Neurotoxic Mechanism Of Sodium Salicylate:

Objective To observe the effect of sodium salicylate(SS)on mRNA and protein of GABAA receptors(GABAAR)after NMDA receptor(NMDAR)activity on spiral ganglion neurons(SGN)of rats.The receptor mechanism of sodium salicylate ototoxicity was further improved.Methods SGN was cultured in vitro in newborn SD rats,and the NR2B subunit expression of NMDAR was detected by immunofluorescence detection.Fluorescence quantitative PCR and Western blot were used to observe the effect of different concentrations of sodium salicylate(0.5 or 5 mM)on the levels of NMDAR mRNA,protein and phosphorylation induced by GABAAR.Results(1)Immunofluorescence showed that NR2B was expressed in both SGN somas and axons.(2)After 30min was treated with 0.5 mm,5 mm SS,10?M muscimol(GABAAR agonist)and 20 ?M bicuculline(GABAAR inhibitor),the results of fluorescence quantitative PCR showed that muscimol down-regulated NR2B mRNA expression by 10.96%(F=43.084,P=0.003<0.05),while sodium salicylate reversed the down-regulation effect of muscimol.Bicuculline increased the expression of NR2B mRNA by 21.40%(P=0.003<0.05),and its combined effect with sodium salicylate increased the expression of NR2B mRNA more significantly(P=0.000<0.05).All the above changes disappeared under the action of the inhibitor PP2 of tyrosine kinase Fyn(F=0.921,P=0.552>0.05).(3)After the same treatment as(2)above,Western blot results showed that muscimol down-regulated the expression of NR2B membrane protein by 7.39%(F=14.501,P=0.005<0.05),and the combined effect of sodium salicylate and muscimol made the down-regulation more obvious(P=0.000<0.05),in which the combined effect of 0.5/5mm sodium salicylate and muscimol reduced 30.82%and 55.33,respectively,compared with that of muscimol alone.Bicuculline upregulation of NR2B membrane protein expression by 15.73%(P=0.005<0.05),while the combined action of sodium salicylate and bicuculline can reverse this process(P=0.012<0.05).Among them,the combined.effect of 0.5/5 mM sodium salicylate and bicuculline reduced by 14.23%and 29.72%,?compared with that of bicuculline alone.There was no significant change in the total protein of the above treatment groups compared with the control group(F=1.111,P=0.401>0.05).The above changes disappeared under the action of PP2(F=0.436,P=0.884>0.05).(4)The phosphorylation levels of kinase and NMDA receptors were observed by Western blot after the same treatment as above.And it turned out that muscimol could down-regulate the expression of Fyn and p-Y 1472 protein,while bicuculline could up-regulate the expression of Fyn and p-Y1472 protein(p<0.05).The co-effects of 0.5mm,5mM SS and muscimol were more significantly down-regulated than those of muscimol alone(P<0.05).Fyn protein was reduced by 17.48%,48.65%,and p-Y 1472 protein by 25.82%and 29.95%,respectively.The combined effects of 0.5mm and 5mM SS and bicuculline could reverse the effects of bicuculline alone(P<0.05).Fyn protein was reduced by 20.91%,33.06%,and p-Y1472 protein by 25.43%and 54.60%,respectively.However,when SS and GABAAR were active,CK2 protein in each treatment group showed no significant change compared with that in the control group(F=0.742,P=0.655>0.05),and p-S1480 protein showed no change(F=1.230,P=0.337>0.05).Conclusions(1)The expression of NMDA receptor NR2B mRNA is down-regulated by the activation of GABAA receptor,and the down-regulation effect can be weakened by sodium salicylate;(2)The activation of GABAA receptor can promote the internalization of NMDA receptor,and sodium salicylate can facilitate the internalization;(3)Fyn-dependent phosphorylation at Y1472 mediates the internalization of NMDA receptors mediated by sodium salicylate and GABAA receptors.