The Affection Of Peperphentonamine On The Expression Of Cell Autophagy And NKCC1mRNA And ET-1 In Cochlear Damaged By Gentamicin Of Guinea Pigs

BackgroundDysaudia is one of the major disabling diseases. As the WHO reported in 2013,about 5.3% of the world’s people,most of which are from the developing country, suffer from hearing damage. As the largest developing country,there are about 27 million hearing disabilities in China. In all types of disabilities,hearing disabilities are the most,in which deaf-mute population is more than 2 million and increases in the number o 30,000 people each year,causing great pain and heavy burden of the disabilities,their families and the society. Therefore,it’s important to reduce the incidence rate of hearing disease for improving the population quality and life quality. To find out the efficient way to prevent and cure deafness is the huge challenge we face at present as well as the common duty of the society.Deafness can be divided into conductive deafness,sensorineural deafness and mixed deafness in clinic,and the sensonneural deafness has the highest incidence and the treatment of sensonneural deafness is the most remarkable problem in otology, sensorineural hearing loss can be also devided into sensory hearing loss and nervous hearing loss, sensorineural hearing loss(SNHL). sensory hearing loss is also called cochloar hearing loss,for the acoustic hair cell of of organ of Corti demaged,which accounts for the disfunction of voice ceptor. And the nervous hearing loss,also called retrocochlear hearing loss,is that the impairment of the auditory nerve or the auditory conduction tract,or the neuron in any level> induces the dysfunction of auditory nerve conduction and auditory cortical dysfunction. sensorineural hearing loss is a common,frequently occurring and complex disease in clinic,including the drug-induced deafness,sudden deafhess,noise deafhess,senile deafness and so on. It might be the auditory ruction damage caused by the oxygen free radicals,mechanical force and the calcium overload,and the pathological changes mainly distributed in cochlear and auditory nerve, nowadays,the most effective treatment of sensorineural deafness is to wear a hearing aid or to implant the artificial cochlea,but both of them can be applied to limited patients, implanting the artificial cochlea is a not bad choice,but it’s limited by the economic condition and surgery condition. Although wearing the hearing aid can improve the hearing effective,but the disease is still not be in therapy. Therefore,drug therapy is the important way to treat the sensorineural hearing loss, sensorieural hearing loss is an acute disease,and drug can improving the hearing loss to some extent,but the traditional drug therapy is limited. So people begin to search the new drug and therapy. So people begin to search the new drug and therapy, nowadays, the most important treatment is to systematic delivery, however, the blood-labyrinth barrier of cochlear of will lower the drug concentrations in the cochlea even stop the drug after systemic administering,so the therapy by systemic administering is not ideal. On the other hand,local injecting of drug into middle ear can eliminate the influence from blood-labyrinth barrier, attained a higher drug concentration.Gentamicin(GM) is a kind of aminoglycoside antibiotics(AmAn),witch has a wide ly application in clinical with its wide antimicrobial spectrum and strong sterilization ability. For the Specific pharmacological effects, a lot of clinical therapy with gentamicin will be hard to replace. In addition, gentamicin has lesser Systemic side effects, so we usd the gentamicin-induced to establish the sensorineural deafness model in guinea pigs.Piperphentonamine hydrochloride (PPTA) is a kind of drug that treatment of ischemia-reperfusion injury in cardiac muscle cell.lt can reduce the oxygen consumption and increase the sensitivity of calcium ion in cardiac muscle cells, also can avoid the happening of calcium overload simultaneously. Has now successfully completed the Phase I clinical trial, the phase II clinical medicine pharmacology experiment was ongoing. Current studies on the mechanism of ischemia reperfusion injury are almost around the cell mitochondrial dysfunction, intracellular calcium overload plasma disorders, oxygen free radicals, etc. These damage mechanism and aminoglycoside antibiotics (AmAn) is similar to the effect of ototoxicity damage, prompt PPTA can also be used for the protection of gentamicin-induced cochlear damage research.The cell death will happening when the external stimulation factors continuous exist. At present, most scholars consider that the death of cells can be divided into three forms, namely, apoptosis, cell autophagy and cell necrosis. Numerous studies have indicated that the apoptosis of hair cells was exist when sensorineural deafness was happened.Early research has shown that PPTA can improve the injury of guinea pig cochlear witch caused by ischemia-reperfusion, and the mechanism is by inhibiting the expression of IL-1β,TNF-α, Caspase 3 and Fas protein, after ischemia-reperfusion was happened. This trial was based on observing the auditory function, cell autophagy and influence factors of cochlear internal environment, then to explore the relationship between the expression of autophagy-related protein LC3, Beclin1,CKCC1 mRNA and endothelin with gentamicin-induced cochlea damage of guinea pigs, and study the protective mechanism of PPTA against the gentamicin-induced cochlea damage, and assessing the basic situation when PPTA was applied locally at the same time. Aim to provide pharmacological basis for the treatment of gentamicin-induced inner ear disease,and to develop a protective drugs to the inner ear injury. This study consists of two parts:Part ⅠResearch on the cochlea damage model of guinea pig induced by gentamicin and learning the ototoxicity and injection method of PPTA when curing the inner ear diseasesObjectiveTo establish the exact sensorineural deafness model caused by gentamicin injury of guinea pigs,finding a right way of PPTA to curing the inner ear diseases, and assessing the basic situation when it was used. then we can further study the curative effect of PPTA on the gentamicin-induced cochlea damage of guinea pigs.MethodsSixty male guinea pigs were randomly divided into control group, surgery group, PPTA group and GM group.Control group don’s do any processingln surgery group,we continuous injected 0.1ml artificial perilymph in otocyst, three times a day for 7 days.Injected 0.1ml 0.2%PPTA in otocyst of PPTA group, three times a day for seven days continuously.Injected gentamicin in GM group continuously,160mg/kg/d,for seven days. All of the guinea pigs were piercing the otocysts and imbedding tubes before injection PPTA and artificial perilymph. Analyzed hearing with ABR in every guinea pig. Two or four hours later after finished the test. Injected 0.1ml 0.2%PPTA once again in three quarters of surgery group and PPTA group. Measuring the concentration of PPTA in perilymph by UPLC after injected PPTA for 15min,30min,60min respectively, also measuring the quarter of the number.Resultsthe ABR threshold among control group, surgery group and PPTA group had no differences (p>0.05),the ABR threshold in GM group were significantly higher than that in control group (p<0.001).It has a quite high concentration of PPTA in perilymph among 15min,30min,60min. and it was higher in 15min and 30min.ConclusionsThe gentamicin-induced cochlea damage of guinea pigs was clearly, it can use to establish sensorineural deafness model of guinea pigs. It’s every easy and convenient to inject drugs by piercing the otocysts and imbedding tubes, and it can attain a high concentration of PPTA in perilymph of guinea pigs, This suggests that PPTA has high permeability to through cochlear window membrane. The ABR threshold was unchanged after used PPTA, Indicated that PPTA was no ototoxic, it can be used to study the protective mechanism of PPTA against the gentamicin-induced cochlea damage.Part Ⅱ The affection of Peperphentonamine on the expression of Cell Autophagy and NKCC1mRNA and ET-1 in Cochlear Damaged by Gentamicin of Guinea PigsObjectiveTo explore the relationship between the expression of autophagy-related protein LC3 and Beclinl and CKCC1 mRNA and endothelin with gentamicin-induced cochlea damage of guinea pigs, and study the protective mechanism of PPTA against the gentamicin-induced cochlea damage.MethodsSixty male guinea pigs were randomly divided into control group, model group, concurrent treatment group, model control group, lately treatment group. We injected NS and artificial perilymph on control group for 7 days, injected GM and artificial perilymph on model group for 7 days, injected GM and PPTA on treatment group for 7 days. In model control group and lately treatment group, we respectively injected artificial perilymph and PPTA for 7 days after we had finished injecting GM in both group for 7days. All of the guinea pigs were piercing the otocysts and imbedding tubes before injection, NS and GM (160mg·kg-1·d-1) applied peritoneal injection, artificial perilymph and PPTA were injected into otocysts. Analyzed hearing with ABR and tested the protein expression of beclinl and LC3 in cochlea tissue by WB,tested the expression of NKCC1 mRNA by RT-PCR and applied IHC to detecting the expression of ET-1.ResultsThe ABR threshold between model group and model control group had no differences (p>0.05),but both were significantly higher than that in other 3 groups (p<0.05),the ABR threshold in concurrent treatment group were significantly lower than that in lately treatment group (p<0.05).Compared with those of the other 4 groups, the expressions of LC3Ⅱ and Beclinl and NKCC1 mRNA increased significantly in model group (p<0.05 in the result of NKCC1 mRNA),and the expressions of LC3Ⅱ, Beclinl and NKCC1 mRNA in lately treatment group were significantly lower than that in model control group (p<0.05 in the result of NKCC1 mRNA). The expression of ET-1 in. the Corti organ, stria vascularis and spiral ganglion of model group was significantly higher than those of the other 4 groups, and the expression of ET-1 in the control group was significantly lower than those of the other 4 groups, and the expression of ET-1 in the lately treatment group, was significantly lower than that in model control group.ConclusionsPPTA can defense the gantamicin—induced damage in the cochlea of guinea pigs by inhibiting the cell autophagy and the expression of NKCC1 mRNA and ET-1, and the efficacy was better when applied PPTA Earlier, which indicated that GM would induce irreversible injury of the auditory cells.