| Objective:Xerostomia is a common symptom caused by disorders of salivation,among which Sjogren’s syndrome and estrogen deficiency are the two main causes.AQP5 is an aquaporin closely related to saliva secretion.Increasing its expression can correct the symptoms of dry mouth.Estrogen and its analogs can bind to hormone receptors in the body to up-regulate the expression of AQP5 and improve xerostomia.Therefore,this study first screened estrogen-like drug monomers that can up-regulate the transcription and expression of the AQP5 gene in vitro,and explored whether they act through estrogen receptors,and then observe their effects through in vivo experiments.Methods:1.Through dual-luciferase reporter gene experiment and western blot experiment to screen 6 kinds of drug monomers to promote the expression of AQP5 gene and protein.2.Through CCK-8 experiment,RT-PCR and western blotting experiment to find the optimal concentration of the screened drugs.3.After determining the screened drug and its optimal concentration,through gene overexpression and silencing and western blotting experiments,combined with the expression of AQP5,explore the relationship between its interaction with different estrogen receptors.4.Administer SS model mice and ovariectomized ICR female mice by gavage for 8 weeks,then detect dry mouth indicators and observe the degree of lesions of the mouse glands by HE and immunohistochemical staining.Results:1.Through the double reporter system and western blotting experiments,it is found that calycosin can significantly promote the transcriptional activity of the AQP5 gene promoter and then up-regulate its protein expression through the translation process.2.Through the CCK-8 experiment,it was found that the proliferation activity of HSG cells can be up-regulated at each concentration after 3 days of calycosin,and the effect is the most obvious in the 1×10-5M concentration group.At the same time, combined with RT-PCR and western blotting experiments,it is found that 1×10-5M can Significantly up-regulate the expression of AQP5 gene and protein.3.After screening the optimal concentration of calycosin,through overexpression and silencing of the two estrogen receptors,it was found that both estrogen receptors can bind to the two receptors to up-regulate the expression of AQP5,and its binding effect to ERαis even greater.4.Based on in vitro experiments,in vivo experiments were selected NOD mice and ovariectomized ICR mice to simulate dry mouth symptoms caused by SS and estrogen deficiency.The results showed that in the SS group,saliva secretion in each group was significantly lower than that of normal mice.However,the difference within the group was not obvious.The submandibular gland index of the calycosin group was significantly lower than that of the model group.At the same time,HE and immunohistochemical staining found that the gland tissue of the SS model group was severely damaged,and a large number of lymphocytes infiltrated and formed infiltration foci.At the same time,the expression of AQP5 was significantly reduced.Both the hydroxychloroquine sulfate and the calycosin group can improve the inflammatory response of the glands and up-regulate the expression of AQP5.The ICR group found that the saliva secretion of the ovariectomized model group was significantly reduced,while the estradiol group and the calycosin group significantly up-regulated its saliva secretion,but the submandibular gland index of each group was not significantly different.HE and immunohistochemical staining found that compared with the ovariectomized group,the integrity of the submandibular gland tissue in the estradiol and calycosin group was higher,and the expression of AQP5 was significantly increased.Conclusion:Calycosin can significantly up-regulate the transcriptional activity of AQP5 gene promoter,and may promote its protein expression by combining with ERa and ERβ,and has a stronger binding with ERa.At the same time,it can improve the dry mouth caused by estrogen deficiency by up-regulating AQP5 in ovariectomized mice,which has a similar protective effect as estrogen,and can improve the inflammatory response caused by Sjogren’s syndrome in NOD model mice.Up-regulating the expression of AQP5,but its influence on SS salivary secretion still needs further study. |
