Inhibition of the NLRP3 inflammasome attenuates spiral ganglion neuron degeneration in aminoglycoside-induced hearing loss

Aminoglycosides are a widely used class of antibacterials renowned for their effectiveness and broad antimicrobial spectrum.However,their use leads to irreversible hearing damage by causing apoptosis of hair cells as their direct target.In addition,the hearing damage caused by aminoglycosides involves damage of spiral ganglion neurons upon exposure.To investigate the mechanisms underlying spiral ganglion neuron degeneration induced by aminoglycosides,we used a C57BL/6J mouse model treated with kanamycin.We found that the mice exhibited auditory deficits following the acute loss of outer hair cells.Spiral ganglion neurons displayed hallmarks of pyroptosis and exhibited progressive degeneration over time.Transcriptomic profiling of these neurons showed significant upregulation of genes associated with inflammation and immune response,particularly those related to the NLRP3 inflammasome.Activation of the canonical pyroptotic pathway in spiral ganglion neurons was observed,accompanied by infiltration of macrophages and the release of proinflammatory cytokines.Pharmacological intervention targeting NLRP3 using Mcc950 and genetic intervention using NLRP3 knockout ameliorated spiral ganglion neuron degeneration in the injury model.These findings suggest that NLRP3 inflammasome-mediated pyroptosis plays a role in aminoglycoside-induced spiral ganglion neuron degeneration.Inhibition of this pathway may offer a potential therapeutic strategy for treating sensorineural hearing loss by reducing spiral ganglion neuron degeneration.

Correlation of PDCD5 and Apoptosis in Hair Cells and Spiral Ganglion Neurons of Different Age of C57BL/6J Mice

This study examined the expression pattern of programmed cell death 5 (PDCD5) in co-chlear hair cells and spiral ganglion neurons (SGNs) and its association with age-related hearing loss in mice.Sixty C57BL/6J (C57) mice at different ages were divided into four groups (3,6,9 or 12 months).PDCD5 expression was detected by using immunohistochemistry,real-time PCR and Western blot.Morphological change of the cochleae was also evaluated by using immunoassay.The results showed that the expression of PDCD5 had a gradual increase with ageing in both protein and RNA levels in C57 mice,as well as gradually increased apoptosis of cochlear hair cells and SGNs.In addition,we also found that caspase-3 activity was enhanced and its expression was enhanced with ageing.It is implied that overexpression of PDCD5 causes the increase in caspase-3 activity and the subsequent increase of apoptosis in cochlear hair cells and SGNs,and thereby plays a role in the pathogenesis of presbycusis.Thus,PDCD5 may be a new target site for the treatment and prevention of age-related hearing loss.

Gamma-aminobutyric acid A receptor and N-methyl-D-aspartate receptor subunit expression in rat spiral ganglion neurons

BACKGROUND： Gamma-aminobutyric acid A （GABAA） and N-methyl-D-aspartate （NMDA） receptors are significant receptors in the central nervous system. An understanding of GABAA and NMDA receptor expression in spiral ganglion neurons （SGN） provides information for the functional role of these receptors in the auditory system. OBJECTIVE： To investigate mRNA expression of GABAA receptor （GABAAR） and NMDA receptor （NMDAR） subunits in the rat SGN. DESIGN, TIME AND SETTING： This in vitro, molecular biological study was performed at the Laboratory of Otolaryngology-Head and Neck Surgery, Guangxi Medical University, China from July 2007 to May 2008. MATERIALS： Reverse Transcriptase Kit and Taq DNA polymerase were purchased from Fermentas Burlington, ON, Canada; GABAAR and NMDAR primers were purchased from Shanghai Sangon, Shanghai, China. METHODS： SGN from 3-5 day postnatal Wistar rats was collected for primary cultures, mRNA expression of GABAAR and NMDAR subunits in the SGN was determined by reverse transcription polymerase chain reaction. MAIN OUTCOME MEASURES： Expression levels of GABAAR and NMDAR subunits were determined by quantitative analysis. RESULTS： GABAAR subunits （αl 6, β1 3, and y1 3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were detected in the SGN. In α subunit genes of GABAAR, α1 and α3 expression was similar （P 〉 0.05） and greater than the other subunits. Of the β subunit genes, β1 subunit mRNA levels were greater than β2 and β3. Of the y subunit genes, y2 subunit mRNA levels were greater than y1 and y3. NR1 mRNA expression was the greatest of NMDAR subunits. CONCLUSION： GABAAR subunits （α1 6, β1-3, and y1-3） and NMDAR subunits （NR1, NR2A, NR2B, NR2C, NR2D, NR3A, and NR3B） were expressed in the rat SGN. Through comparison of GABAAR and NMDAR subunit expression, possible GABAAR combinations, as well as highly expressed subunit combinations, were estimated, which provided information for pharmacological and electrophysiological characteristics of GABAAR in the auditory system.

Adenovirus-mediated human β-nerve growth factor gene transfer has a protective effect on cochlear spiral ganglion after blast exposure

Objective:To study whether adenovirus-mediated human β-nerve growth factor (Ad-hNGFβ) gene has any protective effect on blast hearing impairment. Methods:Deafness was induced by blast exposure (172.0 dB) in 30 healthy guinea pigs. On day 7 of blast exposure, Ad-hNGFβ was infused into the perilymphatic space of 20 animals as the study group (hNGFβ group), and artificial perilymph fluid (APF) was infused into the perilymphatic space of the other 10 animals as the control group. At weeks 1, 4 and 8 after blast exposure, the animals were sacrificed and the cochleae were removed for immunohistochemical and HE stainings. Results: Expression of Ad-hNGFβ protein was detected in each turn of the cochlea at the 1st week, with almost equal intensity in all turns. At the 4th week, the reactive intensity of the expression of Ad-hNGFβ protein decreased. At the 8th week, no expression was detectable. The results of HE staining showed that the amount of spiral ganglions in hNGFβ group was significantly greater than that of the control group at week 4 (P<0.01). Conclusion: Ad-hNGFβ can be expressed at a high level and for a relatively long period in the blast impaired cochlea, suggesting that Ad-hNGFβ has a protective effect on cochlear spiral ganglion cells after blast exposure and the efficient gene transfer into cochlea had been achieved without toxicity.

Gene transfection mediated by polyethyleneiminepolyethylene glycol nanocarrier prevents cisplatininduced spiral ganglion cell damage

Polyethyleneimine-polyethylene glycol （PEI-PEG）, a novel nanocarrier, has been used for trans- fection and gene therapy in a variety of cells. In our previous study, we successfully carried out PEI-PEG-mediated gene transfer in spiral ganglion cells. It remains unclear whether PEI-PEG could be used for gene therapy with X-linked inhibitor of apoptosis protein （XIAP） in the inner ear. In the present study, we performed PEI-PEG-mediated XIAP gene transfection in the cochlea of Sprague-Dawley rats, via scala tympani fenestration, before daily cisplatin injections. Audito- ry brainstem reflex tests demonstrated the protective effects of XIAP gene therapy on auditory function. Immunohistochemical staining revealed XIAP protein expression in the cytoplasm of cells in the spiral ganglion, the organ of Corti and the stria vascularis. Reverse transcription-PCR detected high levels of XIAP mRNA expression in the cochlea. The present findings suggest that PEI-PEG nanocarrier-mediated XIAP gene transfection results in XIAP expression in the cochlea, prevents damage to cochlear spiral ganglion cells, and protects hearing.

Phenotypic differentiation of neonatal rat cochlear spiral ganglion neurons following trypsin dissociation and culture

BACKGROUND： Under laboratory conditions, cochlear spiral ganglion neurons are commonly isolated and cultured by mechanical dissociation. However, these neurons are extremely fragile and survive for only a short time. OBJECTIVE： To establish a trypsin dissociation and culture method for studying neonatal rat cochlear spiral ganglion neurons. DESIGN： A single sample study. SETTING： Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA. MATERIALS： This study was performed at the central laboratory for Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital, Fourth Military Medical University of Chinese PLA from February to May 2006. A total of 40 neonatal Sprague Dawley rats of either gender, aged 2-5 days, were provided by the Laboratory Animal Center of the Fourth Military Medical University of Chinese PLA. Trypsin and neuronal-specific nuclear protein （NeuN） monoclonal antibodies were purchased from Sigma Company, USA. Culture medium was synthesized using Dulbecco＇s modified Eagle＇s medium （DMEM）/F12 （Gibco Company, USA） supplemented with 10% fetal bovine serum （Sigma Company, USA）, 100 000 U/L penicillin, and 1 mol/L NaOH. The following protocol was performed in accordance with ethical guidelines for the use and care of animals. METHODS： After anesthesia, rats were sacrificed by neck dislocation. A complete cochlear axis with spiral ganglion tissue was removed. The cochlear axis was rinsed three times in a culture dish with a diameter of 35 mm using Hank＇s balanced solution. After washings, the tissue was cut into pieces, digested with 0.25% trypsin for about 20 minutes, and incubated in a 37 ℃ water bath. The tissue was centrifuged, then mixed with serum-containing culture medium. Using a transfer pipette, the cell suspension was transferred to polylysine （0.1%）-treated culture dishes with a diameter of 35 mm. The culture dish was incubated at 37 ℃, with a 5% CO2-air environment. Once the cells adhered to the culture dish wall, DMEM/F12 supplemented with 10% fetal bovine serum was added. MAIN OUTCOME MEASURE： Using an inverted microscope, the adherent cultured cells were observed and neurite growth index was calculated. Immunocytochemistry was performed to identify the spiral ganglion neurons, and NeuN-positive cells were analyzed. Following immunofluorescence, cochlear spiral ganglion neurons were identified through a microscope. RESULTS： Observation of cellular morphology： after digestion, inoculated cells exhibited were spherical, well stacked, and had a transparent appearance. Six hours later, some cells adhered to the culture dish wall, and small neurites were detected in a small number of cells. Twelve hours later, the adherent cells developed into polarized cells. Eighteen hours after inoculation, the adherent cells presented an ellipsoidal appearance, clear cell membranes, homogeneous cytoplasm, good refraction, and a transparent cell body surrounded by a marked halation. Twenty-four hours later, most of the cochlear spiral ganglion neurons exhibited a bipolar neuronal morphology with neurite length ranging from 2-5 times the length of a cell body. Some cochlear spiral ganglion neurons exhibited a tripolar neuronal morphology with neurites that stretched in three directions; neurite length was several times greater than the transverse diameter. Forty-eight to seventy-two hours later, the cells further differentiated and exhibited interwoven neurites, with a length that was 7-8 times greater than the cell body length. Seven days later, cells began to degenerate and underwent apoptosis. Identification of cochlear spiral ganglion neurons： immunocytochemical staining revealed whole cochlear spiral ganglion neurons that were green-colored and exhibited an ellipsoidal cell body with clear neurites. Measurement of neurite growth index： neurite growth index was 0.52±0.13, 0.86±0.21, 1.22±0.33, and 1.05 ±0.26 for 24, 48, 72, and 120 hours after inoculation, respectively. CONCLUSION： Under cell culture conditions of serum and trypsin dissociation, neonatal rat cochlear spiral ganglion neurons grow well, survive for long periods in vitro, and exhibit normal phenotypic differentiation.

A study of gentamicin injury mechanisms using cultured mouse cochlear spiral ganglion cells

Objective To study gentamicin injury mechanisms using postnatal mouse cochlear spiral gangcells （SGC）. Methods SGCs were isolated using a combinatorial approach of enzymatic digestion and mechanical separation from P2 - 6 Kunming mouse cochleae. After 4 days, cultured SGCs were fixed with 4% paraformaldehyde at room temperature for immunocytochemical examination using the methods of S-P and the monoclonal antibody against mouse neurofilament protein （Neurofilament-68/200Kda, NF-L＋ H）. SGCs were randomly divided into a blank control group and three gentamicin treatment groups （medium gentamicin concentration at 50 mg/L, 100 mg/L and 150 mg/L respectively）, SGCs were collected and examined under a transmission electron microscope after being cultured for 48 h. Results SGC primary culture was successful. SGC cytoplasm and neurites were dyed brownish yellow by the monoelonal mouse neurofilament protein antibody. SGCs showed classical bipolar neuron appearance. Under the transmission electron microscope,.gentamicin treated SGCs showed morphological features different compared to those in the blank control group, which might indicate apoptosis. Conclusion Our results indicate that gentamicin has direct toxic effects on cochlear SGCs in mice and the injury mechanism is closely related with apoptosis. Damage to mitochor, dria may play an important role in the process.

Salicylate enhances expression and function of NMDA receptors in cochlear spiral ganglion neurons

Objective To study the effect of salicylate on the expression and function of NMDA receptors in spiral ganglion neurons （SGNs）. Methods The mRNA of NR1 subunit of NMDA receptor in modiolus tissues were detected by Real time fluorescence quantitative PCR （FQ-PCR）. NMDA receptor whole-cell currents were recorded using patch clamp in acute isolated SGNs. Results Compared with the control group, salicylate significantly increased the mRNA level of NR1 subunit in SGNs. NMDA of concentrations ranging from 0.1 mM to 10 mM evoked no current in SGNs. NMDA （0. 1mM and 0.5 mM） applied with salicylate （5 mM）, however, induced inward currents （212.6±15.2pA, n=5; 607.9±44.3pA, n=5） in a dose-dependent manner, which could be inhibited by APV. Salicylate alone did not produce any current in SGNs. Conclusion Salicylate increases the expression of NMDA receptors and facilitates the currents mediated by NMDA receptors in SGNs.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.

Injury and protection of spiral ganglion neurons

Cochlear spiral ganglion neurons(SGNs)are bipolar ganglion cells and are the first neurons in the auditory transduction pathway.They transmit complex acoustic information from hair cells to second-order sensory neurons in the cochlear nucleus for sound processing.Injury to SGNs causes largely irreversible hearing impairment because these neurons are highly differentiated cells and cannot regenerate,making treatment of sensorineural hearing loss(SNHL)arising from SGN injury difficult.When exposed to ototoxic drugs or damaging levels of noise or when there is loss of neurotrophic factors(NTFs),aging,and presence of other factors,SGNs can be irreversibly damaged,resulting in SNHL.It has been found that NTFs and stem cells can induce regeneration among dead spiral ganglion cells.In this paper,we summarized the present knowledge regarding injury,protection,and regeneration of SGNs.

Abnormal Innervation,Demyelination,and Degeneration of Spiral Ganglion Neurons as Well as Disruption of Heminodes are Involved in the Onset of Deafness in Cx26 Null Mice

GJB2 gene mutations are the most common causes of autosomal recessive non-syndromic hereditary deafness.For individuals suffering from severe to profound GJB2-related deafness,cochlear implants have emerged as the sole remedy for auditory improvement.Some previous studies have highlighted the crucial role of preserving cochlear neural components in achieving favorable outcomes after cochlear implantation.Thus,we generated a conditional knockout mouse model(Cx26-CKO)in which Cx26 was completely deleted in the cochlear supporting cells driven by the Sox2 promoter.The Cx26-CKO mice showed severe hearing loss and massive loss of hair cells and Deiter’s cells,which represented the extreme form of human deafness caused by GJB2 gene mutations.In addition,multiple pathological changes in the peripheral auditory nervous system were found,including abnormal innervation,demyelination,and degeneration of spiral ganglion neurons as well as disruption of heminodes in Cx26-CKO mice.These findings provide invaluable insights into the deafness mechanism and the treatment for severe deafness in Cx26-null mice.

Cochlear function after selective spiral ganglion cells degeneration induced by ouabain

Background Ouabain, a cardiac glycoside that specifically binds to Na/K-ATPase and inhibits its activity, was applied to gerbils to develop a method for studying auditory neuropathy. Methods Ouabain was applied to the round window of the cochlea in each gerbil by using a piece of gelfoam with 3 μl or 24 μl （1 mmol/L） ouabain solution. The changes of the threshold of auditory brainstem response, cochlear function round window electrocochleography, as well as the morphological changes of the spiral ganglion cells of the cochlea were observed after application of ouabain for 24 hours or 96 hours. Results In ouabain treated gerbils, auditory brainstem response and compound action potential thresholds showed either elevation or no response at all. However, the thresholds of cochlear microphonic and distortion product otoacoustic emissions were not affected. Degeneration and necrosis of some spiral ganglion cells in ears with applications of ouabain （24 hours, 3 μl, 1 mmol/L; 96 hours, 24 μl, 1 mmol/L ouabain）. The number of spiral ganglion cells was decreased （24 hours, 3 μl, 1 mmol/L ouabain） or near to a total loss （96 hours, 24 μl, 1 mmol/L ouabain）.Conclusions These results indicate a high degree of independence between the spiral ganglion ceils and the outer hair cell systems in the cochlear transduction mechanism. The method used in this study would provide a valuable tool for studying auditory neuropathy.

Projection of spiral ganglion glutamate immunoreactive positive neurons into dorsal cochlear nucleus in guinea pigs

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Recent advances in the application of MXenes for neural tissue engineering and regeneration

Transition metal carbides and nitrides(MXenes)are crystal nanomaterials with a number of surface functional groups such as fluorine,hydroxyl,and oxygen,which can be used as carriers for proteins and drugs.MXenes have excellent biocompatibility,electrical conductivity,surface hydrophilicity,mechanical properties and easy surface modification.However,at present,the stability of most MXenes needs to be improved,and more synthesis methods need to be explored.MXenes are good substrates for nerve cell regeneration and nerve reconstruction,which have broad application prospects in the repair of nervous system injury.Regarding the application of MXenes in neuroscience,mainly at the cellular level,the long-term in vivo biosafety and effects also need to be further explored.This review focuses on the progress of using MXenes in nerve regeneration over the last few years;discussing preparation of MXenes and their biocompatibility with different cells as well as the regulation by MXenes of nerve cell regeneration in two-dimensional and three-dimensional environments in vitro.MXenes have great potential in regulating the proliferation,differentiation,and maturation of nerve cells and in promoting regeneration and recovery after nerve injury.In addition,this review also presents the main challenges during optimization processes,such as the preparation of stable MXenes and long-term in vivo biosafety,and further discusses future directions in neural tissue engineering.

红景天苷改善顺铂引起的小鼠耳蜗毛细胞和螺旋神经节神经元损伤的机制

目的探究红景天苷(SAL)改善顺铂(CIS)引起的耳蜗毛细胞(CHC)和螺旋神经节神经元(SGN)损伤的作用及其与环磷腺苷(cAMP)/蛋白激酶A(PKA)/cAMP效应元件结合蛋白(CREB)通路的关系。方法分离新生C57BL/6小鼠的耳蜗基底膜,分为对照组(C组)、CIS组、SAL组、SAL+SQ22536(cAMP抑制剂)组和SAL+H-89(PKA抑制剂)组,每组20条。C组仅加入无血清BME培养液;CIS组在培养液中加入15μmol/L CIS;SAL组在CIS组基础上加入5μmol/L SAL;SAL+SQ22536组在CIS组基础上加入5μmol/L SAL和5μmol/L SQ22536;SAL+H-89组在CIS组基础上加入5μmol/L SAL和30μmol/L H-89。各组在培养箱中孵育48 h后,免疫荧光染色观察各组CHC和SGN损伤;试剂盒检测各组耳蜗基底膜中ROS和cAMP含量;Western blot检测各组PKA、p-CREB、CREB、Bcl-2、BDNF、NF-M蛋白水平。结果CIS组CHC排列混乱、体积肿大,SGN细胞核破碎、神经突缺失,SAL可减轻CHC和SGNs损伤。与C组相比,CIS组CHC、SGN数量较少(P<0.05),ROS、cAMP含量、PKA、BDNF、NF-M、Bcl-2蛋白及p-CREB/CREB水平较高(P<0.05);与CIS组相比,SAL组CHC、SGN数量较多(P<0.05),ROS含量较低(P<0.05),cAMP含量、PKA、BDNF、NF-M、Bcl-2蛋白及p-CREB/CREB水平较高(P<0.05)。SQ22536和H-89均可逆转SAL对CHC和SGN的保护作用。结论SAL可能通过激活cAMP/PKA/CREB通路,促进抗凋亡蛋白和神经保护因子表达,缓解CIS引起的CHC和SGN损伤。

基于自噬途径探讨银杏叶提取物对老年性聋大鼠的保护作用

目的探讨银杏叶提取物对老年性聋大鼠听功能、耳蜗组织形态学和自噬相关蛋白表达的作用。方法将45只大鼠随机分为对照组、模型组和银杏叶提取物低、中、高剂量组(10、20、30 mg/kg),每组9只,采用腹腔注射500 mg/kg D-半乳糖(D-gal)构建老年性聋模型。给药8周后,听性脑干诱发电位(ABR)检测大鼠听阈变化,HE染色观察耳蜗毛细胞、血管纹和螺旋神经节细胞形态变化,免疫荧光染色法检测耳蜗内外毛细胞数量变化,透射电子显微镜观察耳蜗毛细胞超微结构变化,Western blot法检测耳蜗组织中自噬相关蛋白表达。结果与对照组比较,模型组ABR阈值升高(P<0.01),耳蜗内外毛细胞、螺旋神经节细胞和血管纹损伤严重,血管纹厚度、螺旋神经节细胞数、内外毛细胞数和自噬小体数均降低(P<0.01),耳蜗组织Beclin1、LC3Ⅱ蛋白表达和LC3Ⅱ/LC3Ⅰ比值均降低(P<0.01),P62蛋白表达升高(P<0.01);与模型组比较,银杏叶提取物中、高剂量组ABR阈值均降低(P<0.01),耳蜗内外毛细胞及血管纹形态改善,螺旋神经节细胞形态趋向正常,血管纹厚度、螺旋神经节细胞数、内外毛细胞数和自噬小体数均增加(P<0.05,P<0.01),耳蜗组织Beclin1、LC3Ⅱ蛋白表达和LC3Ⅱ/LC3Ⅰ比值均升高(P<0.01),P62蛋白表达降低(P<0.01)。结论银杏叶提取物对老年性聋大鼠的听功能和耳蜗细胞具有保护作用,其机制可能与上调耳蜗组织Beclin1、LC3Ⅱ蛋白表达,下调P62蛋白表达有关。

三组固定方式对应小鼠耳蜗组织染色效果的研究

目的研究不同固定方式对耳蜗结构及细胞形态的影响、探索耳蜗内特定细胞类型的最佳固定方式,最大限度保存耳蜗不同细胞类型以及细胞内细胞器的形态结构,为生理和病理条件下的耳蜗细胞形态学研究提供一种更为个性化的免疫荧光染色方法。方法选择野生型C57BL/6小鼠9只,将小鼠随机分为一组、二组及三组,每组3只。一组采用0.9%氯化钠溶液+4%多聚甲醛(polyformaldehyde,PFA)心脏灌流、二组采用直接4%PFA心脏灌流、三组采用耳蜗4%PFA灌流。对3组固定后的耳蜗采用常规切片免疫荧光染色,激光扫描共聚焦显微镜在低倍镜、高倍镜以及超高分辨率等模式下观察并比较3组耳蜗内不同细胞和细胞内形态结构。结果3种染色方法在不同观察模式下均能清楚显示毛细胞、神经丝形态结构,3组毛细胞、神经丝形态结构比较,差异无统计学意义(P>0.05)。血管纹区域在一组的固定方式下,其形态结构相比于其他两种固定方式保存的更为完整。结论3组耳蜗固定方式均可以用来研究Corti's器、螺旋神经节区域的形态结构,而一组的固定方式更适合用于研究血管纹区域内的细微结构。

当归芍药散调控IKK/IκBα/NF-κB通路对老年性聋小鼠耳蜗螺旋神经节细胞凋亡的影响

目的探究当归芍药散调控核转录因子-κB抑制蛋白激酶(IKK)/核因子κB抑制蛋白(IκBα)/核因子-κB(NF-κB)通路对老年性聋小鼠耳蜗螺旋神经节细胞凋亡的影响。方法将48只C57BL/6J小鼠随机分为老年性聋模型组(Model组)、低剂量当归芍药散组(中药-L组,3.2 g/kg生药)、高剂量当归芍药散组(中药-H组,6.4 g/kg生药)和高剂量当归芍药散+IKK/IκBα/NF-κB信号通路激活剂白藜芦醇(RES)组(中药-H+RES组,6.4 g/kg生药+43.33 mg/kg RES),每组12只小鼠;取12只2月龄的C57BL/6J小鼠作为对照组(Control组)。采用听性脑干反应(ABR)检测各组小鼠听阈值;HE染色观察小鼠耳蜗螺旋神经节的病理变化,并对小鼠耳蜗中回与底回的螺旋神经节细胞的数量进行计数;TUNEL染色法检测小鼠耳蜗螺旋神经节细胞的凋亡。ELISA法检测小鼠耳蜗组织中丙二醛(MDA)与超氧化物歧化酶(SOD)水平。qRT-PCR法检测小鼠耳蜗组织中IL-1β、IL-6与TNF-αmRNA水平。Western blot法检测小鼠耳蜗组织IKK/IκBα/NF-κB通路与凋亡相关蛋白的表达。结果Model组小鼠较Control组小鼠8 kHz、16 kHz、24 kH、32 kHz听阈值、耳蜗螺旋神经节组织TUNEL阳性细胞数、耳蜗组织MDA及IL-1β、IL-6与TNF-αmRNA水平、p-IκBα、凋亡蛋白(Bax、cleaved-Caspase-3)表达水平、IKK、NF-κB p65磷酸化水平均显著升高(均P<0.05),耳蜗螺旋神经节细胞数量、SOD水平、IκBα蛋白表达水平显著降低(均P<0.05),耳蜗螺旋神经节细胞的形态异常、排列疏松紊乱,发生空泡化,出现病理学损伤。中药-L组、中药-H组小鼠较Model组相应指标变化程度较轻(均P<0.05)。RES减弱了当归芍药散对老年性聋小鼠耳蜗螺旋神经节细胞凋亡的抑制作用。结论当归芍药散可能通过下调IKK/IκBα/NF-κB通路抑制老年性聋小鼠耳蜗螺旋神经节细胞凋亡。

PTC-209诱导体外螺旋神经元损伤作用及机制

目的 探讨Bmi1特异性抑制剂PTC-209诱导体外培养耳蜗器官螺旋神经元(SGNs)、听觉神经纤维(ANFs)的损伤作用,并分析其可能的调控机制。方法 选用P3新生SD仔鼠进行耳蜗器官培养,首先与不同浓度PTC-209进行培养,确定损伤模型(PTC-209组),即10μM PTC-209与耳蜗器官共培养48 h。随后将10μM PTC-209与5 mM GSH共培养,确定最佳保护模型(GSH+PTC-209组),即Pre6h 5 mM GSH对10μM PTC-209培养48 h。最后将实验分为Ctrl组、GSH组、PTC-209组及GSH+PTC-209组,培养结束后予MitoSOXRed进行组织染色,激光共聚焦显微镜观察。结果 PTC-209诱导耳蜗SGNs和ANFs损伤随浓度增高和作用时间延长而加重,其损伤作用呈时间和浓度依赖性。不同时间预处理GSH对PTC-209诱导SGNs和ANFs损伤存在差异保护作用,将各组的ANFs进行统计分析发现Pre 6 h GSH 5 mM+PTC-209 10μM组与其余各组ANFs差异均有统计学意义(P<0.01),提示Pre 6 h 5 mM GSH对10μM PTC-209培养48 h诱导SGNs和ANFs的损伤有显著保护作用。MitoSOX Red染色显示Ctrl组、GSH组及GSH+PTC-209组均未见红色荧光标记的ROS在SGNs、支持细胞(SCs)和ANFS蓄积;PTC-209组红色荧光标记的ROS在SGNs、SCs和ANFS大量蓄积。统计分析显示PTC-209组和Ctrl组、GSH+PTC-209组ROS的红色荧光光密度值差异均有统计学意义(q分别为343.5、349.3,P<0.01)。结论 PTC-209可诱导SGNs处细胞内的自由基和线粒体中ROS大量蓄积,进而导致氧化还原失衡,促进SGNs死亡,但是调控机制尚需要进一步探讨。

Inhibition of histone methyltransferase PRMT5 attenuates cisplatininduced hearing loss through the PI3K/Akt-mediated mitochondrial apoptotic pathway

This study aimed to evaluate the therapeutic potential of inhibiting protein arginine methyltransferase 5(PRMT5)in cisplatin-induced hearing loss.The effects of PRMT5 inhibition on cisplatin-induced auditory injury were determined using immunohistochemistry,apoptosis assays,and auditory brainstem response.The mechanism of PRMT5 inhibition on hair cell survival was assessed using RNA-seq and Cleavage Under Targets and Tagment-quantitative polymerase chain reaction(CUT&Tag-qPCR)analyses in the HEI-OC1 cell line.Pharmacological inhibition of PRMT5 significantly alleviated cisplatin-induced damage to hair cells and spiral ganglion neurons in the cochlea and decreased apoptosis by protecting mitochondrial function and preventing the accumulation of reactive oxygen species.CUT&Tag-qPCR analysis demonstrated that inhibition of PRMT5 in HEI-OC1 cells reduced the accumulation of H4R3me2s/H3R8me2s marks at the promoter region of the Pik3ca gene,thus activating the expression of Pik3ca.These findings suggest that PRMT5 inhibitors have strong potential as agents against cisplatininduced ototoxicity and can lay the foundation for further research on treatment strategies of hearing loss.