Mechanism And Protection Of Programmed Necrosis Induced By Sodium Salicylate In Rat Spiral Ganglion

Objective: Sodium salicylate,the active component of the non-steroidal anti-inflammatory drug aspirin is commonly used to manage rheumatoid arthritis.The salicylate-induced tinnitus model has been used to identify the biological mechanism of tinnitus ever since its introduction as a behavioral model.Tinnitus induced by sodium salicylate was reported to be related to damage to cochlear hair cells and spiral ganglion cells.Studies have shown that the receptor-interacting protein kinase 3(RIPK3)-mediated necroptosis pathway was involved in noise-induced outer hair cells injuries and ouabain-induced spiral ganglion neurons injuries.In addition,In addition,previous research by our group found that caspase-3-mediated apoptosis pathways are involved in sodium salicylate-induced tinnitus in guinea pigs.However,with the intervention of caspase inhibitors,the decrease of apoptotic cells in spiral ganglia is not obvious,which there may be a death pathway independent of caspase-3.We postulated that the RIPK3-mediated necroptosis pathway may also be a key pathway in the development of tinnitus.Therefore,this study aimed to explore whether sodium salicylate-induced spiral ganglion neurons injuries induced necroptosis in rats by activating the receptor-interacting protein kinase 1(RIPK1)/RIPK3/mixed lineage kinase domain-like protein(MLKL)pathway,and to investigate the protective mechanism of Necrostatin-1(Nec-1),a specific inhibitor of necroptosis,on the injuries of spiral ganglion neurons induced by sodium salicylate.That further to provide a basis for exploring the mechanism of tinnitus.Methods: All rats underwent auditory brainstem response(ABR)and distortion product otoacoustic emissions(DPOAE)examinations before experiment and before sacrifice.Before the experiment,forty-eight adult male rats with ABR threshold <40d BSPL and DPOAE pass,and were randomly divided into four groups,including blank control group(saline was injected intraperitoneally),artificial perilymph group(left ear was injected with Nec-1artificial perilymph through round window membrane and saline was not intraperitoneally injected),sodium salicylate group(left ear was injected with artificial perilymph through round window membrane,and sodium salicylate was injected intraluminally with 350 mg/kg/d),and Nec-1 group(Intraperitoneal injection of sodium salicylate after injection of Nec-1 through round window membrane).After 7 days of continuous administration,ABR and DPOAE were tested to evaluate cochlear function,and the animals were sacrificed to remove the cochlea.Observe the morphological changes of spiral ganglion cells and calculate the density of spiral ganglion cells by HE staining.Quantitative real-time PCR(q RT-PCR)was used to detect the expression levels of RIPK1,RIPK3 and MLKL m RNA in cochlea of each group rats.Immunohistochemistry was used to detect the expression of RIPK1,RIPK3,and MLKL proteins in spiral ganglion neurons of each group of rats.Results: Before administration,the ABR threshold of both ears in each group was lower than 40 d BSPL.There was no significant difference in threshold between the control and APL groups after administration(P=0.149).Compared with the control and APL groups,the threshold of sodium salicylate group was significantly increased after administration,and the difference was statistically significant(P<0.0001;P<0.0001).Compared with the sodium salicylate group,the threshold of the Nec-1 group was significantly lower than that of the sodium salicylate group(P<0.0001),but the threshold was still increased compared with the control and APL groups(P<0.0001;P<0.0001).Before administration,DPOAE in all groups of rats passed.There was no significant difference in the frequency amplitudes between the control and APL groups(P>0.05).Comparing the sodium salicylate group with the control group,except for 5714 Hz,the amplitudes of other frequencies increased slightly,but the difference was not statistically significant(P>0.05).Compared with the APL group,the amplitude of the sodium salicylate group decreased slightly at4444 Hz,5714Hz,and 8000 Hz.The amplitudes of 2222 Hz,2963Hz,and10000 Hz all increased slightly,but the difference was not statistically significant(P>0.05).Compared with the sodium salicylate group and Nec-1 group,except for the amplitude of the 2222 Hz frequency increased(P>0.05),the amplitudes of other frequencies decreased,and the difference of is statistically significant in4444 Hz,5714Hz and 10000Hz(P=0.001;P<0.0001;P=0.01).Compared with the control group,the amplitude of Nec-1 group except for the amplitude of2222 Hz increased(P>0.05),the amplitude of other frequencies decreased.The differences were statistically significant in 4444 Hz,5714Hz,and 10000Hz(P=0.002;P=0.001;P=0.028).Compared with the APL group,the amplitude of Nec-1 group except 2963 Hz increased(P>0.05),the amplitude of other frequencies decreased.The differences were statistically significant in 4444 Hz,5714Hz,8000 Hz,and 10000Hz(P=0.001;P<0.0001;P=0.049;P=0.013).The HE showed that compared with the control and APL groups,the SGN in the sodium salicylate group had significant necrotic changes after administration(P<0.0001;P<0.0001).Compared with the sodium salicylate,the number of SGN in Nec-1 group necrotic cells decreased(P<0.0001),but compared with the control group and the APL group,the number of necrotic cells in Nec-1group still increased(P<0.0001;P<0.0001).There was no significant difference in the number of necrotic cells between the control and APL groups(P=0.324).The q RT-PCR showed that the expression of RIPK1 m RNA was significantly different between the groups(F=21.303,P<0.0001).It was expressed at the lowest level in the control and APL groups,and strongly increased in the sodium salicylate group(P<0.0001;P<0.0001).Nec-1 group can significantly reduce the increase of RIPK1 m RNA induced by sodium salicylate(P=0.001).However,compared with control and APL group,RIPK1 m RNA levels in Nec-1 group were still elevated(P=0.002;P=0.044).The expression trend of RIPK3 m RNA was consistent with the result of RIPK1 m RNA.It was expressed at the lowest level in the control and APL groups,and strongly increased in the sodium salicylate group(P=0.001;P=0.001).The Nec-1 group can significantly reduce the increased level of RIPK3 m RNA induced by sodium salicylate(P=0.001),but the level of RIPK3 m RNA still increased in the Nec-1 group compared with the control and APL groups(P=0.014;P=0.038).The expression trend of MLKL m RNA was consistent with that of RIPK3 m RNA.It was expressed at the lowest level in the control and APL groups,and strongly increased in the sodium salicylate group(P <0.0001;P <0.0001).The Nec-1 group can significantly reduce the increased level of MLKL m RNA induced by sodium salicylate(P=0.001),but the MLKL m RNA level is still increased in the Nec-1 group compared with the control and APL groups(P=0.009;P=0.018).Immunohistochemistry showed that RIPK1,RIPK3,and MLKL were mainly expressed in the cytoplasm,cell membrane,and some nuclei.In the control and APL groups,there was almost no expression in the SGN region.Sodium salicylate increased the expression of RIPK1,RIPK3,and MLKL in SGN.After Nec-1 treatment,the expression of RIPK1,RIPK3 and MLKL proteins was lower,but the expression of Nec-1 group was still higher than that of control and APL groups.The protein expression pattern is consistent with the m RNA expression pattern.Conclusion: RIPK1/RIPK3/MLKL-mediated programmed necrosis is involved in salicylate-induced SGN injury.Inhibition of necroptosis to protect SGN from death may be a means of treating tinnitus and SGN disease-related hearing loss.