| Background:The ototoxicity of cochlear spiral ganglion neurons is the common molecular mechanism of many sensorineural deafness.Inhibition of GABA_Areceptor response is one of the pathways that sodium salicylate induces excitatory toxicity of SGNs.Studies have shown that dopamine D2-like receptors(DR2)weakens the function of GABA_Areceptors and promotes excitatory toxicity of neurons by interacting with GABA_Areceptors in neurons.The mechanism is still not clear,and the endocytosis of GABA_Areceptor is closely related to the phosphorylation level,and protein kinase A is the main protein kinase regulating the phosphorylation level of GABA_Areceptor.Therefore,it is proposed that DR2 can mediate the regulating effect of sodium salicylate on GABAA receptor through the PKA-dependent phosphorylation pathway.Objective:To observe the effect of DR2 and PKA activity on the mRNA and protein expression of GABA_Areceptor after the intervention of sodium salicylate on SGNs in SD neonatal rats,and to explore the mechanism of sodium salicylate induced ototoxicity injury in SGNs.Method:The expression of GABA_ARβ1 subunit(GABRβ1)and DR2(DRD2)in SGNs was detected by cell immunofluorescence technique.The mRNA and protein levels of GABRβ1 subunit in primary cultured SGNs were observed by fluorescence quantitative PCR and Western Blot techniques under physiological state and sodium salicylate condition.Result:(1)The expression of GABRβ1 and DRD2 in the SGNS cells process were detected by immunofluorescence assay.(2)Under the effect of5m M sodium salicylate,the mRNA expression of GABRβ1 subunit detected by qPCR was increased by 66.5%compared with the control group(P<0.05),while the mRNA expression of DRd2 was increased by 55.5%(P<0.05).Western Blot results showed that the expression of GABRβ1 protein on the cell membrane surface of SGNs was significantly decreased after treatment with DR2 agonists(Quinpirole),sodium salicylate,sodium salicylate and DR2 agonists(P<0.05).The expression ofβ1 subunit protein in the Quinpirole group was 16.34%lower than that in the control group,the expression of GABRβ1 subunit protein in the sodium salicylate group was 19.98%lower than that in the control group,and the expression ofβ1 subunit protein in the SS+Quinpirole group was 22.72%lower than that in the control group.When the DR2 agonist Quinpirole group was added with the DR2 inhibitor Eticlopride,or the combination of Eticlopride and the protein kinase A inhibitor H-89,the expression of GABRβ1 subunit cell membrane protein was not statistically significant compared with the control group.The results showed that Eticlopride and H-89 could decrease the inhibitory effect of sodium salicylate and Quinpirole on GABβ1 subunit membrane protein,that is,after antagonizing the effects of dopamine D2receptor and protein kinase A,they could block the regulatory effect of Quinpirole on GABA_AR.Inhibit the internalization of GABA_Areceptor mediated by SS;The total protein expression of GABRβ1 did not change significantly.Conclusion:After sodium salicylate activates DR2,PKA is activated,GABA_AR enters the cell via an activated protein mediated endocytosis pathway,decreases GABA_AR cell membrane expression,weakening the inhibitory response mediated by GABA_AR,and finally leading to the ototoxicity of SGNs. |
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