Apocynum leaf extract inhibits the progress of atherosclerosis in rats via the AMPK/mTOR pathway.

Apocynum leaf extract is an extract of the dried leaves of Apocynum venetum (a member of the Apocynaceae family) that has many effects on the cardiovascular system. The aim of the present study was to evaluate the protective effects of apocynum leaf extract on the atherosclerosis in rats induced by high-fat diet combined with vitamin D3 intraperitoneal injection. The atherosclerosis in rats were induced with a high-fat diet and an intraperitoneal injection of VD3 once daily for three contiguous days at a total injection dose of 70 U/kg. At the end of the 18th week, serum total cholesterol (TC) and triglyceride (TG) contents were measured. Hydroxyproline content in the aorta were measured by the alkali hydrolysis method. The hematoxylin-eosin (HE) and immunohistochemical staining were applied to evaluate the morphological changes and the collagen I and α-smooth muscle actin expression. The protein expression and the mRNA level of AMPK and mTOR were detected by western blot analysis and reverse transcript PCR. After treatment with apocynum leaf extract, the serum total cholesterol and triglyceride concentration of the atherosclerotic rats were significantly decreased, both the Collagen I expression and the hydroxyproline content in the aorta were significantly reduced, and the α-SMA, a smooth muscle-specific marker, expression were also lower than the untreated atherosclerotic rats. Western blot analyses showed that the apocynum can marked increase the p-AMPK but decrease the mTOR protein expression. The apocynum leaf extract also exhibited higher AMPK and lower mTOR mRNA expression of the aorta in the atherosclerotic rats. We believe that the apocynum leaf extract can effectively reduce blood lipid levels in rats with atherosclerosis, delay atherosclerotic progression by inhibiting excessive collagen synthesis and inhibiting smooth muscle cell over-proliferation. The underlying mechanism may be related to the AMPK/mTOR signaling pathway activity. Our results contribute towards validation of the traditional use of apocynum leaf extract in the treatment of atherosclerosis.

Inhibitory effect of puerarin on vascular smooth muscle cells proliferation induced by oxidised low-density lipoprotein via suppressing ERK 1/2 phosphorylation and PCNA expression.

Puerarin, an isoflavonoid isolated from the traditional Chinese herbal medicine Pueraria lobata (Wild.) Ohwi, has been shown to process antioxidant, anti-inflammatory, anti-cancer, anti-hypercholesterolemic, and anti-hyperglycemic activities in vivo and in vitro. The aim of the present study was to investigate the antiproliferative effects and the possible mechanisms of puerarin in vascular smooth muscle cells (VSMCs) stimulated with oxidised low-density lipoprotein (ox-LDL). VSMCs were cultured and pretreated with different concentrations of puerarin (0, 1, 10, 50 µM) before stimulated by ox-LDL (50 µg/mL). Cell proliferation was evaluated by MTT assay. Flow cytometry was used to study the influence of puerarin on cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK) 1/2 were detected by western blotting analysis. The results indicated that puerarin significantly inhibited VSMCs proliferation induced by ox-LDL and phosphorylation of ERK 1/2. Furthermore, puerarin also blocked the ox-LDL-induced cell-cycle progression at G1/S-interphase and down-regulated the expression of PCNA of VSMCs. The results suggest puerarin inhibits ox-LDL-induced proliferation of VSMCs by suppressing ERK 1/2 phosphorylation and PCNA expression.

Icariin inhibits oxidized low-density lipoprotein-induced proliferation of vascular smooth muscle cells by suppressing activation of extracellular signal-regulated kinase 1/2 and expression of proliferating cell nuclear antigen.

Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to possess anti-inflammatory, anti­oxidant and anti-atherosclerotic activities in vivo and in vitro. The aim of the present study was to investigate the effects of icariin on oxidized low­density lipoprotein (ox-LDL)-induced proliferation of vascular smooth muscle cells (VSMCs) and the possible underlying mechanism. VSMCs were cultured and pre­treated with various concentrations of icariin (0, 10, 20 or 40 µm) prior to stimulation by ox­LDL (50 µg/ml). Cell proliferation was evaluated by an MTT assay. Flow cytometry was used to study the influence of icariin on the cell cycle. Proliferating cell nuclear antigen (PCNA) expression and phosphorylation levels of extracellular signal-regulated kinase (ERK)1/2 were detected by western blot analysis. The results indicated that icariin significantly inhibited ox­LDL­induced proliferation of VSMCs and phosphorylation of ERK1/2. Furthermore, icariin also blocked the ox­LDL­induced cell­cycle progression at G1/S­interphase and downregulated the expression of PCNA in VSMCs. In conclusion, the present study indicated for the first time that icariin reduced the amount of ox­LDL­induced proliferation of VSMCs through suppression of PCNA expression and inactivation of ERK1/2.

[Effects of icariin on proliferation of vascular smooth muscle cell induced by ox-LDL via impacting MAPK signaling pathway].

This paper was aimed to study the effects of icariin (ICA) on the proliferation of vascular smooth muscle cell (VSMC) induced by oxidized low density lipoprotein (ox-LDL), and the molecular mechanism of the expression of proliferating cell nuclear antigen (PCNA) and MAPK signaling pathway. In this study, VSMC was induced by ox-LDL (50 mg•L⁻¹),the effect of ICA on the proliferation of VSMC was detected by MTT assay, Western blot and Real-time PCR. The results showed that after stimulation of ox-LDL, the proliferation activity of VSMC was increased, S phase, G2/M phase cells were increased, G0/G1 phase cells were decreased, PCNA protein expression was enhanced; ICA (40, 20, 10 µmol•L⁻¹) could effectively inhibit ox-LDL-induced VSMC proliferation, S phase and G2/M phase cells were decreased, the percentage of cells in G0/G1 phase were increased, PCNA expression was decreased, p38MAPK and ERK1/2 activation were inhibited. These results indicate that ICA can inhibit the proliferation of VSMC by reducing the expression of PCNA and blocking the p38MAPK and ERK1/2 signaling pathway.

Icariin Attenuates High-cholesterol Diet Induced Atherosclerosis in Rats by Inhibition of Inflammatory Response and p38 MAPK Signaling Pathway.

Icariin is a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim and has been reported to be effective for the treatment of a variety of cardiovascular diseases. The aim of the present study was to investigate the effect and mechanism of icariin on atherosclerosis (AS) using a high-cholesterol diet (HCD)-induced rat model. Seventy male Wistar rats were divided into five groups: 20 in the control group, 20 in the AS group, 10 in the simvastatin group, 10 in the low-dose icariin group, and 10 in the high-dose icariin group. A HCD and vitamin D3 were administered to establish AS rat model. The five groups of rats received daily intragastric administration of normal saline, simvastatin, or icariin (30 mg/kg/d, 60 mg/kg/d) for 4 weeks. The levels of blood lipids, superoxide dismutase (SOD), and malonaldehyde (MDA) were measured. The mRNA levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α were analyzed by real-time RT-PCR, and the serum levels of IL-6 and TNF-α were measured using ELISA kit. In addition, the expression of phosphorylated p38 (p-p38) MAPK was detected by Western blot analysis. The results indicated that AS rat models were successfully constructed. In the AS group, the levels of blood lipids including total cholesterol (TC), triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), and MDA were significantly increased, while high-density lipoprotein-cholesterol (HDL-C) and SOD were significantly decreased, compared with those in the control group. However, icariin succeeded in improving these biochemical parameters towards the normal values in the control group. In the simvastatin group and the icariin groups, the serum levels of IL-6 and TNF-α and the related tissue mRNA levels, as well as the expression of p-p38 MAPK, were markedly reduced compared with the AS group. In conclusion, the present study indicated that icariin inhibited the HCD-induced dyslipidemia in rats, the mechanisms may be associated with the anti-inflammation, anti-oxidative stress, and downregulation of p-p38 MAPK by icariin.

Effects and mechanisms of icariin on atherosclerosis.

PURPOSE: Icariin, a flavonoid isolated from the traditional Chinese herbal medicine Epimedium brevicornum Maxim, has been shown to process anti-inflammatory, antioxidative actions and anti-atherosclerosis activity in vivo and in vitro. The purpose of this study was to investigate the effects and mechanisms of icariin on atherosclerosis by human umbilical vein endothelial cells (HUVECs). METHODS: The effects of icariin on the activity of HUVECs induced by oxidized low-density lipoprotein (ox-LDL) were detected by MTT assay. Then we studied the effects of icariin on the adhesion of monocyte with HUVECs induced by ox-LDL. The secretion of E-selectin, intercellular adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) by HUVECs were measured by enzyme-linked immunosorbent assay (ELISA) method. Finally the mRNA levels of ICAM-1, VCAM-1, E-selectin of HUVECs were analyzed by real time RT-PCR. RESULTS: MTT result indicated that icariin (10, 20, 40 µmol/L) could inhibit HUVECs injury induced by ox-LDL in a concentration-dependent manner (P < 0.05). The adhesion of monocyte with HUVECs induced by ox-LDL was inhibited by icariin in a concentration-dependent manner (P < 0.05). The levels of ICAM-1, VCAM-1, E-selectin of icariin groups were significantly decreased in a concentration-dependent manner compared with ox-LDL-simulated group (P < 0.05). The mRNA expressions of ICAM-1, VCAM-1, E-selectin of icariin groups were also downregulated significantly compared with ox-LDL-simulated group (P < 0.05). CONCLUSIONS: Icariin can prevent atherosclerotic lesion. Its mechanism may be that it can defend against the oxidation damage to HUVECs, inhibit the adhesion of monocyte to HUVECs, and reduce the secretion and expression of adhesion molecules including ICAM-1, VCAM-1, E-selectin.

[Effect and mechanism of icariin on myocardial ischemia-reperfusion injury model in diabetes rats].

To study the therapeutic effect and possible mechanism of icariin on myocardial ischemia-reperfusion injury ( MIRI) model in diabetes rats. The model of diabetic rats were induced by Streptozotocin (STZ), then the model of MIRI was established by ligating the reversible left anterior descending coronary artery for 30 min, and then reperfusing for 120 min. totally 40 male SD were randomly divided into five groups: the control group (NS), the ischemia reperfusion group (NIR), the diabetes control group (MS), the diabetic ischemia reperfusion group (MIR) and the diabetic ischemia reperfusion with icariin group (MIRI). The changes in blood glucose, body weight and living status were observed; the enzyme activity of serum CK-MB, LDH, GSH-Px and myocardium SOD and the content MDA and NO in myocardium were detected; the myocardial pathological changes were observed by HE staining; the myocardial Caspase-3, the Bcl-2, Bax protein expressions were detected by Western blot. The result showed that the diabetes model was successfully replicated; myocardial ischemia-reperfusion injury was more serious in diabetes rats; icariin can increase NO, SOD, GSH-Px, Bcl-2 protein expression, decrease MDA formation, CK-MB and LDH activities and Caspase-3 and Bcl-2 protein expressions and myocardial damage. The result suggested that icariin may play a protective role against ischemia reperfusion myocardial injury in diabetes rats by resisting oxidative stress and inhibiting cell apoptosis.