Protective Effect And Mechanism Of PPTA On Gentamicin Cochleotoxicity In Guinea Pigs

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=389.87Chemical name:(E)-5-[(3,4-methylene2oxo phenethyl) amino] a-1-(4)--pentene-3-hydroxy phenyl-1methadone hydrochloridePepper 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 microcellularObjective 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 radicalsObjective 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 tissueObjective 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.