Protective effects of Schisandrin B against D-GalN-induced cell apoptosis in human hepatocyte (L02) cells via modulating Bcl-2 and Bax.

Schisandrin B is a dibenzocyclooctadiene derivative extracted fromSchisandra chinensis (Turcz.) Baill., that exhibits anti-oxidation, anti-inflammation, anti-tumor and hepatoprotective activities. To understand the hepatoprotective mechanism of schisandrin B, this study investigated the efficacy of schisandrin B on L02 cells after treatment with D-GalN. Following pretreatment with 40 µM schisandrin B, L02 cells were stimulated with 40 mM D-GalN. Cell viability, apoptosis, the expression levels of genes associated with apoptosis, and the intracellular oxidative stress indexes were measured. The viability of L02 cells was determined using MTT assay, and the Annexin V-FITC/PI assay kit was utilized for the assessment of apoptosis. The activities of GSH-Px and SOD, the level of MDA were assessed, separately, using relative detection kits. Moreover, RT-PCR as well as Western blot was applied to measure the mRNA and protein expression of Bax and Bcl-2. The results indicated that schisandrin B significantly prevented D-GalN­induced oxidative damage in L02 cells (P<0.05), decreased GSH-Px and SOD activities (P<0.05), increased MDA content (P<0.05). Furthermore, schisandrin B inhibited D-GalN-induced apoptosis in L02 cells (P<0.05), regulated the expression of Bax and Bcl-2 (P<0.05). The results indicated that schisandrin B decreased the D-GalN-induced intracellular oxidative stress indexes generation, and inhibited the down-regulation of Bcl-2 and up-regulation of Bax induced by D-GalN. In conclusion, schisandrin B was shown to exert protective effect against oxidative damage of L02 cells, which, in part, was achieved by regulating the mRNA and protein levels of Bax and Bcl-2.

Complete chloroplast genome of adonis amurensis (ranunculaceae), an important cardiac folk medicinal plant in east asia.

Adonis amurensis Regel et Radde is an important cardiac folk medicinal plant which endemic to Northeast Asia. We determined the first complete chloroplast genome of A. amurensis using genome skimming approach. The cp genome was 157,032 bp long, with a large single-copy region (LSC) of 86,218 bp and a small single-copy region (SSC) of 18,212 bp separated by a pair of inverted repeats (IRs) of 26,301 bp. It encodes 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 ribosomal RNA genes. We also reconstructed the phylogeny of Adonideae and Isopyreae using maximum likelihood (ML) method, including our data and previously reported cp genomes of related taxa. The phylogenetic analysis indicated that A. amurensis is close related with Adonis sutchuenensis.

Puerarin protects vascular smooth muscle cells from oxidized low-density lipoprotein-induced reductions in viability via inhibition of the p38 MAPK and JNK signaling pathways.

Puerarin belongs to the family of flavonoids extracted from Pueraria lobata (Wild.) Ohwi, which exhibits antioxidative, anti-inflammatory, anti-hyperglycemic, antitumor, anti-hypertensive and anti-atherosclerotic activities. In the present study, the effects of puerarin on oxidized low-density lipoprotein (ox-LDL)-stimulated vascular smooth muscle cells (VSMCs) were explored to understand the mechanisms underlying the anti-atherosclerotic effects of puerarin. VSMCs were treated with various concentrations of puerarin (0, 20, 40 and 80 µM) prior to stimulation with ox-LDL (50 µg/ml). VSMC viability was evaluated by performing MTT and Cell Counting Kit-8 assays. Moreover, superoxide dismutase (SOD) and malondialdehyde (MDA) levels were measured by performing ELISAs. The mRNA expression levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined via reverse transcription-quantitative PCR. Western blotting was conducted to assess the levels of p38-MAPK and JNK phosphorylation. The results indicated that puerarin inhibited ox-LDL-induced VSMC viability. Moreover, puerarin significantly decreased the mRNA expression levels of IL-6 and TNF-α, significantly reduced the production of MDA and significantly increased SOD activity in ox-LDL-stimulated VSMCs. Puerarin also inhibited ox-LDL-induced phosphorylation of p38 and JNK in VSMCs. The results suggested that puerarin reduced ox-LDL-induced VSMC viability via inhibition of the p38 MAPK and JNK signaling pathways. The present study provided theoretical evidence that puerarin may serve as a therapeutic agent to reduce the development of atherosclerosis.

Protective effects of icariin on human vascular endothelial cells induced by oxidized low-density lipoprotein via modulating caspase-3 and Bcl-2.

Icariin belongs to the family of flavonoids that is extracted from Epimedium brevicornum Maxim, and exhibits antioxidative, antitumorigenic, antiosteoporotic, immunoregulatory and antiatherosclerotic properties. To understand the mechanisms underlying the antiatherosclerotic properties of icariin, the present study investigated the effects of icariin on human vascular endothelial cells (HUVECs) following treatment with oxidized low­density lipoprotein (ox­LDL). Thus, following pretreatment with icariin at four various concentrations (0, 10, 20 and 40 µM), HUVECs were stimulated with ox­LDL (100 µg/ml). The viability of cells was evaluated via an MTT assay and flow cytometry was performed to assess apoptosis. Additionally, the protein and mRNA expression levels of apoptosis regulator Bcl­2 (Bcl­2) and caspase­3 were determined by western blotting and reverse transcription­quantitative polymerase chain reaction. The findings of the present study indicated that icariin prevented injury and apoptosis in HUVECs following ox­LDL treatment, in particular via the regulation of protein and mRNA expression levels of Bcl-2 and caspase-3.

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.

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 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.

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.

[Changes of lignans' content in stems of Schisandra chinensis].

OBJECTIVE: To determine lignan content in the vine stem of Schisandra chinensis during 12 months and provide the scientific basis for the development and utilization of the resources. METHOD: Analysis was carried out on an Eclipse XDB C18 column eluted with a mixture of methanol-acetonitrile-water (43: 28: 29) as the mobile phase. The flowrate was 1.0 mL x min(-1), and the detection wavelength was set at 250 nm. Schisandrin, deoxyschizandrin and schisandrin B were used as reference substance, and the external standard method was used. RESULT: The content of three constituents in the vine stem varied under different months. Schisandrin's maximum is 2.3 mg x g(-1) in December, minimum is 1.4 mg x g(-1) in April. A Deoxyschizandrin's maximum is 0.8 mg x g(-1) in November, minimum is 0.4 mg x g(-1) in March; Schisandrin B's maximum is 3.0 mg x g(-1) in January, minimum is 1.1 mg x g(-1) in April. CONCLUSION: The collection seasons for the vine stem of S. chinensis are autumn and winter.