| Xerostomia is a common disease in the mouth,mainly as dry mouth cuased by the reduce the secretion of saliva.Sjogren's syndrome as the represent in clinical,in addition to a variety of reasons are related to the disease concerned.The incidence of patients with Sjogren's syndrome,male to female ratio is 1:9.menopausal women has much higher risk.Sjogren's syndrome is an autoimmune disease,manifested as lymphocytic infiltration and progressive destruction of salivary gland,but clinically more menopausal patients with xerostomia the salivary has no significant inflammatory changes,but the mechanism of its saliva secretion reduction is unknown.In fact,the estrogen sharp decline in menopause is an important stress response,however,the role of stress in regulatory mechanisms in the salivary glands have not been reported.Hypothalamus-pituitary-adrenal axis(hypothalamic-pituitary-adrenal axis,HPA)is activated as the main feature in stress response.Studies have shown that estrogen reduction is one of the ways to promote feedback of HP A activation,whether HPA change is a key factor in xerostomia occurs,which urgent need to clarify.Xerostomia is associated with the water secretion barrier in salivary gland.The water transport inside and outside the cell is mediated by the water channel protein aquaporins(aquaporins,AQPs),and the secretion of water in saliva was mainly mediated by AQP5 in salivary glands.The paper used AQP5 as a key molecular target for salivary gland function.Using a lot of dexamethasone simulate the physiological changes of the body during the stress reaction in vivo to study AQP5 expression in rat submandibular gland tissue and steroid hormone receptor changes on stress response.The paper revealed the effect and regulation mechanism of stress response on the secretion of salivary gland,established the association between steroid hormone receptor signaling and the expression of AQP5 gene.This study also.screened the Chinese herbal medicine with AQP5 transcription activation,and was expected to treat Sjogren's syndrome.Firstly,we studied AQP5 expression and steroid hormone expression changes in animal models.The group adopted the way of high-dose intramuscular Dexamethasone in rats for 18 weeks to construct the rat stress model.By detecting the amount of physical signs in each group of rats,we found that the water intake of Dexamethasone stress model rats group is significantly higher than the control group,and the static salivary flow rate of Dexamethasone stress model rats group was significantly lower than control group.Next,we study the changes of the organizational structure of the rat submandibular gland.After HE staining of submandibular glands of rats,we found that connective tissue proliferation existed significantly between stress model rats submandibular gland acinar and duct.After AQP5 submandibular gland tissue immunohistochemistry staining,We found by Immunohistochemical staining that AQP5 staining intensity was significantly decreased,on dexamethasone stress model group rat submandibular gland acinar cells,suggestted that stress response reduced the submandibular gland AQP5 expression' and induced salivary secretion disorders.We further examined the changes of various steroid receptors in submandibular glands.After immunohistochemical staining of various steroid hormone receptors in submandibular gland,we found that compared with saline control group,estrogen receptor a(estrogen receptor alpha,ERa)expression in dexamethasone stress model group was significantly lower,glucocorticoid receptor(glucocorticoid receptor,GR)expression was higher,and expression of androgen receptor(androgen receptor,AR)and estrogen receptor ?(estrogen receptor,ER?)had no significant differences.These results suggest that steroid hormone receptor expression is associated with AQP5.These results showed steroid receptors and AQP5 expression.may be related.On the basis,we studied the relationship between steroid hormone receptor and AQP5 expression in cellular level.Our research group recombined AQP5 promoter sequences from-852 to +111,and successfully constructed the cell model of luciferase reporter gene of AQP5 promoter.We then use the target to establish the cell model and study the relationship between a variety of with hormone receptor and AQP5 in cellular level.we found,ERa over-expression had significant activation of the transcriptional level of AQP5 promoter,followed by ER?,GR and AR had no significant effect.Then we use shRNA interference experiments to show that ERa silence had significant activation of the transcriptional level of AQP5 promoter,followed by ER?,GR and AR had no significant effect.Then,we used the human salivary gland(HSG)cells to study the effect in four kinds of steroid hormone receptor signaling on AQP5 protein expression levels,found ER alpha overexpression enhances the glycosylation of AQP5 protein expression level,and enhanced the modification of protein(phosphorylation).The results prove that ER alpha is the key signal of AQP5 regulation.And then we stucy the relevance between ERa signaling and GR signaling pathway.We over-expressed GR on the basis of Era over-expression and found AQP5 could reverse the activation of AQP5 expression by Era in a dose-dependent manner.It was suggested that GRcould inhibit the activation of AQP5 expression by ER?.By zoology and cytology results show that increasing the expression of GR and decreasing the expression of ERa are the important factors in reducing the AQP5 expression,they may be involved in the pathogenesis of Sjogren's syndrome.Then on the basis,we screened out ginseng and astragalus in the cellular level and found that they could promote AQP5 expression.Using the cell model which regard AQP5 promoter as a target,we screened for more than 200 kinds of traditional Chinese medicine and found that ginseng and astragalus could activate AQP5 promoter in transcriptional level,and increase AQP5 expression in translation level.On the basis,we over-expressed and silenced four kinds of steroid hormone receptors and found that ginseng and astragalus promoted AQP5 transcription and translation mainly by ERa pathway.Then we use dexamethasone stress model rats as a target to study the effective Chinese medicine of Sjogren's syndrome.We force feeded rats in epimedium,astragalus,ginseng separately when they were given dexamethasone.Firstly,after the detection of the physical signs,we found it has an obvious increase of water intake and static salivary flow rate in ginseng and astragalus group of rats,in which ginseng group was closer to the saline control group.But there was no significant difference between Epimedium group and dexamethasone stress model group.HE staining was found that proliferation level in the connective tissue of the submandibular gland acinar and duct was decreased obviously in ginseng and astragalus group.The immunohistochemical staining of AQP5 found AQP5 expression of rat submandibular gland acinar cells was significantly increased in ginseng group and astragalus group,indicating that ginseng and astragalus can increase the AQP5 expression of rat submandibular gland acinar cells.Our further study of four kinds of hormone receptor staining found ERa expression of rats submandibular gland was significantly increased in the ginseng and astragalus group of rats,approaching normal.But the level of AR,ERP,GR has no significant changes.These results proved that ginseng and astragalus could improve AQP5 expression of submandibular gland in dexamethasone stress model,and the function was regulated by ERa signaling pathways.This study demonstrates that:large and long-term use of dexamethasone can successfully cause an animal model where the constructed and secretion of salivary gland structure changed,decreasing submandibular gland AQP5 expression by upregulating the expression of GR and decreasing the expression of ER?.ER?expression is a key signaling in activated AQP5 expression,overexpression of GR in stress response activation may reverse the effects of ERa in AQP5 expression,and inhibiting salivary secretion and causing dry mouth.On the basis of traditional Chinese medicine screening showed that American ginseng and astragalus through direct regulation of the expression of ERa,corrected the inhibitory effect of dexamethasone/GR signal in AQP5 expression,restore submandibular glands secrete saliva,may become important in the future treatment of drug clinical xerostomia... |
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