Effects Of Estrogen On Genioglossus Activity And Its Potential Mechanism

AimsObstructive sleep apnea hyponea syndrome (OSAHS) is a breathing disordered phenomenon characterized by the recurrent interruption of breathing during sleep. Patients always present excessive sleepiness, headaches, leading to cardiovascular, cerebrovascular diseases which will consequently result in death. The pathogenesis of OSAHS might attribute to anatomical narrow and dilator muscle dysfunction in the upper airway. Epidemiological investigations showed that the incidence of male OSAHS was twice to three times higher than that of the female. However, some clinical research indicated that the incidence of female OSAHS increased significantly after their menopause, but it could be reduced by using estrogen replacement therapy; meanwhile, the activity of genioglossus muscle was remarkably enhanced in these cases. These results suggested that the estrogen might play a very important role in the prevention of the occurrence of OSAHS, while the detailed mechanism is still unclear. Therefore, the aim of our study is to investigate the effects of estrogen on biological functions of genioglossus muscle in vivo and in vitro and to explore the molecular mechanisms involved.Methods(1) The methods of ovariectomy and estrogen replacement were applied to establish experimental animal models at different levels of estrogen; then, the effects of estrogen on the function of genioglossus were detected by using electromyograph. The expression of estrogen receptorαandβin genioglossus muscles were detected by using immunohistochemistry.(2) The primary culture of rats'genioglossus myoblasts was established by using mechanical dissociation combined with collagenase digestion. Cells were purified by using the different speed-attaching method.The myoblasts were identified by immunofluorescence staining.(3) The distribution and location of estrogen receptorαandβin genioglossus myoblast were detected by immunofluorescence staining.(4) The effects of estrogen on intracellular Ca2+ of genioglossus myoblast were observed using the laser confocal microscope system(5) The real time PCR technology was employed to investigate the effects of estrogen on the mRNA expression level of different myosin heavy chain isoforms of genioglossus myoblast under different concentrations of 17β-estradiol.Results(1) Estrogen has significant effects on the function of rats'genioglossus. The activity of genioglossus muscle enhanced as a result of the increase of serum estrogen levels. (2) The results of immunohistochemical staining showed that both ER-αand ER-βwere expressed in the rats'genioglossus tissue with weakly positive staining. The expressions of ER-αand ER-βdidn't show much difference among different experimental groups.(3) The immunofluorescence staning indicate the positive expression ofα-sarcomeric actin and skeletal muscle myosins in genioglossus myoblasts. The percentage of positive staning was more than 95%, implying the sufficient purity of the primary culture cells for subsequent studies.(4) The results of immunofluorescence showed that both ER-α, ER-βwere expressed in nuclei and cytoplasm. There was no significant difference between ER-αand ER-βexpression in cytoplasm; however, the expression of ER-αis stronger than ER-βin the nucleus.(5) The results from the laser confocal microscope system showed that the effect of estrogen on the intracellular Ca2+ of genioglossus myoblasts was not significant in normal condition; however, in the acid condition, estrogen can reduce the concentration of cytoplasmic Ca2+ in the genioglossus myoblasts through reducing the accumulation of Ca2+ in cytoplasma.(6) The real time PCR showed that estrogen increased the expression of MHC I mRNA and decreased the expression of MHC IIb significantly. The effects of estrogen on MHC IIa and IIx were not significant.Conclusions(1) The animal models with different levels of estrogen were successfully established via ovariectomy and estrogen replacement. The electromyography confirmed that estrogen could affect the function of genioglossus. (2) The primary culture of rats'genioglossus myoblasts with high purity were successfully generated, which could serve as a cell model for biological finction studies in vitro.(3) The immunofluorescenc stydy has confirmed the existence and also distribution of estrogen receptorα,βin genioglossus myoblasts, which implyed that genioglossus might be the target tissue of estrogen.(4) Estrogen can affect the function of genioglossus by modulating the concentration of cytoplasmic Ca2+ in genioglossus myoblast cytoplasm.(5) Estrogen can affect the function of genioglossus by regulating mRNA expression level of different myosin heavy chain insoforms of genioglossus myoblast.