Inhibitory Effect Of Reinioside C On Vascular Smooth Muscle Cell Proliferation Induced By Angiotensin Ⅱ

BACKGROUND:Hypertension and its complications are one of the main death causes of cardiovascular disease. The development of hypertension and its complications are closely related to vascular remodeling. Many studies have established that vascular smooth muscle cell (VSMC) proliferation play an important role in the major pathological process in vascular remodeling. It has been discovered that angiotensin II (Ang II) could induce VSMC proliferation by enhance nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase activity and reactive oxygen species (ROS) generation and then activated the extracellular signal-regulated kinases 1/2 (ERK1/2) pathway in VSMC. ERK1/2 signaling pathway was the classic mitoten activated protein kinase (MAPK) signal transduction pathway, which could activate downstream targets such as nuclear fact or-κB (NF-κB), activator protein-1 (AP-1), c-myc, promoting proliferation of VSMC and acceleratiing vascular remodeling process.Polygala fallax Hemsl, a commonly used Chinese medicinal herb, has been used to treat some diseases such as infective inflammation and hypercholesterolemia. Reinioside C is the main active ingredient of Polygala fallax Hemsl. Our previous study found that reinioside C could not only inhibit the monocyte-endothelial cells adhesion but also the elevated expression of LOX-1 in endothelial cells induced by oxidized low-density lipoprotein (ox-LDL). And these actions of reinioside C are related to its antioxdation properties. So in the present study, we therefore test the effect of reinioside C on Ang II induced VSMCs proliferation and explore the possible mechnsim.METHODS:VSMC proliferation was measured by BrdU cell proliferation analysis for cell DNA synthesis ability and flow cytometry for cell cycle. Real-time PCR was used for testing NADPH oxidase subunits:p22phox and gp91phox; AP-1 subunits:c-fos and c-jun; c-myc mRNA expression. Changes in intracellular ROS level were determined by measuring the oxidative conversion of DCFH-DA to DCF in fluorospectrophotometer. The phosphorylation status of ERK1/2 and the level of IκB-αprotein were determined by western blot analysis. The NF-κB DNA-binding activity wag determined by Electrophoretic Mobility Shift Assay (EMSA).RESULTS:(1) Ang II (10-6M) treatment for 24 h markedly enhanced the DNA synthesis ability and cell cycle of VSMC. Pretreatment with reinioside C (3,10 or 30μM) dose-dependently inhibited the proliferative effect of AngⅡ. These effects were also inhibited by ROS specificity inhibitor DPI (10μM). Reinioside C (30μM) itself had no effect on the proliferation of VSMCs.(2) AngⅡ(10-6M) treatment for 2 h markedly enhanced NADPH oxidase subunits:p22phox and gp91phox mRNA expression and ROS production in the VSMC. Pretreatment with reinioside C (3,10 or 30μM) dose-dependently inhibited the NADPH oxidase and intreacellular ROS production induced by AngⅡ. These effects were also inhibited by DPI (10μM). Reinioside C (30μM) itself had no effect on intracellular NADPH oxidase and ROS level in VSMCs.(3) AngⅡ(10-6 M) treatment for 2 h markedly increased phosphorylation status of ERK1/2. Pretreatment with reinioside C (3,10 or 30μM) dose-dependently inhibited ERK1/2 phosphorylation induced by AngⅡ. This effect was also inhibited by DPI (10μM) and ERK1/2 specificity inhibitor PD98059 (40μM). Reinioside C (30μM) itself had no effect on ERK1/2 phosphorylation.(4) AngⅡ(10'6 M) treatment for 24 h markedly enhanced NF-κB DNA-binding activity,AP-1 subunit:c-fos and c-jun and proto-oncogene c-myc mRNA expression in the VSMC. Pretreatment with reinioside C (3, 10 or 30μM) dose-dependently inhibited the above effects induced by AngⅡ. These effects were also inhibited by NF-κB specificity inhibitor PDTC (10μM) and PD98059 (40μM). Reinioside C (30μM) itself had no effect on activiry or expression of NF-κB, AP-1 and c-myc. CONCLUSIONS:Reinioside C attenuated Ang II-induced VSMC proliferation via inhibiting NADPH oxidase-ROS-ERK1/2-NF-KB/AP-1 pathway.