Rutaecarpine Prevents Ox-LDL-induced Upregulation Of Cx43 And Vascular Smooth Muscle Cell Phneotypical Transformation And Migration Through TRPV1/[Ca²⁺]_i/NF-?B Pathway

Objective: Atherosclerosis?AS?is a dyslipidemia disease that causes vascular wall remodeling.The phenotypic transformation,hyperplasia,migration to the intima and lipids phagocytosis of the vascular smooth muscle cells?VSMC?are important pathological pathologies for AS vascular wall thickening.The connexin 43?connexin43,Cx43?is the major Cxs that make up the gap between VSMCs.Recent research found that Cx43 has been involved in promoting the phenotypic transformation,migration and proliferation of vascular smooth muscle,and accelerating AS vascular disease.Rutaecarpine?Rut?is the main active ingredient of traditional Chinese medicine Evodia rutaecarpine,which activates the capsaicin receptor?TRPV1?to produce a variety of cardiovascular protective effects.Our preliminary study on the rat VSMC cell line A7r5 found that Rut inhibited Ox-LDL-induced Cx43 expression and inhibited VSMC proliferation,and its mechanism was associated with activation of TRPV1.In this study,the effects of Rut on the phenotypic transformation and migration of VSMC induced by Ox-LDL were further observed in cultured primary cultured rat vascular smooth muscle cells.And the inhibition of Rut on the expression of Cx43 was detected whether through TRPV1/ [Ca2+] i pathway to inhibit Ox-LDL-induced NF-?B activation.Methods:?1?Primary culture of VSMC: culture rat primary thoracic aortic vascular smooth muscle cells by tissue explants adherent method,immunohistochemical ?-SMA for vascular smooth muscle cell identification,take 4to 10 generations of cells for experiments.?2?Effects of Rut on Ox-LDL-induced migration and phenotypic transformation of VSMC: Ox-LDL?50 mg/L?-induced VSMC were used to simulate atherosclerotic cell models,and using different concentrations of Rut?1 ?mol/L,3 ?mol/L,10 ?mol/L?to determine whether the effect of Rut on TRPV1 pathway and Cx43 specific antagonist Gap26?100?mol/L?to determine the effect of Cx43 on vascular smooth muscle migration and phenotypic transformation.Western blot was used to detect the expression of ?-SMA andCalponin in VSMC to observe the phenotype of smooth muscle cells.The migration of VSMC was detected by scratch healing and Transwell chamber.?3?To determine that Rut inhibit the expression of Cx43 through TRPV1/ [Ca2+] i signaling pathway:Rut was administered to VSMCs incubated with Ox-LDL,premedient with TRPV1 blocker Capsazepine?CAPZ,10?mol/L?,intracellular calcium chelator BAPTA-AM?10?mol/L?or calmodulin antagonist W-7?10?mol/L?.The expression of Cx43 was detected by Western blot.The effect of Rut on intracellular calcium was detected by fluorescence microscopy and fluorescent microplate reader.?4?The effect of Rut on the activation of NF-?B induced by Ox-LDL: The nucleoprotein was extracted from VSMC cells incubated with Ox-LDL at different time points?2h,4h,8h,12 h and24h?.Western blot was used to detect the expression level of p65 to identify NF-?B activation time point.On the bais of which,Rut,Capsazepine?10?mol/L?,intracellular calcium chelator BAPTA-AM?10?mol/L?or calmodulin antagonist W-7?10?mol/L?were given to the VSMC,Western blot was used to detect nucleoprotein Expression of NF-?B p65.Results: The results of Western blot showed that Ox-LDL could significantly inhibit the expression of ?-SMA and Calponin in VSMC,which indicated that VSMC was transformed from contractile to synthetic type,while Rut could restore the expression of two kinds of contracting proteins.At the concentration of 3?mol / L effect shows the most significant effect,which can be blocked by TRPV1 blocker CAPZ.Scratch wound healing experiments and Transwell chambers showed that Rut dose-dependent inhibition of Ox-LDL-induced migration of VSMCs,which was abolished by CAPZ.In addition,the specific blocker Gap26 of Cx43 increased the expression of ?-SMA and Calponin and inhibited the migration of Ox-LDL-induced VSMC.These results suggest that Cx43 is involved in the regulation of phenotypic transformation and migration of Ox-LDL-induced VSMCs,and Rut inhibits VSMC phenotype transformation and migration through the TRPV1 pathway.?2?The results of Western blot showed that Rut could significantly inhibit the upregulation of Cx43 induced by Ox-LDL.TRPV1 blocker CAPZ,intracellular calcium chelator BAPTA-AM and calmodulin antagonist W-7 can abolish the inhibitory effect of Rut on Cx43.Fluorescence microscopy or fluorescencemicroplate reader showed that 10?mol / L Rut was significant promote the flow of calcium ions into the cell.Indicating that Rut can inhibit the expression of Cx43 by activating TRPV1/[Ca²⁺]_i signaling pathway.?3?Our preliminary study in the laboratory found that elevated Cx43 expression induced by Ox-LDL was blocked by NF-?B-specific blockers,suggesting that Ox-LDL up-regulates Cx43 expression through activating NF-?B.The results of Western blot showed that the level of NF-?B p65 was significantly increased when VSMC was incubated with Ox-LDL for 8 hours,indicating that Ox-LDL could significantly promote NF-?B nuclear translocation.Western blot results showed that the expression of p65 was decreased after adding Rut,while continuously adding CAPZ,BAPTA-AM and W-7 can eliminate this effect.These results suggest that Rut inhibits Ox-LDL-induced NF-?B activation through the TRPV1/[Ca²⁺]_i pathway,which up-regulates Cx43 expression.Conclusion: Rutaecarpine inhibits the up-regulation of Cx43 expression induced by Ox-LDL and inhibits the transformation and migration of vascular smooth muscle cells.The mechanism of which involves the activation of TRPV1/[Ca²⁺]_i signaling pathway to inhibit Ox-LDL-induced NF-?B activation.