Rutaecarpine Prevents Endothelial-monocyte Adhesion And Gap Junction Remodeling Induced By Ox-LDL Via TRPV₁

Objective: Gap junction (GJ) is common existence between adjacent cells, gapjunction is constituted of the connexin proteins (Cxs) on the adjacent cellmembranes that allows ions and small molecules through, and mediates the transferof matter and energy between adjacent cells, namely gap junction intercellularcommunication (GJIC). Monocytes and endothelial cells adhesion, migration is asignificantly pathophysiological process of vascular inflammation pathologicaldiseases such as atherosclerosis. A large number of researches suggest that gapjunction protein is involved in mediated the interaction betweenmonocytes/macrophages and endothelial cells. Rutaecarpine (Rut) is the effectiveingredient of traditional Chinese medicine Evodia rutaceae plants which activates thecapsaicin receptor (Transient receptor potential vanilloid,TRPV1) and shows a widerange of cardiovascular effects. The cycle monocytes are adhesion to the damageendothelial cells, the stimulating monocytes then migrates to vascular intima andeventually develops into the foam cells, which is a key link in the process ofatherosclerosis plaque progression, so reduce the cell adhesion, the cell migrationbetween monocytes and the endothelial cells is an important strategy to treatatherosclerosis vascular lesions diseases. Our lab early studies have found that Rutinhibits LPC (The main component of the Ox-LDL) to induce endothelial cell injury,and this protection action involved in the mechanism that raised the resistantAtherosclerosis (AS) protiens of Cx37and Cx40expression levels, also improvedendothelial GJIC. This experiment use Ox-LDL incubation endothelial cells andmonocytes to establish the damage models, this study intends to further research theinteraction between monocytes and endothelial cells in the process, the focusobservation of Rut on the process of monocytes-endothelial cells adhesion whichinvolved in the Cxs proteins expression levels, adjust the half channel and GJfunction to determine the mechanism whether are mediated by TRPV1way.Methods: Using Ox-LDL (100μg/ml) respectively incubation endothelial cells and monocytes24hours to construct the cell damage model for simulationarteriosclerosis, pre-adding different concentrations of Rut (10-6mol/L,3x10-7mol/L,10-7mol/L) to deal with these cells, and then use the TRPV1antagonists Capsazepine(10-5mol/L) to determine the effect of Rut whether is involved in TRPV1way. Bymeans of Western blot to detect the protein expression levels of Cx37, Cx40andCx43, the Lucifer Yellow and Propidum Iodide scratches load experiments to test thecells communication functions of gap junctions, detection the ATP levels ofmonocytes to reflect the half channel functions. Testting cells’ vitalities (determinedby MTT method) and Nitric oxide (NO), Lactic dehydrogenase (LDH) levels of cellcultures to evaluate the endothelial cells’ function, and the adhesion ratios ofmonocytes-endothelial cells to evaluate the interactions of endothelial cells andmonocytes.Results: The gap junction remodeling of endothelial cells and monocytes afterincubated by Ox-LDL are expressed in a specific that the original protein expressionlevels of Cx37and Cx40are declined significantly, the protein expression levels ofCx43are increased moderately, the monocytes source protein expression levels ofCx37express declined significantly, the protein expression levels of Cx43areincreased significantly, scratch load experiments showed that the endothelial cells’GJIC functions are impaired, the reduced ATP levels of monocytes are indicated thatthe half a channel functions of gap junctions are declined. Different concentrations ofRut can significantly improve the Ox-LDL induced gap junction proteins remodeling,restore the expressions of the original Cx37and Cx40, also the expressions of themonocytes source Cx37, suppress the expressions of the original and the monocytessource Cx43, significantly improve the endothelial GJIC function, and increased theATP levels of monocytes. In addition, the Rut significant inhibition of the Ox-LDLinduced endothelial injury, characterized by increase the levels of endothelial cellsviability and NO, inhibit the release levels of LDH, and significantly decrease theadhesion ratios of monocytes-endothelial cells which induced by Ox-LDL. Inadvance dosing the Capsazepine (Capsazepine is one of the capsaicin receptorblockers) can block these effects of the Rut.Conclusion: Rut inhibits the endothelial cells injury and the adhesion of monocytes-endothelial cells induced by Ox-LDL, the mechanism is relevant to theactivation of TRPV1(Capsaicin receptor) and then improve the gap junctionremodeling of endothelial cells and monocytes.