Study On Natural Medicine Screening Against Sjogren's Syndrome Based On The Target Of Aquaporin-5 Genes And The Underline Mechanism

Sjogren's syndrome(SS)is an autoimmune disease,it has a higher prevalence in menopausal women.lt is mainly characterized by lymphocytes infiltration of exocrine glands resulting in reduced glandular secretion and thus lead to the patients'dry mouth,thin saliva and other clinical symptoms.In dental medicine,retention of complete denture in the mouth has an important relationship with the quality and quantity of saliva.Sjogren's syndrome patients show less and sticky saliva,have a dry mouth state,it is not only difficult for the denture retention,but also easy to stimulate the mucosa causing discomfort and pain.It seriously affects the quality of daily life of patients.Numerous studies show that there is an assoication between aquaporin 5(AQP5)abnormal expression and SS,and its decreased expression is an important factor to SS patients with salivary gland abnormal secretion of saliva.And now AQP5 has become a new targeted gene on SS diseases research.But the sensitivity of AQP5 antibody on the market is not high,that creates obstacles to the study.So preparing a high purity and high specific antibodies becomes the key step of the experiment.Because Sjogren's syndrome pathogenesis is not very clear,there is no effective treatment at this stage.We use AQP5 specific promoter screen kinds of Chinese medicine,that provides a new idea for drug development.In addition,SS animal models currently used are mostly immune induction,that fails to fully reflect the environmental factors and pathological reactions of SS.In particular,it's easy to ignore the effects of estrogen to the SS.Therefore,we combined estrogen deprivation with the immune induction method to establish the clinical manifestation of the Sjogren syndrome SS animal model.And it has important significance for the study of the treatment of SS.For the preparation of high sensitivity and specificity of the antibody,we used AQP5 extracellular domain prepared AQP5 antibody.We amplified AOP5(183-205)amino acid fragment,and connected with the plasmid pGEX-4T-1 which expressed the glutathione-S-transferase(GST),got the pGEX-4T-1-AQP5 recombinant.Then we transformed the digested recombinant into E.coli BL21,accessed GST-AQP5 fusion protein induced by IPTG,immunized rabbits as antigen after purification to prepare AQP5 polyclonal antibodies.Then we used indirect ELISA assay AQP5 antibody titer.Experimental results showed that we had successfully prepare AQP5 polyclonal antibodies,provided the necessary tools for the immunohistochemical staining and western blot.Chinese herbal is the treasure of traditional Chinese medicine,the natural ingredients has many potential applications.We used luciferase reporter gene system filters more than 200 kinds of Chinese herbs,Ginseng and American ginseng was found to activate the transcriptional activity of AQP5 promoter.To further study the role of Chinese herbal medicine for the treatment of Sjogren's syndrome.In this study,we used different concentrations of American ginseng,Ginseng,and the main active ginsenosides Rbl to stimulate AQP' promoter cell model and tested AQP5 promoter activity with luciferase reporter gene methods,looking drug concentration which can significantly activate the transcriptional activity of AQP5.The results showed that Rbl,Amercan ginseng,Ginseng activated AQP5 promoter activation in a dose-dependent manner,and the activation of Rbl was more obvious.After that,we used Rb1,Amercan ginseng,Ginseng stimulated human salivary gland cells(HSG),testing the effects of drugs on AQP5 protein expression by western blot,further confirmed the regulation of drugs on AQP5 expression activity.The results showed that Rbl significantly increased the expression level of AQP5 protein.Based on Panax drugs upregulation of AQP5 protein expression in cell level,we used SS animal model which set up by the immune induction combined with estrogen deprivation method to study Rbl,American ginseng,ginseng and other drug effect in vivo.First,emulsified mouse submandibular gland with complete freund's adjuvant was injected into homogenates ovariectomized mice,then pertussis combination vaccine booster,Sjogren's syndrome mouse model was successfully constructed.Thereafter,Rb1,ginseng,ginseng and estradiol intervene SS mouse model to study the mechanism of drug action in mice.Eight weeks after administration,the mice was observed and recorded changes in body weight,the amount of saliva,water intake.The results showed that,compared with the normal group,model mice showed significantly reduced saliva secretion,increased water intake,inflammatory submandibular gland enlargement,clinical symptoms of dry mouth are more significant.HE staining showed submandibular glands of mice in the model group has a large number of lymphocytes,form an island structure.Immunohistochemical staining showed that the expression of SS model AQP5 positioned lower than the normal mice mice.That proved reduced AQP5 expression in submandibular gland is the key forreduced secretion of saliva of SS mice model.Compared with the model group,Rb1,American ginseng,Ginseng intervened SS model mice saliva secretion increased,reduced water consumption,decreased lymphocytes in submandibular gland and inflammatory infiltration was ameliorated,and Rbl correct dry mouth symptoms was better than the other drug groups.Especially submandibular gland AQP5 expression levelsof Rbl,American ginseng,Ginseng group had different degrees of recovery,wherein Rbl on expression of AQP5 protein promoting effect was more obvious and closer to the normal group.These results show that American ginseng,Ginseng,especially Rbl improve the SS mice submandibular gland AQP5 expression levels and alleviate the symptoms of dry mouth in SS mice.Research shows that American ginseng and Ginseng have similar effects with steroid hormones.Steroid hormones play their biological effects by specific binding to intracellular receptor.In this study,immunohistochemical staining used to detect ER?,ER?,AR,GR in mouse submandibular glands of different groups.We strive to find a receptor pathway by which Rbl and other drugs regulated AQP5 expression.Experimental results show that Compared with the normal mice,the expression of ER?,ER ?reduced in the model mice submandibular gland,and AR,GR had no obvious change.And compared with the model group,Rb1 drug group has high expression of ER?,ER? in submandibular glands of mice,but the regulation of AR,GR is not obvious.In this study,though drug screening,we found that Rb1,American ginseng and Ginseng components activated AQP5 promoter reporter gene activity,enhanced the expression of AQP5 in HSG cells.In animal models,we further confirmed the regulation of Rb1,American ginseng and Ginseng components on the submandibular.It was found that the three drugs,especially Rb1 up-regulated the expresssion of AQP5 in mouse submandibular,up-regulated the expression of ER?,ER?,promoted the secretion of saliva glands,decreased water intake,relieved the symptoms of glandular inflammation.Our study found Rbl may upregulate submandibular gland AQP5 expression by ER?,ER? pathway,relieve and treat dry mouth symptoms of SS mice.Our study provides a new idea for the SS in the drug research and development,and shows the broad application prospect.