| Objective: Atherosclerosis(atherosclerosis,AS)plaque formation refers to the whole blood vessel pathological changes,including that the abnormal proliferation vascular smooth muscle cell(VSMC)is the important pathology foundation which causes the AS vessel wall to be stenosis and spasm.In recent years,the role of connexin43(connexin43,Cx43)has been aroused widespread concern in atherosclerotic disease.In the animal models,we found that the expression of Cx43 increased first,furthermore accompanying with the change of Cx43 phosphorylation in the early AS plaque formation.The aim of this study was to examine the time-and dose-effects of Ox-LDL on Cx43 and phosphorylation level in cultured rat vascular smooth muscle cells and explored its mechanism preliminary.A large number of cases showed that Cx43 promoted VSMC proliferation which regulated SMC proliferation as an important target.Rutaecarpine(Rut)is the main active ingredients of traditional Chinese medicine Evodia rutaecarpa,a quinazoline indole alkaloid.Rut activated transient receptor potential vanilloid 1(TRPV1)to mediate the effects of cardiovascular protection.The goal of this study was to investigate the influences of Rut on Ox-LDL-induced Cx43 expression and proliferation of SMCs and elucidated whether the mechanism involved in TRPV1 pathway.Methods: Adding different concentrations of Ox-LDL(25 ~ 100mg/L)into cultured rats vascular smooth muscle cell line A7r5,and then we used western bolt to detecte the time-and dose-effects of Ox-LDL on Cx43 expression and phosphorylation(ser279/282)level.Pre-adding oxygen free radical scavenger NAC,antioxidant PDTC and NF-?b antagonist BAY11-7082 to deal with VSMCs,and we observed whether Ox-LDL up-regulation of Cx43 expression involved in ROS or NF-kappa B pathway.Pretreatment with different concentrations of Rut(1 ×10-5mol/L,3x10-6mol/L,1×10-6mol/L)to treat with VSMCs,and then we used the TRPV1 antagonist Capsazepine(1×10-5mol/L)to determine whether the effects of Rut involved in TRPV1 way.Adding Gap junction(GJ)antagonist 18?-GA intoVSMCs,we observed the effect of 18?-GA on Ox-LDL-induced VSMC proliferation.We untilized western blot to detect the expression of Cx43,CCK8 assay to test Cell viability,Ed U kit to evaluate the DNA replication,flow cytometry to evaluate the cell cycle,fluorescence plate reader to detect intracellular ROS generation.Results: Western Blot results showed that different concentrations of Ox-LDL(25~100mg/L)could significantly increased Cx43 expression,of which 100mg/L was most significant,but the effect of Ox-LDL on the phosphorylation level of Cx43 was not obvious.This result showed that the expression of Cx43 significantly increased after 12 hours induced by Ox-LDL,of which 48 h was most significant.Oxygen free radical scavenger NAC can not cancel the effect of Ox-LDL,but antioxidants PDTC and NF-?b antagonist BAY11-7082 could reverse the role of Ox-LDL,suggesting that the effect of Ox-LDL may be not associated with oxidative stress pathway but related to activating NF-?b pathway.We used CCK8 assay,flow cytometry and Ed U to found that Ox-LDL could promote the proliferation of VSMCs,but GJ blocker 18?-GA can cancel the effect of Ox-LDL.Pretreatment with Rut dose-dependently inhibited the upregulation of Cx43 and VSMC proliferation induced by Ox-LDL,characterized by reducing the number of VSMCs(CCK8 assay),inhibiting the number of Ed U positive cells,decreasing the percentage of proliferative phase(S and G2/M)in VSMCs.These effects of Rut were attenuated by capsazepine,an antagonist of transient receptor potential vanilloid subtype 1(TRPV1).Conclusion: Ox-LDLsignificantly increased Cx43 expression,but had no significant impact on phosphorylation of Cx43.Rut inhibited the up-regulation of Cx43 and VSMCs proliferation induced by Ox-LDL,which involved in TRPV1 way.These protective effects of Rut were remarkably attenuated by pretreatment with capsazepine,a competitive antagonist of transient receptor potential vanilloid subtype1(TRPV1). |
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