| Background and Objectives:Cisplatin is a potent antineoplastic agent which is widely used for a variety of cancertypes including germ cell tumors，nasopharyngeal carcinoma, lung cancer, ovarian cancer,endometrial cancer and testicular cancer. Unfortunately, its use leads to side effects such asnephrotoxicity, neurotoxicity and ototoxicity. Up to93%of patients receiving cisplatinchemotherapy will develop progressive and irreversible sensorineural hearing loss whichleads to a decreased quality of life in cancer survivors. No treatment is currently availablefor cisplatin-induced ototoxicity. Therefore, the mechanism of ototoxicity caused bycisplatin is important to study. Researchers found that cisplatin was took in by hair cells atfirst and the ability of cochlear support cells to uptake cisplatin was relatively weak, whichsuggested that hair cells are the major target cells.IHCs are the genuine sensory cells transmitting information and intensity of sound tothe central nervous system (CNS). The IHC synapse like an analog system with minimalsensory information loss. Indeed, neurotransmitter release of the mature IHC synapse istriggered by graded membrane potential variations, increasing with sound intensity.Studying the molecular mechanisms underlying the specific functional features of IHCsynapses remains a major challenge. Synapses formed by inner hair cells (IHCs) containspecialized structures called synaptic ribbons. it has been proposed that the transmembraneprotein otoferlin is a functional protein in IHCs that regulates hair cell neurotransmitterrelease by accelerating embrane fusion. It can bind synaptotagmin I, cytosolicphospholipase A2, Ras-related GTP-binding protein, which plays an important role inmembrane transport, signal transduction and neurotransmitter release, etc. In the adultmouse cochlea, otoferlin mRNA was detected only in the IHCs. Otoferlin might function asa calcium sensor in cochlear IHCs and is closely related to the exocytosis of IHCs. It couldcause deafness without otoferlin. Cisplatin (cis-diamminedichloroplatinum(II)) is a highly reactive molecule. Onceinside the cell, it is transformed to its more active form as water molecules replace thechloride groups by an aquation reaction. The aquated form can then bind a variety ofmacromolecules including RNA, proteins (such as superoxide dismutase), membranephospholipids, microfilaments and DNA, which is its primary target. Cisplatin also inducesoxidative stress. The reactive oxygen species form hydrogen peroxide or react with nitricoxide resulting in peroxynitrite formation. Peroxynitrite can interact with proteins and formnitrotyrosine while hydrogen peroxide can lead to the production of4-hydroxynonenal as aresult of catalysis by iron and posterior interaction with polyunsaturated fatty acids in cellmembranes. The oxidative environment results in cytochrome c release from themitochondria which leads to an increase in calcium release from the endoplasmic reticulum.The calcium release then causes a massive cytochrome c release activating the apoptoticcascade. Previous experimental studies of cisplatin were focus on outer hair cells and spiralganglion cell damage, but researches of the inner hair cells were rarely. To explore themechanism of hearing loss caused by cisplatin, and whether the damage of inner hair cellsrelate to otoferlin, we carried out the experiment. We established a stable and effectivelow-dose cisplatin hearing loss model, including hair cells had no significant change. Thenwe observed how otoferlin protein expressed in inner hair cells. On the basis of the resultsto verify the mechanism of injury to inner hair cells caused by cisplatin. What’s more, wehope to provide new evidence to protect patients from cisplatin damage and provid adamage model for subsequent research of otoferlin protein regulation mechanisms.Methods:1. Male C57mice(8weeks to12weeks old) were randomly divided into seven groups:normal group（first group）; others were given different dose or period of cisplatin: thesecond group:0.75mg/kg×4d, the third group：0.75mg/kg×7d, the forth group：1.5mg/kg×4d, the fifth group：1.5mg/kg×7d；the sixth group：3.0mg/kg×4d；the seventhgroup：3.0mg/kg×7d。2. Measure the auditory brainstem response threshold(ABR) of the mice, and using theprogram spss18.0to compare the variation of the groups. We chose the t-test and ANOVAanalysis method, it was statistically significant when P＜0.05;3. The morphology of the IHCs: Stainned the inner hair cells by using5%AgNO3and observe the difference of each group;4.To detect the expression of otoferlin protein of each group by using the technologyof IF、RT-PCR.Results:1. Hearing (the threshold of ABR): After intraperitoneal injecting for4days,1.5mg/kgand3.0mg/kg were obviously different from the normal group, the threshold was bigger inhigh dose group. After using ciaplatin for7days, all of the experimental groups wereobviously decreased in hearing compared to normal group. With the same dose, the hearingresults of0.75mg/kg×7d group were worse than0.75mg/kg×4d group, other groups had notime difference. As a result, we successfully constructed a mouse model with hearing lossby injecting low dose cisplatin.2. AgNO3staining: The IHCs in the normal group were round, the size wereuniform and the gaps of the IHCs were clear. We could observe the cilium. The size and cellgaps of elliptic IHCs were regular in0.75mg/kg×4d group, and we could observe the cellmembrane was smooth and could not see the cilium.1.5mg/kg×4d group: The IHCs wereelliptic, the size was normal, the cell gap decreased, the membrane was not smooth, and thecilium was not be found.3.0mg/kg×4d group: The IHCs were elliptic, the size was reducedobviously, the cell gap decreased, the membrane was shrinking, and the cilium was not befound.0.75mg/kg×7d group: The IHCs were elliptic, the size was reduced lightly, the cellgap decreased, and the cilium and membrane was not be found.3.0mg/kg×7d group: TheIHCs were round, the size was reduced obviously, the cell gap decreased obviously, and thecilium and membrane was not be found.3. IF: In normal group, the expression of otoferlin was uniformly. The expression wasdifferent in each exprimental group, but there were no deficiency of IHCs in every group.To compare the gray value of the fluorescence, we found that3.0mg/kg×7d group wasobviously lower and others were not different from normal group. The1.5mg/kg×4d groupand1.5mg/kg×7d group were higher than others.4. RT-PCR: The0.75mg/kg group and3.0mg/kg group were lower than otherexperimental groups, others had no statistics difference.Conclusions:1.We successfully established a stable hearing loss mouse model by low-dose cisplatin, and hearing loss changed with different times or dose. With increasing time anddose of cisplatin, hearing loss of mice gradually increased; But the ABR threshold changedsimilarly in0.75mg/(kg×d)×7d group and1.5mg/(kg×d)×7d group. We thought thatsome damage might active the self-protection of mice cochlea;2. Cisplatin could damage the IHCs in different degree.3. The expression of otoferlin was different because of the different time and the doseof injecting cisplatin, and the changes in protein expression is not synchronized with theIHCs’ morphology.4. In summary, hearing loss caused by cisplatin related to the IHCs, the extent of thedamage related to the time and dose; the hearing loss caused by cisplatin might relate tootoferlin within IHCs, but the extent of the damage was not proportional to the time anddose, neither synchronized with the IHCs’ morphology. We thought the mRNA stored inIHCs might be active by1.5mg cisplatin, then otoferlin increased to prevent from damage.The hearing loss were badly in3.0mg/kg groups and the expression decreased, maybe that’sbecause of overstepping the ability of self-protection, and cisplatin possibly combined withotoferlin in DNA, RNA, protein levels, resulting in otoferlin decreased. |
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