The Study Of Thromboeiastography In Predicting The Prognosis Of Sudden Sensorineural Hearing Loss And The Mechanism Of Hearing Loss Induced By GSDME Mutation

ObjectiveSudden sensorineural hearing loss（SSNHL）is one of the common emergencies in otorhinolaryngology,which is characterized by sudden and unexplained hearing loss with poor prognosis.The etiology and pathogenesis of SSNHL are still unclear.SSNHL can be caused by vascular diseases,infectious diseases,tumors and so on.Because most of the inner ear vessels are terminal,and there is no collateral circulation,inner ear thrombosis or bleeding will lead to the reduction of cochlear blood flow,which will cause serious damage to the cochlea.Therefore,inner ear microcirculation disorders are closely related to the occurrence and development of SSNHL.Studies have shown that hematological parameters can reflect the prognosis of SSNHL.Thromboelastography（TEG）is an indicator of dynamic changes in blood coagulation,which can more sensitively reflect the coagulation state of the body,and may have guiding significance in predicting the prognosis of SSNHL.However,few studies have clarified the relationship between TEG and the prognosis of SSNHL.In order to better evaluate SSNHL,this study aims to explore the relationship between TEG and clinical features and prognosis of SSNHL.MethodsPatients with SSNHL hospitalized in our hospital from March 2020 to September 2020 were retrospectively analyzed.The inclusion and exclusion criteria were as follows:first onset and course of disease≤2 weeks;Age 18-70 years,no blood or immune system related diseases;Retrocochlear lesions,middle ear lesions,acoustic neuroma,genetic factors and other pathogenic factors were excluded.No contraindication of glucocorticoid use,non-pregnant women;He received no treatment prior to admission.The patient’s medical history,audiological examination,vestibular function test,auxiliary examination results and efficacy were collected.Audiological examination included pure tone audiometry,acoustic immittance,distortion product otoacoustic emission and auditory brainstem response.Vestibular function tests included caloric test,vestibular-evoked myogenic potential,head impulse test and vestibular autorotation test.Ancillary tests included routine blood tests,TEG,and MRI of the inner ear.TEG has three types of results:normal,hypercoagulable,and hypocoagulable.Hypercoagulation was manifested as decreased reaction time（R）,decreased kinetic time（K）,increased α-angle,increased G value and maximum amplitude（MA）.Hypocoagulation was manifested by increased R,increased K,decreased a angle,and decreased G and MA.For patients with hypercoagulable or hypocoagulable TEG,the TEG results of reexamination one week later were also collected,and the difference between the two results was expressed as ΔTEG.SPSS 20.0 software was used to analyze the relationship between TEG,TEG parameters（R value,K value,α Angle,G value and MA value）,ΔTEG,ΔTEG parameters（ΔR,ΔK,Δα,ΔG and ΔMA）and clinical characteristics and efficacy,A P value of less than 0.05 was considered statistically significant.ResultsA total of 133 patients were included in the study.TEG correlated with prognosis（P=0.049）,degree of hearing loss（P=0.030）and type of hearing loss（P=0.013）in patients with SSNHL.R（P=0.002）and α-angle value（P=0.010）correlated with prognosis.MA value（P=0.022）and G value（P=0.020）correlated with degree of hearing loss.R（P=0.033）correlated with inner ear MRI.ΔG（P=0.010）correlated with fibrinogen.ΔTEG（P=0.032）correlated with the prognosis of patients with abnormal TEG.Logistic regression analysis showed that TEG correlated with prognosis（P=0.013）,and ΔTEG correlated with the prognosis（P=0.013）and vertigo（P=0.016）.ConclusionsIn general,TEG is an independent risk factor for the prognosis of SSNHL.Abnormal R value and Δ angle often indicate poor prognosis,while abnormal MA value and G value predict more severe hearing loss.The prognosis of SSNHL is correlated with the change of TEG.Therefore,TEG can be used as a prognostic indicator for SSNHL.Objective:GSDME（DFNA5）gene is a known non-syndromic sensorineural hearing loss related gene,which is an autosomal dominant inheritance.GSDME is a postlingual deafness-related gene.Patients with deafness mutations in GSDME show high frequency hearing loss at about the age of 10,and then gradually involve the full frequency.In our previous study,we performed genetic diagnosis of 45 postlingual deafness families by whole exome sequencing,and found that 4 families were clearly caused by GSDME gene mutation.So far,14 mutations of GSDME gene have been reported,all of which are located near exon 8,and the mechanism of deafness is still unclear.GSDME is a member of the gasdermin family（GSDMA,GSDMB,GSDMC,GSDMD,GSDME and PJVK）.All proteins except PJVK,which contains a truncated C-terminal domain,contain an N-terminal cytotoxic domain（poreforming/pyroptosisinducing activity）and a C-terminal inhibitory domain（inhibiting N-terminal activity）.Gasdermin cleavage by activated caspases,granzyme B or other proteases liberates the GSDME-N domain,which mediates pyroptosis by forming pores in the plasma membrane.The GSDME deaf-inducing mutation site results in skip splicing of mRNA at exon 8,resulting in a truncated protein,which leads to a shortened C-terminal domain of GSDME.The inhibition of N-terminal activity became weaker.Therefore,we speculate that the mechanism of deafness caused by GSDME gene mutation may be related to pyroptosis.The aim of this study is to explore the molecular mechanism of deafness induced by GSDME gene mutation at the animal and cellular levels.Methods:1.To collect the basic information of the families with hearing impairment caused by GSDME mutations,and investigate the changes in mRNA levels caused by GSDME mutations.2.The pcDNA3.1 expression plasmids of GSDME gene wild type（GSDME-WT）and exon 8 deletion mutant（GSDME-Mut）were constructed.3.Animal experiments:（1）GSDME-WT,GSDME-Mut and pcDNA3.1 empty vectors encapsulated by in vivo-jetPEI were delivered into the cochlea of 10-day-old C57BL/6 mice by round window injection separately;（2）Four weeks after round window injection,Auditory brainstem responses（ABR）and Distortion product optoacoustic emission（optoacoustic emission）were used to detect the hearing level of mice.（3）The cochlear basilar membrane of mice was dissected and the number of hair cells was observed.4.Cell experiments:（1）GSDME-WT,GSDME-Mut and pcDNA3.1 empty vectors were transfected into 293T cells by Lipo-fectamine 3000,respectively.（2）Cell morphology was observed under a microscope,and the level of lactate dehydrogenase（LDH）was detected.（3）The expression levels of GSDME protein and inflammatory factor IL-1β were detected by western blot（WB）;（4）The level of cell apoptosis was detected by immunofluorescence and WB.（5）293T cells were treated with different concentrations of disulfiram（DIS）and dimethyl fumarate（DMF）for 2 hours,and then transfected with GSDME-Mut.The effect of drugs on alleviating the cytotoxicity of GSDME-Mut cells was analyzed by cell morphology and WB at 48 hours after transfection.Results:1.Among the 45 postlingual deafness families,4 families were caused by GSDME gene mutation,and 3 of them were caused by GSDME gene c991-15₉91-13del mutation and the other family was caused by c.1183+4 A>G mutation.2.In animal experiments,it was found that after injection of GSDME-Mut into the round window of the cochlea,the ABR threshold and DPOAE threshold of the mice were significantly increased,and almost all the basal and intermediate hair cells（inner hair cells and outer hair cells）of the cochlear basilar membrane were lost,and only a small number of inner hair cells survived at the apical turn.3.Through cell experiments,it was found that GSDME-Mut could cause pyroptosis,mainly accompanied by the increase of GSDME-N terminal protein level,LDH level and inflammatory factor IL-1β level.4.The GSDME mutant was not only localized to the cell membrane,but also distributed in the mitochondria by immunofluorescence,and the GSDME-mut group showed positive TUNEL staining.Further WB analysis showed that the expression of Cyt C and activated caspase3 was up-regulated in the GSDME-mut group.These results indicate that GSDME-Mut can lead to the occurrence of apoptosis.5.DIS and DMF can effectively reduce pyroptosis and alleviate the cytotoxicity of GSDME-Mut.Conclusions:The deafness mutation of GSDME gene leads to skip splicing of mRNA.The truncated protein loses the inhibitory effect of C terminus on N terminus,which has pore-forming effect,and triggers pyroptosis and apoptosis.The cytotoxicity of GSDME gene mutants could be effectively alleviated by using DIS or DMF.This study preliminarily reveals the molecular mechanism of deafness caused by GSDME gene mutation,and provides a new perspective for the treatment of deafness caused by GSDME.