Calcium imaging in gentamicin ototoxicity: increased intracellular calcium relates to oxidative stress and late apoptosis

Objectives

To estimate intracellular calcium changes in gentamicin (GM) ototoxicity using calcium imaging. To investigate GM-induced physiologic changes in auditory cells including cell viability, apoptosis, and oxidative stress.

Methods

Varying concentrations of GM were applied to the HEI-OC1 cochlear cell line. Calcium imaging tracked changes in intracellular calcium concentration during GM cytotoxicity. Cell viability and intracellular reactive oxygen species (ROS) levels also were measured.

Results

Little change in calcium levels occurred in HEI-OC1 cells exposed to less than 35 mM GM. However, calcium rose continuously in cells exposed to more than 60 mM GM. With administration of intermediate concentrations of 40 or 50 mM GM, calcium increased variably in different cells, returning to baseline in some cases, or rising continuously in others. Upon increase of GM concentration, intracellular calcium concentration and ROS were increased, and cell viability was decreased due to late apoptosis.

Conclusion

This study shows that GM increased intracellular calcium, ROS, and late apoptosis of HEI-OC1 cells derived from cochlear tissue. Increase of intracellular calcium is related to GM-induced apoptosis and oxidative stress. Calcium imaging can be used to determine change of intracellular calcium concentrations and apoptosis in GM ototoxicity.     

活性氧诱导离体大鼠耳蜗Corti器毛细胞凋亡的观察

目的探讨氧自由基中毒后耳蜗毛细胞有无凋亡及其变化规律。方法选用新生1～5d龄SD大鼠24只,随机分为4组。每组6只（12耳）：①无血清培养液组（H2O2浓度为零）;②0．05mmol／LH2O2组;③0．1mmol／LH2O2组;①0．5mmol／LH2O2组。分离Cord器,并用尖刀按顶回、中回、底回将其分为3段,分别放人相应浓度的H2O2培养液中培养．培养结束后用丫啶橙／碘化丙啶双重染色技术检测并计数凋亡的毛细胞。结果不同浓度H2O2组均检测到凋亡毛细胞,外毛细胞（OHC）是H2O2攻击的主要靶细胞,而支持细胞无凋亡。底回毛细胞损伤明显重于顶回和中回,各回外毛细胞损伤明显高于内毛细胞;随H2O2浓度增加,各回凋亡细胞增加。结论本实验条件下,外源性H2O2可直接诱导离体培养大鼠耳蜗Corti器毛细胞凋亡。支持细胞无凋亡。     

灯盏花素对顺铂所致豚鼠耳蜗损伤的拮抗作用

目的探讨灯盏花素对顺铂致豚鼠耳毒性的拮抗作用。方法将30只豚鼠分为三组:正常对照组(A组)、实验对照组(B组)、实验组(C组),每组10只。B组和C组一次性腹腔注顺铂10 mg/kg,同时C组连续10天腹腔注射灯盏花素15 mg.kg-1.d-1,B组等时程腹腔注等量生理盐水。每组豚鼠在实验前后均行听性脑干反应(ABR)检测。在豚鼠顺铂致耳毒性模型完成并检测ABR反应阈后,用免疫组织化学方法检测4-羟基-2-壬烯醛(4-HNE)在各组动物耳蜗的表达,同时用扫描电镜观察各组豚鼠耳蜗形态。结果实验前三组豚鼠ABR反应阈差异无统计学意义(P>0.05),实验后B组和C组豚鼠ABR反应阈分别为50.12±18.45、36.32±15.63 dB SPL,均明显高于实验前,且B组显著高于C组(P<0.05),B组豚鼠听功能损伤明显重于C组。4-HNE在A组表达呈阴性,在B组和C组耳蜗表达呈阳性,且在B组的表达明显强于C组。B组耳蜗外毛细胞的损伤明显较C组重。结论 4-HNE在顺铂所致豚鼠耳蜗损伤中呈阳性表达。灯盏花素对4-HNE的形成有明显抑制作用,且能拮抗顺铂对豚鼠的耳蜗毒性,表明活性氧(ROS)在顺铂所致豚鼠耳蜗损伤中起重要作用。     

Protective Effect of Metformin Against Cisplatin-Induced Ototoxicity in an Auditory Cell Line

Metformin, an antidiabetic drug with potent anticancer activity, is known to prevent oxidative stress-induced cell death in several cell types through a mechanism dependent on the mitochondria. In the present study, we investigated the influence of metformin on cisplatin ototoxicity in an auditory cell line. Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (Sigma, St. Louis, MO, USA) cell proliferation assay. Oxidative stress and apoptosis were assessed by flow cytometry analysis, Hoechst 33258 staining, reactive oxygen species (ROS) measurement, and western blotting. Intracellular calcium concentration changes were detected using calcium imaging. Pretreatment with 1 mM metformin prior to the application of 20 μM cisplatin significantly decreased the frequency of late apoptosis in HEI-OC1 cells and also significantly attenuated the cisplatin-induced increase in ROS. In addition, metformin inhibited the activation of caspase-3 and levels of poly-ADP-ribose polymerase (PARP). Pretreatment with metformin prevented the cisplatin-induced elevation in intracellular calcium concentrations. We propose that metformin protects against cisplatin-induced ototoxicity by inhibiting the increase in intracellular calcium levels, preventing apoptosis, and limiting ROS production.     

Protective Effect of Hexane and Ethanol Extract of Piper Longum L. on Gentamicin-Induced Hair Cell Loss in Neonatal Cultures

Objectives

Gentamicin (GM) is a commonly used aminoglycoside antibiotic that generates free oxygen radicals within the inner ear, which can cause vestibulo-cochlear toxicity and permanent damage to the sensory hair cells and neurons. Piper longum L. (PL) is a well-known spice and traditional medicine in Asia and Pacific islands, which has been reported to exhibit a wide spectrum of activity, including antioxidant activity. In this study, we evaluated the effect of hexane:ethanol (2:8) PL extract (subfraction of PL [SPL] extract) on GM-induced hair cell loss in basal, middle and apical regions in a neonatal cochlea cultures.

Methods

The protective effects of SPL extract were measured by phalloidin staining of cultures from postnatal day 2-3 mice with GM-induced hair cell loss. The anti-apoptosis activity of SPL extract was measured using double labeling by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and myosin-7a staining. The radical-scavenging activity of SPL extract was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay.

Results

SPL extract at a concentration of 1 µg/mL significantly inhibited GM-induced hair cell loss at basal and middle region of cochlea, while 5 µg/mL was effective against apical region hair cell loss. The protective effect of SPL extract was concentration dependent and hair cells retained their stereocilia in explants treated with SPL extract prior to treatment with 0.3 mM GM. SPL extract decreased GM-induced apoptosis of hair cells as assessed by TUNEL staining. The outer hair and inner hair counts were not decreased in SPL extract treated groups in compare to GM treated explants. Additionally, SPL extract showed concentration dependent radical scavenging activity in a DPPH assay.

Conclusion

An anti-apoptosis effect and potent radical scavenger activity of SPL extract protects from GM-induced hair cell loss at basal, middle and apical regions in neonatal cochlea cultures.     

卡那霉素对三种小鼠耳毒性比较及对血管纹Na-K-2Cl联合转运子1表达的影响

目的建立小鼠氨基糖甙类抗生素（aminoglycoside antibiotics，AmAn）耳毒性模型，探讨在不同种小鼠中的AmAn耳毒易感性及其对耳蜗血管纹Na—K-2Cl联合转运子-1（Na-K-2Cl cotransporter-1，NKCC1）表达的影响。方法将72只C57BE／6J、CBA／CaJ、NKCC1＋／-小鼠各自随机分为A、B、C、D4组。A组：卡那霉素组；B组：卡那霉素＋2，3-二羟基苯甲酸组；C组：2，3-二羟基苯甲酸组；D组：生理盐水组。各组连续用药14d。各组动物在用药前、用药后第14天及用药后第35天行脑干诱发电位（auditory brainstem response，ABR）检测听功能；耳蜗琥珀酸脱氢酶组织化学染色观察耳蜗形态学变化；免疫组化法观察血管纹NKCC1表达的变化。结果①A组小鼠ABR闽值明显提高（P〈0．01）并伴随外毛细胞的减少；②B组小鼠ABR阈值变化明显小于A组（P〈0．01），外毛细胞的损害也明显减轻；③A组小鼠耳蜗血管纹NKCCl表达减弱，与D组比较有明显差异（P〈0．01），而B组小鼠血管纹NKCC1表达较A组增强（P〈0．01）；④3种小鼠中CBA／CaJ小鼠对AmAn最敏感，C57BE／6J和NKCC1＋／-小鼠对AmAn的易感性无明显差异。结论应用卡那霉素可建立小鼠AmAn耳毒性模型；卡那霉素可抑制血管纹NKCC1的表达；2，3-二羟基苯甲酸拮抗AmAn耳毒性的途径之-可能是通过减轻AmAn对血管纹NKCC1的抑制作用；具有年龄相关性听力损失特性的小鼠对AmAn耳毒作用并不易感。     

褪黑素在豚鼠耳蜗内的抗氧化作用研究

目的 通过检测豚鼠耳蜗中活性氧(reactive oxygen species,ROS)含量研究褪黑素(melatonin,MLT)在豚鼠耳蜗内的抗氧化作用.方法 雄性杂色豚鼠40只,随机分为4组,每组10只.第2组肌肉注射庆大霉素,且肌肉注射褪黑素.第1组只注射褪黑素,注射褪黑素的时间与剂量与第2组相同.第3组注射庆大霉素,且肌肉注射与褪黑素同等剂量的生理盐水,注射时间与第2组相同.第4组单纯注射庆大霉素.第2、3、4组每天都注射庆大霉素,连续10天.所有动物均于注射庆大霉素前及注射庆大霉素后立即检测听性脑干反应(ABR)阈,庆大霉素注射结束后,测完ABR立即断头处死,取出豚鼠双侧耳蜗,并测定其活性氧含量.结果 注射庆大霉素前,4组实验动物的ABR阈值无显著性差异,本实验的庆大霉素注射剂量可以使听阈提高.注射褪黑素能使ABR阈值降低.注射庆大霉素可以引起耳蜗内活性氧含量增加,注射褪黑素能使耳蜗内活性氧含量降低.结论 褪黑素可以抵抗豚鼠耳蜗中增多的活性氧.     

褪黑素对噪声性聋的防护作用

目的通过检测耳蜗中活性氧(reactive oxygen species,ROS)的含量研究褪黑素(melatonin,MLT)对噪声性聋的防护作用.方法雄性杂色豚鼠40只,随机分为4组,每组10只.第1组为正常对照组,不接触噪声,仅肌肉注射褪黑素,注射褪黑素的时间及剂量与第2组相同.第2、3、4组每天接触倍频程连续噪声(中心频率为4kHz,强度为100dB SPL)8小时,连续3天.第2组于噪声暴露前24h、即刻及噪声暴露中肌肉注射褪黑素3天.第3组在相同时间肌肉注射与第2组相同剂量的生理盐水.第4组为单纯接触噪声对照组.所有动物均于噪声暴露前及暴露后立即检测听性脑干反应(ABR)阈值,噪声暴露3天测完ABR阈值后,立即断头处死豚鼠,取出双侧耳蜗,并测定其活性氧含量.结果噪声暴露后,第2组的ABR阈移及活性氧含量分别与第3、4组比较,差异均有显著性.结论褪黑素能够减少噪声暴露后耳蜗中增多的活性氧水平,可能对噪声性耳蜗损伤具有一定的防护作用.     

Effects of caffeic acid on cisplatin-induced hair cell damage in HEI-OC1 auditory cells

IntroductionCisplatin is a widely used anticancer chemotherapeutic agent. However, it is notorious for its ototoxicity and nephrotoxicity due to induction of reactive oxygen species (ROS). Caffeic acid is a naturally occurring polyphenol present in honey that is known to reduce the generation of oxygen-derived free radicals. The objective of the present study was to evaluate the protective effects and mechanism underlying the effect of caffeic acid on cisplatin-induced ototoxicity in HEI-OC1 auditory cell lines.MethodsCell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by Hoechst 33258 staining and Annexin V-fluorescein isothiocyanate/propidium iodide double staining. Cell cycle stages were analyzed by flow cytometry. The radical-scavenging activity of caffeic acid was assessed using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The expression levels of caspase-3, -8, and -9, as well as the activity of caspase-3, were evaluated.ResultsCaffeic acid showed a protective effect against cisplatin-induced HEI-OC1 cell damage as demonstrated by the MTT assay. Caffeic acid decreased cell death by apoptosis and necrosis. Caffeic acid showed strong scavenging activity against the radical DPPH and decreased intracellular ROS production. Caffeic acid decreased the expression of caspase-3 and -8 and increased the activity of caspase-3.ConclusionsCaffeic acid attenuated cisplatin-induced hair cell loss in HEI-OC1 cell lines; these effects were mediated by its radical scavenging activity and inhibition of apoptosis.     

氧化应激与噪音性聋发病机制及治疗进展

噪音性聋属常见职业病,其致病机制的研究涉及组织、分子及基因等方面,衍生出机械学说、代谢学说、血管学说、钙离子平衡失调、神经因子缺乏及钾离子循环途径等学说。就氧化应激机制在噪音性聋发病中的作用及治疗进行综述。     

Apoptosis in the OC-k3 Immortalized Cell Line Treated with Different Agents: Apoptosis en linea celular OC k3 inmortalizada,tratada con diferentes agentes

The aim of this study is to outline the mechanisms leading cochlear cells to die. We utilized an immortalized cell line (OC-k3 cells) derived from the organ of Corti of transgenic mice in order to perform in-depth biochemical studies with no limitations on sample size and number. We probed these cells with cisplatin and gentamicin, two drugs which display in vivo undesired ototoxic side-effects. We investigated cell viability, reactive oxygen species (ROS) production and glutathione (GSH) levels and tested the effects of different concentrations of cisplatin and gentamicin from 0 to 48 h. Results show that cells undergo a dose-and treatment-time-dependent apoptosis characterized by nuclear fragmentation, integrity of the cell membrane and mitochondria, and absence of DNA endonuclease activity. During the early part of treatment, ROS production increases and intracellular GSH decreases, probably due to the activation of protein kinase Ca. Use of antioxidants such as acetylcysteine, GSH and vitamin C rescues cells from apoptosis almost completely. Overall, these data indicate that ROS generation might play a central role in inducing inner ear cell apoptosis and may have an additive role in the ageing process.     

葛根素对噪声性聋的防治作用

目的探讨葛根素对噪声性聋的预防及治疗作用。方法 18只白化雄性豚鼠随机分为三组,每组6只,单纯噪声组每日腹腔注射生理盐水2 ml,噪声+葛根素组每日腹腔注射葛根素150 mg/kg,空白对照组每日腹腔注射生理盐水2 ml。三组动物均持续给药10日。单纯噪声组和葛根素组于给药第3日给予噪声刺激,噪声模式采用4 kHz纯音,强度128 dB SPL,时间6小时。并于实验第1、4、7、10日分别对各组豚鼠进行ABR检测。10日后随机抽取各组豚鼠进行耳蜗基底膜铺片、免疫组化染色、硝基自由基含量测定。结果三组豚鼠噪声刺激前ABR阈值无明显差异(P>0.05);实验第4、7、10日时,噪声+葛根素组ABR反应阈较单纯噪声组显著降低(P<0.01),但比空白对照组明显升高(P<0.01)。耳蜗基底膜铺片结果显示,单纯噪声组外毛细胞缺失率约72.65%,噪声+葛根素组外毛细胞缺失率约21.48%,空白对照组外毛细胞无缺失。免疫组化染色的结果显示,噪声+葛根素组的硝基自由基表达较单纯噪声组弱,但比空白对照组强。结论葛根素对噪声性聋有一定的防治作用,可以减少听力下降的幅度,降低毛细胞损伤的比例,但不能完全逆转噪声所致的听力下降。     

庆大霉素对豚鼠耳蜗橄榄束传出神经影响的研究

目的探讨庆大霉素对豚鼠耳蜗橄榄束传出神经的损害。方法采用AchE组织化学染色,全耳蜗铺片观察对照组和庆大霉素组停药后不同时间耳蜗橄榄束传出神经的分布特征,并测量耳蜗橄榄束传出神经纤维的数量。实验前后行畸变产物耳声发射对侧声抑制测试。结果肌注庆大霉素2周后耳蜗AchE终末反应物减少,神经纤维连续性中断,耳声发射对侧声抑制幅度下降,损害随观察时间延长而加重,8周后损害最为严重,耳声发射未引出。结论庆大霉素可对豚鼠内侧橄榄耳蜗束传出神经系统产生不可逆性的损害。     

负向调控鼻咽癌微小RNA-9表达促进紫外线介导的活性氧损伤

目的 探讨负向调控鼻咽癌细胞微小RNA-9(micro RNA-9,miR-9)表达对紫外线介导的活性氧损伤的调节作用.方法 应用脂质体Lipofectamine 2000转染抑制鼻咽癌细胞miR-9表达,转染抑制对照剂作为参照.应用紫外线照射45 min后,二氯二氢荧光素双醋酸盐检测两组细胞紫外线诱导的活性氧变化;Annexin V-FITC法检测细胞凋亡和碱性彗星实验检测细胞DNA损伤差异;并观察谷胱甘肽在调节紫外线活性氧损伤中的作用.结果 抑制CNE-2细胞miR-9表达后,miR-9抑制组和抑制对照组细胞在紫外线照射的活性氧水平平均(-x±s,下同)分别为26 895 ±218和15 765±927,两者差异有统计学意义(t=39.754,P＜0.001);紫外线照射后miR-9抑制组DNA损伤和凋亡率分别为28.0％±10.0％和8.0％±0.9％,显著大于抑制对照组的23.6％±9.2％和4.5％±0.8％;并且miR-9抑制组细胞中谷胱甘肽的表达低于抑制剂对照组,含量分别为(1.87±0.15) μmol/L和(9.85 ±0.15) μmol/L(t=-48.832,P＜0.001).负向调控CNE-1细胞表达抑制了谷胱甘肽的表达和增加了紫外线介导的活性氧水平.结论 鼻咽癌细胞中,负向调控miR-9能够促进肿瘤细胞对紫外线介导的活性氧损伤.     

Comparison of the effects of lipoic acid and glutathione against cisplatin-induced ototoxicity in auditory cells

ObjectivesThe aims of this study were to examine lipoic acid (LA)- or glutathione (GSH)-mediated protection against cytotoxicity following cisplatin exposure in HEI-OC1 auditory cells and measure the potential of LA and GSH to scavenge reactive oxygen species (ROS). This study also compares their protective effects and discusses the determination of a preventive or therapeutic dose.MethodsHEI-OC1 cells were pretreated with LA or GSH for 24 h and then exposed to 15 μM cisplatin for 48 h. The resulting cytotoxicity was measured using a cell counting kit-8, and intracellular ROS level was measured using flow cytometry. The protective or anti-ROS effects of LA and GSH were compared. Measurement of caspase 3, 8, 9 activity and Western blot analysis of PARP were performed.ResultsPretreatment with LA at 300 μM and GSH at 3 mM protected HEI-OC1 cells against cisplatin-induced cytotoxicity and significantly reduced the cisplatin-induced increase in ROS. LA showed a significantly more effective protection against cisplatin-induced ototoxicity compared to that shown by GSH (85.4% vs. 73.1% cell viability). Both LA and GSH showed the maximal protective effect at different concentrations in normal or cisplatin-induced cytotoxic conditions. The preventive or therapeutic dose for harmful conditions is quite different for the two drugs and needs careful adjustments.ConclusionThis comparative study on the protective effects of LA and GSH against cisplatin-induced ototoxicity in an auditory cell line posed many challenges. Although LA and GSH showed a significant protective effect against cisplatin, the LA's effect was superior. The concentration at which the maximal protective effect of LA or GSH was noted was 3 times higher in cytotoxic conditions than in normal conditions, which suggests the need for drug dose adjustments based on the purpose (preventive or therapeutic).     

鼻咽癌患者血清中活性氧、超氧化物歧化酶和谷胱甘肽过氧化物酶活性测定

目的 :测定鼻咽癌患者血清中活性氧 (ROS)、超氧化物歧化酶 (SOD)和谷胱甘肽过氧化物酶 (GSH PX)的活性水平 ,以探讨鼻咽癌患者体内自由基代谢状态及其在鼻咽癌发生、发展中的作用。方法 :分别采用Fenton反应显色法 ,黄嘌呤氧化酶法和二硫代二硝基苯甲酸法对 4 0例鼻咽癌患者 (鼻咽癌组 )和 2 0例健康体检者 (对照组 )血清中ROS、SOD和GSH PX水平进行检测。结果 :鼻咽癌组血清中ROS活性 [(11 32 7.835±4 83.777) /(U·ml-1) ]显著高于对照组 [(10 2 6 2 .712± 5 2 5 .2 87) /(U·ml-1) ](P <0 .0 5 ) ,SOD和GSH PX活性[(76 .6 19± 2 2 .2 83) /(NU·ml-1) ,(98.6 5 3± 4 6 .374 ) /AU ]显著低于对照组 [(15 4 .6 0 3± 2 7.2 4 1) /(NU·ml-1) ,(30 7.872± 116 .2 75 ) /AU](P <0 .0 5 ) ;不同病理类型鼻咽癌患者血清中ROS、SOD和GSH PX活性间的差异均无显著性意义 (P >0 .0 5 )。结论 :鼻咽癌患者体内自由基代谢紊乱 ,抗氧化损伤能力显著下降 ,给予抗氧化治疗有利于抑制鼻咽癌的发展     

庆大霉素对豚鼠血管纹黑色素的影响及其机制

目的了解庆大霉素(gentamicin,GM)对豚鼠血管纹黑色素的影响及其作用机制.方法豚鼠杂色40只和白色20只每日肌内注射庆大霉素150 mg/kg体重7 d后用脑干诱发电位仪检测两种豚鼠的听阈变化,以透射电镜观察杂色豚鼠用药前后血管纹内黑色素含量及酪氨酸酶活性的变化,并用免疫组化法观察血管纹增殖细胞核抗原(proliferating cell nuclear antigen,PCNA)表达的变化.结果庆大霉素作用后,杂色和白色豚鼠的听阈与用药前比较,差异有非常显著性意义(P<0.001),且两组豚鼠间的阈移差异也有非常显著性意义(P<0.001).杂色豚鼠对照组和实验组(各10只)血管纹中间细胞内平均(±s,以下同)每个观测区域(300 μm2)的黑素体含量分别为(19.83±2.74)个和(58.33±16.22)个,差异有非常显著性意义(P<0.001);酪氨酸酶活性反应产物面积与测定区域面积之比从1.65%±0.40%增加为3.45%±0.41%,差异有非常显著性意义(P<0.001);但对照组与实验组PCNA阳性中间细胞分别为(14.08±2.76)个和(13.58±2.09)个,差异无显著性意义(P>0.05).结论庆大霉素作用后血管纹内黑色素含量的增加,可能是由于药物促进了酪氨酸酶活性的增强,而不是促进黑素细胞的增殖能力增强.黑色素可保护豚鼠内耳免受庆大霉素的耳毒性作用.     

Effect of antioxidant supplementation on the auditory threshold in sensorineural hearing loss: a meta-analysis

Introduction

Hearing loss is conceptualized as any impairment of the ability to hear and/or detect speech or environment sounds, regardless of cause, type, or degree. It may occur at different stages of life; during pregnancy or childbirth, in childhood, adulthood or old age. It should be noted that aging is the most common cause of sensorineural hearing loss followed by noise-induced hearing loss, and both are closely related to the formation of reactive oxygen species. Dietary antioxidant supplementation has been employed as a therapeutic strategy to prevent and/or delay the risks of major human diseases.