盐酸椒苯酮胺对庆大霉素豚鼠耳蜗损伤保护作用及机制
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摘要
听力障碍是一类常见疾病,严重影响着人类的身体健康和生活质量,据世界卫生组织(WHO)估计,全世界有轻度听力损失的人近6亿,2.5亿人患有中度以上听力损失,其中三分之二在发展中国家。而每出生700—1000个新生儿中,就有一个听障患儿。中国是世界上最大的发展中国家,13亿人口中听力障碍残疾人就有2780万,为各类残疾之首。能否找到有效的方法预防和治疗听力障碍,是我们共同面临的巨大挑战。听力障碍分传导性、感音神经性和混合性,绝大部分为感音神经性。感音神经性聋是耳鼻咽喉头颈外科学中常见病,是内耳及其神经传导通路一系列病变所致听力损失的总称,发病原因有遗传性、缺血性、耳毒性、感染性、老年性、噪声性、创伤性、代谢性、自身免疫、突发或特发性、中枢性、听神经病、肿瘤、功能性等,其中以缺血、病毒感染、耳毒性药物中毒及老年性退行性变等最常见。病理改变主要是耳蜗毛细胞损伤、螺旋神经节、支持细胞及神经末梢的器质性改变以及耳蜗神经、脑干听觉通路及听觉中枢病变或受损。耳蜗内包含两种类型感觉细胞：内毛细胞(IHC)和外毛细胞(OHC),它们负责将声音转化为电信号进行传递,这些信号经过螺旋神经节神经元(SGC)换元传递到听觉脑干通路。而这些IHC、OHC和SGC都缺乏再生的能力,因此,耳蜗的任何损伤都会导致不可逆的听力损失。在耳蜗损伤所导致的听力障碍里,耳毒性药物所致损伤是当前临床治疗上的难题,众多研究围绕其发病机制及治疗展开,希望可以为临床治疗耳毒性药物损伤提供更好的方法。关于药物性损伤的机制,目前多认为与自由基损伤、钙离子超载等有关[2],其中氨基糖苷类所致的耳毒性感音神经性聋属于临床中常见的类型。
氨基糖苷类抗生素(Aminglycosides, AmAn)具有抗菌谱广、杀菌抑菌作用强、价格便宜等许多适用于临床使用的优点,但因其具有耳毒性、肾毒性等严重的不良反应,限制了临床应用,在短期应用AmAn治疗的病例中,耳毒性发生率为20%,而在治疗结核及其他严重细菌(尤其是革兰阴性细菌)感染的长期应用中,耳毒性发生率可高达80%[3]。近年来由于结核和其他新的感染性疾病如抗艾滋病的发生率增高,使AmAn又有新的应用。有研究表明AmAn可与HIV中的RNA的逆转录病毒蛋白应答因子(RRE)结构域结合,使HIV的RRE. Rev(逆转录病毒蛋白)相互作用产生抑制,从而具有阻遏HIV复制的活性[4],使得AmAn的很多应用不可替代。属于氨基糖苷类抗生素的庆大霉素(gentamicin, GM)是由绛红小单孢菌、棘孢小单孢菌等发酵产生的一种杀菌力较强的广谱抗菌素,目前被广泛用于临床。GM由于廉价抗菌谱广,在临床广泛应用,以至出现了不少致感音神经性聋的病例。同时,因GM的肝肾毒性较其他AmAn小,使其成为是实验研究中制造感音神经性聋动物模型的理想药物。
GM在机体内的药理学分布特点是不易进入胞内,不易透入关节腔,也不易透过血脑屏障,其主要集中在细胞外液,特别是内耳淋巴液。庆大霉素的内耳淋巴液的蓄积[7]构成了其耳毒性的生理基础,机制较为复杂,一直是国内外众多学者研究的热点。目前认为内耳毛细胞受损与氧有自由基、钙超载和启动凋亡等相关。氧自由基主要通过一系列的过氧化反应造成组织细胞的损伤,往往损害细胞膜磷脂分子中不饱和脂肪酸的过氧化,最终使生物膜受到严重损伤。郭玉芬等证实聚天冬氨酸酶对庆大霉素致耳蜗组织氧自由基的产生有抑制作用,从而拮抗了庆大霉素所致耳蜗毒性的发生。McFadden等已证实一种超氧化物歧化酶的类似物,可防止由庆大霉素引起的毛细胞损伤。氧自由基和钙超载除直接造成细胞损伤外还可导致细胞的凋亡[11]。也有学者证明,细胞凋亡是氨基糖苷类抗生素致聋的主要方式,细胞凋亡的“瀑布式”级联反应主要有两条途径：①细胞外途径,即死亡受体途径。②细胞内途径,研究比较多的是线粒体途径。但两种途径最终都依赖于Caspase-3的活化,caspase-3在凋亡级联反应中处于核心地位,是凋亡的最终执行者。Shimizu等通过研究发现凋亡抑制剂—钙蛋白酶和半胱天冬酶抑制剂可以阻止AmAn耳毒性的产生,促进前庭毛细胞的存活,表明凋亡与AmAn前庭耳毒性有着紧密关系。
目前临床上仍然缺乏治愈感音神经性听力障碍和保护听觉功能的有效的方法。虽然近年来一些研究报道了哺乳动物甚至人的内耳前庭及耳蜗毛细胞在受伤后可能再生,但哺乳动物OHC、IHC和SGC再生的在体试验尚未取得成功。有研究提出耳聋基因概念,认为耳聋与线粒体DNA突变有关,发现tRNAIleA4317G同质性突变,确认A1555G和C1494T突变与感音神经性耳聋和氨基糖甙类耳聋有关。上述研究停留在实验阶段,尚未展开临床应用。感音神经性耳聋多数治疗效果不佳,大多数药物治疗无效的患者只能选择配戴助听器或行人工耳蜗植入术。当前人工耳蜗植入术可以赝复重度的感音神经性耳聋,但价格昂贵。所以,探索对各种因素导致内耳损伤的早期干预和有效治疗药物就显得尤为重要。
由中国医学科学院药物研究所自主创新研制的化合物盐酸椒苯酮胺(peperphentonamine hydrochloride,PPTA),3个发明专利已获授权,2个国内和1个国际PCT发明专利己申请。2008年获11类化学药品临床试验批件并已完成127例Ⅰ期临床试验。临床前研究提示其具有清除再灌注损伤时自由基,抑制脂质过氧化反应的作用。徐江平研究PPTA对脑细胞受损所产生的氧自由基具有清除作用,可增加SOD活力和GSH含量。也有实验表明PPTA有抗钙超载的作用,对心肌损伤线粒体有明显的保护作用。PPTA也可使线粒体膜流动性保持正常,明显减轻线粒体的超微结构损伤。而氧自由基和钙超载往往导致机体大多数组织和细胞损伤,而线粒体的损伤又可活化Caspase-3启动细胞凋亡。有研究表明PPTA具有显著降低Caspase-3mRNA的表达,表现出良好的抗调亡作用。这为PPTA用于感音神经性耳聋的干预和治疗奠定了基础。
本研究以庆大霉素的耳毒性制造耳蜗损伤豚鼠作为实验动物,采用鼓阶开窗微孔注入技术和腹腔注射方法,观察PPTA对耳蜗的保护作用,通过听性脑干反应(ABR)、免疫组化(IHC)、脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(ternial-deoxynucleotidyl transferase mediated nick end labeing, TUNEL)、蛋白质免疫印迹技术(Western blot)、扫描电镜(SEM)等方法,透射电镜(TEM)等方法检测ABR RT、PL、T-SOD、GSH、Caspase-3等指标及凋亡和坏死等形态学变化,探讨GM的耳毒性机理及PPTA对耳蜗损伤的保护机制,为感音神经性聋药物治疗提供新的实验依据。
本研究分为以下三个部分：
第一部分庆大霉素致豚鼠耳蜗损伤模型和鼓阶开窗微孔注入技术的建立
目的建立庆大霉素制造豚鼠的耳蜗损伤模型和鼓阶开窗微孔注入技术,探讨在模型豚鼠上ABR的应用及听力学特征,并研究经鼓阶开窗手术对听力和耳蜗组织的影响。方法听力正常实验级豚鼠30只,随机分为三组：A组(Control group):10只,生理盐水,等量,每日称重,按体重计算用量,肌注,28d；B组(GM group):10只,硫酸庆大霉素注射液,120mg/(kg-d),每日称重,按体重计算用量,肌注,28d。C组(Fenestration group):10只,左耳行鼓阶开窗微孔注入手术,注入人工外淋巴液,正常饲养7d。A、B两组GM前1h及第28天测ABR;A、C两组术前及术后7d测ABR。数据以X±S表示,采用SPSS13.0软件包进行统计学分析。对实验前后数据进行协方差分析,若实验前ABR反应阈对试验后ABR无影响,则用独立样本t-test检验两组前后差异,用配对样本t-test检验同一组前后差异。结束后将A,B,C组豚鼠断头取听泡,行扫描电镜(SEM)形态学观察、免疫组化(IHC)细胞染色观察。结果用药28天后,GM组比正常组ABR RT显著升高(t=18.108,P<0.001)；经鼓阶开窗微孔注入术7天后,C组和正常组ABR RT无显著差异(t=0.000, P=1.000)。IHC及SEM观察见GM组毛细胞纤毛倒伏、断裂、细胞缺失,细胞核出现固缩、棕染等,A组及C组未见这些现象。结论庆大霉素可以建立稳定的感音神经性聋动物模型；庆大霉素耳毒性体现在RT提高,耳蜗OHC、IHC、SGC、SV细胞坏死及凋亡；通过豚鼠耳蜗鼓阶钻孔开窗,微孔注入手术后对豚鼠听力及耳蜗组织没有产生明显影响。
第二部分鼓阶微孔注入PPTA对庆大霉素豚鼠耳蜗损伤的保护作用及相关机制研究
目的将PPTA经鼓阶开窗微孔注入技术注入豚鼠耳蜗,研究其对庆大霉素耳毒性的拮抗作用及氧自由基机制。方法听力正常豚鼠45只,分为三组：A组(Control group)15只,以等量生理盐水肌肉注射,连续3d；B组(GM group)15只,以GM160mg/kg.d,肌肉注射,连续3d；C组(PPTA+GM group)15只,以GM160mg/kg.,3d,每日GM1小时前,行双侧鼓阶开窗微孔技术注入PPTA (浓度2mg/ml)10μl。(注：每组都以10只做统计学分析,额外为扫描电镜做形态学观察用)。三组动物分别在用药前,第3天用药后测ABR。在最后一次测ABR每组取10只迅速断头取听泡取左侧耳蜗(n=10)行T-SOD活力检测,取右侧耳蜗(n=10)行GSH含量检测；各组ABR RT、T-SOD、GSH统计使用SPSS13.0分析软件,数据以X±S表示,用协方差分析处理数据,若实验前ABR反应阈对实验后无影响,对试验后数据进行单因素方差分析。每组剩余5只豚鼠耳蜗行扫描电镜(SEM)形态学观察。结果用药3d后,GM组ABR RT显著高于正常组(P<0.05),GM组T-SOD活力、GSH含量显著显著低于GM+PPTA组(P<0.05)；经鼓阶开窗微孔技术注入PPTA3d后,PPTA+GM组ABR RT明显高于Control组(P<0.05),但显著低于GM组(P<0.05)。PPTA+GM组比GM组及正常组T-SOD活力、GSH含量显著低于正常组(P<0.05),但显著高于GM组(P<0.05)。SEM观察见：GM后毛细胞纤毛倒伏、断裂、细胞缺失等,PPTA+GM组亦有此现象,但较GM组轻微,正常组未见这些现象。结论促耳蜗组织内产生过量OFR造成耳蜗组织细胞损伤,是GM耳毒性的一个重要原因。经鼓阶开窗微孔技术注入PPTA可拮抗GM所致听力和耳蜗组织损伤；PPTA可以通过清除耳蜗组织内氧自由基和增强抗氧化能力从而发挥耳蜗保护作用。
第三部分腹腔注入PPTA对庆大霉素豚鼠耳毒性拮抗作用及对耳蜗组织Caspase-3表达的影响
目的研究PPTA全身给药对庆大霉素耳毒性的拮抗作用及相关机制。方法听力正常豚鼠45只,分为三组：A组(Control group)15只,以等量生理盐水肌肉注射,连续14d；B组(GM group)15只,GM120mg/kg.d,肌肉注射,连续14d；C组(PPTA+GM group)15只,PPTA10mg/kg.d腹腔注射+GM120mg/kg.d (1h后),肌注,连续14d。三组动物在用药前1h,第14天用药后测ABR。在最后一次测ABR后所有动物迅速断头取听泡,以左侧耳蜗(n=10)行Western blot检测Caspase-3表达,统计使用SPSS13.0分析软件,数据以X±S表示,用协方差分析处理数据,若实验前ABR反应阈对实验后无影响,对试验后数据进行单因素方差分析。以右侧耳蜗行TUNEL染色,每组剩余5只一侧耳做扫描电镜另一侧耳做透射电镜观察。结果GM组、PPTA+GM组ABR RT显著高于Control组(P<0.05), PPTA+GM组ABR RT显著低于GM组；Western blot结果表明用药后GM组豚鼠caspase-3表达显著增加(P<0.001); PPTA+GM组caspase-3的表达显著高于Control组但显著低于GM组(P<0.001)。SEM/TEM和TUNEL观察见：GM组毛细胞损伤严重,耳蜗Corti器、侧壁的血管纹和螺旋神经节存在阳性细胞,出现了凋亡和坏死的形态学特征；而PPTA+GM组损伤较GM组明显减轻；Control组未见阳性细胞。而结论庆大霉素耳毒性所致耳蜗组织损伤中,凋亡与坏死并存,GM通过caspase-3途径参与了耳蜗细胞凋亡；PPTA具有拮抗GM耳毒性所致的耳蜗功能和结构损伤的作用；PPTA通过降低caspase-3蛋白表达,抑制凋亡,从而对豚鼠耳蜗组织起到保护作用。
Hearing disorder is a common disease, severely affecting the healthy and living quality of human race. According to WHO, there are about250million people having moderate hearing loss, and among2/3in the developing countries. One of700to1,000new born babies has hearing disorder. China is the biggest developing country, with a population of1.3billion, of which,20million have hearing disorder, accounting for16.79%o. Our nation faces the big challenge whether we can find the effective method to prevent and cure hearing disorder.There are conductive, sensorineural and combinative hearing losses, and most are sensorineural hearing loss.
Sensorineural hearing loss is common in Otorhinolaryngology Head and Neck Surgery, which is the general term for hearing loss caused by a series of lesions of internal ear and its nerves. Reasons are hereditary, ischemic, ototoxic, sensorineural, age-related, noise-induced, traumatic, metabolic, autoimmunity, sudden or idiopathic, central, auditory neuropathy, tumor, functional and so on, among which, ischemic ischemic, sensorineural and age-related are the most common reasons. Pathological changes basically due to the damage to hair cells of cochlea, the physical changes of spiral ganglion, support cells and nerve endings, and lesions or damage of cochlear nerve and auditory pathway and center of brainstem. Cochlear sensory cell contains two types:inner hair cells (IHC) and outer hair cells (OHC). They are responsible for transferring sound signals into electrical signals, which are passed through the spiral ganglion neurons (SGCs) to the auditory brainstem pathways. And these IHCs, OHCs and SGCs lack of regeneration ability, therefore, any damage to cochlea can lead to irreversible hearing loss. Among the cochlear damages, the damage caused by ototoxic drugs is the most difficulty. Many researches on its pathogenesis and treatment have been conducted, with a hope for a better way. Drug-induced damage mechanism related to free radical damage, calcium overload and so on, among which, the sensorineural deafness caused by aminoglycoside ototoxicity is common clinical type.
Aminoglycoside antibiotics (Aminglycosides, AmAn) are suitable for clinical use for advantages wide antimicrobial spectrum, strong sterilization bacteriostasis, cheap, etc, but because of nephrotoxicity, ototoxicity and other serious adverse reactions, limit the clinical application。 In the short-term treatment cases using AmAn, the rate of ototoxicity incidence was20%, and in the long-term application treatment of tuberculosis and other serious bacterial infection (especially gram-negative bacteria), the rate of ototoxic incidence can be as high as80%. But in recent years, due to application in tuberculosis and other new areas, such as AIDS, studies have shown that aminoglycoside antibiotic line (gentamicin such antibiotics) can combine with RRE (retrovirus protein response factor) of RNA in HIV, create restrain effect between RRE and Rev (retrovirus protein) of HIV, and repress HIV replication activity. All these make many applications of AmAn can not be replaced.As one of aminoglycoside antibiotics, gentamicin (gentamicin, GM) is a strong sterilization produced by the fermentation of the color of small single spore bacteria, spines spore small single spore bacteria and such as, which has been widely used in clinical. The widely use of GM as its cheap advantage caused a large number of clinical cases of sensorineural deafness. At the same time, because the ototoxicity is more serious than the hepatorenal toxicity, compared to other aminoglycoside antibiotics, GM is the ideal drug causing the sensorineural deafness in the study of animal models.
The pharmacology characteristics of GM distribution in the body is not easy to enter the intracellular, not easy to penetrate into articular cavity, also not easy through the blood brain barrier, but mainly concentrates in the extracellular fluid, especially in the inner ear lymph. The accumulation of lymph of Gentamicin in the inner ear constitutes the physiological basis of its ototoxicity, and its mechanism is more complicated. Many researches focuses on this at home and abroad, mainly on the free radicals, calcium overload and start the apoptosis, etc. Many researches on GM ototoxicity show that the damage of hair cells of inner ear is associated with overload of oxygen free radicals and calcium, peroxidation of unsaturated fatty acid causes the severe damage to the biofilm. Guo Yufen and so have confirmed, Poly (aspartic acid enzymes) can restrain the overload of oxygen free radicals, so to reduce the incidence of cochlea toxicity induced by gentamicin. McFadden and so on has confirmed a kind of similar content, superoxide dismutase (sod) can prevent the damage to hair cells induced by gentamicin. Overload of oxygen free radicals and calcium can directly cause cell damage, even worse, lead to cell apoptosis. Also some scholars proved that cell apoptosis is the main way of deafness induced by aminoglycoside antibiotic,"waterfall" cascade of apoptosis mainly has two ways:(1) extracellular approach, namely the death of receptor.(2) inner cell approach, most refer to the death of mitochondrial pathway. But the two ways are ultimately dependent on the activation of Caspase3, Caspase3is the most important, acting as the end executo. Shimizu, such as found that calcium protease and apoptosis caspase inhibitors can block the ototoxicity induced by AmAn, facilitate the survive of vestibular hair cells. It showed that apoptosis has close relationship with vestibular ototoxicit induced by AmAn y.
At present, there is still no any effective ways to cure the sensorineural hearing impairment and protect the auditory capabilities in clinical. Although some researchers recently shows that, in the mammals' or even the human beings' body, the vestibular system of the inner ear and the cochlear hair cells are likely to regenerate after wounded, there are still no successful results in researches about the regeneration of OHC、IHC and SGCs during the experiment of mammals. In recent years, with the improvemt of the knowledge about biomechanism of the loss molecular in listening, some researchers put forward the concept of Deafness Gene, but all these new technology are in the experimental stage, which cannot be used in the clinical treatment. Many therapeutic efficacies of sensorineural deafness not work so well and the patients, who suffer from the ineffective drug therapy, have had to choose wearing the hearing aids or receive the artificial cochlea implantation. Though the artificial cochlea implantation can cure the severe sensorineural deafness at present, the price of it is quite expensive. Therefore, it's more important to explore the effective medicines in clinical which are useful for the early prevention from the factors causing the damage of inner ear and then provide the effictive treament.
The new compound named peperphentonamine hydrochl-oride,PPTA, which is first synthesized creatively by China, is the synthesized medicine with completely independent intellectual property screened by the Materia Medica Institute in Chinese Academy of Medical Sciences. This new synthesized medicine has alreasy authorized three invention patent and also two China invention patents and one international PCT invention patent are under application. In2008, PPTA had won11kinds of chemical medicines clinical test approved documents and even completed127clinical phase I tests.
Molecular formula:C21H24O4NCl或C21H23O4N·HCl,
Molecular Weight:MW=389.87
Chemical name:(E)-5-[(3,4-methylene2oxo phenethyl) amino] a-1-(4)--pentene-3-hydroxy phenyl-1methadone hydrochloride
Pepper phenyl ketone amine of the single crystal X-ray diffraction molecular structure as shown in figure1.
PPTA as the advanced international calcium sensitizer original innovation chemical medicine, preclinical research proves it can clear free radical of injury cells, as well restrain the overload of peroxidatic reaction of lipid. According to the research by Xu Jiangping, PPTA has a clear-up function to oxygen free radicals produced by the damage of brain cells; as well increase SOD activity and GSH content. Experiment results show that the PPTA has the function of resisting calcium overload, as well obvious protective effects on mitochondria of myocardial injury. PPTA can also remain the membrane fluidity of mitochondria normal, significantly reduce the damage to the ultrastructure of mitochondria. Overload Oxygen free radicals and calcium often leads to the damage of major tissue and cell, and mitochondrial injury can activate the Caspase-3to start the apoptosis of cell. Studies have shown that PPTA can significantly reduce the expression of Caspase-3mRNA, exhibit good resistance to apoptosis. So we consider introduce PPTA this innovative drug to prevent and treat sensorineural hearing loss.
PPTA as a state-level research and development new drugs for calcium sensitization agent drugs for cardiovascular, is on phase I clinical trials, not yet entered the clinical application. The protective effect and mechanism for the inner ear damage of PPTA has not been reported.
In this study, the guinea pig as experimental animal, on the foundation of the animal model of cochlear damage in manufacturing using the gentamicin ototoxicity of scala tympani fenestration, application of microporous injection and intraperitoneal injection, through using the innovative drug PPTA on cochlea, auditory brainstem response (ABR), immunohistochemistry (IHC), DNA end nick end labeling transferase mediated (ternial-deoxynucleotidyl transferase mediated nick end labeing, TUNEL), protein immunoblotting technique (Western blot), scanning electron microscopy (SEM) and other methods, to detect the changes in ABR RT, T-SOD, GSH, Caspase-3index and apoptosis and necrosis of morphology, and study the ototoxicity mechanism and protection in clinical application of antibiotics GM in-depth, and research on the protective effect of PPTA for cochlear injury and its mechanism, in order to explore the feasibility and specific mechanisms of national calcium sensitizer cardiovascular innovative drug on prevention of sensorineural deafness, further expand the scope of application and find a new drug for the treatment.
This study is divided into three parts:
Part one:Research on the cochlea damage model of guinea pig induced by gentamicin and injection technology on the tympani window of microcellular
Objective Base on the cochlea damage model of guinea pig induced by gentamicin, to discuss the application o and characteristics ABR of guinea pig model; to discuss the application and the effect to hearing and cochlear of technology on the tympani window of microcellular; and to observe changes of cochlear tissues inside the cell morphological using TUNEL and scanning electron microscopy techniques. Method30guinea pigs with normal hearing were randomly divided into three groups:Group A (control group)10, Group B (GM1)10, Group C (GM2)10. Group A (control group):amount of normal saline, weigh daily, give calculation of the dosage according to the weight, total duration with group B; Group B (GM1) with gentamycin sulfate injection120mg/(kg/d), weigh daily, give calculation of the dosage according to the weight for28days. Group C (GM2) with gentamycin sulfate injection160mg/(kg/d), weigh daily, give calculation of the dosage according to the weight for3days. Collect the RT and Ⅰ, Ⅲ PL, Ⅰ-Ⅲ IPL and Ⅲ AP within one hour before injection and within24hours after injection on7th,14th,21st and28th days. Collect the RT within one hour before injection and within24hours after the last injection.After collection, carried the injection technology on the tympani window of microcellular on10guinea pigs of Group A; collected ABR7days after surgery. After all the inspection, took otocyst beheaded of guinea pigs of Group A and B, scanned electron microscope and observed the immunohistochemical staining and TUNEL staining. Result ABR RT had significant difference after28days' injection between Group A and Group B; ABR RT had significant difference after3days' injection between Group C and Group B; ABR RT had no significant difference after7days before and after surgery. Observation by TUNEL and SEM, there were cilia lodging, fault, loss of cells on GM groups, while there was no these phenomena on Group A and surgery group. normal group of those who did and did not see these phenomena. Conclusion Gentamicin applied in guinea pigs, can lead to severe sensorineural deafness; medicine deafness animal model induced by gentamicin is a simple and reliable method; there were individual differences of ototoxicity induced by gentamicin in the short term, the damage became similar in a long time application; long-term application of gentamicin could lead guinea pigs to death easily, as it has hepatorenal toxicity; as ABR waveforms easy to recognized and recorded, it is an objective evaluation for hearing change of guinea pigs; under appropriate conditions, the ABR is relatively simpler and more accurate when guinea pigs are at waking than narcosis; Technology of injection on the tympani window of microcellular did not have significant effects on the mechanism of inner ear or the hearing the characteristics of hair cells poisoned by gentamicin:gradually reduce from the bottom to upward, the damage of outer hair cell is relatively serious than that of inner hair cells, among which, the most outside hair cells is the worst; the mechanism of ototoxicity induced by gentamicin could be related to the overload of oxygen free radicals and calcium, and activation of Caspase3which induces apoptosis.
Part Two:The protection of PPTA injected through the tympani window of microcellular on the damage cochlea induced by gentamicin, and the mechanism of oxygen free radicals
Objective Inject the PPTA through the tympani window of microcellular to the damage cochlea induced by gentamicin, observe the changes of hearing through ABR, and to study the effect and protective mechanism of PPTA for oxygen free radicals and the damage of cochlea, through the detection on the changes of T-SOD, GSH, and the shape change of hair cells by scanning electron microscope. Method45guinea pigs with normal hearing were randomly divided into three groups:Group A (control group)15, Group B (Deafness model induced by gentamicin, GM Gruop)15, Group C (Injected PPTA through the tympani window of microcellular, PPTA+GM Group)15. Group A (control group):give calculation of amount of normal saline for3days, the spot times of tests on hearing were the same as Group C. Group B (GM1) with gentamycin sulfate injection160mg/(kg/d) for3days. Group C with GM injection of160mg/(kg/d) for3days. One hour before injected with gentamicin, injected PPTA (concentration of2mg/ml)101into the lymph in the scala tympanianesthesia. Tested ABR one hour before injection and after24hours after injection the3rd day. After the last test on the ABR, took all the animals' otic vesicles by decapitation quickly, carried out the vitality test of T-SOD on one side cochlea (n=10) randomly, and content test of GSH on the other side cochlea (n=10); carried scanning electron microscope test on one side of the remain cochlea the TUNEL on other side cochlea. Result After3days' injection, there were significant difference on ABR, vitality of T-SOD and the content of GSH between GM Group and normal Group; After3days' injection of PPTA through the tympani window of microcellular, there were significant difference n ABR, vitality of T-SOD and the content of GSH between PPTA+GM Group and normal Group, as well the GM Group; observation by SEM, there were cilia lodging, fault, loss of cells on GM Group, PPTA+GM Group had less phenomenon, while no on normal Group. Conclusion Injection of PPTA through the tympani window of microcellular, can enhance the vitality of SOD in cochlea tissue, increase the content of GSH, the antagonism of PPTA to GM ototoxicity can protect the cochlea by eliminate the oxygen free radicals in cochlea tissue and enforce the antioxidant ability.
Part Three:The influence of PPTA on the antagonism to oxicity in the GM guinea pigs and the Caspase-3in the Cochlear tissue
Objective To study on the project that whether PPTA has the influence on the ototoxicity antagonism in the GM guinea pigs and then investigate its mechanism by using the ABR, testing on the Caspase-3through Western blot, staining the TUNEL and observing the scanning electron microscope. Method45normal hearing guinea pigs are divided into three groups:Group A(control group,15), Group B(GM group,15), Group C(PPTA+GM group,15). Group A were intramuscular injected by equivalence saline for continuous14days. The time-point of Group A for listening test on ABR is the same with Group C. Group B were intramuscular injected by gentamycin sulfate120mg/kg.d for continuous14days. Group C were intraperitoneal injected by PPTA10mg/kg and then after1hour were intramuscular injected by gentamycin sulfate120mg/kg.d for continuous14days. All the three groups are testing1hour earlier before using the medicine. On the seventh day and the fourteenth day, all the groups should test the ABR after using the medicine24hours ago. After the last test on the ABR, took all the animals' otic vesicles by decapitation quickly, carried out the Western blot to test the expression of Caspase-3on one side ears (n=10) and TUNEL (n=10)the other side.SEM and TEM on another5guinea pigs. Result ABR threshold shift in the GM Group increases obviously. Compared with the GM Group, the ABR threshold shift in PPTA+GM Group decreases apparently, but higher than the Control Group. Observing the GM Group through TUNEL, it shows that those hair cells are injuried badly. There are some TUNEL positive cells existing in the the organ of Corti, the lateral wall of the vascularis stria and the spiral ganglion, which now emerge the morphological characteristics of apoptosis. Compared with GM Group, the injury in PPTA+GM Group relief clearly. Besides, there are no positive cells in the Control Group. The result of Western blot shows that the expression of caspase-3increases obviously in the GM Group after using the medicine. The expression of caspase-3in the PPTA+GM Group rise slightly, but lower than that of GM Group. Conclusion PPTA showed antagonism effect with GM ototoxicity, which express the anti-effect. The mechanismis likely to restrain the expression of Caspase-3and then realizes its effect.
氨基糖苷类抗生素(Aminglycosides, AmAn)具有抗菌谱广、杀菌抑菌作用强、价格便宜等许多适用于临床使用的优点,但因其具有耳毒性、肾毒性等严重的不良反应,限制了临床应用,在短期应用AmAn治疗的病例中,耳毒性发生率为20%,而在治疗结核及其他严重细菌(尤其是革兰阴性细菌)感染的长期应用中,耳毒性发生率可高达80%[3]。近年来由于结核和其他新的感染性疾病如抗艾滋病的发生率增高,使AmAn又有新的应用。有研究表明AmAn可与HIV中的RNA的逆转录病毒蛋白应答因子(RRE)结构域结合,使HIV的RRE. Rev(逆转录病毒蛋白)相互作用产生抑制,从而具有阻遏HIV复制的活性[4],使得AmAn的很多应用不可替代。属于氨基糖苷类抗生素的庆大霉素(gentamicin, GM)是由绛红小单孢菌、棘孢小单孢菌等发酵产生的一种杀菌力较强的广谱抗菌素,目前被广泛用于临床。GM由于廉价抗菌谱广,在临床广泛应用,以至出现了不少致感音神经性聋的病例。同时,因GM的肝肾毒性较其他AmAn小,使其成为是实验研究中制造感音神经性聋动物模型的理想药物。
GM在机体内的药理学分布特点是不易进入胞内,不易透入关节腔,也不易透过血脑屏障,其主要集中在细胞外液,特别是内耳淋巴液。庆大霉素的内耳淋巴液的蓄积[7]构成了其耳毒性的生理基础,机制较为复杂,一直是国内外众多学者研究的热点。目前认为内耳毛细胞受损与氧有自由基、钙超载和启动凋亡等相关。氧自由基主要通过一系列的过氧化反应造成组织细胞的损伤,往往损害细胞膜磷脂分子中不饱和脂肪酸的过氧化,最终使生物膜受到严重损伤。郭玉芬等证实聚天冬氨酸酶对庆大霉素致耳蜗组织氧自由基的产生有抑制作用,从而拮抗了庆大霉素所致耳蜗毒性的发生。McFadden等已证实一种超氧化物歧化酶的类似物,可防止由庆大霉素引起的毛细胞损伤。氧自由基和钙超载除直接造成细胞损伤外还可导致细胞的凋亡[11]。也有学者证明,细胞凋亡是氨基糖苷类抗生素致聋的主要方式,细胞凋亡的“瀑布式”级联反应主要有两条途径：①细胞外途径,即死亡受体途径。②细胞内途径,研究比较多的是线粒体途径。但两种途径最终都依赖于Caspase-3的活化,caspase-3在凋亡级联反应中处于核心地位,是凋亡的最终执行者。Shimizu等通过研究发现凋亡抑制剂—钙蛋白酶和半胱天冬酶抑制剂可以阻止AmAn耳毒性的产生,促进前庭毛细胞的存活,表明凋亡与AmAn前庭耳毒性有着紧密关系。
目前临床上仍然缺乏治愈感音神经性听力障碍和保护听觉功能的有效的方法。虽然近年来一些研究报道了哺乳动物甚至人的内耳前庭及耳蜗毛细胞在受伤后可能再生,但哺乳动物OHC、IHC和SGC再生的在体试验尚未取得成功。有研究提出耳聋基因概念,认为耳聋与线粒体DNA突变有关,发现tRNAIleA4317G同质性突变,确认A1555G和C1494T突变与感音神经性耳聋和氨基糖甙类耳聋有关。上述研究停留在实验阶段,尚未展开临床应用。感音神经性耳聋多数治疗效果不佳,大多数药物治疗无效的患者只能选择配戴助听器或行人工耳蜗植入术。当前人工耳蜗植入术可以赝复重度的感音神经性耳聋,但价格昂贵。所以,探索对各种因素导致内耳损伤的早期干预和有效治疗药物就显得尤为重要。
由中国医学科学院药物研究所自主创新研制的化合物盐酸椒苯酮胺(peperphentonamine hydrochloride,PPTA),3个发明专利已获授权,2个国内和1个国际PCT发明专利己申请。2008年获11类化学药品临床试验批件并已完成127例Ⅰ期临床试验。临床前研究提示其具有清除再灌注损伤时自由基,抑制脂质过氧化反应的作用。徐江平研究PPTA对脑细胞受损所产生的氧自由基具有清除作用,可增加SOD活力和GSH含量。也有实验表明PPTA有抗钙超载的作用,对心肌损伤线粒体有明显的保护作用。PPTA也可使线粒体膜流动性保持正常,明显减轻线粒体的超微结构损伤。而氧自由基和钙超载往往导致机体大多数组织和细胞损伤,而线粒体的损伤又可活化Caspase-3启动细胞凋亡。有研究表明PPTA具有显著降低Caspase-3mRNA的表达,表现出良好的抗调亡作用。这为PPTA用于感音神经性耳聋的干预和治疗奠定了基础。
本研究以庆大霉素的耳毒性制造耳蜗损伤豚鼠作为实验动物,采用鼓阶开窗微孔注入技术和腹腔注射方法,观察PPTA对耳蜗的保护作用,通过听性脑干反应(ABR)、免疫组化(IHC)、脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术(ternial-deoxynucleotidyl transferase mediated nick end labeing, TUNEL)、蛋白质免疫印迹技术(Western blot)、扫描电镜(SEM)等方法,透射电镜(TEM)等方法检测ABR RT、PL、T-SOD、GSH、Caspase-3等指标及凋亡和坏死等形态学变化,探讨GM的耳毒性机理及PPTA对耳蜗损伤的保护机制,为感音神经性聋药物治疗提供新的实验依据。
本研究分为以下三个部分：
第一部分庆大霉素致豚鼠耳蜗损伤模型和鼓阶开窗微孔注入技术的建立
目的建立庆大霉素制造豚鼠的耳蜗损伤模型和鼓阶开窗微孔注入技术,探讨在模型豚鼠上ABR的应用及听力学特征,并研究经鼓阶开窗手术对听力和耳蜗组织的影响。方法听力正常实验级豚鼠30只,随机分为三组：A组(Control group):10只,生理盐水,等量,每日称重,按体重计算用量,肌注,28d；B组(GM group):10只,硫酸庆大霉素注射液,120mg/(kg-d),每日称重,按体重计算用量,肌注,28d。C组(Fenestration group):10只,左耳行鼓阶开窗微孔注入手术,注入人工外淋巴液,正常饲养7d。A、B两组GM前1h及第28天测ABR;A、C两组术前及术后7d测ABR。数据以X±S表示,采用SPSS13.0软件包进行统计学分析。对实验前后数据进行协方差分析,若实验前ABR反应阈对试验后ABR无影响,则用独立样本t-test检验两组前后差异,用配对样本t-test检验同一组前后差异。结束后将A,B,C组豚鼠断头取听泡,行扫描电镜(SEM)形态学观察、免疫组化(IHC)细胞染色观察。结果用药28天后,GM组比正常组ABR RT显著升高(t=18.108,P<0.001)；经鼓阶开窗微孔注入术7天后,C组和正常组ABR RT无显著差异(t=0.000, P=1.000)。IHC及SEM观察见GM组毛细胞纤毛倒伏、断裂、细胞缺失,细胞核出现固缩、棕染等,A组及C组未见这些现象。结论庆大霉素可以建立稳定的感音神经性聋动物模型；庆大霉素耳毒性体现在RT提高,耳蜗OHC、IHC、SGC、SV细胞坏死及凋亡；通过豚鼠耳蜗鼓阶钻孔开窗,微孔注入手术后对豚鼠听力及耳蜗组织没有产生明显影响。
第二部分鼓阶微孔注入PPTA对庆大霉素豚鼠耳蜗损伤的保护作用及相关机制研究
目的将PPTA经鼓阶开窗微孔注入技术注入豚鼠耳蜗,研究其对庆大霉素耳毒性的拮抗作用及氧自由基机制。方法听力正常豚鼠45只,分为三组：A组(Control group)15只,以等量生理盐水肌肉注射,连续3d；B组(GM group)15只,以GM160mg/kg.d,肌肉注射,连续3d；C组(PPTA+GM group)15只,以GM160mg/kg.,3d,每日GM1小时前,行双侧鼓阶开窗微孔技术注入PPTA (浓度2mg/ml)10μl。(注：每组都以10只做统计学分析,额外为扫描电镜做形态学观察用)。三组动物分别在用药前,第3天用药后测ABR。在最后一次测ABR每组取10只迅速断头取听泡取左侧耳蜗(n=10)行T-SOD活力检测,取右侧耳蜗(n=10)行GSH含量检测；各组ABR RT、T-SOD、GSH统计使用SPSS13.0分析软件,数据以X±S表示,用协方差分析处理数据,若实验前ABR反应阈对实验后无影响,对试验后数据进行单因素方差分析。每组剩余5只豚鼠耳蜗行扫描电镜(SEM)形态学观察。结果用药3d后,GM组ABR RT显著高于正常组(P<0.05),GM组T-SOD活力、GSH含量显著显著低于GM+PPTA组(P<0.05)；经鼓阶开窗微孔技术注入PPTA3d后,PPTA+GM组ABR RT明显高于Control组(P<0.05),但显著低于GM组(P<0.05)。PPTA+GM组比GM组及正常组T-SOD活力、GSH含量显著低于正常组(P<0.05),但显著高于GM组(P<0.05)。SEM观察见：GM后毛细胞纤毛倒伏、断裂、细胞缺失等,PPTA+GM组亦有此现象,但较GM组轻微,正常组未见这些现象。结论促耳蜗组织内产生过量OFR造成耳蜗组织细胞损伤,是GM耳毒性的一个重要原因。经鼓阶开窗微孔技术注入PPTA可拮抗GM所致听力和耳蜗组织损伤；PPTA可以通过清除耳蜗组织内氧自由基和增强抗氧化能力从而发挥耳蜗保护作用。
第三部分腹腔注入PPTA对庆大霉素豚鼠耳毒性拮抗作用及对耳蜗组织Caspase-3表达的影响
目的研究PPTA全身给药对庆大霉素耳毒性的拮抗作用及相关机制。方法听力正常豚鼠45只,分为三组：A组(Control group)15只,以等量生理盐水肌肉注射,连续14d；B组(GM group)15只,GM120mg/kg.d,肌肉注射,连续14d；C组(PPTA+GM group)15只,PPTA10mg/kg.d腹腔注射+GM120mg/kg.d (1h后),肌注,连续14d。三组动物在用药前1h,第14天用药后测ABR。在最后一次测ABR后所有动物迅速断头取听泡,以左侧耳蜗(n=10)行Western blot检测Caspase-3表达,统计使用SPSS13.0分析软件,数据以X±S表示,用协方差分析处理数据,若实验前ABR反应阈对实验后无影响,对试验后数据进行单因素方差分析。以右侧耳蜗行TUNEL染色,每组剩余5只一侧耳做扫描电镜另一侧耳做透射电镜观察。结果GM组、PPTA+GM组ABR RT显著高于Control组(P<0.05), PPTA+GM组ABR RT显著低于GM组；Western blot结果表明用药后GM组豚鼠caspase-3表达显著增加(P<0.001); PPTA+GM组caspase-3的表达显著高于Control组但显著低于GM组(P<0.001)。SEM/TEM和TUNEL观察见：GM组毛细胞损伤严重,耳蜗Corti器、侧壁的血管纹和螺旋神经节存在阳性细胞,出现了凋亡和坏死的形态学特征；而PPTA+GM组损伤较GM组明显减轻；Control组未见阳性细胞。而结论庆大霉素耳毒性所致耳蜗组织损伤中,凋亡与坏死并存,GM通过caspase-3途径参与了耳蜗细胞凋亡；PPTA具有拮抗GM耳毒性所致的耳蜗功能和结构损伤的作用；PPTA通过降低caspase-3蛋白表达,抑制凋亡,从而对豚鼠耳蜗组织起到保护作用。
Hearing disorder is a common disease, severely affecting the healthy and living quality of human race. According to WHO, there are about250million people having moderate hearing loss, and among2/3in the developing countries. One of700to1,000new born babies has hearing disorder. China is the biggest developing country, with a population of1.3billion, of which,20million have hearing disorder, accounting for16.79%o. Our nation faces the big challenge whether we can find the effective method to prevent and cure hearing disorder.There are conductive, sensorineural and combinative hearing losses, and most are sensorineural hearing loss.
Sensorineural hearing loss is common in Otorhinolaryngology Head and Neck Surgery, which is the general term for hearing loss caused by a series of lesions of internal ear and its nerves. Reasons are hereditary, ischemic, ototoxic, sensorineural, age-related, noise-induced, traumatic, metabolic, autoimmunity, sudden or idiopathic, central, auditory neuropathy, tumor, functional and so on, among which, ischemic ischemic, sensorineural and age-related are the most common reasons. Pathological changes basically due to the damage to hair cells of cochlea, the physical changes of spiral ganglion, support cells and nerve endings, and lesions or damage of cochlear nerve and auditory pathway and center of brainstem. Cochlear sensory cell contains two types:inner hair cells (IHC) and outer hair cells (OHC). They are responsible for transferring sound signals into electrical signals, which are passed through the spiral ganglion neurons (SGCs) to the auditory brainstem pathways. And these IHCs, OHCs and SGCs lack of regeneration ability, therefore, any damage to cochlea can lead to irreversible hearing loss. Among the cochlear damages, the damage caused by ototoxic drugs is the most difficulty. Many researches on its pathogenesis and treatment have been conducted, with a hope for a better way. Drug-induced damage mechanism related to free radical damage, calcium overload and so on, among which, the sensorineural deafness caused by aminoglycoside ototoxicity is common clinical type.
Aminoglycoside antibiotics (Aminglycosides, AmAn) are suitable for clinical use for advantages wide antimicrobial spectrum, strong sterilization bacteriostasis, cheap, etc, but because of nephrotoxicity, ototoxicity and other serious adverse reactions, limit the clinical application。 In the short-term treatment cases using AmAn, the rate of ototoxicity incidence was20%, and in the long-term application treatment of tuberculosis and other serious bacterial infection (especially gram-negative bacteria), the rate of ototoxic incidence can be as high as80%. But in recent years, due to application in tuberculosis and other new areas, such as AIDS, studies have shown that aminoglycoside antibiotic line (gentamicin such antibiotics) can combine with RRE (retrovirus protein response factor) of RNA in HIV, create restrain effect between RRE and Rev (retrovirus protein) of HIV, and repress HIV replication activity. All these make many applications of AmAn can not be replaced.As one of aminoglycoside antibiotics, gentamicin (gentamicin, GM) is a strong sterilization produced by the fermentation of the color of small single spore bacteria, spines spore small single spore bacteria and such as, which has been widely used in clinical. The widely use of GM as its cheap advantage caused a large number of clinical cases of sensorineural deafness. At the same time, because the ototoxicity is more serious than the hepatorenal toxicity, compared to other aminoglycoside antibiotics, GM is the ideal drug causing the sensorineural deafness in the study of animal models.
The pharmacology characteristics of GM distribution in the body is not easy to enter the intracellular, not easy to penetrate into articular cavity, also not easy through the blood brain barrier, but mainly concentrates in the extracellular fluid, especially in the inner ear lymph. The accumulation of lymph of Gentamicin in the inner ear constitutes the physiological basis of its ototoxicity, and its mechanism is more complicated. Many researches focuses on this at home and abroad, mainly on the free radicals, calcium overload and start the apoptosis, etc. Many researches on GM ototoxicity show that the damage of hair cells of inner ear is associated with overload of oxygen free radicals and calcium, peroxidation of unsaturated fatty acid causes the severe damage to the biofilm. Guo Yufen and so have confirmed, Poly (aspartic acid enzymes) can restrain the overload of oxygen free radicals, so to reduce the incidence of cochlea toxicity induced by gentamicin. McFadden and so on has confirmed a kind of similar content, superoxide dismutase (sod) can prevent the damage to hair cells induced by gentamicin. Overload of oxygen free radicals and calcium can directly cause cell damage, even worse, lead to cell apoptosis. Also some scholars proved that cell apoptosis is the main way of deafness induced by aminoglycoside antibiotic,"waterfall" cascade of apoptosis mainly has two ways:(1) extracellular approach, namely the death of receptor.(2) inner cell approach, most refer to the death of mitochondrial pathway. But the two ways are ultimately dependent on the activation of Caspase3, Caspase3is the most important, acting as the end executo. Shimizu, such as found that calcium protease and apoptosis caspase inhibitors can block the ototoxicity induced by AmAn, facilitate the survive of vestibular hair cells. It showed that apoptosis has close relationship with vestibular ototoxicit induced by AmAn y.
At present, there is still no any effective ways to cure the sensorineural hearing impairment and protect the auditory capabilities in clinical. Although some researchers recently shows that, in the mammals' or even the human beings' body, the vestibular system of the inner ear and the cochlear hair cells are likely to regenerate after wounded, there are still no successful results in researches about the regeneration of OHC、IHC and SGCs during the experiment of mammals. In recent years, with the improvemt of the knowledge about biomechanism of the loss molecular in listening, some researchers put forward the concept of Deafness Gene, but all these new technology are in the experimental stage, which cannot be used in the clinical treatment. Many therapeutic efficacies of sensorineural deafness not work so well and the patients, who suffer from the ineffective drug therapy, have had to choose wearing the hearing aids or receive the artificial cochlea implantation. Though the artificial cochlea implantation can cure the severe sensorineural deafness at present, the price of it is quite expensive. Therefore, it's more important to explore the effective medicines in clinical which are useful for the early prevention from the factors causing the damage of inner ear and then provide the effictive treament.
The new compound named peperphentonamine hydrochl-oride,PPTA, which is first synthesized creatively by China, is the synthesized medicine with completely independent intellectual property screened by the Materia Medica Institute in Chinese Academy of Medical Sciences. This new synthesized medicine has alreasy authorized three invention patent and also two China invention patents and one international PCT invention patent are under application. In2008, PPTA had won11kinds of chemical medicines clinical test approved documents and even completed127clinical phase I tests.
Molecular formula:C21H24O4NCl或C21H23O4N·HCl,
Molecular Weight:MW=389.87
Chemical name:(E)-5-[(3,4-methylene2oxo phenethyl) amino] a-1-(4)--pentene-3-hydroxy phenyl-1methadone hydrochloride
Pepper phenyl ketone amine of the single crystal X-ray diffraction molecular structure as shown in figure1.
PPTA as the advanced international calcium sensitizer original innovation chemical medicine, preclinical research proves it can clear free radical of injury cells, as well restrain the overload of peroxidatic reaction of lipid. According to the research by Xu Jiangping, PPTA has a clear-up function to oxygen free radicals produced by the damage of brain cells; as well increase SOD activity and GSH content. Experiment results show that the PPTA has the function of resisting calcium overload, as well obvious protective effects on mitochondria of myocardial injury. PPTA can also remain the membrane fluidity of mitochondria normal, significantly reduce the damage to the ultrastructure of mitochondria. Overload Oxygen free radicals and calcium often leads to the damage of major tissue and cell, and mitochondrial injury can activate the Caspase-3to start the apoptosis of cell. Studies have shown that PPTA can significantly reduce the expression of Caspase-3mRNA, exhibit good resistance to apoptosis. So we consider introduce PPTA this innovative drug to prevent and treat sensorineural hearing loss.
PPTA as a state-level research and development new drugs for calcium sensitization agent drugs for cardiovascular, is on phase I clinical trials, not yet entered the clinical application. The protective effect and mechanism for the inner ear damage of PPTA has not been reported.
In this study, the guinea pig as experimental animal, on the foundation of the animal model of cochlear damage in manufacturing using the gentamicin ototoxicity of scala tympani fenestration, application of microporous injection and intraperitoneal injection, through using the innovative drug PPTA on cochlea, auditory brainstem response (ABR), immunohistochemistry (IHC), DNA end nick end labeling transferase mediated (ternial-deoxynucleotidyl transferase mediated nick end labeing, TUNEL), protein immunoblotting technique (Western blot), scanning electron microscopy (SEM) and other methods, to detect the changes in ABR RT, T-SOD, GSH, Caspase-3index and apoptosis and necrosis of morphology, and study the ototoxicity mechanism and protection in clinical application of antibiotics GM in-depth, and research on the protective effect of PPTA for cochlear injury and its mechanism, in order to explore the feasibility and specific mechanisms of national calcium sensitizer cardiovascular innovative drug on prevention of sensorineural deafness, further expand the scope of application and find a new drug for the treatment.
This study is divided into three parts:
Part one:Research on the cochlea damage model of guinea pig induced by gentamicin and injection technology on the tympani window of microcellular
Objective Base on the cochlea damage model of guinea pig induced by gentamicin, to discuss the application o and characteristics ABR of guinea pig model; to discuss the application and the effect to hearing and cochlear of technology on the tympani window of microcellular; and to observe changes of cochlear tissues inside the cell morphological using TUNEL and scanning electron microscopy techniques. Method30guinea pigs with normal hearing were randomly divided into three groups:Group A (control group)10, Group B (GM1)10, Group C (GM2)10. Group A (control group):amount of normal saline, weigh daily, give calculation of the dosage according to the weight, total duration with group B; Group B (GM1) with gentamycin sulfate injection120mg/(kg/d), weigh daily, give calculation of the dosage according to the weight for28days. Group C (GM2) with gentamycin sulfate injection160mg/(kg/d), weigh daily, give calculation of the dosage according to the weight for3days. Collect the RT and Ⅰ, Ⅲ PL, Ⅰ-Ⅲ IPL and Ⅲ AP within one hour before injection and within24hours after injection on7th,14th,21st and28th days. Collect the RT within one hour before injection and within24hours after the last injection.After collection, carried the injection technology on the tympani window of microcellular on10guinea pigs of Group A; collected ABR7days after surgery. After all the inspection, took otocyst beheaded of guinea pigs of Group A and B, scanned electron microscope and observed the immunohistochemical staining and TUNEL staining. Result ABR RT had significant difference after28days' injection between Group A and Group B; ABR RT had significant difference after3days' injection between Group C and Group B; ABR RT had no significant difference after7days before and after surgery. Observation by TUNEL and SEM, there were cilia lodging, fault, loss of cells on GM groups, while there was no these phenomena on Group A and surgery group. normal group of those who did and did not see these phenomena. Conclusion Gentamicin applied in guinea pigs, can lead to severe sensorineural deafness; medicine deafness animal model induced by gentamicin is a simple and reliable method; there were individual differences of ototoxicity induced by gentamicin in the short term, the damage became similar in a long time application; long-term application of gentamicin could lead guinea pigs to death easily, as it has hepatorenal toxicity; as ABR waveforms easy to recognized and recorded, it is an objective evaluation for hearing change of guinea pigs; under appropriate conditions, the ABR is relatively simpler and more accurate when guinea pigs are at waking than narcosis; Technology of injection on the tympani window of microcellular did not have significant effects on the mechanism of inner ear or the hearing the characteristics of hair cells poisoned by gentamicin:gradually reduce from the bottom to upward, the damage of outer hair cell is relatively serious than that of inner hair cells, among which, the most outside hair cells is the worst; the mechanism of ototoxicity induced by gentamicin could be related to the overload of oxygen free radicals and calcium, and activation of Caspase3which induces apoptosis.
Part Two:The protection of PPTA injected through the tympani window of microcellular on the damage cochlea induced by gentamicin, and the mechanism of oxygen free radicals
Objective Inject the PPTA through the tympani window of microcellular to the damage cochlea induced by gentamicin, observe the changes of hearing through ABR, and to study the effect and protective mechanism of PPTA for oxygen free radicals and the damage of cochlea, through the detection on the changes of T-SOD, GSH, and the shape change of hair cells by scanning electron microscope. Method45guinea pigs with normal hearing were randomly divided into three groups:Group A (control group)15, Group B (Deafness model induced by gentamicin, GM Gruop)15, Group C (Injected PPTA through the tympani window of microcellular, PPTA+GM Group)15. Group A (control group):give calculation of amount of normal saline for3days, the spot times of tests on hearing were the same as Group C. Group B (GM1) with gentamycin sulfate injection160mg/(kg/d) for3days. Group C with GM injection of160mg/(kg/d) for3days. One hour before injected with gentamicin, injected PPTA (concentration of2mg/ml)101into the lymph in the scala tympanianesthesia. Tested ABR one hour before injection and after24hours after injection the3rd day. After the last test on the ABR, took all the animals' otic vesicles by decapitation quickly, carried out the vitality test of T-SOD on one side cochlea (n=10) randomly, and content test of GSH on the other side cochlea (n=10); carried scanning electron microscope test on one side of the remain cochlea the TUNEL on other side cochlea. Result After3days' injection, there were significant difference on ABR, vitality of T-SOD and the content of GSH between GM Group and normal Group; After3days' injection of PPTA through the tympani window of microcellular, there were significant difference n ABR, vitality of T-SOD and the content of GSH between PPTA+GM Group and normal Group, as well the GM Group; observation by SEM, there were cilia lodging, fault, loss of cells on GM Group, PPTA+GM Group had less phenomenon, while no on normal Group. Conclusion Injection of PPTA through the tympani window of microcellular, can enhance the vitality of SOD in cochlea tissue, increase the content of GSH, the antagonism of PPTA to GM ototoxicity can protect the cochlea by eliminate the oxygen free radicals in cochlea tissue and enforce the antioxidant ability.
Part Three:The influence of PPTA on the antagonism to oxicity in the GM guinea pigs and the Caspase-3in the Cochlear tissue
Objective To study on the project that whether PPTA has the influence on the ototoxicity antagonism in the GM guinea pigs and then investigate its mechanism by using the ABR, testing on the Caspase-3through Western blot, staining the TUNEL and observing the scanning electron microscope. Method45normal hearing guinea pigs are divided into three groups:Group A(control group,15), Group B(GM group,15), Group C(PPTA+GM group,15). Group A were intramuscular injected by equivalence saline for continuous14days. The time-point of Group A for listening test on ABR is the same with Group C. Group B were intramuscular injected by gentamycin sulfate120mg/kg.d for continuous14days. Group C were intraperitoneal injected by PPTA10mg/kg and then after1hour were intramuscular injected by gentamycin sulfate120mg/kg.d for continuous14days. All the three groups are testing1hour earlier before using the medicine. On the seventh day and the fourteenth day, all the groups should test the ABR after using the medicine24hours ago. After the last test on the ABR, took all the animals' otic vesicles by decapitation quickly, carried out the Western blot to test the expression of Caspase-3on one side ears (n=10) and TUNEL (n=10)the other side.SEM and TEM on another5guinea pigs. Result ABR threshold shift in the GM Group increases obviously. Compared with the GM Group, the ABR threshold shift in PPTA+GM Group decreases apparently, but higher than the Control Group. Observing the GM Group through TUNEL, it shows that those hair cells are injuried badly. There are some TUNEL positive cells existing in the the organ of Corti, the lateral wall of the vascularis stria and the spiral ganglion, which now emerge the morphological characteristics of apoptosis. Compared with GM Group, the injury in PPTA+GM Group relief clearly. Besides, there are no positive cells in the Control Group. The result of Western blot shows that the expression of caspase-3increases obviously in the GM Group after using the medicine. The expression of caspase-3in the PPTA+GM Group rise slightly, but lower than that of GM Group. Conclusion PPTA showed antagonism effect with GM ototoxicity, which express the anti-effect. The mechanismis likely to restrain the expression of Caspase-3and then realizes its effect.
引文
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