电码针对庆大霉素耳中毒的防治作用及其机制的研究

目的：观察电码针对豚鼠庆大霉素（GE）耳毒性的防治作用,方法：测定脑干听觉诱发电位（BAEP）和用组织化学方法测定耳蝇毛细胞及血管纹的琥珀酸脱氢酶（SDH）。结果：电针能降低CE引起的BAEP反应阈的上升幅度,缩小BAEP波峰潜伏期及波峰间期的延长;能保护毛细胞及耳蜗血管纹细胞线粒体呼吸酶的活性。结论：电针能降低GE5的耳毒性,保护毛细胞及耳蜗血管纹细胞线粒体酶的活性。保证这些细胞能量代谢,维持细胞所需要能量的各种功能的活动。减少细胞的损伤,可能有是电针防治GE耳毒性的机制之一。     

电针对庆大霉素耳毒性的预防作用

目的和方法 :本实验用耳廓反射 (PR)、脑干听觉诱发电位 (BAEP)、组织化学方法 ,观察电针疗法对豚鼠庆大霉素 (GE)耳毒性的防治作用 ,并探讨其机制。结果 :电针疗法能降低GE引起的PR阈值和BAEP反应阈的上移幅度及BAEP波峰潜伏期和波峰间期的延长 ,能维持毛细胞溶酶体的完整 ,防止毛细胞的自溶性破坏。结论 :电针疗法有对抗GE耳毒性作用。维持耳蜗毛细胞内溶酶体完整 ,防止溶酶体内水解酶的逸出而发生细胞自溶 ,可能是电针疗法的作用机制之一。     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗血管纹NOS活性的影响

目的：研究丹参注射液（SM）对庆大霉素（GM）耳中毒豚鼠耳蜗血管纹一氧化氮合酶（NOS）活性的影响及其与听阈的关系,探讨SM对GM耳毒性损伤的保护作用。方法：应用NADPH－黄递酶（NADPH-d）组织化学染色以及图象分析技术,并结合听性脑干反应（ABR）测试。结果：SM－GM组耳蜗血管纹NOS活性和ABR阈值均明显低于GM组（P＜0.01）;且各组NOS活性变化与ABR阈移高庆相关（rcontrol=-0.9464;rGM=-0.9117;rSM GM=-0.8958,P＜0.01）。结论：SM可通过降低耳蜗血管纹NOS活性以减轻GM的耳毒性损伤,从而改善听功能。     

顺铂耳毒性大鼠耳聋模型的研究

摘要 目的：探讨顺铂对大鼠造成的听力损伤及耳蜗细胞形态学变化。方法：体内实验,运用顺铂腹腔注射的方法,连续七天注射,通过听性脑干反应检测,观察顺铂对不同日龄的大鼠听力损伤情况;测听后取耳蜗,通过基底膜铺片和冰冻切片的免疫荧光染色,观察听力损伤后对耳蜗毛细胞和螺旋神经元的影响。体外实验,耳蜗器官培养免疫荧光染色,观察顺铂对耳蜗毛细胞和螺旋神经元的影响。结果：顺铂具有耳毒性,会对大鼠听力造成损伤,高频听力损伤更加严重,而且对不同日龄的大鼠造成的听力损失不同,小日龄的大鼠对顺铂耳毒性更加敏感。体内实验,顺铂耳毒性造成听力损失,会引起大鼠耳蜗毛细胞的缺失,但未观察到明显的螺旋神经元缺失,也没有观察到明显的Cleaved caspase-3阳性螺旋神经元细胞。体外实验,可以观察到顺铂同时引起毛细胞和螺旋神经元产生明显的损伤。结论：体、内外实验,都可以建立稳定的顺铂耳毒性大鼠耳聋模型,对研究顺铂损伤耳蜗毛细胞的发生机制和保护奠定了实验基础。     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗NOS异构体表达的影响

目的：研究丹参注射液（SM）对庆大霉素（GM）耳中毒豚鼠耳蜗一氧化氮合酶（NOS）异构体表达的影响,探讨SM对GM耳毒性的防护机制。方法：40只豚鼠随机分成对照组、GM组、SM组和GM＋SM组,应用SABC免疫组织化学方法及显微图像分析技术,观察NOS三型异构体在豚鼠耳蜗的表达;同时结合听脑干反应（ABR）测试,观察用药前后豚鼠听阈的变化。结果：GM＋SM组豚鼠耳蜗诱导型NOS（iNOS/NOSⅡ）表达和ABR阈值均明显低于GM组（P〈0.01）;且iNOS表达变化与ABR阈值改变高度相关（｜r｜〉0.7,P〈0.01）;而各组豚鼠耳蜗神经元型NOS（nNOS/NOSⅠ）和内皮型NOS（eNOS/NOSⅢ）表达均无显著性差异。结论：SM对GM耳中毒后豚鼠耳蜗nNOS和eNOS表达无影响,但可通过抑制GM所致iNOS高表达,以减少NO的过量生成,从而对GM的耳毒性损伤发挥防护作用。     

丹参注射液对庆大霉素耳中毒豚鼠耳蜗氧自由基生成的影响

目的:研究丹参注射液(SM)对庆大霉素(GM)耳中毒豚鼠耳蜗氧自由基生成的影响,探讨SM对GM耳毒性损伤的保护作用及其机制.方法:检测豚鼠耳蜗组织中超氧化物歧化酶(SOD)活力和丙二醛(MDA)含量,结合听性脑干反应(ABR)测试及透射电镜技术.结果:经GM处理的耳蜗组织中SOD活力明显下降,MDA含量则明显增加(P＜0.01),且与ABR阈值升高高度相关(|r|＞0.8,P＜0.05).同时接受SM的动物,其耳蜗组织中SOD活力明显升高(P＜0.01),MDA含量则明显减少(P＜0.05),且听功能显著改善.电镜观察显示耳蜗形态学改变与听力变化相一致.结论:氧自由基及其引发的脂质过氧化参与了GM耳中毒过程,SM可通过提高耳蜗组织中SOD活力,防止脂质过氧化,减轻GM的耳蜗毒性,改善听功能.     

甲状腺激素对豚鼠卡那霉素中毒性耳聋的预防作用

卡那霉素、庆大霉素等抗生素常引起耳聋,目前尚无较好的防治方法。卡那霉素对内耳的毒性作用,主要先影响有关的酶功能,继而破坏毛细胞而致聋。甲状腺激素具有促进蛋白质合成、增强细胞生物氧化的功能。因此可能具有减轻卡那霉素耳毒性的作用。本实验以耳廓反射、内耳生物电及耳蜗铺片为指标,观察甲状腺激素对卡那霉素耳中毒的预防。实验豚鼠分两组,各13只,对照组每天注射卡那霉素300mg/kg,共10天;甲状腺素组先隔天服甲状腺片20mg共四次,以后给予与对照组相同剂量卡那霉素,同时仍隔天服甲状腺片20mg直至停药后16天,前后总共服17次。结果:(1)耳廓反射阈变化,对8、4、2KHz三个频率听力均下降的耳,对照组为11只耳,甲状腺素组为3只耳,两者差异显著。听力下降的频率范围及程度,对照组比甲状腺素组更大。对照组听力下降开始出现的时间明显早于甲状腺素组;(2)内耳生物电,0～80dβ不同程度短声引起的耳蜗微音器电位与听神经动作电位幅值甲状腺素组动物均高于对照组;(8)耳蜗铺片,对照组大部分动物耳蜗各回的毛细胞严重变性缺损,甲状腺素组耳蜗病变仅局限在底回。以上结果表明甲状腺激素能减轻卡那霉素的耳毒性,为耳毒性抗生素致聋的防治提供了一条新的研究途径。     

刺五加注射液对豚鼠庆大霉素耳毒性拮抗作用的实验研究

目的：探讨中药刺五加注射液（ASS）对庆大霉素（GM）耳毒性作用的影响及其机制。方法：豚鼠随机分成对照组、GM组、ASS4-GM组和ASS组。采用听性脑干反应（ABR）、透射电镜技术（TEM）、Western blot方法观察用药前后豚鼠的ABR阈值、形态学变化及caspase-3的表达情况。结果：用药后GM组ABR阈值明显升高;ASS组ABR阈值与对照组相比无显著性差异,与GM组和ASS4-GM组相比明显降低。透射电镜观察用药后GM组毛细胞损伤严重,出现了凋亡的形态学特征,而ASS4-GM组损伤较轻。Western blot结果表明用药后GM组豚鼠caspase-3表达明显增加;ASS4-GM组caspase-3的表达稍有升高。结论：刺五加注射液对庆大霉素耳毒性具有拮抗作用,其机制可能是通过抑制caspase-3的表达来实现的。     

川芎嗪抗链霉素耳毒性作用及其对豚鼠耳蜗外毛细胞钾通道的影响

本文旨在研究川芎嗪（tetramethylpyrazine,TMP）拮抗链霉素耳毒性作用及其对豚鼠耳蜗外毛细胞K^＋通道的影响,探讨TMP拈抗链霉素耳毒性的离子通道机制。60只豚鼠随机分为6组,应用听觉脑干反应（auditory brainstem response,ABR）技术检测豚鼠ABR听阈,观测TMP的抗链霉素耳毒作用;并采用全细胞膜片钳技术观察TMP对耳蜗外毛细胞Ca^2＋敏感艮电流的影响。结果显示,TMP明显降低链霉素导致的豚鼠ABR听阈升高,提示TMP具有抗链霉素耳毒性作用;TMP能明显增大豚鼠耳蜗外毛细胞Ca^2＋敏感艮电流,并呈浓度依赖关系。结果提示,TMP通过增大艮通道电导而拮抗链霉素耳毒性作用。     

硫酸卡那霉素对成年大鼠的耳毒性效应

本研究旨在观察硫酸卡那霉素(kanamycin sulfate,KM)对成年大鼠的耳毒性效应。6～7周龄的雄性Sprague-Dawley(SD)大鼠40只,随机分为2组:实验组,每天腹腔注射KM(500mg/kg)2周;对照组,注射等量生理盐水2周。通过检测脑干听觉诱发电位(auditory brainstem response,ABR)观察大鼠听力改变。ABR检测结束后,分离出耳蜗进行基底膜铺片、耳蜗冰冻切片,观察耳蜗螺旋神经节细胞(spiral ganglion cells,SGCs)的密度和耳蜗形态学改变。结果显示,注射KM2周后,大鼠在各频率的听觉阈值均有明显升高,其上升幅度超过60dB;随着时间推移,KM组SGCs密度逐渐降低,Corti器结构尚存,但外毛细胞及内毛细胞均有不同程度的缺失,以外毛细胞为甚;内毛细胞缺失与SGCs的密度下降相平行。以上结果表明,6～7周龄大鼠经过KM作用2周后,听力会明显下降,达到重度耳聋甚至全聋。KM的耳毒性作用与SGCs和内外毛细胞的损伤密切相关。     

Gentamicin rapidly inhibits mitochondrial metabolism in high-frequency cochlear outer hair cells

Aminoglycosides (AG), including gentamicin (GM), are the most frequently used antibiotics in the world and are proposed to cause irreversible cochlear damage and hearing loss (HL) in 1/4 of the patients receiving these life-saving drugs. Akin to the results of AG ototoxicity studies, high-frequency, basal turn outer hair cells (OHCs) preferentially succumb to multiple HL pathologies while inner hair cells (IHCs) are much more resilient. To determine if endogenous differences in IHC and OHC mitochondrial metabolism dictate differential sensitivities to AG-induced HL, IHC- and OHC-specific changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH) fluorescence during acute (1 h) GM treatment were compared. GM-mediated decreases in NADH fluorescence and succinate dehydrogenase activity were observed shortly after GM application. High-frequency basal turn OHCs were found to be metabolically biased to rapidly respond to alterations in their microenvironment including GM and elevated glucose exposures. These metabolic biases may predispose high-frequency OHCs to preferentially produce cell-damaging reactive oxygen species during traumatic challenge. Noise-induced and age-related HL pathologies share key characteristics with AG ototoxicity, including preferential OHC loss and reactive oxygen species production. Data from this report highlight the need to address the role of mitochondrial metabolism in regulating AG ototoxicity and the need to illuminate how fundamental differences in IHC and OHC metabolism may dictate differences in HC fate during multiple HL pathologies.     

Involvement of calpain-I and microRNA34 in kanamycin-induced apoptosis of inner ear cells

Inner ear cells, including hair cells, spiral ganglion cells, stria vascularis cells and supporting cells on the basilar membrane, play a major role in transducing hearing signals and regulating inner ear homoeostasis. However, their functions are often damaged by antibiotic-induced ototoxicity. Apoptosis is probably involved in inner ear cell injury following aminoglycoside treatment. Calpain, a calcium-dependent protease, is essential for mediating and promoting cell death. We have therefore investigated the involvement of calpain in the molecular mechanism underlying ototoxicity induced by the antibiotic kanamycin in mice. Kanamycin (750 mg/kg) mainly induced cell death of cochlear cells, including stria vascularis cells, supporting cells and spiral ganglion cells, but not hair cells within the organ of Corti. Cell death due to apoptosis occurred in a time-dependent manner with concomitant up-regulation of calpain expression. Furthermore, the expression levels of two microRNAs, mir34a and mir34c, were altered in a dose-dependent manner in cochlear cells. These novel findings demonstrated the involvement of both calpain and microRNAs in antibiotic-induced ototoxicity.     

Death-associated protein kinase as a sensor of mitochondrial membrane potential: role of lysosome in mitochondrial toxin-induced cell death

We have investigated here the mechanism of dephosphorylation and activation of death-associated protein kinase (DAPK) and the role of lysosome in neuroblastoma cells (SH-SY5Y) treated with mitochondrial toxins, such as MPP(+) and rotenone. Mitochondrial respiratory chain inhibitors and uncouplers decreased mitochondrial membrane potential leading to DAPK dephosphorylation and activation. The class III phosphoinositide 3-kinase inhibitors attenuated DAPK dephosphorylation induced by mitochondrial toxins. Complex I inhibition by mitochondrial toxins (e.g. MPP(+)) resulted in mitochondrial swelling and lysosome reduction. Inhibition of class III phosphoinositide 3-kinase attenuated MPP(+)-induced lysosome reduction and cell death. The role of DAPK as a sensor of mitochondrial membrane potential in mitochondrial diseases was addressed.     

Notch Signaling Limits Supporting Cell Plasticity in the Hair Cell-Damaged Early Postnatal Murine Cochlea

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.     

骨髓间充质干细胞对大鼠急性肾损伤的修复作用

目的:观察外源性骨髓间充质干细胞(Mesenchymal stem cells,MSCs)对庆大霉素(Gentamycin,GM)诱导的大鼠急性肾损伤是否具有治疗作用,并初探其机制。方法:建立腹腔注射庆大霉素致大鼠急性肾损伤模型实验分为正常对照组、模型组、MSCs治疗组(模型+MSCs)、生理盐水组(模型+生理盐水)。于不同处理后4d分别检测血尿素氮(BUN)和肌酐(Scr)水平,观察肾组织病理改变,免疫印迹及RT-PCR法检测肾组织肝细胞生长因子(Hepatocyte growth factor,HGF)水平。结果:模型组大鼠的BUN及Scr较正常对照组显著升高,且肾小管组织病理损伤严重;而MSCs治疗组大鼠的BUN及Scr水平较生理盐水组显著降低,肾小管组织病理损伤明显减轻。此外,促肾小管损伤修复的肝细胞生长因子(HGF)表达在MSCs治疗组显著高于生理盐水组。结论:MSCs输注可促进庆大霉素所致急性肾小管损伤的修复,改善肾功能,其作用机制可能是与上调肾组织中肝细胞细胞生长因子的表达有关。     

Effects of neuroactive steroids on cochlear hair cell death induced by gentamicin

As neuroactive steroids, sex steroid hormones have non-reproductive effects. We previously reported that 17β-estradiol (βE2) had protective effects against gentamicin (GM) ototoxicity in the cochlea. In the present study, we examined whether the protective action of βE2 on GM ototoxicity is mediated by the estrogen receptor (ER) and whether other estrogens (17α-estradiol (αE2), estrone (E1), and estriol (E3)) and other neuroactive steroids, dehydroepiandrosterone (DHEA) and progesterone (P), have similar protective effects. The basal turn of the organ of Corti was dissected from Sprague-Dawley rats and cultured in a medium containing 100 μM GM for 48 h. The effects of βE2 and ICI 182,780, a selective ER antagonist, were examined. In addition, the effects of other estrogens, DHEA and P were tested using this culture system. Loss of outer hair cells induced by GM exposure was compared among groups. βE2 exhibited a protective effect against GM ototoxicity, but its protective effect was antagonized by ICI 182,780. αE2, E1, and E3 also protected hair cells against gentamicin ototoxicity. DHEA showed a protective effect; however, the addition of ICI 182,780 did not affect hair cell loss. P did not have any effect on GM-induced outer hair cell death. The present findings suggest that estrogens and DHEA are protective agents against GM ototoxicity. The results of the ER antagonist study also suggest that the protective action of βE2 is mediated via ER but that of DHEA is not related to its conversion to estrogen and binding to ER. Further studies on neuroactive steroids may lead to new insights regarding cochlear protection.