| Benign paroxysmal positional vertigo(BPPV)is the most common peripheral vertigo and accounts for a high proportion of vertigo patients in outpatient and emergency department of neurology.Vast majority of patients with primary BPPV are perimenopausal women.Decreased estrogen level is an independent risk factor for BPPV,but the specific mechanism of otoconia loss remains unclear.In previous studies,we applied the rat ovariectomy(OVX)model and found that:Estradiol plays an important role in maintaining the normal structure of otoconia in the inner ear.The number of otoconia in the inner ear of OVX rats were decreased and the structure of that was loose under the electron microscope.Moreover,estradiol is involved in the expression of OC90 by binding to the heterodimer formed by ER?/?.Therefore,estradiol may regulate the expression of OC90 in the inner ear and participate in the occurrence of BPPV through the transcription-factor-like action of estrogen receptor.However,the formation and maintenance of otoconia are dynamic regulatory processes based on the comprehensive effect of many otolith regulatory proteins.Whether estradiol is also relevant to otolith regulatory proteins has not been studied.plasma membrane Ca2+-ATPase isoform2(PMCA2)and Pendrin are two major otolith regulatory proteins.PMCA2 is expressed at the tip of the inner ear's static cilia,which can pump calcium ions from hair cells into the otolith microenvironment to provide materials for otoconial formation while Pendrin is expressed at transitional cells of membranous labyrinth and plays a main role in regulating the PH of otolith microenvironment.In addition,mice with Atp2b2 gene(expression product is PMCA2)mutation or Slc26a4 gene(expression product is Pendrin)mutation showed morphological and structural abnormalities of otoconia and related balance disorders.In this study,we explored the effect of estradiol on the expression of PMCA2 and Pendrin by using the rat ovoectomized model and the basal membrane culture method,to further expand the pathogenesis and provide theoretical support for the prevention,diagnosis and treatment of BPPVPART1 The study of the expression levels of PMCA2 and Pendrinapplying the ovariectomy rat modelObjective:To evaluate whether the decreased serum level of estradiol(or progesterone)could impact the expression of PMCA2 and Pendrin by using the ovariectomy rat model.Methods:(1)twenty-five 3-month-old female sprague-dawley(SD)rats were randomly divided into five groups:E2 group,P group,E2+P group,Veh group and Sham group.Sham group was performed Sham operation and the remaining four groups were performed bilateral ovariectomy.One month After the operation,each group was injected subcutaneously into the neck of estradiol(E2 group),progesterone(P group),estradiol and progesterone(E2+P group),and sesame oil(Veh group and Sham group).Drug treatments that applied to the rats daily were last for one month.At the end of the second month after surgery,all rats were executed and bilateral inner ears were obtained;(2)Serum estradiol and progesterone levels were measured by enzyme-linked immunosorbent assay(ELISA).(3)Western Blot(WB)was used to detect the expression of total PMCA2 and Pendrin in the inner ear of rats in each group.(4)Quantitative real-time polymerase chain reaction(qRT-PCR)was used to detect the mRNA transcription levels of Atp2b2 gene.Results:(1)before bilateral ovariectomy and sham operation,there were no significant difference in serum estradiol and progesterone between the five groups(F values were 0.232,0.484 and P values were 0.917,0.747).Before injection(at the end of one month after surgery),serum estradiol and progesterone levels in each group after bilateral ovariectomy were significantly lower than those in Sham group,with statistically significant differences(all P values were less than 0.001).There were no significant difference in serum estradiol levels between the E2 group and Sham group,and between E2+P group and Sham group before the execution(at the end of two months after the operation),with P values of 0.993 and 0.725,respectively.Compared with Sham group,the other two groups showed significant differences(all P values were less than 0.001)before the execution.Compared with Sham group,progesterone levels of rats in group P and E2+P were significantly increased,with statistically significant differences(P=0.001,P<0.001)while Veh group and E2 group were significantly decreased with P values less than 0.001 before the execution.(2)There were statistically significant differences in the relative expression levels of total PMCA2 protein and mRNA in the inner ear of the rats in each group(F=5.480,P=0.004 and F=71.486,P<0.001).The relative expression levels of PMCA2 protein in E2 group,E2+P group,P group and Veh group were compared with Sham group,and the P values were 1.000?0.895?0.008 and 0.042,respectively.mRNA transcription levels of the four groups were compared with that of the Sham group,and the results were P=0.829,P=0.676,P<0.001 and P<0.001,respectively.(3)the relative expression levels of total Pendrin protein in the inner ear of E2 group,P group,E2+P group,Veh group and Sham group showed no statistical significance(F=0.911,P=0.477).Conclusions:The decrease of estradiol level may lead to a decline in the expression level of total PMCA2 mRNA and protain in the inner ear of rats,and this change can be reversed by supplementation of estradiol.The change of serum progesterone level has no effect on the expression level of PMCA2 protain in the inner ear of rats.Changes of estradiol and progesterone levels had no significant impact on Pendrin protein expression in rats' inner ears.PART2 Study on the mechanism of estradiol regulating PMCA2Objective:,To further explore the mechanism underlying estradiol regulates the expression of PMCA2 by tissue culture on the basis of the first part of animal experiments.Methods:(1)The 3-day-old SD rats from the same nest were sacrificed.The basilar membranes of bilateral inner ears were taken under the microscope and then divided into four groups for tissue culture,namely DMSO group,DMSO+E2 group,Fu1+E2 group and XCT+E2 group.After 24 hours of culture,DMSO,DMSO and estradiol,Fulvestrant(ER antagonist)and estradiol,XCT790(ERR ? reverse agonist)and estradiol were added for interference separately once a day.qRT-PCR was used to compare the transcription levels of PMCA2 mRNA in each group after 2 days of continuous culture.(2)The basilar membranes of bilateral inner ears obtained from the 3-day-old SD rats were used for tissue culture,the methods of obtaining specimens and tissue culture as described in the(1).Basilar membranes were divided into DMSO,DPN,XCT,PPT,Ful,PPT+XCT,DPN+XCT seven groups,and after culturing for 24 hours,drugs as each group name were added respectively,once per day.qRT-PCR was applied after 2 days' culture to compare PMCA2 mRNA transcription levels between groups.(3)Basilar membranes of the inner ears of 3-day-old SD rats were taken and ChIP assay was used to verify whether there was ERE sequence in the upstream of Atp2b2 gene that could specifically bind to estrogen receptor.Results:(1)The relative translation levels of PMCA2 mRNA in the basilar membranes of the inner ear from the four groups of SD rats were not completely equal(F=58.333,P<0.001).Compared with the DMSO group,the relative transcription levels of PMCA2 mRNA in the XCT+E2 group and the Ful+E2 group showed no statistical difference(P=0.472,P=0.307),while that in the DMSO+E2 group was significantly higher than that in the DMSO group(P<0.001).(2)The relative transcription levels of PMCA2 mRNA in the basilar membrane of the inner ear from the seven groups of SD rats were not completely equal(F=33.945,P<0.001).Compared with the DMSO group,the relative transcription levels of PMCA2 mRNA in the DPN group and the PPT group were significantly increased,(P<0.001,P<0.001).The relative transcription levels of PMCA2 mRNA in XCT group,Ful group,XCT+PPT group and XCT+DPN group were lower than that of DMSO group,with statistically significant differences(P values were 0.006,0.008,0.008 and 0.005,respectively).The transcription level of PMCA2 mRNA in DPN group was significantly higher than that in XCT+DPN group(P<0.001),while that in PPT group was significantly higher than that in XCT+PPT group(P<0.001).(3)Atp2b2 gene does have specific nucleic acid sequences that can bind to ER gene and ER gene,namely ERE sequence.These results indicated that the classical estrogen signaling pathway mediated by ER?/? is an action mode of E2 regulating the expression of PMCA2 in the inner ear of rats.Conclusions:ER?,ER? and ERR ? all play a role in process that E2 regulats the expression of PMCA2 in the inner ear of rats.There is ERE sequence on Atp2b2 gene that can bind E2-ERs complex.Combined with the conclusion of previous experiments,E2 participates in the regulation of otolith regulatory protein PMCA2 expression through the classical direct genotypic signaling pathway,that is,E2 binds ER?/? to allosterically form a dimer,and binds to the ERE sequence of Atp2b2 gene to regulate the expression of PMCA2 at the transcriptional level.ERR? plays a key role in the processes above.A significant decrease in serum E2 level may affect the expression of PMCA2 in the inner ear through the above-mentioned ER/ERR mediated regulatory pathway,resulting in abnormal calcium metabolism,leading to decreased structural stability of otoconia and easier to fall off,thus triggering BPPV,which may be one of the mechanisms of perimenopausal women's susceptibility to BPPV. |
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