Effect Of Isoimperatorin On Ang-Ⅱ-Induced VSMC Proliferation And Its Potential Mechanisms

【Objective】To study the effect of isoimperatorin(ISOIMP)on VSMCs proliferation induced by Ang-Ⅱ and explore its involvement in MAPK/ERK signaling pathway.Our study is to provide more experimental basis for the research and treatment of cardiovascular diseases.【Methods】1.The effects of Ang-Ⅱ on VSMCs proliferationThe CCK-8 method was used to detect the proliferation of VSMCs treated with different concentrations of Ang-Ⅱ(0,0.01,0.05,0.1,0.5,1.0μmol/L)from various time up to 72 hours.Meantime,the effect of the VSMCs proliferation induced by Ang-Ⅱ was monitored dynamiclly by Real time cellular analysis(RTCA),by do so the optimal concentration and time course of Ang-Ⅱtreatment were determined.2.The effects of ISOIMP on VSMCs proliferationVSMCs were exposed to different concentrations of ISOIMP(0,0.05,0.1,0.5,0.1,0.5 μmol/L)in the presence or absence of Ang-II.The proliferation of VSMCs and Ang-Ⅱ-induced VSMCs after treated with ISOIMP was detected by CCK-8,and monitored dynamiclly by RTCA.These methods were allowed us to identify the optimal concentration of ISOIMP on VSMCs proliferation induced by Ang-Ⅱ.3.The effects of ISOIMP on Ang-Ⅱ-induced VSMCs apoptosisHoechst 33342 staining,Fluorescence double staining flow cytometry Annexin V-FITC/PI staining and Mitochondrial membrane potential assay kit with JC-1 were used to detect the effects of ISOIMP on Ang-Ⅱ-induced VSMCs apoptosis.The expression of apoptosis related proteins Bax,Bcl-2 were detected by Western Blot in Ang-Ⅱ-induced VSMCs.of Ang-Ⅱ-induced VSMCs proliferation 4.The effects of MAPK/ERK signaling pathway on ISOIMP-mediated inhibitionFirst,Western Blot analysis was used to measure the expression of MAPK/ERK signaling pathway key proteins p-c-Raf,p-MEK and p-ERK1/2 in Ang-Ⅱ-induced VSMC followed by treated with ISOIMP.The inhibitory effect of MAPK inhibitor PD98059 on the proliferation of VSMCs was determined by RTCA and cck-8 assays,and the pharmacologic concentration of PD98059 was also determined.Western Blot analysis was further used to compare the effect of MAPK inhibitor PD98059,MAPK agonist Isoproterenol(ISO)as well as ISOIMP on the expression of MAPK/ERK signaling pathway related proteins.Using Hoechst 33342 staining,Fluorescence double staining flow cytometry Annexin V-FITC/PI staining and Mitochondrial membrane potential assay kit with JC-1,the effects of ISOIMP,PD98059 and ISO on Ang-Ⅱ-induced VSMCs apoptosis were measured.Lastlly,the expression of apoptosis related proteins Bax,Bcl-2 were measured on Ang-Ⅱ-induced VSMCs followed by treated with ISOIMP.【Results】1.The effects of Ang-Ⅱ on the VSMCs proliferationThe results of RTCA and CCK-8 measurements showed that the proliferation of VSMC was significantly promoted by the different concentrations of Ang-Ⅱ treatment,and the best dosage and time point were treatment with 0.1μmol/L Ang-Ⅱ for 24h(P<0.01).2.The effects of ISOIMP on VSMCs proliferationThe results of RTCA and cck-8 measurements showed that the proliferation ratio of VSMC was significantly at different levels and by different concentrations of ISOIMP followed 24 h treatment compared with the control.As comparison with Ang-Ⅱ group,the proliferation of Ang-Ⅱ-induced VSMC was significantly inhibited by ISOIMP.Interestingly,the low concentration of 0.05 μmol/L had achieved strongest inhibitory effect(P<0.01).3.The effects of ISOIMP in Ang-Ⅱ-induced VSMCS apoptosisHoechst 33342 staining,Fluorescence double staining flow cytometry Annexin V-FITC/PI staining and Mitochondrial membrane potential assay kit date showed that both ISOIMP and Ang-Ⅱ had certain inhibition on apoptosis of normal VSMC(P<0.05 or P<0.01).But Ang-Ⅱ-induced VSMC apoptosis was promoted to a certain extent(P<0.01).Western Blot analysis showed that the expression of bcl-2 was increased compared with DMSO group,while the expression of Bax decreased in normal VSMC cells after treated with Ang-Ⅱ and ISOIMP alone(P<0.05 or P<0.01).As comparison with Ang-Ⅱ group,ISOIMP treatment increased the expression of pro-apoptotic protein Bax in Ang-Ⅱ-induced VSMC,while the expression of anti-apoptotic protein bcl-2 was decreased significantly(P<0.01).4.The effects of MAPK/ERK signaling pathway in ISOIMP mediated inhibition of Ang-Ⅱ-induced VSMC proliferationWestern blot analysis showed that Ang-Ⅱ and ISOIMP treatment significantly increased the expression of MAPK/ERK signaling pathway related proteins p-c-Raf and p-MEK.As compared with Ang-Ⅱ group,ISOIMP significantly reduced the expression of p-c-Raf、p-MEK、p-ERK1/2 in Ang-Ⅱ-induced VSMC(P<0.05 or P<0.01).The RTCA analysis showed that MAPK/ERK inhibitor PD98059 had a significantly inhibition on Ang-Ⅱ-induced VSMC proliferation.As compared with Ang-Ⅱ group,ISOIMP and PD98059 inhibited the expression of p-c-Raf、p-MEK、p-ERK1/2 in Ang-Ⅱ-induced VSMC(P<0.05 or P<0.01).Hoechst 33342 staining,Fluorescence double staining flow cytometry Annexin V-FITC/PI staining and Mitochondrial membrane potential assay kit studies showed that both PD98059 and ISOIMP significantly promoted apoptosis in Ang-Ⅱ-induced VSMC(P<0.01).Western blot analysis showed that both PD98059 and ISOIMP reduced the expression of p-c-Raf、p-MEK、p-ERK1/2(P<0.05 or P<0.01),suppressed the expression of Bcl-2 protein,but increase the expression of protein Bax(P<0.01)in Ang-Ⅱ-induced VSMC(P<0.01).【Conclusions】Treatment with ISOIMP inhibited Ang-Ⅱ-induced VSMC proliferation,promote Ang-Ⅱ-induced VSMC apoptosis.The MAPK/ERK signal path way may be important in this process.