Effects Of TRPV1on Inflammation And The Role Of Evodiamine In Regulation Of TRPV1

Background: It is well-known that several cardiovascular diseases arecharacterized by the dysfunction of vascular endothelial cells (ECS) associated withchronic inflammation. An intervention for inhibiting inflammatory response will be apotential therapy for cardiovascular diseases. Several studies suggest that the transientreceptor potential vanilloid type1(TRPV1) channel plays a protective role ininflammation, but the mechanisms undelying TRPV1-mediated anti-inflammation arestill unclear. Evodiae has been used in treating cardiovascular disease for hundreds ofyears. Several studies indicate that Evodiamine extracted from Evodiae may be aTRPV1agonist. To explore the role of TRPV1in ECS-mediated inflammation andintervention of Evodiamine will provide a basis for future study on animalexperiments and clinical application.Objectives: To determine the molecular mechanisms undrelying TRPV1andEvodiamine-mediated anti-inflammation, we explored the effects of TRPV1on LPS-inducedproduction of cytokines, chemical chemokines and adhesion molecules in the culture ofcultivated human umbilical vein endothelial cell (HUVEC), and interaction betweenmonocytes and endothelial cells, we also determined the role of TRPV1inEvodiamine-mediated anti-inflammation.Methods:1.Experimental groups①control group (normal HUVECs)②vehicle group (5%DMSO)③LPS-induced group (1μg／ml）④CAP+LPS group (Capsincin10umol／L, LPS1μg／ml)⑤CAPZ+CAP+LPS group (Capsazepine10umol／L, Capsincin10umol／L, LPS 1μg／ml)⑥EVO+LPS group (Evodiamine10umol／L, LPS1μg／ml)⑦CAPZ+EVO+LPS group (Capsazepine10umol／L, Evodiamine10umol／L,LPS1μg／ml)2.Cell growth curve drawing by MTT to select the cell density used in theexperiment.3.Cell viability of HUVECs exposed to different concentrations of Capsaicin for24h and48h was determined by MTT assay．4.Effect of TRPV1on TNF-α、IL-6and MCP-1was detected by cell ELISA，sodid the evodiamine on TNF-α.5.The expression of ICAM-1and VCAM-1mRNA and membrane protein weredetected by real time-PCR and western blot，respectively．6.Effect of TRPV1on adhesion of THP-1cells to endothelial cells induced byLPS was measured by Calcein-AM assayResults:1.Cell growth curve drawing by MTT to select the cell density used in theexperiment.After4days cultivation,3×104/㎝2HUVEC got a smooth and stable growthcurve.2.Cell viability of HUVECs exposed to different concentrations of Capsaicin for24h and48h was determined by MTT assay．Compared with normal control group, concentrations of Capsaicin to1μM,3μMand10μM cell survival rate has no obvious change.3.Effect of TRPV1on TNF-α、IL-6and MCP-1was detected by cell ELISA.After LPS stimulation, the protein concentration of TNF-α, Il-6and MCP-1aresignificantly increased, activating TRPV1can significantly reduce theseinflammatory mediators, the effect would be restrained by blocking TRPV1.4.The expression of ICAM-1and VCAM-1mRNA and membrane protein weredetected by real time-PCR and western blot，respectively．After LPS stimulation,both ICAM-1and VCAM-1mRNA and membrane proteinexpression were significantly elevated, activating TRPV1can significantly reduceadhesion molecules gene and membrane protein expression level, this function isrestrained after blocking TRPV1.5.Effect of TRPV1on adhesion of THP-1cells to endothelial cells induced by LPS was measured by Calcein-AM assayAfter LPS stimulation, the adhesive ability of HUVEC and THP-1cells enhanceda lot, activating TRPV1can obviously reduce the adhesion between endothelial cellsand monocytes, this role has been restricted after blocking TRPV1.Conclusions:1. The release of cytokines, chemokines and adhesion molecules induced by LPS inHUVEC cell culture was inhibited by the activation of TRPV1, and it also attenuated theadhesion of monocytes and vascular endothelial cells. The results suggest TRPV1expressedin ECS plays a inhibitory role in inflammation.2. Evodiamine-induced inhibition of cytokine production induced by LPS in HUVECcell culture was attenuated partially by the TRPV1antagonist. The data suggest thatEvodiamine may inhibit inflammatory response partially via the activation of TRPV1.