| Objective: Large doses of sodium salicylate can cause hearing damage at different levels of the hearing system from the cochlea to the central nervous system,especially the cochlear spiral ganglia(SGN),causing tinnitus,hearing loss,and speech cognition.Recent studies have found that neuroinflammation is a new mechanism of sodium salicylate-induced hearing loss.Sodium salicylate can cause up-regulation of multiple inflammatory factors TNF-α,IL-1β,and IL-1 in the cochlea.Inflammasome may be responsible for the upregulation of inflammatory factors in SNG cells,although it is not clear whether they are actually involved in the development of tinnitus.In addition,NLRP3,as a sensor protein of ROS,may help assembly of inflammasome and subsequent cochlear inflammation.Drugs that act as NLRP3 blockers may represent innovative strategies for treating tinnitus.We investigated the activation of inflammasome and related pathways in the cochlea of rats,explored the key molecular mechanisms of sodium salicylate-induced SGN injury,and provided new directions for prevention and treatment of tinnitus that SS may cause.Methods: SD rats were used as research objects in this study.80 rats were randomly divided into 4 groups: control group,artificial external lymph fluid(APL)group,sodium salicylate(SS)group and NLRP3 antagonist(SS +MCC950)group.The control group was intraperitoneally injected with the same amount of normal saline;the SS group was intraperitoneally injected with sodium salicylate for 7 days;the SS + MCC950 group was administered with MCC950 through a round window before pretreatment with sodium salicylate,and the NLRP3 irreversible inhibitor(MCC950)block NLRP3 inflammasome formation directly and act at the NLRP3 signaling pathway upstream.In the APL group,artificial external lymph fluid was administered instead of the MCC950 through round window.We used ABR,DPOAE,and other electrophysiological methods to detect the animals to confirm the degree of hearing damage;morphological methods such as HE staining were used to observe the pathophysiology of cochlea SGN;real-time quantitative PCR was used to detect the expression levels of inflammasome and related inflammatory factors of the cochlea tissue of each group.Finally,the protein expression levels of sodium salicylate-induced cochlear inflammasome and related inflammatory factors and their localization in the cochlea were evaluated by immunohistochemistry.Results: The average hearing threshold of ABR in the rats before administration was 31.79 ± 4.13 d B SPL,and on the7 th day of modeling,the ABR threshold of the control group animals was 32.5(26.25,35)d B SPL,and the APL group was 32.5(30,35)d B SPL,the ABR threshold of the SS group was increased to 50(45,55)d B SPL,and the SS + MCC955 group was40(35,40)d B SPL.Through DPOAE detection,we found that there was no difference among the groups at low frequency of 2k Hz(P> 0.05);under 4k Hz,6k Hz,and 8k Hz frequency stimulation,there was no significant difference in DP amplitude between the control group and the APL group(P> 0.05),the amplitude of DP in SS group was significantly lower than that in control group and APL group(P < 0.001,P < 0.05,P < 0.001),suggesting that high-dose sodium salicylate administration caused rats cochlear injury.At 3k Hz,4k Hz,8k Hz,and 10 k Hz frequencies,the SS + MCC950 group with MCC950(NLRP3 inhibitor)pretreatment was significantly different from the SS group(P<0.001),and compared with the control and APL groups,there are little difference.The SGN damage caused by high-dose sodium salicylate was observed by HE staining.We observed that the cochlear structure of the control group and the APL group was normal;while in the SS group and the SS +MCC950 group cochlear SGN cells showed varying degrees of deformation,displacement,unclear boundaries,partial dissolution disappeared,significantly cell body shrink,edge set of chromatin in cell nucleus,nuclear condensation,nuclear lysis,with the characteristics of cell death.Quantitative analysis of the damage of SGN cells,we found that compared with the control group and the APL group,the SGN damage of the SS group was significantly changed(P<0.001,P <0.001);the SGN damage pretreated by MCC950 was more improved significantly than that of the SS group(P <0.01),and there is no significant difference compared with the control group and the APL group.The differences among the three groups of the SS + MCC950 group,the control group,and the APL group were not statistically significant(P> 0.05).The results of immunofluorescence quantitative PCR were calculated,we got that(1)the △ CT expression of each group of NLRP3 m RNA was: control group 7.438 ± 0.483,APL group 7.254 ± 0.848,SS group 5.41 ± 0.622,and SS + MCC950 group6.378 ± 0.479;(2)the △ CT expression of each group of Caspase-1 m RNA was:control group 6.811 ± 0.05,APL group 6.76 ± 0.401,SS group 4.035 ± 0.26,SS+ MCC950 group 5.602 ± 0.157;(3)the △ CT expression of IL-1β m RNA in each group was 8.547 ± 0.157 in control group,8.363 ± 0.375 in APL group,5.831 ± 0.059 in SS group,and 7.271 ± 0.296 in SS + MCC950 group.The immunohistochemical results showed that the three proteins were expressed in the SGN region of the control group and the APL group with very little positive expression;a large number of dark brown particles in the cytoplasm of a large number of SNG cells in the SS group showed strong positive expression;the expression of the target protein in the SNG region in the SS + MCC950 group was positively expressed with degree between the above two.In this study,rt-PCR and immunohistochemistry corroborated each other,demonstrating that in the SS-induced rat hearing impairment model,the transcriptional expression levels of NLRP3,IL-1β,and caspase-1 in the SNG region were significantly increased,causing neurotoxicity,leading to hearing impairment,and the increase of these factors may further upregulate the expression of other inflammatory factors.MCC950 pretreatment significantly weakened this process.Through electrophysiological examination and morphological observation,we found that the NLRP3 inhibitor MCC950 can effectively offset the pathophysiology of SGN associated with tinnitus.Conclusion: Based on animal experiments,we found that the expression level of NLRP3 in SGN cells was very low when not activated.The m RNA transcription levels of NLRP3,caspase-1,and IL-1β were significantly amplified in SGN cells after sodium salicylate administration.In the SGN region,the NLRP3 inhibitor MCC950 can reduce the expression of NLRP3 and its downstream caspase-1,IL-1β,and effectively offset the pathophysiology of SGN associated with tinnitus.Our results reveal that sodium salicylate ototoxicity may involve triggering NLRP3 inflammasome activation,which is a novel inflammatory immune mechanism.Sodium salicylate can activate the NLRP3 receptor-mediated inflammasome in the cochlea.This During the process,there may be NLRP3 inflammatory body signal transduction pathways,which promote the large release of IL-1β through the NLRP3-ASC-caspase-1 pathway,and may indirectly promote the up-regulation of many other inflammatory factors,exacerbate the intra-cochlear inflammatory response,leading to important intra-cochlear lesions.The mechanism of SGN damage may also be an important cause of tinnitus caused by sodium salicylate.In addition,we demonstrated that NLRP3 specific inhibitor MCC950 can improve SS-induced hearing damage,and direct inhibition of NLRP3 is a new target for the treatment of SS-induced hearing damage.In conclusion,inflammation induced by activation of inflammasome plays an important role in the pathological process of sodium salicylate-induced hearing impairment,and may be involved in the complex pathogenesis of tinnitus.MCC950 is a NLRP3-specific inhibitor,which can improve the degree of hearing damage in rats administered with salicylate sodium.Direct inhibition of NLRP3 may be a suitable strategy for treating tinnitus.In addition,the NLRP3 inflammasome signaling pathway can cause pyroptosis,and in combination with the cell death situation observed by HE staining,we speculate that the pyroptosis caused by NLRP3 inflammasome activation may also be involved in the mechanism of SGN injury induced by sodium salicylate.Follow-up studies are needed to confirm. |
PDF Full Text Request