Rutaecarpine Prevents The Dysfunction Of GJIC Induced By High Glucose In HUVECs

Objective: The vascular complications of diabetes, including diabetic cardiovascular disease, nephropathy, retinopathy, etc., are the leading cause of high mortality and disability by diabetes. Among them, vascular endothelial cells dysfunction was thought to play an important role in vascular complications of diabetes. Gap junctions(GJs) mediate direct cell-to-cell communication. This form of communication is referred to as gap junctional intercellular communication(GJIC),which allows the passage of metabolites, ions and other messenger molecules between cells in contact. GJs consist of connexins(Cxs), a large family of transmembrane proteins. The main expression of vascular endothelial cells is Cx37,40 and 43.which play an important role in maintaining the function of cells. Changes in the expression pattern of vascular Cxs, a condition called “GJ remodeling”, have been observed in animal model studies. during Microvascular endothelial cells in rat and Coronary artery endothelial cells in mice.and then inducing GJIC dysfunction and endothelial cells injury. In the present study, we observe changes the expression of Cx37, Cx40 and Cx43 and function of GJIC in HUVECs in different concentrations of high glucose medium. Rutaecarpine(Rut), one of the effective ingredient of Evodia, which is traditional Chinese medicine, activates the capsaicin receptor(transient receptor potential vanilloid,TRPV1) and shows cardiovascular protective effect. Our previous study found that Rut inhibits endothelial cells injury by Ox-LDL,which was involved in activation of TRPV1 and improve gap junction remodeling.The goal of this experiment is to observe recent discoveries regarding the role of Rut in protect of endothelial, and recent studies which support a role in regulation of connexins(Cxs)of endothelials and gap junctional intercellular communication(GJIC)among them.Methods: Human umbilical vein endothelial cells(HUVECs), were immersed in glucose(5.5, 11, 22, 33, 44 m M) and incubated 48 hours. By means of Western blot to detect the protein expression levels of Cxs of endothelial cells and the intercellularcommunication functions of gap junctions. 33 m M concentration of glucose, using it respectively incubation endothelial cells 48 hours to construct the cell damage model for simulation diabetes, pretreatment with different concentrations of Rut(0.1,0.3,1μM), detect the protein expression levels of Cxs by western blot and then observe the protein expression levels of Cxs of endothelial cells and the communication functions of gap junctions around them. use the TRPV1 antagonists Capsazepine(10-5mol/L) to determine the effect of Rut whether is involved in TRPV1 way,After experimental treatment, the protein expression levels of Cx37、Cx40、Cx43 were detected by means of Western blot, Use the Lucifer Yellow and Propidum Iodide scratch load experiment to detect the cells communication functions of gap junctions around them, cell viability was measured by Methyl thiazolyl tetrazolium(MTT) method, Nitric oxide(NO) were detected by Nitric Oxide assay kit from cell culture medium.and intracellular ROS level was measured by fluorescece probe(DCFH-DA) method to evaluate the extent of damage to endothelial cells.Results: The dose-dependent effect of glucose show that, when the glucose concentration reached 33 mmol/L, Cx37 expression was significantly reduced, but the expression of Cx40 and Cx43 remained unchanged, and expression of Cxs were unaffected by the mannitol(33 mmol / L) treatment(hypertonic control group).Scratch load experiment show that the endothelial cells GJIC functions were suppressed(can be delievery GJ that formed Cx37), but LY(can be delievery GJ that formed Cx40 and Cx43) inter-cellular communication without significant changes.In addition,The Rut with different doses inhibited remodeling formed gap junction protein that induced by hyperglycemia(33mmol / L); restored endothelial’s Cx37 expression and improved the function of GJIC, and relieve endothelial cells injury significantly;characterized by increase the levels of endothelial cells viability and promote the production of NO, inhibiting the release ROS; In advance dosing the Capsazepine, one of the capsaicin receptor blockers TRPV1 can block these effects of Rut.Conclusion: High glucose inhibited the expression of Cx37 in HUVECs, which caused dysfunction of GJIC; Rut prevents endothelial injury and gap junction dysfunction induced by high glucose, which is related to regulation of connexinexpression patterns via TRPV1 activation.