The Study Of Acetaldehyde Dehydrogenase And Its Related Metabolites In The Pathogenesis Of Meniere’s Disease

BACKGROUND: Meniere’s disease(MD)is an idiopathic inner ear disease.According to data from the National Institute on Deafness and Other Communication Disorders under the National Institutes of Health,the prevalence of Meniere’s disease in the United States is(50-200)/100,000,while there is a lack of clinical multicenter epidemiological surveys with large samples in China.Studies have shown that there are fewer male MD patients than female MD patients,with a prevalence ratio of about 1:1.89,and the prevalence of MD increases with age,while it is most common between the ages of 40 and 60.MD is not only a common disease,but also a disabling disease because it often manifests with balance dysfunction and falls,hearing impairment,anxiety and depression,etc.It affects the quality of life of patients especially their work and study,and brings a heavy burden of disease to patients’ families and society,so the treatment and research of MD has been receiving a lot of attention and concern.The clinical manifestations of Meniere’s disease include fluctuating sensorineural hearing loss with aural fullness,tinnitus and episodic vertigo.A typical episode of MD is characterized by vertigo lasting from several minutes to several hours,with positional vertigo during the episode.Numerous medical and surgical treatments have shown that there is no effective treatment to cure this disease.Endolymphatic hydrops(EH)is considered to be the predominant histopathological findings of MD.Previous studies have suggested that oxidative stress is closely related to endolymphatic hydrops,while cell damage and apoptosis may also contribute to the development of sensorineural hearing loss in late stage of MD,but the relevant molecular mechanisms underlying the development of MD are still unclear.Aldehyde dehydrogenase-2(ALDH2)is an isoenzyme of mitochondrial aldehyde dehydrogenase and a major contributor in alcohol elimination.As an important aldehyde oxidase in vivo,ALDH2 plays a key role in scavenging endogenous aldehydes,which can effectively reduce the toxic effects of harmful aldehydes on cells,accelerate the clearance of harmful aldehydes,and play a protective role in oxidative emergency damage.However,whether ALDH2 involved in the regulation of oxidative stress plays a role in MD and its exact mechanism underlying the development of EH is still unclear.In this study,we collected peripheral blood samples from a total of96 MD cases and 140 normal subjects,as well as clinical data from 96 patients with MD to establish a comprehensive clinical database and extract peripheral blood genomic DNA.Genome Polymorphism Array Analysis was used to investigate the relationship between ALDH2 gene polymorphisms and MD.Subsequently,we used a mouse model of endolymphatic hydrops established from ALDH2 knockout mouse model to identify a new candidate gene-ALDH2 for possible involvement in the pathogenesis of MD.The findings of this study may provide a new theoretical basis and candidate target for the diagnosis and treatment of MD.OBJECTIVE: a)Establishing a clinical data and genetic database for MD;b)Screening the polymorphic site of ADH1 B,ADH1C and ALDH2 in patients with MD to clarify the relationship between gene polymorphisms and clinical phenotype in patients with MD;c)Exploring the molecular mechanisms involved in the development of MD using mouse model of endolymphatic hydrops established from ALDH2 knockout mouse.METHOD: Peripheral blood from patients with MD and normal subjects and clinical data from patients with MD were collected for establishment of MD clinical data and genetic database.After peripheral blood genomic DNA was extracted,The genetic polymorphisms of ADH1 B,ADH1C and ALDH2 in MD patients were clarified using a genetic polymorphism site screening,and then the relationship between the polymorphisms of these genes and the clinical phenotype was statistically analyzed.An ALDH2 knockout mouse was established by CRISPR/Cas9 technology,and an endolymphatic hydrops mouse model was constructed by intratympanic injection of lipopolysaccharide(LPS)in ALDH2 knockout mouse.Immunohistochemistry,immunofluorescence,enzyme-linked immunosorbent assay,immunoblotting,real-time quantitative fluorescence PCR,transcriptomic and metabolomic analyses were used to explore the role of candidate gene--ALDH2 and its related metabolites involved in the progress of the development of EH.RESULTS: a)By analyzing the clinical sample data obtained from 96 patients with MD and the gene polymorphisms of ADH1 B,ADH1C and ALDH2 established using Mass ARRAY molecular weight array technology from the peripheral blood samples of 96 patients with MD and 140 normal subjects,who were recruited from the Department of Otolaryngology-Head and Neck Surgery,the Second Xiangya Hospital,Central South University,we found that the incidence of ALDH2 mutant phenotype(*1/*2+*2/*2)was elevated in MD patients comparing with that in normal subjects,and ALDH2 mutant phenotype(ALDH2*2/*2 and ALDH2*1/*2)might be an independent risk factor for MD.b)An ALDH2 knockout mouse model was successfully constructed by using CRISPR/Cas9 technology and a mouse model of endolymphatic hydrops was successfully established by intratympanically injecting LPS into the middle ear of the ALDH2 knockout mouse.This success mouse model of endolymphatic hydrops from ALDH2 knockout mouse may lay the foundation for further investigation of the functional role of ALDH2 involoved in the mechanism of MD.c)Using the metabolomics combined with transcriptomic analysis,we found that an increased oxidative stress in inner ear resulted in by the ALDH2 deficiency was involved in the progress of developing EH when a local inflammatory response was induced in the middle ear.CONCLUSION: In this study,we established clinical data and genetic database for the patients with MD recruited from our hospital,performed screening of gene polymorphisms for ADH1 B,ADH1C and ALDH2,and identified that ALDH2 was closely linked to genetic cause for the development of MD.By establishment of the mouse model of endolymphatic hydrops from ALDH2 knockout mouse,ALDH2 deficiency was confirmed to increase oxidative stress in inner ear resulting in EH when a local inflammatory response was induced in the middle ear.