Rutaecarpine Prevents The Disfunction Of Gap Junction Inter-cellular Communication Induced By LPC In Endothelial Cell

Objective: Gap junction （GJ） is a hydrophilic channel constituted of theconnexin proteins（Cxs） on the adjacent cell membranes. Gap junction intercellularcommunication（GJIC） mediate the transfer of matter and energy between adjacentcells. Numerous researchs show that dysfunction of intercellular communicationcaused by changes in the types、numbers and spatial distributions of the vesselwall Cxs is an important mechanism of AS. Rutaecarpine（Rut） is the effectiveingredient of traditional Chinese medicine Evodia which has lots of cardiovasculareffects. Vascular endothelial injury is the initiating part of artherosclerosis （AS）,Protection of the vascular endothelial is an important strategy for anti-AS vasculardisease. Early experiments of our lab find the Rut can inhibit apoptosis ofendothelial cells induced by LPC （the main effective ingredient of Ox-LDL）,itsmechanism is relevant with the activation of the capsaicin receptor （TRPV1）,promoting release and expression of calcitonin gene-related peptide （CGRP）.WhenAS occurs, inflammation and oxidative stress can change the levels of Cxs.As CGRPis an important inflammation regulatory factor, In this study, further study on theimpact of RUT to.the Cx37and Cx40and endothelial GJIC function,determinewhether the TRPV1/CGRP way plays a role in affecting gap junction intercellularcommunication function.Methods: LPC-induced endothelial cell injury model,pre-adding differentconcentrations of Rut (10^-6,3x10^-6,10^-5M) for10min,then add the LPC （10mg/l）co-incubated for24h.detected the endothelial cell viability （MTT） and Lactatedehydrogenase （LDH） of cell culture medium,ROS （streaming） and the adhesion ratioof monocyte-endothelial-cell in order to evaluate endothelial cell injury. In order toinvestigate whether the protective effect of Rut on endothelial cells involving TRPV1ways, applying the TRPV1antagonist Capsazepine (10^-5M) analysis, Fluorescencequantitative PCR detect the mRNA levels of CGRP andCx37、Cx40.Western blotdetect the expression levels of Cx37、Cx40protein.Lucifer Yellow （fluorescent yellow） scratches load experiment measures the function of GJIC.Results: Endothelial cell viability was reduced significantly after incubated byLPC, The levels of LDH、ROS and the adhesion ratio of monocyte-endothelial-cellwere significantly increased, the mRNA and protein levels of Cx37、Cx40weredown-expressed, the distance of gap junction dye migration was reduced. Rut canincrease the cell viability, inhibit the increase levels of ROS which induced by LPCand the adhesion ratio of monocyte-endothelial-cell, while up-regulate the mRNAlevels of CGRP and the mRNA and protein levels of Cx37、Cx40, increase thedistance of gap junction dye migrationt, and all the effect can be blocked byCapsazepine.But the effect of Rut that improves mRNA expression of Cx37and40can’t be blocked by CGRP8-37.Exogenous CGRP can’t restore expression of the injuryCxs.Conclusion: Rut can reduce the endothelial cell injury and monocyte, It also canup-regulate the expression of CGRP and improve the GJIC by restoring expressionsof Cx37and40.Its mechanism is relevant to the activation of TRPV1.