Rutaecarpine Prevents Ang ?-induced Dysfunction Of Vascular Smooth Muscle Cells And Related Mechanisms

Background: Angiotensin II(AngII)induced abnormal proliferation,migration and phenotypic transformation of vascular smooth muscle cells(VSMC),which is an important pathological basis for the development of vascular remodeling diseases such as hypertension and atherosclerosis.Recently,it has been reported that the proliferation and migration of VSMC induced by AngII are related to the activation of NF-?B pathway to increase the expression of Connexin43(Cx43).Rutaecarpine(Rut),an effective component of traditional Chinese medicine,has been found to have inhibitory effect on vascular remodeling as antihypertensive drug.Our previous studies found that Rut could inhibit VSMC proliferation induced by Ox-LDL,and its mechanism was related to activation of Transient receptor potential vanilloid 1(TRPV1)to inhibit the expression of Cx43.Recently,the role of AngII induced VSMC senescence in various vascular remodeling diseases has also attracted wide attention,and its mechanism is related to the downregulation of the expression of SIRT1.We have found that Rut can increase the expression of SIRT1 by inhibiting the aging of endothelial cells which high glucose induced by activating TRPV1.Objective: To explore the effects of Rut on VSMC proliferation,migration and phenotypic change induced by AngII and whether its mechanism involves activation of TRPV1 inhibited the expression of Cx43 increased,and further investigate whether the inhibition of VSMC aging by Rut via TRPV1/SIRT1 pathway.Methods:(1)The primary VSMC was cultured with tissue explants adherent method.?-SMA for vascular smooth muscle cell identification was by immunocytochemical,and take 3 to 8 generations of cells for experiments.(2)Effects of Rut on AngII induced VSMC proliferation,migration and phenotype conversion: VSMC were incubated with AngII(1 ?mol/L)for 24 h,pre-treated with different concentrations of Rut(0.3 ?mol/L,1 ?mol/L,3 ?mol/L),TRPV1 antagonist Capsazepine(CAPZ,10 ?mol/L)was applicated to identify whether it is mediated in the effection of Rut.CCK-8 and EdU were used to detect cell viability and proliferation;distribution of cell cycle was detected by flow cytometry,the expression of Cyclin D1 was detected by Western blot;The migration of VSMC was detected by scratch healing and Transwell chamber;Western blot was used to detect the expression of ?-SMA and Calponin in VSMC to observe the phenotype of smooth muscle cells.Western blot was used to detect Cx43 expression in VSMC.Cytoplasmic and nuclear separation techniques were used to detect P65 level of NF-?B subunit,and immunocytochemistry was used to determine P65 nuclear translocation.(3)The effect of Rut on AngII induced VSMC senescence: incubated VSMC for 72 h with AngII(1 ?mol/L)was to induced VSMC senescence,the dose of Rut and CAPZ were the same as above.Fluorescence probe DCFH-DA was used to detect ROS production.Senescence ?-galactosidase staining detected the number of senescent cells.Western blot was used to detect SIRT1 and aging related protein P53 and P21 expressions.Western blot detected the expression of phosphorylated AMPK,application of AMPK inhibitor Compound C(1 ?mol/L)to determine whether Rut upregulates the expression of SIRT1 through activate AMPK.Results:Rut inhibited proliferation of VSMC induced by AngII,which represented as inhibit DNA synthesis and cell viability of VSMC,reduced the proportion of cells in proliferative phase(S+G2/M)and Cyclin D1 expression.The results of the scratch healing experiment and Transwell chambers showed that Rut dose-dependent inhibition of Ang II-induced migration of VSMC.Rut also inhibited the phenotypic transformation of VSMC induced by AngII and partially restored the expression of two kinds of contracting proteins.The above effects of Rut in VSMC can be blocked by CAPZ.Western blot results showed that Rut could significantly inhibit the up regulation of Cx43 induced by AngII;incubated VSMC for 4 h in AngII could increase the P65 expression of nucleoprotein and reduced the cytoplasmic P65 level.Different concentrations of Rut could inhibit the nuclear translocation of P65,and which effects could be abolished by CAPZ.The immunocytochemical results also showed that Rut inhibited P65 nuclear translocation induced by AngII.Rut also inhibited AngII induced VSMC senescence: the results of ?-galactosidase staining showed that AngII significantly increased the number of senescent cells in VSMC,while Rut could decrease cell senescence in a dose-dependent manner,and CAPZ could abolish its effect.Fluorescence microscopy results showed that Rut could inhibit ROS generation of cells induced by AngII;Western blot results showed that Rut could significantly restore down-regulation of SIRT1 and inhibit P53 and P21 expressions induced by AngII,CAPZ could cancel the effect of Rut.Further studies have found that incubation of VSMC with AngII could significantly inhibit the phosphorylation of AMPK,and pretreated in Rut can restore AMPK phosphorylation,which can be blocked by CAPZ.Pretreated with AMPK blocker Compound C could cancel the effect of Rut on the expression of SIRT1.Conclusion: Rut prevented the proliferation,migration and phenotypic transformation of VSMC induced by AngII,and its mechanism was related to activation of TRPV1/NF-?B pathway,prevented upregulation of Cx43 expression.In addition,Rut inhibited VSMC senescence induced by AngII,and its mechanism was related to activation of TRPV1/AMPK/SIRT1 pathway,and inhibition of P53 and P21 expression.