Study On Hypoglycemic And Hypotensive Effects And Mechanisms Of Total Glycosides Of Plantaginis Semen

Plantaginis Semen,the dried and ripe seeds of Plantago asiatica L.or Plantago Depressa Willd,has been commonly used for diuretic,antitussive and antipyretic properties with a long history as a traditional medicine,and applied to the treatment of hypertension,diabetes,oedema,etc.Total glycosides of Plantain Semen（TGPS）is an extract of Plantain Semen,which is enriched in iridoids,phenylethanoid and alkaloid glycosides or aglycones prepared by our group.In this study,islets damage models in vivo or in vitro and the insulin secretion experiment were used to investigate the effect and mechanism of TGPS and major components on diabetes.Meanwhile,the antihypertensive effect and mechanism of TGPS were studied on spontaneous hypertensive rats（SHR）model.This paper provides the basis for the clinical development and application of TGPS in the treatment of diabetes mellitus and hypertension.1.Pharmacodynamic evaluation and mechanism of TGPS on islets damageIslets damage models induced by streptozocin（STZ）in vivo and in vitro were employed to evaluate the effect of antidiabetics and islets damage protection,including diabetic mice induced by multiple low doses of STZ（MLDS）,isolated primary islets and RINm5F islet tumor cells.Results revealed that TGPS significantly reduced fasting blood glucose level and increased insulin level of MLDS-induced diabetic mice,increased islets area and the expression of insulin in islets,and decrease the expression ratio of glucagon and insulin.In the primary islet cell damage model,TGPS markedly improved islet cells degranulation and insulin secretion function after STZ intervention,and up-regulated the pancreatic developmental gene Pdx1 expression and lowered the apoptosis gene Caspas3and Parp expression.In RINm5F cell damage model,TGPS and acteoside significantly reversed STZ-induced cells viability loss and inhibited the activation of Caspase3 and Parp1.TGPS and acteoside obviously activated the transcriptional level of antioxidant response element（ARE）in RINm5F cells transfected ARE-luciferase reporter gene and markedly inhibited the release of ROS after STZ stimulation.Western blot,cell immunofluorescence and Q-PCR were used to measure the expression of Nrf2 in cell nucleus.Obtained results demonstrated that TGPS and acteoside promoted Nrf2 transfer to the nucleus,then advanced the gene and protein expression of antioxidant enzymes Homx1 and Nqo1 to playing the role of antioxidant protection against islet damage.Meanwhile,TGPS and acteoside observably activated the signaling pathway of PI3K/AKT and JNK to promote Nrf2 transfer to the nucleus.TGPS and acteoside unsuccessfully improved the morphological changes and secretion function of Nrf2^-/-islet cells after STZ stimulation,indicated that Nrf2 plays a key role against islet damage in the protection of TGPS and acteoside.In summary,TGPS played a key role in the treatment of diabetes by protecting islets damage.TGPS and acteoside protect islets damage islet damage through nrf2-mediated antioxidation.2.Pharmacodynamic evaluation and mechanism of TGPS on insulin secretionTGPS and geniposidic acid promoted insulin secretion under high glucose stimulation by insulin secretion experiment on islets.TGPS significantly increased the gene expression of Cacna1c,Creb1,Glp1r,Prkaca,Prkca and Vamp2 in insulin secretion pathway by Q-PCR.The results of proteomics demonstated TGPS and geniposidic acid were involved in three metabolic pathways,pancreatic secretion,fat digestion and absorption and glycerolipid metabolism.The enriched proteins in three pathways were mainly lipase and protease.In summary,TGPS and geniposidic acid perhaps promote insulin secretion through GLP1R-mediated calcium channel.3.Antihypertensive effect and mechanism of TGPS on SHRThe antihypertensive effect of TGPS was estimated on spontaneous hypertensive rats（SHR）.After administration for 2 weeks,the minimum dose of TGPS for antihypertension was 12 mg/kg and TGPS at 24mg/kg could maintain blood pressure stability for 12 days after withdrawal.TGPS treatment for 6 weeks effectively reduced systolic and diastolic blood pressure of SHR,alleviated left ventricular index and left ventricular wall thickness,and improved thoracic aortic and left ventricular interstitial and perivascular fibrosis.Enzyme linked immunosorbent assay（Elisa）and UPLC-MS technique were used to detect the serum level of several key components in RAS system,and it was found that TGPS obviously reduced Angiotensin II（Ang II）and increased the content of Angiotensin I（Ang I）and Angiotensin 1-7（Ang 1-7）.Meanwhile,TGPS significantly reduced Ang II expression in the thoracic aorta,heart and kidney.In summary,TGPS plays a role in lowering blood pressure by inhibiting angiotensin converting enzyme.