盐酸椒苯酮胺对庆大霉素致豚鼠耳蜗细胞自噬、NKCC1mRNA及ET-1表达的影响

发布时间：2018-02-25 19:23

  本文关键词： 听泡注射 庆大霉素 耳蜗 椒苯酮胺 听力损伤 耳蜗 自噬 Na+-K+-2Cl-联合转运体 内皮素　出处：《南方医科大学》2016年硕士论文　论文类型：学位论文

【摘要】：研究背景听力障碍是主要的致残疾病,据2013年WHO报道,全球约5.3%的人口患有听力类疾病,其中大部分为发展中国家公民。中国作为最大的发展中国家,听力残疾人有2700万,居各类残疾首位,其中聋哑人有200多万,并以每年3万人的数量增长,造成个人、家庭和社会的巨大痛苦和沉重负担。因此,降低人群中听力障碍疾病的发病率对提高人口素质、改善生活质量有着重大意义,能否找到防聋治聋的有效方法,是我们目前面临的巨大挑战,也是全社会的共同责任。临床上将耳聋分为传导性聋、感音神经性聋及混合性聋三类,其中以感音神经性聋的发病率最高且治疗难度最大,为耳科最大难症之一。感音神经性聋(sensorineural hearing loss, SNHL)又可细分为感音性聋(sensory hearing loss),其病变部位在耳蜗,主要为螺旋器毛细胞损伤致声音的感受功能异常,也称耳蜗性聋(cochlear hearing loss);以及神经性聋(nervous hearing loss),为听神经、听觉传导径路及其各级神经元受损害,致听觉神经冲动传导障碍以及听皮层功能减退者,也称蜗后聋(retrocochlear hearing loss)。感音神经性聋是临床常见、多发、疑难之疾病,包括药物性聋、突发性聋、噪声性聋、老年性聋等。其机制可能为通过氧自由基、机械力、钙超载等致使听觉功能区损害,病理改变主要分布于耳蜗和听神经。目前,感音神经性聋最有效的治疗方法为配戴助听器或植入人工耳蜗,但是这两种方法对听力损害有所改善的病人使用是受限的,人工耳蜗植入术虽能获得很好的效果,但受经济条件、手术适应症的限制；佩戴助听器虽然有助于改善耳聋患者的听力功能,但其本质上并未对疾病本身进行有效的治疗。因此,药物仍然是治疗感音神经性聋的重要手段。虽然感音性耳聋发生在急性期,药物有时可以改善听力下降,但传统药物治疗有一定的限制,因此人们开始寻找新的药物和治疗方法。目前临床用药仍以全身给药方式为主,然而,由于耳蜗血-迷路屏障对药物通透性的影响,全身给药后耳蜗中药物浓度较低,甚至存在不能通过该屏障的问题,导致感音神经性聋选择全身给药治疗时效果不理想。而采用耳内局部用药可有效解决耳蜗血-迷路屏障对药物有效浓度的影响。庆大霉素(Gentamicin,GM)是常见的杀菌力强抗菌谱广的氨基糖苷类抗生素(Aminoglycoside antibiotics, AmAn), I临床应用广泛,且因其特有药理作用,很多临床应用难以替代。此外,GM全身毒性较小,故本实验采用GM建立感音神经性聋的动物模型。盐酸椒苯酮胺(Piperphentonamine hydrochloride, PPTA)是一类治疗心肌缺血再灌注保护药,可降低心肌耗氧量,增加心肌细胞对钙离子的敏感性,同时不引发钙超载,目前已顺利完成I期药物试验,正在进行II期临床药药理实验中。目前对于缺血再灌注损伤发生的机制研究多围绕着细胞线粒体功能障碍、胞内钙超载等离子紊乱、氧自由基等方面进行,这些损伤机制与氨基糖苷类抗生素(AmAn)耳毒性损伤的作用方面十分相似,提示PPTA亦可用于对GM耳蜗损伤的保护研究。当外部刺激因素持续存在时,细胞将发生死亡。目前大多数学者将细胞的死亡分为3种形式,即细胞凋亡、细胞自噬和细胞坏死。众多研究已表明感音性耳聋存在着内耳毛细胞的凋亡,但是否存在自噬尚无文献报道。先期研究表明PPTA可改善豚鼠耳蜗缺血再灌注损伤,并抑制缺血再灌注后IL-1β、TNF-α,Caspase-3及Fas蛋白的表达。本次试验通过观察豚鼠听觉功能、耳蜗内环境的影响因素、细胞自噬等方面对豚鼠耳蜗庆大霉素损伤及椒苯酮胺的保护作用和局部用药情况进行研究,探讨椒苯酮胺对耳蜗庆大霉素损伤保护过程中细胞自噬相关蛋白LC3和Beclin1、NKCC1 mRNA、ET-1表达的影响,以期为椒苯酮胺用于感音神经性聋的治疗提供药理学依据。本研究分为如下两个部分：第一部分豚鼠GM耳蜗损伤模型的建立及PPTA给药途径和耳毒性的评估目的建立确切的由庆大霉素损伤所致的豚鼠感音神经性聋模型,寻找PPTA合适的给药途径,并评估PPTA局部给药治疗内耳疾病的一些基本情况,以便进一步研究PPTA对豚鼠耳蜗GM损伤的治疗情况。研究内容及方法健康成年纯白红目雄性豚鼠32只,体质量300-350g,随机分为4组,每组8只：对照组(control group,A组)、手术组(surgery group,B组)、PPTA组(PPTA group,C组)庆大霉素组(GM group,D组)。对照组不做处理；手术组行听泡外侧壁置管、听泡内注入人工外淋巴液0.1ml/次、3次/d、连续7d;PPTA组行听泡外侧壁置管、听泡内注入0.2%PPTA液0.1ml/次、3次/d、连续7d；GM组腹腔注射庆大霉素(GM),160mg/kg/d,连续7d。各组完成给药后进行听性脑干反应(ABR)测定,观察ABR各波形的阈值及形态变化。ABR测定2-4小时后,手术组与PPTA组各随机挑选2只豚鼠断头处死,剩余各6只老鼠再予听泡内注入0.2%PPTA液0.1ml,2组分别于给药后15min、30min、60min各断头处死2只豚鼠,所有断头处死豚鼠均迅速取出听泡,即各时间点8个听泡,快速提取耳蜗外淋巴液,并采用超高效液相色谱-质谱技术(UPLC(?))检测其PPTA的药物浓度。实验数据均以均数土标准差(贾士S)表示,采用SPSS19.0软件进行one-way AVONA方差分析,一组与多组之间比较时采用LSD法,P0.05时差异有统计学意义。结果ABR检测反应阈：手术组、PPTA组ABR反应阈与对照组无明显差异(P0.05)；GM组豚鼠ABR阈值明高于对照组(P0.001)。未给药豚鼠耳蜗外淋巴液中为检测出PPTA,给药后15min、30min、60min的豚鼠耳蜗外淋巴液PPTA药物浓度分别为(ug/ml):29.83±3.11、37.65±3.36、12.25±3.77。结论庆大霉素致豚鼠听力损失作用明确,表明其可用于建立豚鼠损伤的动物模型；听泡注射简单方便,经听泡注射PPTA后豚鼠耳蜗外淋巴液中药物浓度较高,同时豚鼠听力未见损失,说明PPTA对豚鼠园窗膜的通透性良好,且其本身不具有耳毒性,可采用局部给药应用于豚鼠耳蜗损伤保护的研究。第二部分PPTA对豚鼠耳蜗庆大霉素损伤细胞自噬相关蛋白LC3和Beclin1、NKCC1 mRNA、ET-1表达的影响目的研究PPTA对庆大霉素致耳蜗损伤的保护作用及其与自噬蛋白LC3、beclin1、内皮素(Endothelin 1 ET-1)、Na+-K+-2C1-共同转运体(Na+-K+-2Cl-cotransporter NKCC1) mRNA表达的关系。方法健康成年纯白红目雄性豚鼠60只,体质量300-350g,随机分为5组、每组12只：对照组(control group,A组)、模型组(model group,B组)、同期治疗组(concurrent treatment group,C组)、造模后对照组(model control group,D组)、后期治疗组(lately treatment group, E组)。所有豚鼠行听泡置管手术处理后进行给药：对照组行腹腔注射生理盐水十听泡注入人工外淋巴液连续7d；模型组腹腔注射GM (160mg·kg-1·d-1)+听泡注入人工外淋巴液连续7d；治疗组腹腔注射硫酸庆大霉素注射液(160mg·kg-1·d-1)+听泡注射PPTA连续7d；造模后对照组腹腔注射GM连续7d后,再行人工外淋巴液听泡内连续注入7d；后期治疗组腹腔注射硫酸庆大霉素注射液(160mg·kg-1·d-1)连续7d后,再行PPTA听泡内连续注入7d。各组豚鼠完成给药后进行听性脑干反应测定(auditory brainstem response, ABR),分析各组ABR阈值变化及相关关系。所有豚鼠完成ABR结果测定后断头处死,迅速取出双侧听泡并分离耳蜗,免疫印迹法检测beclinl和LC3表达、PCR检测NKCC1 mRNA的表达、免疫组织化学染色法检测ET-1的表达情况。实验数据以均数士标准差(X±s)表示,采用SPSS 19.0软件进行one-way AVONA方差分析,一组与多组之间比较时采用LSD法,验水准α为0.05。结果ABR反应阈：治疗组豚鼠ABR阈值明显低于于模型组(P0.001),但明显高于对照组(P0.001),后期治疗组AB值明显低于模型对照组(P0.001),但明显高于治疗组(P0.001),模型组、模型后对照组ABR反应闽差异无统计学意义(P0.05)。自噬相关蛋白LC3和Beclinl的表达：模型组LC3Ⅱ(与actin的比值)及Beclinl的表达量较其余四组明显升高,后期治疗组LC3 II(与actin的比值)及Beclinl的表达量较造模后对照组低。模型组NKCClmRNA表达较余四组明显升高(p0.05),后期治疗组NKCClmRNA表达明显低于造模后对照组(p0.05),造模后对照组NKCClmRNA表达较对照组明显升高(p0.05)。ET-1蛋白免疫组化表达结果：模型组耳蜗各部位ET-1表达较其余四组明显增高,对照组各部位ET-1表达低于其余四组,后期治疗组各部位ET-1表达较造模后对照组降低。结论PPTA可通过抑制耳蜗内ET-1表达和NKCC的激活,维持耳蜗内环境的稳态,维持耳蜗的正常生理功能,保护耳蜗组织免受损伤,保护听力；通过调节自噬程序的发生来减轻耳蜗自噬性细胞损伤,进而减少听力损失。PPTA对豚鼠庆大霉素的耳蜗损伤保护主要体现为拮抗作用,其抗损伤作用需早期及时应用。
[Abstract]:On the background of hearing disorder is a major cause of disability, according to a 2013 WHO report, about 5.3% of the world's population suffers from hearing diseases, most of which are developing countries Chinese citizens. As the largest developing country, there are 27 million hearing disabilities, among all kinds of disabilities, the deaf people have about 2000000, and growth in the number of 30 thousand people each year, resulting in individual, family and society of great suffering and heavy burden. Therefore, to reduce the incidence of disease prevalence of hearing impairment to improve the quality of the population, is of great significance to improve the quality of life, to find effective methods to prevention and treatment of hearing loss, is a huge challenge facing us, but also the common responsibility of the whole society. Clinically, deafness divided into conductive deafness, sensorineural hearing loss and mixed deafness in three categories, with sensorineural hearing loss and the highest incidence of treatment is the most difficult, most difficult for otology disease A. Sensorineural hearing loss (sensorineural hearing, loss, SNHL) can be subdivided into sensorineural deafness (sensory hearing loss), the lesions in the cochlea, mainly for spiral hair cells injury induced by sound feeling dysfunction, also known as cochlear deafness (cochlear hearing loss); and sensorineural hearing loss (nervous hearing, loss) for the auditory nerve, auditory pathway and neuron damage, induced auditory nerve impulse conduction disorders and auditory cortex dysfunction, also known as retrocochlear deafness (retrocochlear hearing loss). Sensorineural hearing loss is a common clinical disease, multiple problems, including drug sudden deafness. Hearing loss, noise deafness, presbycusis. The possible mechanism is through mechanical force, oxygen free radical, calcium overload resulting in the damage of auditory function, pathological changes were mainly distributed in the cochlea and auditory nerve. At present, the sense of treatment of sensorineural hearing loss is the most effective For hearing aids or cochlear implants, but the use of these two methods to improve the hearing impairment of patients is limited, cochlear implantation can get good results, but because of economic conditions, indications of surgery; while wearing hearing aid helps to improve deaf hearing function, but the essence of the disease itself is not treated effectively. Therefore, the drug is still an important method in the treatment of sensorineural hearing loss. Although sensorineural hearing loss occurred in the acute stage, drugs can sometimes improve hearing loss, but the traditional drugs have certain limitations, so people began to search for new medicines and methods of treatment. At present, the clinical medication still to the systemic administration mode, however, because of the cochlear blood labyrinth barrier effects on drug permeability, after systemic administration of drug concentration in the cochlea is low, even can't pass the screen Avoidance problems lead to sensorineural hearing loss choose systemic drug treatment effect is not ideal. And the use of topical ear can effectively solve the cochlear blood labyrinth barrier effect of effective drug concentration. Gentamicin (Gentamicin, GM) is a common strong bactericidal antibacterial spectrum of aminoglycoside antibiotics widely (Aminoglycoside antibiotics, AmAn, I) is widely used in clinic, and because of its unique pharmacological effects, clinical application of many difficult to replace. In addition, the GM body is less toxic, so we adopt GM to establish the animal model of sensorineural hearing loss. Peperphentonamine hydrochloride (Piperphentonamine hydrochloride PPTA) is a kind of treatment of myocardial ischemia-reperfusion injury drugs can reduce myocardial oxygen consumption, increase the sensitivity of myocardial cells to calcium ion, and does not lead to calcium overload, has successfully completed phase I trial, the ongoing phase II clinical pharmacological experiment.. Study on the mechanism for the injury of the ischemia reperfusion around cell mitochondrial dysfunction, intracellular calcium overload in plasma disorders, oxygen free radicals and other aspects, the mechanism of injury and aminoglycoside antibiotics (AmAn) on the ototoxicity of injury is very similar, suggesting that PPTA can be used for the protection of research on GM cochlear injury. When external the incitants persist, cell death occurs. Most scholars will be cell death is divided into 3 forms, namely cell apoptosis, autophagy and cell death. Many studies have shown that sensorineural hearing loss is the apoptosis of hair cells. But whether autophagy has not been reported. Previous studies showed that PPTA can improve the guinea pig cochlea ischemia reperfusion injury after ischemia reperfusion, and inhibition of IL-1 beta, TNF- alpha, Caspase-3 expression and Fas protein. This experiment through the observation of auditory function in guinea pigs, Influence factors of inner environment, autophagy aspects of protective effect on injury of guinea pig cochlea of gentamicin and JBAT and topical, explore JBAT on autophagy of Cochlea Injury in gentamicin related protein LC3 and Beclin1, NKCC1, mRNA, ET-1 expression, in order to provide pharmacological basis. The treatment of sensorineural hearing loss as JBAT used. This study is divided into two parts as follows: the first part is evaluation and establishment of PPTA GM in the guinea pig cochlea injury model of the route of administration and to establish the exact ototoxicity induced by gentamicin injury in guinea pigs with sensorineural hearing loss model, looking for PPTA suitable route of administration the evaluation and some basic PPTA topical treatment of inner ear diseases, in order to further study the treatment of PPTA GM on cochlear damage in guinea pigs. The research contents and methods of health 鎴愬勾绾�鐧界孩鐩�闆勬，

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