The Protective Mechanism Of Piperphentonamine Against Cochlear Ischemia-reperfusion Injury In Guinea Pig

Hearing impairment is the important factor that affects public health and quality of life, which threatens hundreds of millions of people. On February27,2013, according to World Health Organization in Geneva, there were360million people with deafness or hearing impairment all over the world, accounting for5%of the world’s population and two-thirds of which in developing countries. As the world’s largest developing country, China has20.57million hearing-disabled people, accounting for16.79‰of the population of1.3billion; the vast majority of them are sensorineural deafness.The main pathological changes of sensorineural deafness are the damage to hair cells, spiral ganglion, sertoli cells and physical changes of nerve endings, as we all know, the reasons include ischemia of cochlear,virus infection, ototoxic drugs poisoning and senile degeneration etc, especially ischemia is the most common factor. Blood disorders cause by only a few of isolated ischemic damage, mostly by reperfusion injury after ischemia. Sudden deafness, for example, now thought to be caused by the inner ear microcirculation of sensorineural deafness. So to explore the mechanism of inner ear ischemia reperfusion injury will be very beneficial to the research of inner ear disease.piperphentonamine hydrochloride (PPTA)is chemical innovation drug, synthetized and screened by the Chinese academy of medical sciences institute of drug synthesis. Related studies show that PPTA has the protective effect of myocardial ischemia-reperfusion injury, is Calcium sensitization agent cardiac medicine and myocardial protective agent, there are studies show that protective effect to cerebral ischemic reperfusion injury. At home and abroad, there are not studies about PPTA protect the inner ear research of ischemia-reperfusion injury.The study was to explore PPTA on the protective effects of inner ear ischemia/reperfusion injury and the possible mechanism, and to expand the clinical indications of PPTA by the methods of auditory sense,cell morphology,inflammation factor and cell apoptosis. The indexes include evoked auditory brainstem responses(ABR),scanning electron microscope(SEM),Fas protein,TUNEL method, and mRNA expression of IL-1β,TNF-α and Caspase-1with RT-PCR skill.The research aim to provide pharmacological basis for the treatment of ischemic inner ear disease,and to develop a protective drugs to the inner ear ischemia reperfusion injury. This study consists of four parts:Part Ⅰ Cochlear Ischemia/Reperfusion (I/R) Model in Guinea PigsObjectiveThe purpose of this study was to establish an animal model of cochlea ischemia/reperfusion(I/R) injury through vertebrobasilar artery ischemia reperfusion and to investigate the changes of auditory function in guinea pigs.MethodsTwenty-four healthy guinea pigs with normal Preyer’S reflex weighed from200to250g were used in this study. They were randomly divided into3groups:the normal control group、blank control group、and ischemia reperfusion groups. The ischemia/reperfusion model by transient blocking bilateral vertebral artery and unilateral cephalic artery for60min,the cochlear blood flow(CoBF)was monitored continuously with Laser Doppler flowmeter. The auditory function was evaluated by evoked auditory brainstem responses(ABR).Each animal was measured before the operation and after ischemia-reperfusion(6h、24h、48h).ResultsThe threshold of ABR of the normal control group and blank control group, which was not significant change before and after every experiment (P>0.05).The changes of ABR during the ischemia-reperfusion group consisted of a prolonged in all wave latencies and interpeak latencies Ⅰ-Ⅲ, an increase of the auditory threshold, especially24hours after I/R, threshold declined when48h,but a statistically significant difference of the auditory threshold shift compared with pre-operation(P<0.05). A statistically significant difference of the auditory threshold shift before and after ischemia/reperfusion(P<0.05).ConclusionWe had successfully established an animal model by blocking bilateral vertebral artery and unilateral cephalic artery in guinea pigs.This model could increase the threshold.The highest auditory threshold of Ischemia reperfusion injury was measured at24h. Part2PPTA Protects Cochlea from Ischemia/Reperfusion Injury and the Change of Histomorphology under Scanning Electron Microscope and Transmission Electron MicroscopeObjectiveTo investigate the effects of PPTA on auditory brainstem responses(ABR) and the change of cochlear histomorphology in cochlea of guinea pigs encounted ischemia/reperfusion injury.MethodsUsed the ischemia reperfusion model, thirty-two healthy guinea pigs with normal Preyer’S reflex weighed from200to250g were used in this study. They were randomly divided into four groups:the normal control group、blank control group、 and ischemia reperfusion control group、ischemia reperfusion group treated with PPTA. Four guinea pigs in each group were randomly assigned to SEM, others were assigned to TEM. Ischemia-reperfusion treated with PPTA group immediately injected PPTA via femoral vein after reperfusion, ischemia reperfusion control group inject same dose of NaCl. Threshold of auditory brainstem response(ABR)was measured before and after every experiment.Killed and taken cochlea quickly when24h after reperfusion, observe the change of histomorphology of cochlear under SEM and TEM.Results1、The threshold of ABR of the normal control group and blank control group,which was not significant change before and after every experiment (P>0.05).The changes of ABR during the ischemia/reperfusion control group consisted an increase of the auditory threshold, especially24hours after ischemia/reperfusion, a statistically significant difference of the auditory threshold shift compared with pre-operation(P<0.05). A statistically significant difference of the auditory threshold shift of ischemia reperfusion group treated with PPTA before and after I/R(P<0.05),but better than ischemia/reperfusion.2、We observed the change of cochlear histomorphology of guinea pigs. Three rows of outer hair cells(OHCs),one row of inner outer hair cells in basical membrane arranged neatly,the stereocilium of hair cells arranged neatly,without loss. Outer hair cell swollen,loss, stereocilium which arranged disorder, collapsed of ischemia reperfusion control group.Ischmia reperfusion group treated with PPTA better than ischemia reperfusion control group.3. In normal group and sham group, membranes of hair cell nuclei were clearly and completely,however,membranes of hair cell were incompletely and typical changes of chromatin condensation and aggression were observed in ischemia reperfusion group.It is much better in PPTA treated group compared with ischemia reperfusion group.The changes of demyelinations in spiral ganglion of ischemia reperfusion group were much more than PPTA treated group. Congestion was observed in stria vascularis in ischemia reperfusion group,but it is not obvious in PPTA treated group,normal group and sham group.ConclusionIntravenous application of PPTA in the early period of cochlear ischemia/reperfusion can protect hearing in guinea pigs, reduced the damage of cochlea. Part3Effect of PPTA on mRNA expression of IL-1β and TNF-α in Guinea Pigs injured by ischemia reperfusionObjective1.To investigate the relationship between inflammatory response and ischemia reperfusion injury of cochlear.2.To investigate the relationship between inflammatory response and mRNA expression of IL-1βand TNF-α of cochlear.3.To investigate the protective effects of piperphentonamine hydrochloride on cochlear of guinea pigs.MethodsThe ischemia reperfusion injury model is the same as mentioned above.24guinea pigs were randomly divided into the normal group, the sham group,the ischemia reperfusion group and the PPTA treated group(10mg/kg). Guinea pigs of PPTA treated group were subjected to ischemia for1hour induced by bilateral vertebral arteries and unilateral carotid artery and then injected piperphentonamine hydrochloride from femoral vein after reperfusion immediately. Ischemia reperfusion group were treated with the same volume of physiological saline instead. Cochlear were removed after24hours of reperfusion.The mRNA expression of IL-1β and TNF-α were measured by Real Time PCR. All data was analyzed by one-way ANOVA using SPSS13.0. LSD test was used if equal variances are assumed. Dunnett’s T3were used if not. A value of P<0.05was considered as statistically significant.ResultsIschemia reperfusion group significantly increased the transcription level of IL-1β mRNA as compared with that of normal group and sham group (P<0.001). In comparison with that in PPTA treated group, the expression levels of IL-1βmRNA were significantly higher (P<0.001). In addition, there is no significant difference between PPTA treated group and normal group(P=0.056>0.05). Compared with normal and sham group, a significantly higher expression of TNF-amRNA was detected in ischemia group(P<0.001). Compared with PPTA treated group, ischemia reperfusion group also had a significantly higher expression of TNF-a mRNA (P<0.001). However, the expression of TNF-amRNA in PPTA treated group is higher than that of normal group (P=0.009<0.05)Conclusions1.Piperphentonamine hydrochloride(PPTA) has an antagonism action toward cochlear ischemia.2.The antagonism action of PPTA towards cochlear ischemia may perform with inhibiting inflammatory reaction. Part4Effect of Fas Protein and mRNA Expression of Caspase-1on Cochlear Ischemia Reperfusion Injury in Guinea Pigs and Protection of PPTAObjective:1. To observe the cell apoptosis in cochlear after ischemia reperfusion in different time.2. To analysis the relationship between expression of Fas protein and transcription of Caspase-1and the cell apoptosis in cochlear after ischemia reperfusion. 3. To investigate the application of PPTA.Methods:48healthy guinea pigs were devided into the normal group, negative control group, IRI control group after the application of0.9%NaCl,Group with PPTA (10mg/kg) were given by intraperitoneal injection after onset of ischemia-reperfusion(60min after ischemia). Every groups have12guinea pigs and we would take samples24h after reperfusion.The Fas protein immunoactivity in cochlear was studied by immunohistochemisty and labeling ratio of Fas protein expression was studied by image analysis. The cell apoptosis was studied by TUNEL method. Statistical analysis Was performed with one-way ANOVA of SPSS13.0statistical package and statistical significance was defined as P<0.05.Result:Immunohistochemical staining showed Fas protein expression of cochlear stria, spiral ganglion, Corti’s organ for weakly positive in normal group, control group and the ischemia reperfusion group with the PPTA.Fas protein expression in Ischemia reperfusion group significantly enhanced. IOD values of the normal group, control group, ischemia-reperfusion PPTA protection group there was no statistically significant difference between (P>0.05), ischemia reperfusion group was significantly enhanced than other three groups (P<0.05).No or only individual cell apoptosis appeared in the each part of cochlear in the normal group, control group and the ischemia-reperfusion group with the PPTA with TUNEL method.In the ischemia-reperfusion control group the cochlea apoptotic cells increased obviously.PPTA could significantly reduce apoptotic cells.The expression of Caspase-1mRNA of cochlear in ischemia-reperfusion group is significantly higher than normal group, control group and ischemia-reperfusion group with PPTA (P<0.001).In ischemia reperfusion group with the PPTA the expression rate of Caspase-1mRNA of cochlear was significantly higher than it in normal group (P<0.001). The difference of expression of Caspase-1mRNA of cochlear tissue is not significant between the normal group and the control group (P=0.825).Conclusion:There is slight positive expression of Fas protein in the cochleas of normal group and control groups. In the ischemia group and reperfusion groups, labeling ratio of Fas protein expression and transcription level of Caspase-1mRNA increased significantly compared with the normal group and the control groups. Compared with the model groups, the labeling ratio of Fas system expression and transcription level of Caspase-1mRNA in cochlear decreased in drug(PPTA) groups. There were almost no cell apoptosis in the normal group and the control groups. But there were more cell apoptosis in the ischemia group and the reperfusion groups(P<0.05). The numbers of apotosis cells in the drug(PPTA) groups were smaller than the groups without drug(PPTA). All of above indicates that Fas protein may get involved in the cochlear injury caused by the ischemia reperfusion injury. The ischemia reperfusion injury can cause the cell apoptosis in cochlear. The therapy of PPTA may prevent the cochlear from cellapoptosis in the ischemia reperfusion injury through blocking the expression of Fas protein and transcription of Caspase-1.