Sodium Danshensu(SDSS) Alleviate Sodium Salicylate Induced Tinnitus By Decreasing The Activity Of NMDA Receptor In Inferior Colliculus

ObjectiveTinnitus,without ideal cure method,is a common medical symptom that can be distressing by the subsequent mental disorders.The increased neural activity along the auditory pathway are most commonly thought to be related to tinnitus.The imbalance in the excitatory and inhibitory inputs to neurons in the the inferior colliculus(IC),an important station in auditory pathway,is a important reason why tinnitus happen.The higher excitability of glutamate systerm or the lower inhibition of GABA systerm in IC can be amplified by the local circuit and then causing tinnitus.We find that sodium Danshensu(SDSS),the extractive of danshen,could decrease the activity of NMDA receptor of inferior colliculus in our research.We speculate that SDSS could prevent SS induced tinnitus by inhibiting the NMDA receptor.Methods1.Electrophysiological experiment.Prepare inferior colliculus slices,pharmacologically separate NMDA receptor and GABAa receptor rspectively,then whole cell patch clamp is used to record the NMDAR current with and without perfusion of SS(1 mM),with perfusion of SS and SDSS,as well as the NMDAR and GABAAR current with and without appearance of SDSS(100 mM)respectively.2.Cultured IC neurons(10-13DIV)are diveded into eight groups,Control,SS,SS+APV,SS+SDSS,NMDA,NMDA+APV,NMDA+SS,NMDA+SS+SDSS.After treated with medicine accordingly,apoptotic neuronal death was determined by visualizing neurons stained with Hoechst-33342,and the culture medium after drug treatment are collected to test released lactate dehydrogenase(LDH).3.Sixty three C57BL/6 male mice aged 6-7weeks with normal hearing are radomly divide into Control group,SS group and SS+SDSS group.15 mice are used for emotional evaluation and 6 for ABR test.SS is intraperitoneal injection in 400mg/kg for 9 consecutive days,and the SDSS is used half an hour before SS,and the mice in Control group are given saline of the same quantity.Tests for emotional evaluation included open field the elevated puls maze and force swimming,as well as ABR test are performed two days before the first and after the last drug injection for each mouse.4.The IC is harvest after last emotional evaluation and ABR test to extract proteins.The expression of four types of NR2 subunits and cleaved caspass3 are detected by western blotting.Results1.Compared with Control,the amplitude are increased to 139.6±5.5%after perfusion of SS,while decresed to 67.6±3.59%after perfusion of SDSS.When perfusing SS and SDSS togethter,the amplitude is almost the same with control(96.5±4.3%).SDSS shows little effect on GABAA current(98.2±2.9%of Control).2.The increased neuron death(apoptosis from 8±0.5%to 34.6±1.6%and relative LDH release is 1.3)induced by SS can be supressed by APV(apoptosis 14.4±0.3%and LDH 1.1),and SDSS(apoptosis 18.0±1.8%and LDH 1,1).The excitation toxicity of NMDA(apoptosis 41.6± 1.2%and LDH 1.4)can be increased by SS(apoptosis 53.1±1.1%and LDH 1.7).And the SDSS can protect cultured IC neurons from damage caused by SS and NMDA together(apoptosis from 53.1±1.1%to 17.7±0.7%and LDH to 1.3).3.Compared with the state,the model mice spend less time(from80.6±7.5s to 47.1±8.5s)and has less activity distance(775.0±68.5cm to 429.6±68.8cm)in the centure area of open field,and spend less time(from 33.1 ±6.0s to 10.8±3.8 s)and less entrance(from 6.0±0.9s to 2.3±0.6)in the open arm,while become more immobile(time from 100.5±13.3s tol38.7±14.5s)in water.Mice in SDSS group and Control group has little different activity in open filed,the open arm and water.SDSS effectively eliminate the anxity and depression induced by SS releated tinnitus.4.SDSS protect IC neurons from increased cleaved caspass3 and four types of NR2 subunits which induced by SS.Conclusion1.Incressing the hyperactivity of NMDA receptor while inhibiting the GABAA receptor,breaking the balance of the excitatory and inhibitory in IC,and indeusing the hyperactivity of IC,that's why SS induce tinnitus.2.SDSS can inhibit the activity of NMDAR partly,while has no influence on the GABAA receptor.3.SDSS can inhibit SS-induced tinnitus and hering loss by decresing hyperactivity of NMDA receptor,remitting imbalance of the excitatory and inhibitory in inferior collicious,and protecting neurons from damage caused by SS.