| IntroductionProliferation and migration of vascular smooth muscle cells (VSMCs) and endothelial injury play a pivotal role in the development of hypertension and in the progression of atherosclerosis. A vital stimulus for VSMC proliferation and hypertrophy is angiotensinⅡ(AngⅡ), the main effector of the renin-angiotensin system. AngⅡis an important contributing factor to many vascular diseases, in part through its effects on VSMCs and vascular endothelial cells (VECs).Provious studies indicate that many signal molecular, such as mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2 and P38 have been shown to be implicated in the hypertrophic respense of VSMC to AngⅡ.Heme oxygenases (HOs) are the rate-limiting enzymes in the degradation of heme into carbon monoxide, iron, and biliverdin. To date, three distinct members of the HO-family have been identified. HO-1 is highly inducible by a variety of compounds and different physiologic and pathophysiologic stimuli. Data from current studies suggest that HO-1 plays a vital role in control of cell growth and differentiation.Neferine is a bis-benzylisoquinoline alkaloid extracted from the green seed embryo of Nelumbo nucifera Gaertn, which is effective in preventing the onset of reentrant ventricular tachyarrhythmias. Neferine can inhibit very low density lipoprotein oxidation and platelet aggregation, protect VECs from damage induced by oxygen free radicals. Neferine was shown to exert biological effects on the cardiovascular system consistant with a Calcium channel blocker. However, the precise mechanism of anti-artherosclerosis by which this drug affects the growth of VSMCs and VECs remains obscure. No studies exist that address the effect of neferine on VSMC and VEC proliferation or the interference of neferine with Ang II-induced signaling pathway.Therefore, the aim of this study was to test whether neferine affected the growth of HUVSMCs and HUVECs as well as to identify the underlying signaling pathways in HUVSMCs and HUVECs.To test this assuption, this study was as follows:1) to characterize the effect of neferine on cell proliferation in HUVSMCs and HUVECs; 2) to explore the effect of neferine on cell proliferation induced by AngⅡin HUVSMCs and HUVECs; 3) The role of HO-1 and ERK1/2 signal pathway in the cardioprotective effect of neferine was also investigated.Material and Methods1,The two cell lines:HUVSMC and HUVEC were cultured. Cell proliferation were detected by the MTT assay and flow cytometry analysis. HUVSMCs or HUVECs were respectively pretreated with neferine, ZnPPⅨ, PD98059 for 1h followed by a stimulation with AngⅡfor 24h.2,Cell cycle status of HUVSMC or HUVEC was analyzed with a FACScan flow cytometer by measuring fluorescence from cells stained with propidium iodide.The percentages of cells in the different phases of the cell cycle was analyzed using ModFit 3.0 software. The percentage of apoptosis of HUVSMCs was detected by Annexin V-FITC and PI staining with flow cytometry.3,The gene expressions of p21 WAF1/CIP1 and CyclinDl were detected by RT-PCR at 24h after neferine and or AngⅡin the treated HUVSMC. 4,The phosphorylated and total ERK1/2 proteins and HO-1 protein expression were analyzed by Western Blot at 24h in the treated HUVSMC and HUVEC. For normalization, blots were reprobed with antibodies to detect total amounts of GAPDH.5,NO concentrations in the HUVEC supernatants were detected by using NO enzyme-immunoassay kit based on nitrate reductase method.6,Statistical analysis:Analysed were performed using the SPSS(13.0) statistical software package. Results are expressed as means±SE. Data were analyzed using one-way analysis of variance (ANOVA) followed by the Student Newman-Keuls post hoc Tukey test. Differences between groups were considered to be significant at P<0.05.Result1. Neferine inhibited AngⅡ-induced proliferation Of HUVSMCCompared to the control group, neferine decreased the HUVSMC proliferation in a concentration-dependent manner. Neferine did not increase the percentage of apoptosis of HUVSMC.The proliferation of HUVSMC incubated with AngⅡwas significantly attenuated in the presence of neferine. The antiproliferative effect of neferine was confirmed by cell cycle analysis and the cell percentage of S and G2/M phase decreased. Neferine(0.5-5.0μmol/L) arrested HUVSMCs at the G0/G1 phase through suppressing CyclinDl and promoting p21WAF1/CIP1 mRNA expression.2. Neferine increased HO-1 protein expression and inhibited p-ERKl/2 in HUVSMC stimulated by AngⅡNeferine remarkably induced the expression of HO-1 in a concentration-dependant manner when compared to the control in HUVSMC. Coapplication of neferine(5.0μmol/L) with ZnPP IX significantly inhibited neferine-induced HO-1 expression in cells treated with AngⅡ. The inhibitory effect of neferine on AngⅡ-induced cell proliferation and increased p-ERK1/2 was significantly reversed by ZnPP IX. Moreover, CoPP, a HO-1 inducer, was also capable of preventing AngⅡ-induced proliferation in HUVSMCs, whereas ZnPP IX increased it. Perincubation with PD98059 also decreased of AngⅡ-induced cell proliferation in HUVSMCs. PD98059 inhibited the increase in cell proliferation induced by ZnPP IX.3. Neferine induced HUVEC proliferation and increased the secretion of NO in HUVEC.Compared to the control group, neferine(0.1,0.5,1.0,5.0μmol/L) increased HUVEC proliferation on 12h,24h,48h and promoted the cell cycle in a concentration-dependent manner. Neferine caused the up-regulation of HO-1 expression and increased the secretion of NO in HUVEC. ERK1/2 phosphorylation was significantly increased compared with the control in HUVEC. Coapplication of neferine with ZnPP IX significantly inhibited neferine-induced HO-1 expression and proliferation in HUVEC. PD98059 and ZnPP IX did not affect neferine-induced HO-1 expression,but reversed up-regulated ERK1/2 phosphorylation induced by neferine.Conclusion1,Neferine is capable of inhibiting HUVSMC proliferation and AngⅡ-induced HUVSMC proliferation in vitro. Neferine inhibits the gene expression of CyclinDl mRNA and promotes p21WAF1/CIP1 mRNA expression in AngⅡ-induced HUVSMC.2,AngⅡinhibites the growth of HUVEC in vitro. Neferine promotes HUVEC proliferation and against AngⅡ-induced inhibitory proliferation.3,Neferine up-regulates HO-1 expression and inhibits the phosphorylation of ERK1/2 in HUVSMC.4,Neferine up-regulates HO-1 expression and the phosphorylation of ERK1/2 in HUVEC.5,Neferine can attenuate the decreased the secretion of NO induced by AngⅡin HUVEC.
|
PDF Full Text Request