Effects Of Jinxueyuan And Its Iconic Monomers On M3-Ca²⁺-AQP5 Pathway Of Salivary Gland In Sjogren Syndrome Model Mice

Purpose:To explore the traditional chinese medicine theory and material basis for the treatment of Sjogren's syndrome with Jinxueyuan.To investigate the effects of Jinxueyuan and its iconic monomers on the M3-Ca2+-AQP5 pathway at the protein and gene levels,this will help us understand the difference of AQP5 expression in different parts of submandibular gland cells and to further understand its action site.It also aims to study the effect of Jinxueyuan and its iconic monomers on calcium and potassium channels of submandibular gland cell membrane and to explore the mechanism of increasing extracellular calcium influx and promoting intracellular calcium release.Method:The theoretical study was divided into two parts,one was the research progress of Sjogren syndrome,the other was the analysis of the theory of body fluid and blood from the same source,the analysis of the composition of Jinxueyuan and clinical research foundation.Also,the experimental research was divided into four parts.The first part was to observe the effect of Jinxueyuan and its iconic monomers on the distribution of Acinar cell membrane and secretory tubules in rat submandibular gland cells of acute xerostomia.First,we made an animal model of acute xerostomia.Then we divided the rats into normal group,atropine model group,4-DAMP model group,atropine/4-DAMP+ cevimeline group,atropine/4-DAMP+Jinxueyuan group,atropine/4-DAMP+ salvianolic acid A group,atropine/4-DAMP+paeoniflorin group and atropine/4-DAMP+ophiopogonin D group.The distribution of AQP5 on the surface of the submandibular acinar cell membrane and in secretory tubules were observed in each group by immunohistochemistry.In this way,we can understand the relationship between the receptor type(M3 receptor)and the dynamic changes of cell surface and secretory canal AQP5 of Jinxueyuan and its iconic monomers.The second part studied the effects of Jin-Xueyuan and its iconic monomers on the expression of AQP5 protein on apical membrane(APM),basal membrane(BLM)and cell Intima(ICM)in rat submandibular gland acinar cells.We used the same way as the first part to make and group animal models.The effect of Jin-Xueyuan and its iconic monomers on the M3-AQP5 in the different membrane components of the acinar cell membrane was detected with Western blot method.The third part studied the effect of Jin-Xueyuan and its iconic monomers on the expression of AQP5mRNA in different membrane components of rats' submandibular gland acinar cells.The rats were grouped into normal group,4-DAMP group,Staurosporine group,TMB-8 group,atropine group,H-7 group,4-DAMP+ Jinxueyuan group,H-7/4-DAMP/TMB-8/Staurosporine+cevimeline/ophiopogonin D/paeoniflorin/salvianolic acid A group and H-7/TMB-8/staurosporine+ Jinxueyuan/cevimeline group.The expression of AQP5mRNA on Apical membrane(APM)?Basolateral membrane(BLM)and Intracellular membrane(ICM)in submandibular gland cells of different groups was detected by PCR.The influence of M3-Ca2+-AQP5 pathway on the translocation process of AQP5mRNA was determined.The fourth part:We researched the effect of the iconic monomers on the calcium,potassium channel of submandibular gland cells.Patch clamp technique was used to observe the effect of ophiopogonin D?paeoniflorin?salvianolic acid A on calcium and potassium channels,and the effects of three kinds of monomers on ion channels in M3-Ca2+-AQP5 pathway were analyzed.Results:1.Theoretical findingsThere is no record of Sjogren's syndrome in TCM.According to the etiology and pathogenesis,clinical manifestations,clinical experience of mentors and the research foundation of clinical curative effect,the theory of "body fluid and blood from the same source"can be used to guide the diagnosis and treatment of Sjogren's syndrome in TCM.Based on previous studies,it was suggested that the efficacy of Jinxueyuan and its iconic monomers based on the theory of " body fluid and blood from the same source" may be related to the M3-Ca2+-AQP5 pathway which affects the metabolism of salivary gland and body fluid.2.Experimental results2.1 From the morphological point of view,it was revealed that Jin-Xueyuan and its iconic monomers can significantly increase the AQP5 expression in acinar membrane and secretory tubule whose M receptor(Atropine model)and selective M3 receptor(4-DAMP model)were blocked.The effect was the strongest in paeoniflorin,followed by ophiopogonin D.Both of them were stronger than cevimeline,the agonist of M3 receptor and the effect of Jin-Xueyuan was weaker than that of cevimeline.2.2.Jin-Xueyuan,paeoniflorin,saponin D and cevimeline could significantly increase the expression was of AQP5 protein on the apical plasma membrane of submandibular gland cells with the blockade of M receptor and selective M3 receptor blockade.The expression of AQP5 protein was the highest in paeoniflorin group,followed by Ophiopogonin D group,cevimeline group and Jin-Xueyuan group.2.3 Under normal conditions,the expression of AQP5mRNA on the APM of glandular cells was the highest.The second was the BLM.The ICM was the least.Jin-Xueyuan,paeoniflorin,saponins D and cevimeline could significantly up-regulate the expression of AQP5mRNA on the Apical membrane(APM)in submandibular gland cells of the control group with inhibition of M3 receptor,non-selective M receptor,inhibition of protein kinase C and intracellular calcium release.The second is the BLM.The increase on the ICM is not obvious.2.4100 Mol/L saponins D and 100 mol/L paeoniflorin can increase the peak current of cells and promote the opening of calcium channel in the submandibular gland cells.Paeoniflorin also decreased the calcium dependent potassium current.Conclusion:Theoretically,the transformation,abnormal distribution,and the pathological factors between body fluid and blood are related to the pathogenesis and prognosis of primary Sjogren's syndrome.It can guide the differentiation and treatment of Sjogren's syndrome which mainly involves exocrine glands such as dry mouth and eye.In the experimental study,under normal condition or blocking M receptor and inhibiting intracellular calcium ion,the expression of AQP5 on APM of glandular cells was the highest.Jinxueyuan,paeoniflorin,saponins D and cevimeline could obviously up-regulate the expression of AQP5mRNA on APM,which suggested that the translocation process involved activation of M3 receptor,opening of calcium channel in submandibular gland cells and increase of intracellular Ca2+ concentration.Thus,the expression of AQP5 in the APM increased,resulting in saliva secretion of salivary gland increased.