ten NSML/NS probands were diagnosed via the identification of pathogenic variants of PTPN11, which accounted for ~0.67% of the congenital SNHL cases. In mice cochlea, Shp2, which is encoded by Ptpn11, is distributed in the spiral ganglion neurons, hair cells and supporting cells of the inner ear. In zebrafish, knockdown of ptpn11a and overexpression of mutant PTPN11 were associated with a significant decrease in hair cells and supporting cells. We concluded that congenital SNHL could be a major symptom in PTPN11-associated NSML or NS. Other features may be mild, especially in children. CONCLUSION: Screening for PTPN11 in patients with congenital hearing loss and variant-based diagnoses are recommended.
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Perda Auditiva Neurossensorial/congênito , Síndrome de Noonan/genética , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Adolescente , Animais , Povo Asiático/genética , Criança , Pré-Escolar , Estudos de Coortes , Feminino , Técnicas de Silenciamento de Genes , Perda Auditiva Neurossensorial/complicações , Perda Auditiva Neurossensorial/epidemiologia , Humanos , Incidência , Lactente , Masculino , Camundongos , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , Peixe-Zebra , beta Catenina/metabolismoRESUMO
Dominant deafness-onychodystrophy (DDOD) syndrome is a rare, autosomal dominant inherited disorder with no concrete therapies in human. We previously identified c.1516 C > T (p.Arg506*) in ATP6V1B2 as cause of DDOD syndrome, accounting for all cases of this genetic disorder. The induced pluripotent stem cell (iPSC) line was generated using the non-integrating episomal vector method from peripheral blood mononuclear cells (PBMCs) of a 10-month-old female DDOD patient with heterozygous ATP6V1B2 c.1516 C > T variant. This cell line may serve as a useful model for studying the pathogenic mechanisms and treatment of DDOD syndrome.
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Células-Tronco Pluripotentes Induzidas , ATPases Vacuolares Próton-Translocadoras , Linhagem Celular , Feminino , Heterozigoto , Humanos , Lactente , Leucócitos Mononucleares , Mutação , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
BACKGROUND: Dominant deafness-onychodystrophy (DDOD) syndrome is a rare disorder mainly characterized by severe deafness, onychodystrophy and brachydactyly. We previously identified c.1516Câ¯>â¯T (p.Arg506X) in ATP6V1B2 as cause of DDOD syndrome, accounting for all cases of this genetic disorder. Clinical follow-up of DDOD syndrome patients with cochlear implantation revealed the language rehabilitation was unsatisfactory although the implanted cochlea worked well, which indicates there might be learning and memory problems in DDOD syndrome patients. However, the underlying mechanisms were unknown. METHODS: atp6v1b2 knockdown zebrafish and Atp6v1b2 c.1516Câ¯>â¯T knockin mice were constructed to explore the phenotypes and related mechanism. In mutant mice, auditory brainstem response test and cochlear morphology analysis were performed to evaluate the auditory function. Behavioral tests were used to investigate various behavioral and cognitive domains. Resting-state functional magnetic resonance imaging was used to evaluate functional connectivity in the mouse brain. Immunofluorescence, Western blot, and co-immunoprecipitation were performed to examine the expression and interactions between the subunits of V-ATPases. FINDINGS: atp6v1b2 knockdown zebrafish showed developmental defects in multiple organs and systems. However, Atp6v1b2 c.1516Câ¯>â¯T knockin mice displayed obvious cognitive defects but normal hearing and cochlear morphology. Impaired hippocampal CA1 region and weaker interaction between the V1E and B2 subunits in Atp6v1b2Arg506X//Arg506X mice were observed. INTERPRETATION: Our study extends the phenotypic range of DDOD syndrome. The impaired hippocampal CA1 region may be the pathological basis of the behavioral defects in mutant mice. The molecular mechanism underlying V-ATPases dysfunction involves a weak interaction between subunits, although the assembly of V-ATPases can still take place.
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Surdez/genética , Deficiência Intelectual/genética , ATPases Vacuolares Próton-Translocadoras/genética , Adulto , Animais , Cóclea , Surdez/fisiopatologia , Modelos Animais de Doenças , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lactente , Deficiência Intelectual/fisiopatologia , Masculino , Camundongos , Camundongos Transgênicos , Linhagem , Fenótipo , Peixe-Zebra/genéticaRESUMO
BACKGROUND: Follicle-stimulating hormone (FSH) has multiple biological functions. It is currently considered that FSH can inhibit cervical cancer, and our aim was to explore the underlying molecular mechanisms. MATERIALS AND METHODS: An in vivo experiment using nude mice injected with HeLa cells was performed. Flow cytometry, western blotting, and real-time quantitative PCR analyses were done. RESULTS: Twenty one days after injection of HeLa cells, the subcutaneous tumor mass was significantly lower (P<0.01) in mice treated with 20 mIU/mL FSH, but did not disappear. In vitro observations indicated that FSH might inhibit cell proliferation and activate cell apoptosis to induce the reduction of HeLa cells. The mRNA and protein levels of Cyclin D1, Cyclin E1, and Caspase 3 changed accordingly as expected in vivo and in vitro. Moreover, FSH inactivated the nuclear factor-kappa B (NF-κB) pathway in subcutaneous tumors; the NF-κB(p65) activity in HeLa cells was significantly decreased using 20 mIU/mL FSH and was increased when FSH was administered along with lipopolysaccharide, accompanied by the same change of cell number. Further, FSH accelerated protein kinase A (PKA) activity, but inactivated glycogen synthase kinase 3 beta (GSK-3ß) activity. Specific inhibition of PKA and/or GSK-3ß provided in vitro evidence that directly supported the FSH-mediated inhibition of GSK-3ß to inactivate NF-κB via the promotion of PKA activity. CONCLUSION: Our data are the first description of the molecular regulatory mechanisms of FSH-mediated inhibition of the development of cervical cancer by decreasing the cell cycle and activating cell apoptosis via the PKA/GSK-3ß/NF-κB pathway.
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In order to increase application of cross-linked enzyme aggregates (CLEAs) in industry production, a novel micro-reactor system that included a rotating magnetic field (RMF), a micro-reactor and CLEAs with magnetic nanoparticles (M-CLEAs) was designed to synthesize butyl oleate. Result showed that the presence of RMF significantly increased the yield of butyl oleate and the maximum increment was 23%. The yield of butyl oleate was impacted by the dosage and distribution of M-CLEAs in micro-reactor. M-CLEAs showed good reusability, since the morphology and the second structure of protein of M-CLEAs did not show evident change after 4 operative cycles. Although the three-dimensional fluorescence of M-CLEAs showed shift in fluorescence intensity and the maximum emission wavelengths, the yield of butyl oleate was not affected. This study provides a novel design that realized efficient, convenient and continuous application of CLEAs in biosynthesis, and M-CLEAs also show good promises in industry production.
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Proteínas Fúngicas/química , Lipase/química , Nanopartículas de Magnetita/química , Ácidos Oleicos/química , Catálise , Reagentes de Ligações Cruzadas/química , Glutaral/química , Fenômenos Magnéticos , Estrutura Secundária de Proteína , Rhizopus/enzimologiaRESUMO
Irreversible hearing loss induced by aminoglycoside in human through local or systemic administration route negatively impacts quality of life. The aim of this work was to develop and validate an analytical method suitable for the detection and quantification of neomycin in cochlear perilymph of guinea pig after local application. The SupelMIP SPE column was used for the pre-treatment of matrix. Chromatographic separation was conducted by a reversed phase ODS column (100â¯×â¯2.1â¯mm, 3⯵m) at 40⯰C in gradient mode with 0.2 (v/v) HFBA in water and 0.2 (v/v) HFBA in acetonitrile as mobile phase, at a flow rate of 0.30â¯mL/min, with retention time of 3.50 and 3.62â¯min for internal standard tobramycin and analyte neomycin, respectively. The MS was performed with positive ionization mode, with data acquisition in Multi Reaction Monitor (MRM) mode. This method was proved to be specific, accurate (97.1-115% of nominal values) and precise (CV%â¯<â¯15%). Calibration curves for matrix matched standard of neomycin ranged from 1.25 to 200⯵g/mL, with LOD and LLOQ of 0.625 and 1.25⯵g/mL in blank matrix. The matrix effect was corrected to (-0.1) - 1.33 by adding internal standard. The relative SPE recovery values were ≥98.9% in low, medium and high QC samples. Neomycin in matrix proved to be stable under room temperature - and -20⯰C, or under three freeze-thawing cycles, or under processing as well. Finally, the proposed method was successfully applied to a toxicokinetics study of neomycin in perilymph after round window membrane (RWM) administration, which was in accordance with threshold shift of auditory brainstem response (ABR) test related to hearing loss.
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Cromatografia Líquida de Alta Pressão/métodos , Neomicina/análise , Perilinfa/química , Extração em Fase Sólida/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Cobaias , Limite de Detecção , Modelos Lineares , Neomicina/isolamento & purificação , Reprodutibilidade dos Testes , Janela da Cóclea/metabolismoRESUMO
OBJECTIVE: To investigate whether a novel compound heterozygous mutations c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) in GJB2 result in hearing loss. METHODS: Allele-specific PCR-based universal array (ASPUA) screening and sequence analysis were applied to identify these mutations. 3D model was built to perform molecular dynamics (MD) simulation to verify the susceptibility of the mutations. Furthermore, WT- and Mut-GJB2 DNA fragments, containing the mutation of c.257C>G and c.176del16 were respectively cloned and transfected into HEK293 and spiral ganglion neuron cell (SGNs) by lenti-virus delivery system to indicate the subcellular localization of the WT- and Mut-CX26 protein. RESULTS: A novel compound heterozygous mutation c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) in GJB2 was identified in a Chinese family, in which 4 siblings with profound hearing loss, but the fifth child is normal. By ASPUA screening and sequencing, a compound heterozygote mutations in GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) were identified in these four deaf children, each of the mutated GJB2 gene were inherited from their parents. There is no mutation of GJB2 gene identified in the normal child. Besides, the compound heterozygous mutation GJB2 c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) could lead to the alterations of the subcellular localization of each corresponding mutated CX26 protein and could cause the hearing loss, which has been predicted by MD simulation and verified in both 293T and SGNs cell line. CONCLUSION: The c.257C>G (p.T86R)/c.176del16 (p.G59A fs*18) compound mutations in GJB2 detected in this study are novel, and which may be associated with hearing loss in this Chinese family.
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Povo Asiático/genética , Conexinas , Perda Auditiva Neurossensorial/genética , Mutação/genética , Animais , Células Cultivadas , Estudos de Coortes , Conexina 26 , Conexinas/química , Conexinas/genética , Conexinas/metabolismo , Feminino , Gânglios Espinais/citologia , Células HEK293 , Humanos , Masculino , Camundongos , Simulação de Dinâmica MolecularRESUMO
Congenital cytomegalovirus (CMV) infection is the most common infectious cause of sensorineural hearing loss in children. While the importance of CMVinduced SNHL has been described, the mechanisms underlying its pathogenesis and the role of inflammatory responses remain elusive. The present study established an experimental model of hearing loss after systemic infection with murine CMV (MCMV) in newborn mice. Auditory brainstem responses were tested to evaluate hearing at 3 weeks, expression of inflammasome-associated factors was assessed by immunofluorescence, western blot analysis, reverse transcriptionquantitative polymerase chain reaction and ELISA. MCMV sequentially induced inflammasomeassociated factors. Furthermore, the inflammasomeassociated factors were also increased in cultured spiral ganglion neurons infected with MCMV for 24 h. In addition, MCMV increased the content of reactive oxygen species (ROS). These results suggest that hearing loss caused by MCMV infection may be associated with ROSinduced inflammation.
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Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Gânglio Espiral da Cóclea/citologia , Animais , Células Cultivadas , Orelha Interna/citologia , Orelha Interna/fisiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Perda Auditiva/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Espécies Reativas de Oxigênio/metabolismoRESUMO
Background Hereditary sensorineural hearing loss is a genetically heterogeneous disorder. Objectives This study was designed to explore the genetic etiology of deafness in a large Chinese family with autosomal dominant, nonsyndromic, progressive sensorineural hearing loss (ADNSHL). Methods Whole exome sequencing and linkage analysis were performed to identify pathogenic mutation. Inner ear expression of Ifnlr1 was investigated by immunostaining in mice. ifnlr1 Morpholino knockdown Zebrafish were constructed to explore the deafness mechanism. Results We identified a cosegregating heterozygous missense mutation, c.296G>A (p.Arg99His) in the gene encoding interferon lambda receptor 1 (IFNLR1) - a protein that functions in the Jak/ STAT pathway- are associated with ADNSHL Morpholino knockdown of ifnlr1 leads to a significant decrease in hair cells and non-inflation of the swim bladder in late-stage zebrafish, which can be reversed by injection with normal Zebrafish ifnlr1 mRNA. Knockdown of ifnlr1 in zebrafish causes significant upregulation of cytokine receptor family member b4 (interleukin-10r2), jak1, tyrosine kinase 2, stat3, and stat5b in the Jak1/STAT3 pathway at the mRNA level. ConclusionIFNLR1 function is required in the auditory system and that IFNLR1 mutations are associated with ADNSHL. To the best of our knowledge, this is the first study implicating an interferon lambda receptor in auditory function.
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Predisposição Genética para Doença , Perda Auditiva Neurossensorial/genética , Receptores de Citocinas/genética , Receptores de Interferon/genética , Animais , Técnicas de Silenciamento de Genes , Ligação Genética , Perda Auditiva Neurossensorial/fisiopatologia , Heterozigoto , Humanos , Janus Quinase 1/genética , Camundongos , Morfolinas , Mutação de Sentido Incorreto/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais , Sequenciamento do Exoma , Peixe-Zebra/genéticaRESUMO
OBJECTIVE: To understand the crucial role of the klotho gene in hearing development in mouse models. METHODS: PCR was used to identify CBA mice with different genotypes, i.e. WT, heterozygous (klothoâ¯+/-) or homozygous (klothoâ¯-/-). Mice phenotype and weight were recorded postnatal 25 days (P-25) and auditory brainstem responses (ABR) were used to determine auditory function at P-60. RESULTS: klothoâ¯-/- mice tended to have smaller size, lighter weight and higher ABR thresholds at P-60, showing early onset age-related hearing loss (ARHL). CONCLUSION: Heterozygous and homozygous klotho deficient mice exhibit different degrees of hearing loss at young age, with homozygous mice (klothoâ¯-/-) showing more severe hearing loss. Our results indicate that persisted expression of klotho protein in the inner ear may potentially delay the onset of ARHL and play an important role in the protection of auditory function.
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Age-related hearing loss (ARHL) or presbyacusis is a progressive loss of hearing sensitivity that is predominately associated with sensory or transduction neuro-cell degeneration in the peripheral and central auditory systems. Increased production of reactive oxygen species (ROS) and inflammatory response were frequently found in aging cochleae. In addition, inflammasomes are likely responsible for the accumulation of ROS in immune cells, although whether they are in fact involved in the development of ARHL is unknown. In this study, Q-PCR, WB and ELASA demonstrated significantly increased levels of activated Caspase-1, interleukin-1ß and interleukin-18 and even NLRP3 in the inner ears of aging mice compared to younger one. In addition, NLRP3, as a sensor protein of ROS, may contribute to inflammasome assembly and subsequent inflammation in the cochleae. In conclusion, inflammation triggered by the activation of inflammasomes in the cochleae of aging mice appears to be playing an important role in the pathological process of ARHL and may be a potential cause of presbyacusis.
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OBJECTIVE: To investigate the membrane localization function of the CX26 protein when its 86th amino acid is Thr, Ser or Arg, and its relations to deafness. METHODS: CX26-GFP protein with either Thr, Ser or Arg as the 86th amino acid was expressed in mouse SGN cells via the GFP fusion type lenti-virus expression system. The membrane localization of the fusion protein was observed under a fluorescence microscope. RESULTS: The mutated protein of CX26 T86S was localized to cell membrane and form gap conjunction structures, showing no difference to the wild type CX26 protein (with Thr as the 86th amino acid). However, the gap conjunction structure disappeared when the mutation was CX26 T86A. CONCLUSION: These results indicate that the CX26 T86R mutation may be a cause of hearing loss, but CX26 T86S as a non-pathogenic polymorphism mutation does not affect functions of the CX26 protein. The results are in accordance with the results of clinical screening.
RESUMO
OBJECTIVE: To report detection of vestibular-evoked myogenic potentials (VEMPs) in the miniature pig. METHODS: Potentials evoked by 1000 Hz tone bursts were recorded from neck extensor muscles and the masseter muscles in normal adult Bama miniature pigs anesthetized with 3% pentobarbital sodium and Carbachol II. RESULTS: The latency of the first positive wave P from neck extensor muscles was 7.65 ± 0.64 ms, with an amplitude of 1.66 ± 0.34 uv and a rate of successful induction of 75% at 80 dB SPL. The latency of potentials evoked from the masseter muscles was 7.60 ± 0.78 ms, with an amplitude of 1.31 ± 0.28 uv and a rate successful induction of 66% at 80 dB SPL. CONCLUSION: The latencies and thresholds of VEMPs recorded from the neck extensor muscle and the masseter muscle appear to be comparable in normal adult Bama miniature pigs, although the amplitude recorded from the neck extensor muscle seems to be higher than that from the masseter muscle. However, because of their usually relatively superficial and easily accessible location, as well as their large volume and strong contractions, masseter muscles may be better target muscles for recording myogenic potentials.