| BackgroundMeniere's Disease(MD)is a kind disorder involving inner ear,characterized by recurrent vertigo,fluctuant hearing loss,tinnitus,as well as feelings of fullness or pressure in inner ear,with unknown etiology and increased prevalence.It is acknowledged that endolymphatic hydrop(ELH)is the pathologic basis of MD as a result of increased secretion and/or decreased reabsorption of endolymph,both closely associated with anion and liquid exchange.The solute carrier family(SLC),the second largest membrane protein family in humans,is responsible for the absorption and transportation of membranous amino acids,sugars,inorganic ions,etc.,and is critic for maintenance of physical conditions.Briefly,SLC4 and SLC26 families mainly transport inorganic ions,e.g.bicarbonate ion,while the SLC22 family focuses on transporting organic cations.The Solute carrier family 26 member 4(SLC26A4)protein of the SLC26 family is mainly expressed in the cochlea,vestibule and ELS(Endolymphatic sac,ELS),leading to increased endolymph fluid and enlarged membrane labyrinth structure due to overloaded ion and fluid transportation in the inner ear.We previously analyzed the transcriptome profile of human peripheral blood mononuclear cells via RNAsequencing and found that solute carrier family 4 member 1(SLC4A1)and solute carrier family 4 member 10(SLC4A10)were significantly down-regulated compared with the normal control.Evidences from other studies illustrate that mutations in SLC4A1 not only affect the exchange of HCO3-and Cl-in kidneys,but also cause hearing loss.SLC4A10 shares similar structure with SLC4A1 and is expressed in the inner ear being involved in the development of murine hearing.SLC4A10 is also recommended as a screening gene for hearing loss in late murine embryos.Therefore,the above three HCO3exchange proteins are closely related to inner ear function,but their relationship with ELH is still uncertain..ObjectiveThis study aimed to explore the relationship between SLCs(SLC4A1,SLC4A10,and SLC26A4)and ELH and possible molecular mechanisms via detecting the expression changes of SLCs caused by ELH modeling,assessing the pathologic developments after SLCs down-regulation,and transcriptome analysis of ELH.MethodsThis research is divided into 3 parts.1.SLCs expression in inner ear and endolymphatic sac(ELS)of endolymphatic hydrop-modeled mice.1)Mice were randomly divided into control group and Arginine vasopressin(AVP)group.The control group accepted no treatment and AVP group was intraperitoneally injected with AVP for 8 days.2)Temporal tissues were dissected from mice of two groups,fixed,dehydrated,embedded,and sectioned for hematoxylin-eosin(HE)staining.Morphological analysis was performed to detect changes in cochlea and endolymphatic sac(ELS)between the two groups.3)Total RNA was extracted from cochlea and ELS of mice in two groups.Real-time quantitative fluorescent PCR(qRT-PCR)was performed to observe the changes in the mRNA expression levels of SLC4A1,SLC4A10,SLC26A4 in the cochlea and ELS of treated mice.4)Temporal tissues were dissected from mice of two groups,fixed,dehydrated,embedded,and sectioned.Immunofluorescence staining was performed to probe location changes of SLC4A1,SLC4A10,and SLC26A4 in inner ear and ELSbetween the two groups.5)Protein was extracted from cochlea and ELS of mice in two groups.Western blot(WB)was performed to observe the changes in the protein expression levels of SLC4A1,SLC4A10,SLC26A4 in the cochlea and ELS of the two groups of mice.2.Pathological progress induced by SLCs down-regulation1)Mice were randomly divided into control group,AVP group,AVP+DIDS group(DIDS as the inhibitor of SLC4A1,SLC4A10,and SLC26A4).The control group accepted no treatment,AVP group was intraperitoneally injected with AVP for 8 days,and AVP+DIDS group was intraperitoneally injected with AVP and DIDS for 8 days.2)Total RNA was extracted from cochlea and ELS of mice in three groups.Real-time quantitative fluorescent PCR(qRT-PCR)was performed to observe the changes in the mRNA expression levels of SLC4A1,SLC4A10,SLC26A4 in the cochlea and ELS of treated mice.3)Temporal tissues were dissected from mice of three groups,fixed,dehydrated,embedded,and sectioned.Immunofluorescence staining was performed to probe location changes of SLC4A1,SLC4A10,and SLC26A4 in inner earand endolymphatic sac(ELS)between groups.4)Protein was extracted from cochlea and ELS of mice in three groups.Western blot(WB)was performed to observe the changes in the protein expression levels of SLC4A1,SLC4A10,SLC26A4 in the cochlea and ELS of mice.5)Temporal tissues were dissected from mice of three groups,fixed,dehydrated,embedded,and sectioned for hematoxylin-eosin(HE)staining.Morphological analysis was performed to detect changes in cochlea and endolymphatic sac(ELS)between groups.3.Transcriptome analysis of experimental endolymphatic hydrop1)The mice were randomly divided into control group,AVP group,and AVP+DIDS group.According to different groups,they were given no treatment,AVP intraperitoneal injection,and AVP+DIDS intraperitoneal injection for 8 days each.2)Toke some mice from each of the three groups,take the cochlea,perform RNA-seq,analyze the mouse cochlear transcriptome profile,and screen the differentially expressed genes between the control group and AVP group,AVP group and AVP+DIDS group;use gene ontology(Gene ontology,GO)functional analysis method and Kyoto encyclopedia of genes and genomes(KEGG)pathway method to analyze the functional characteristics and signal transduction pathways of differentially expressed genes.3)Analyze the SLCs in the differentially expressed genes between the control group and AVP group,AVP group and AVP+DIDS group,and analyze their expression location and function.Results1.SLCs expression in endolymphatic hydrop-modeled inner ear1)ELH manifestations in the cochlea and ELS.Compared with the control group,the inner vestibular membrane of the cochlea in AVP group was significantly longer,the cochlea and stria vascularis were swollen,the hair cells were vacuolated,and the cytoplasm of marginal cells protruded significantly.The vestibular membrane length(IR-L)and the cross-sectional area of the cochlear duct(IR-S)in the AVP group were significantly larger than those in the control group;the middle and distal parts of the ELS in the AVP group were significantly enlarged,the lumen of the ELS was also significantly expanded,and the epithelial cells were thin with collapsed lateral intercellular space(LIS).After calculating the ELS cross-sectional area(ELS-S)and statistical analysis,it was found that the AVP group was significantly larger than the control group.2)SLCs mRNA expression.The results showed that compared with the control group,the mRNA expression level of SLCs,SLC4A1,SLC4A10,and SLC26A4,in the cochlea and ELS of the AVP group was significantly decreased.3)The localization and expression changes of SLC4A1 protein.The results of IF staining showed that SLC4A1 was expressed in ELS,stria vascularis,spiral organs,and membranous semicircular canals of inner ear in the control group of mice.The expression of SLC4A1 in ELS and inner ear of AVP group mice was significantly decreased.WB results showed that the ACTIN bands in the ELS of the control group and the AVP group mice were basically the same,while the SLC4A1 band became lighter;the gray value of the band was calculated and statistical analysis found that the SLC4A1 in the ELS of the AVP group mice was obviously decreased;the expression of SLC4A1 in the cochlea of the AVP group mice also decreased,and the trend was similar to the change of SLC4A1 expression in the ELS of the AVP group.4)The localization and expression changes of SLC4A10 protein.Structurally,SLC4A10 is basically the same as SLC4A1.IF staining results show that SLC4A10 was also expressed in ELS and stria vascularis,spiral organs,and membranous semicircular canals of inner ear in the control group of mice.Similarly,the expression of SLC4A10 in ELS and inner ear of the AVP group was also decreased.WB results showed that the expression of ELS and SLC4A10 in the cochlea of mice in the AVP group was also decreased,and the trend was the same as that of SLC4A1.5)The localization and expression changes of SLC26A4 protein.The results of IF staining showed that SLC26A4 was expressed in the ELS and membranous semicircular canals of the control group of mice,with observed expression around but not in the central area of the stria vascularis and the spiral organ.In the ELS and inner ear of the AVP group mice,the expression of SLC26A4 also decreased.WB results showed that the expression of ELS and SLC26A4 in the cochlea of mice in the AVP group decreased,and the trend was the same as that of SLC4A1 and SLC4A10.2.SLCs down-regulation exacerbated the pathological progress of ELH1)DIDS decreased expression of SLCs mRNA.The results showed that compared with control group,the mRNA expression of SLCs in the cochlea and ELS of the AVP group decreased,and the expression of SLCs mRNA in the cochlea and ELS of the AVP+DIDS group further decreased.2)DIDS decreased the expression of SLC4A1 protein.The results of IF staining showed that compared with the control group,the expression of SLC4A1 in the ELS and inner ear of mice in the AVP group was significantly decreased.The collapse into the intercellular space was further aggravated than the AVP group.WB results showed that the ELS of mice in the control group,AVP group,and AVP+DIDS group had the same brightness as the ACTIN band in the cochlea,while the band of SLC4A1 gradually became lighter;the gray value of the band was calculated and statistically analyzed and found that,The expression of SLC4A1 protein in the AVP group decreased,and the expression of SLC4A1 in the cochlea of the AVP+DIDS group was further decreased than that in the AVP group.3)DIDS decreased the expression of SLC4A10 protein.The results of IF staining showed that the expression of SLC4A10 in ELS and inner ear of mice in the AVP group was significantly lower than that in the control group,and the expression of SLC4A10 in the AVP+DIDS group was further downregulated than that in the AVP group.WB results showed that the expression of ELS and SLC4A10 in the cochlea of mice in the AVP group and AVP+DIDS group also gradually decreased,and the trend was the same as that of SLC4A1.4)DIDS decreased the expression of SLC26A4 protein.The results of IF staining showed that the expression of SLC26A4 in ELS and inner ear of mice in the AVP group was significantly lower than that in the control group,and the expression of SLC26A4 in the AVP+DIDS group was further downregulated than that in the AVP group.WB results showed that the expression of ELS and SLC26A4 in the cochlea of mice in the AVP group and AVP+DIDS group also gradually decreased,and the trend was the same as that of SLC4A1 and SLC4A10.5)SLCs down-regulation aggravated the pathological progress of ELH.The results of HE staining showed that compared with the AVP group,the cochlear vestibular membrane of the AVP+DIDS group was longer,the edema was more obvious,and the middle and distal parts of the mouse ELS increased significantly.Statistical analysis found that the IR-L,IR-S,ELS-S of the AVP+DIDS group mice were significantly greater than those of the control group and AVP group.3.Transcriptome analysis of experimental endolymphatic hydrop1)Fragments per kilobase of exon per million fragments mapped(FPKM)metrics was used to calculate the expression level of transcripts.Among the AVP and control samples,32,753 genes were detected and 92 genes were found to be differentially expressed.DEGs were further analyzed using GO function enrichment,and results showed that the biological processes with higher expression levels included "cell process","cell composition","biological regulation" and "cell connection".These DEGs involved many signal transduction pathways,such as "longevity signal pathway","calcium pathway","estrogen signal pathway","PI3K-Akt signal pathway" and so on.Further analysis of SLCs in DEGs between the control group and the AVP group showed that,SLC4A1,SLC4A10,SLC26A4,SLC4A5,and SLC4A3 in the AVP group decreased significantly,which were mainly involved in the exchange of bicarbonate ions.Meanwhile,SLC22A4 and SLC38A3 are significantly increased,which were mainly responsible for the transport of cations.SLC25A18 and SLC2A9 were also increased.The former is mainly involved in the process of protein translation,and the latter is involved in glucose transport.2)The above methods were applied and detected 32670 genes in the AVP+DIDS and AVP samples with 25 DEGs screened.GO functional enrichment analysis showed that the biological processes with higher expression of differentially expressed genes included immune system processes,cell components,and cell connections.The signal transduction pathways involved in these DEGs included protein digestion and absorption pathways,C-type lectin receptor signaling pathways,and cell adhesion molecule pathways.Further analysis of SLCs in DEGs between the AVP group and the AVP+DIDS group was performed.Results showed a significant decrease in SLC4A1,SLC4A10,SLC26A4,a significant increase in the cation transport gene SLC22A15,and a significant increase in SLC25A45 and SLC2A6 in AVP+DIDS group.The former mainly participates in the protein translation process,and the latter involves glucose transport.SLC6A3,SLC16A3 and SLC15A3 were also significantly increased,all of which can participate in the DNA templated process.Conclusion1.After AVP caused the mouse membrane labyrinth hydrops,the expression levels of the anionic proteins SLC4A1,SLC4A10 and SLC26A4 of the solute carrier family decreased significantly in ELS and inner ear of mice.2.DIDS further decreased the expression of SLC4A1,SLC4A10 and SLC26A4 in the inner ear and ELS of AVP mice,and increased the degree of membrane labyrinth.3.Among the DEGs between the control group and AVP group,AVP group and AVP+DIDS group,the majority of down-regulated DEGs included SLC4A1,SLC4A10,and SLC26A4 genes,which also suggests that the bicarbonate ion transport family is important for membrane labyrinth hydrops. |
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