Puerarin attenuates high-glucose-and diabetes-induced vascular smooth muscle cell proliferation by blocking PKCbeta2/Rac1-dependent signaling.
ABSTRACT Oxidative stress has been implicated in several steps leading to the development of diabetic vascular complications. The purpose of this study was to determine the efficacy and the possible mechanism of puerarin on high-glucose (HG; 25 mM)-induced proliferation of cultured rat vascular smooth muscle cells (VSMCs) and neointimal formation in a carotid arterial balloon injury model of obese Zucker rats. Our data demonstrated that puerarin significantly inhibited rat VSMC proliferation as well as reactive oxygen species (ROS) generation and NADPH oxidase activity induced by HG treatment. Further studies revealed that HG treatment resulted in phosphorylation and membrane translocation of PKCbeta2 as well as Rac1, p47phox, and p67phox subunits, leading to NADPH oxidase activation. Puerarin treatment remarkably disrupted the phosphorylation and membrane translocation of PKCbeta2 as well as Rac1, p47phox, and p67phox subunits. Blocking PKCbeta2 by infection with AdDNPKCbeta2 also abolished HG-induced phosphorylation and membrane translocation of Rac1, p47phox, and p67phox subunits as well as ROS production and NADPH oxidase activation in VSMCs. In vivo neointimal formation of obese Zucker rats evoked by balloon injury was evidently attenuated by the administration of puerarin. These results demonstrate that puerarin may exert inhibitory effects on HG-induced VSMC proliferation via interfering with PKCbeta2/Rac1-dependent ROS pathways, thus resulting in the attenuation of neointimal formation in the context of hyperglycemia in diabetes mellitus.
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ABSTRACT: AIMS: Vascular inflammation is a key factor in the pathogenesis of diabetes-related vascular complications. Our previous study showed that (-)-epigallocatechin-3-gallate (EGCG) inhibits high glucose-induced vascular smooth muscle cells proliferation, thus may have beneficial effects in diabetes and its complications. However, the effect of EGCG on inflammation in diabetes is not known. In the present study, we investigated whether EGCG suppresses the vascular inflammation induced by high glucose in human umbilical vein endothelial cells (HUVECs). MAIN METHODS: The inhibitory effect of EGCG on high glucose-induced up-regulation of the expression of vascular cell adhesion molecule 1 (VCAM-1) was measured using enzyme-linked immunosorbent, RT-PCR, immunoblotting and cell adhesion assays. The effect of EGCG on high glucose-induced nuclear factor-kappa B (NF-κB) activation was investigated by immunoblotting, immunofluorescence and electrophoretic mobility shift assays. KEY FINDINGS: High glucose increased VCAM-1 expression and enhanced the adhesion of monocytes to HUVECs. Pretreatment with EGCG in a concentration-dependent manner (1.0-50 μM) significantly attenuated these effects. High glucose (25 mM)-mediated vascular inflammation was blocked by PKC pseudosubstrate (PKC inhibitor 19-31) or the NF-κB inhibitor pyrrolidine dithiocarbamate (PDTC). Stimulation with high glucose increased the NF-κB translocation from the cytoplasm to the nucleus, and increased IκB-α phosphorylation, decreased its expression, and in the presence of EGCG, the effect of high glucose on NF-κB and IκB-α were blocked. SIGNIFICANCE: EGCG suppresses high glucose-induced vascular inflammatory process via the inhibition of PKC and NF-κB activation in HUVECs, suggesting that EGCG may be a potential candidate for the treatment and prevention of diabetic vascular complications.Life sciences 02/2013; · 2.56 Impact Factor
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ABSTRACT: BACKGROUND: Vaspin has insulin-sensitizing effects, as well as additional beneficial effects on metabolic diseases. However, little is known about the direct effects of vaspin on vascular complications mediated by diabetes. The objective of this study is to determine the efficacy and mechanism of vaspin on hyperglycemia-induced vascular smooth muscle cells (VSMCs) proliferation, chemokinesis and cell signaling. METHODS: Rat VSMCs proliferation was determined with 5-ethynyl-2'-deoxyuridine cell proliferation assays, chemokinesis was monitored with scratch assays, and reactive oxygen species (ROS) production was assessed using H2DCFDA and SOD-inhibited reduction of ferricytochrome c assay. Luciferase activity is assayed using a Dual Luciferase Reporter Assay System. Cell signaling is assessed by immunblotting. RESULTS: Vaspin significantly inhibited VSMCs proliferation and chemokinesis, as well as ROS generation and NADPH oxidase activity, induced by high glucose (HG) treatment. Compared with HG, vaspin significantly decreased VSMCs proliferation by 40 ± 8% at 100 ng/ml. Vaspin also decreased ROS production by 16 ± 8% at 100 ng/ml and 30 ± 8% at 300 ng/ml (all P < 0.01). Vaspin significantly abolished HG-induced phosphorylation of oxidase subunits p47phox, Akt, p38, and JNK1/2 without affecting their total levels, and attenuated HG-induced phosphorylation of insulin receptor and its downstream IRS-1 and IRS-2. For downstream targets, NF-κB activity and IκBα phosphorylation were both enhanced significantly after HG stimulation, and these effects were inhibited by vaspin. Vaspin also significantly abolished HG-induced PCNA and cyclin D1 expression. CONCLUSIONS: Vaspin inhibits HG-induced VSMCs proliferation and chemokinesis by preventing ROS activation and MAPK, PI3K/Akt, and NF-κB signaling.Atherosclerosis 02/2013; · 3.71 Impact Factor
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ABSTRACT: Several studies demonstrate that estradiol can prevent arterial calcification. However, little is known regarding the effect of puerarin, a phytoestrogen extracted from Radix Puerariae, on arterial calcification. The aim of the present study was to determine whether puerarin reduced osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs). The CVSMCs were isolated from mice aorta and treated with different concentrations of puerarin. The alkaline phosphatase (ALP) activity, osteocalcin secretion and Runx2 expression were determined. To examine whether estrogen receptors (ERs) PI3K and Akt play a role in this effect, ICI182789, phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) was used. Our results showed puerarin could inhibit ALP activity, osteocalcin secretion and Runx2 expression in CVSMCs. Puerarin could induce the activation of Akt. Furthermore, pretreatment of ICI182780, LY294002, HIMO could abolish the effect of puerarin on ALP activity in CVSMCs. Our experiment demonstrated that puerain could attenuate the osteoblastic differentiation of VSMCs through the ER/PI3K-Akt signal pathway.The American Journal of Chinese Medicine 01/2014; 42(2):337-47. · 2.28 Impact Factor