The Study Of PPTA’s Protective Effect On Hairs And Spiral Ganglion Cells Injury Of Guinea Pigs Treated With Gentamicin

BackgroundHearing disorder is a common disease in Otorhinolaryngology Head and Neck Surgery, according to the world health organization (who) reported in2013, there were360million people suffer from crippling hearing loss wordwise, accounting for5.3%of the world’s population, and two-thirds of them in developing countries, this is a huge number. China is the largest developing country, hearing disabled person has20.57million, accounts for the population of16.79‰, for all kinds of disabilities, severely affecting the healthy and living quality of human race. So, now,we are facing the challenge that can we find effective methods of prevention and treatment of hearing impairment.Hearing loss include conduction deafness, sensorineural hearing loss and mixed deafness, the vast majority of patients are sensorineural hearing loss. Now as we know that reasons are hereditary, ischemia, ototoxicity, genetic, senile, noise, virus infection, autoimmune disease, auditory neuropathy, nerve centre disease, tumor, etc. Sensorineural hearing loss caused by the lesion of the inner ear and auditory neural pathway. 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.There are two types of sensory cells in inner ear:inner hair cells (IHCs) and outer hair cells (OHCs).which can convert mechanical energy (sound energy, gravity or acceleration) to electrical energy (cell receptor potential), which are passed through the spiral ganglion neurons (SGCs) to the auditory brainstem pathways, spiral ganglion cells is afferent neurons. Inner hair cells and outer hair cells connect Ⅰ, Ⅱ afferent neuron dendrites of SGCs, respectively. Inner hair cells transform different frequency and intensity voice signal into electrical signal, which can stimulate the presynaptic membrane and release neurotransmitters into the corresponding spiral neurons, signal is transmited to central nervous system by spiral ganglion. Outer hair cells can magnify the voice signal,so, the cochlea has high sensitivity and selectivity to the sound frequency, OHCs assist IHCs to transmit the voice signals.The abnormal structure、function and shape of afferent synapses all can lead to significant sensorineural hearing loss. Any injury of hair cells and afferent nerve will result in sensorineural hearing loss. Cochlea is energy intensive organization, ischemia and hypoxia will cause hair cells necrosis and afferent nerve swelling, which result in hearing impairment, finally;noise is one of the reasons.Animal experiments found that aminoglycoside antibiotics(AmAn) can damage outer hair cell cilia or OHCs itself, and there is a report says that AmAn can directly or indirectly lead to spiral ganglion cells apoptosis or necrosis. Ischemia/Reperfusion, noise, drug are the common causes of sensorineural hearing loss, but there are reports say that afferent nerve was swelling after ischemia5mins, but afferent nerve gradually returned to be normal after reperfusion;the feature of AmAn’s toxicity is persistence, it can continue to damage the cochlea even if the drug withdrawal, so it’s better to observe PPTA how protect the cochlear afferent nerve injury. We establish drug-induced sensorineural hearing loss animal models by using the common aminoglycoside antibiotics—gentamycin.We found that the drug-induced hearing loss was20%by AmAn’s short-term use, and80%by the long-term use in clinical, specially in children, the older people and low immunity ones,meanwhile the damage is irreversible. Toxic effects limited AmAn’s clinical application, but there is still special value of AmAn in the treatment of serious infections,such as sepsis, urinary tract infection, gram-negative bacteria caused intra-abdominal infections, endocarditis which treated with beta lactam, etc. Studies had shown that AmAn can be combined with HIV RNA’s RRE structure, makeing HIV RRE. Rev interact to produce inhibition, and thus inhibit HIV replication activity,Gentamycin (GM) is a strong wide-spectrum antibiotics produced by small single bacillus, clinical application more widely, because of its smaller kidney toxicity than other AmAn, so it is the ideal drug that make the drug-induced sensorineura hearing loss animal model.Gentamicin is mainly concentrated in the endolymphe of cochlear, it act on hair cells and afferent neurons. Now, the mechanisms of GM’s ototoxicity are centered on mitochondrial damage, apoptosis, oxygen free radicals and calcium overload. GM damage mitochondria lead to protein synthesis disorder by blocking70s ribosome cycle, destroy the function of mitochondrial membrane, causing mitochondrial energy barrie; scathing mitochondria reduce membrane’s ability in conversion electron,which generate a large number of oxygen free radicals, induce the cochlea hair cells that produce a large number of intracellular reactive oxygen species (reactive oxygen species,ROS). Oxygen free radicals and calcium overload not only cause cellular damage directly but also lead to cell apoptosis. Studies have pointed out that the main way of aminoglycoside antibiotics cause cochlear damage is cell apoptosis, the way can be achieved through multiple channels, but most of which ultimately rely on the activation of Caspase-3way, Caspase-3,which is an important eventually practitioner, reports have said that apoptosis inhibitor can reduce the damage of AmAn internal ear cells, it sat at the heart of cell’s apoptosis. AmAn has longer half-life in endolymph, hair cells take it into the intracellular, leading to apoptosis necrosis through the above mechanisms. Hair cells and dendrites of spiral ganglion form synaptic connections, the hair cells can release neurotrophic factors and neurotransmitters (glutamic acid) to keep the health of nerve.So, hair cells damaged by AmAn lead to the delayed death of SGCs.AmAn’s tosicity result in the death and loss of IHCs and SGCs,which is the morphology basis of hearing loss and abnormal location function. Hair cells’s regeneration are difficult after injury,now,we try our best to treat sensorineural hearing loss by the regeneration and cochlear stem cell transplantation. Afferent nerve injury is an important pathological changes lead to sensorineural hearing loss, the afferent nerve system including synaptic complexes, nerve endings and spiral ganglion cells,et. Synaptic structure is vulnerable to affected when the environment change, spiral ganglion cells can also be difficult to repair after damage. Reports show that the nerve fibers may be renewable,and contact with hair cells after afferent nerve injury; Schwann cell around spiral ganglion cells can be proliferation, but it can’t differentiate into normal function neurons to play a role. Afferent nerve system is the basis of sensorineural hearing loss patients get part of audioty by hearing aid or artificial cochlear,which have great significance for treatment of sensorineural hearing loss. At present, there is less person recept hearing aid and artificial cochlea. therefore,drug therapy is the main typy of treatments for people with hearing loss. 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 PPTA had won11kinds of chemical medicines clinical test approved documents and even completed127clinical phase I testsPPTA 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 say that, PPTA has a clear-up function to oxygen free radicals produced by the damage of brain cells,so it protect cranial nerve and improve animal cognitive impairment; 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. Like cardio and cerebral tissue damage, calcium overload and free radical, apoptosis are common causes of afferent nerve damage, this research is base on protection mechanism of PPTA.In this study, the guinea pig as experimental animal.In the first part,we establish Ischemia Reperfusion injury animal model and gentamicin damage cochlear animal model,respectively. Observe changes in the inner ear of two models in by auditory brainstem response (ABR) succinate dehydrogenase in hair cells, scanning electron microscopy (SEM). In the end,we found that gentamicin caused cochlear injury model is more suitable for observing cochlear injury;In the second part,we use DNA end nick end labeling transferase mediated (ternial-deoxynucleotidyl transferase mediated nick end labeing, TUNEL),SDH, protein immunoblotting technique (Western blot),transmission electron microscopy (TEM)and other methods, to detect the changes in ABR RT, 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 two parts:Part one:Establish the model on the guinea pigs’s cochlear injuryObjectiveChoose the right model to observe that PPTA protecte guinea pigs’s cochlear injury.Method1.1Select animals for experiment:Randomly divided the thirty-six health guinea pigs into three groups,each group of12guinea pigs,8animals for experiment,4ones reserved:the normal group,:I/R group and GM group.1.2Set up the models:We sat up I/R hearing loss model by blocking bilateral vertebral artery and right common carotid artery for lh,then reperfusion.GM group with muscle injection of gentamicin (120mg/kg/d,7d).Control group don’s do any processing.1.3Main review index:Analyzed the hearing changes with ABR, Succinate dehydrogenase and SEM were performed to observe the morphological changes of hair cells.Result1%The threshold in ABR:There were not significantly different between three times(P>0.05);there were significantly different between three times in B group and C group(P<0.05). The RT of ABR were persistent decline in group C, but it improved at7d after I/R in group B.2、Serious damages were seen in the hair cells by examination of N.B.T and SEM in I/R group and GM group:the hair are normal,no missing, Succinate dehydrogenase colored clearly.But there were annormal in hair cells; Outer hair cells’s cilia lodging, cracks between cells, cells’s shape were not clear. Part of the hair cells were death in group C.ConclusionGentamicin-induced guinea pig’s cochlea damage model is suitable for observing that PPTA protect cochlea injury. Part two:The study of PPTA’s protective effect on hair cells and spiral ganglion cells of guinea pigs treated with gentamicinObjectiveTo study the protective effect of peperphentonamine hydrochloride (PPTA) on hairs and spiral ganglion cells injury of guinea pigs treated with gentamicin.Method1.1Groups:Randomly divided the thirty-six health guinea pigs into three groups,each group of12guinea pigs:the normal group,:GM group and PPTA group.1.2Set up drug-induced cochlear animal model:GM group with muscle injection of gentamicin (100mg/kg/d,14d).Applied intravenous injection of PPTA(10mg/kg/d,14d) on the guinea pigs models of gentamicin-induced cochlear damage.Control group with muscle inj ection of saline(4ml/kg/d,14d).1.3Main review index:Analyzed the hearing changes with ABR.Then killed animals,and took the cochlears quaikly. Succinate dehydrogenase for hairs.By TUNEL staining,wo could count the nember of apoptotic cells of spiral ganglion cells, tested the protein expression of Caspase-3in cochlea tissue by Western blot; SDH> TUNEL staining and TEM were performed to observe the morphological changes.ResultIn The threshold in ABR:After trial, the threshold of control group,GM group and PPTA group were14.58±1.16dB、65.95±1.17dB、36.13±1.17dB, the rate of RT’s change was significantly different between three groups (p<0.05).PL of I:1.094±0.028、1.667±0.028、1.254±0.028; there was significantly different between three groups (p<0.05).2、The apoptosis index of three group were0.02±0.00、0.45±0.03、0.16±0.02,there were significantly different of these group (p<0.05). 3、The expression of Caspase-3tested by Western blot:1.09±0.11、2.55±0.20、1.67±0.07, respectively.4、Serious damages were seen in the hair cells by examination of SDH、TUNEL and TEM in GM group:There was a great amount of cell apoptosis existing in spiral ganglion. The organelles of presynaptic and nerver fibers were swelling,the nember of these were less;the hurt were less seriously in PPTA group.ConclusionPPTA plays a protective role on gentamicin-induced hairs and SGCs’s damage of guinea pigs.