Hsieh-Hsun Ho

Effects of mulberry (Morus alba L.) extracts on lipid homeostasis in vitro and in vivo.

The objective of this study was to investigate the lipid-lowering effects of mulberry water extracts (MWEs). To evaluate the hypolipidemic effect of MWEs, hamsters were fed with either high fat/cholesterol diets (HFCD) or HFCD supplemented with 1 and 2% MWEs for 12 weeks. Plasma total cholesterol (TC) and triglyceride (TG) levels of hamsters fed HFCD with MWEs were significantly reduced by about 30-37% and 16-35%, respectively, as compared to those without MWEs. Similar results were also measured in hepatic TC and TG of hamsters fed HFCD with MWEs. Low-density lipoprotein receptor (LDLR) gene expression and the uptake ability of low-density lipoprotein (LDL) in HepG2 cells were also upregulated by additions of MWEs. MWEs also decreased the gene expressions of enzymes involved in the TG and TC biosyntheses. Results suggest that hypolipidemic effects of MWEs are via an enhancement of LDLR gene expression and the clearance ability of LDL and a decrease in the lipid biosynthesis. Therefore, MWEs could be used as a natural agent against hyperlipidemia.

Mulberry Leaf Extract Inhibits Vascular Smooth Muscle Cell Migration Involving a Block of Small GTPase and Akt/NF-κB Signals

Mulberry, the fruit of Morus alba, is commonly used in Chinese medicines because of its many pharmacologic effects. Mulberry leaves contain many phenolic antioxidants that can reduce cardiovascular disease. Atherosclerosis involves proliferation and migration of vascular smooth muscle cell (VSMC). Thus, we investigated the mechanisms by which mulberry leaf extract (MLE) might inhibit migration of VSMC. MLE was rich in polyphenols (44.82%), including gallic acid, protocatechuic acid, catechin, gallocatechin gallate, caffeic acid, epicatechin, rutin, and quercetin. MLE could inhibit the migration of A7r5 cells in a dose- and time-dependent manner. MLE also inhibited the activities of matrix metalloproteinases (MMPs) MMP-2 and MMP-9, protein expressions, and phosphorylation of FAK and Akt, and protein expressions of small guanosine triphosphatases (GTPases: c-Raf, Ras, Rac1, Cdc42, and RhoA) in a dose-dependent manner. NF-kappaB expression was also inhibited by MLE. MLE could effectively inhibit the migration of VSMC by blocking small GTPase and Akt/NF-kappaB signals.

Extract from the leaf of nucifera reduced the development of atherosclerosis via inhibition of vascular smooth muscle cell proliferation and migration.

Nelumbo nucifera GAERTN, a perennial aquatic plant, has been used as a medicinal herb in China and India. We have previously reported that consumption of nucifera leaf extract (NLE) reduced the development of atherosclerosis in cholesterol-fed rabbits; however, the molecular mechanisms involved were unclear. Atherosclerotic plaque is generated partly by proliferation and migration of vascular smooth muscle cells (VSMC). Herein, we demonstrated that VSMC treated with NLE-triggered apoptosis and affected the JNK and p38 Mitogen-Activated Protein Kinase (MAPK) pathways. Pre-treating VSMC with inhibitors of JNK, p38, and p53 reduced NLE-induced apoptosis. Non-cytotoxic doses of NLE also abolished secretion of MMP-2/9 and inhibited cell migration via restraining the FAK/PI 3-kinase/small G protein pathway. Histopathological examination showed that 1.0% of NLE reduced neointima formation conspicuously and inhibited smooth muscle cell proliferation and MMP-2 secretion in the blood vessel of rabbits fed with a high cholesterol diet (HCD). We also verified that the extracts total phenolic acids and the total flavonoids were approximately about 70%. In conclusion, our results shed light on the molecular mechanisms whereby the polyphenol-rich water extract of nucifera leaves could inhibit both proliferation and migration of VSMC, and it might serve as a potential anti-atherogenic agent.

Mulberry extract inhibits oleic acid-induced lipid accumulation via reduction of lipogenesis and promotion of hepatic lipid clearance

BACKGROUND Mulberries are a traditional edible food used to treat hepatic disease. The anti-obesity and hypolipidemic effects of mulberry water extracts (MWE) have attracted increasing interest. In the present study, MWE were assessed for their hepatic lipid-lowering potential when administered in fatty acid overload conditions in HepG2 cells. RESULTS We found that MWE significantly reduced lipid accumulation, suppressed fatty acid synthesis, and stimulated fatty acid oxidation. Furthermore, MWE also inhibited acetyl coenzyme A carboxylase activities by stimulating adenosine monophosphate-activated protein kinase (AMPK). MWE attenuated the expression of sterol regulatory element-binding protein-1 (SREBP-1) and its target molecules, such as fatty acid synthase. Similar results were also measured in the expressions of enzymes involved in triglyceride and cholesterol biosyntheses including glycerol-3-phosphate acyltransferase, 3-hydroxy-3-methylglutaryl-CoA reductase, and SREBP-2. In contrast, the lipolytic enzyme expression of peroxisome proliferator activated receptor α and carnitine palmitoyltransferase-1 were increased. CONCLUSIONS Our study suggests that the hypolipidemic effects of MWE occur via phosphorylation of AMPK and inhibition of lipid biosynthesis. Therefore, the mulberry extract may be active in the prevention of fatty liver.

Polyphenol-rich extract from mulberry leaf inhibits vascular smooth muscle cell proliferation involving upregulation of p53 and inhibition of cyclin-dependent kinase.

This study was carried out to investigate the impact of polyphenol-rich extract from mulberry leaf on the proliferation of vascular smooth muscle cell (VSMC) and verify its mechanism in vitro. VSMC proliferation is an important pathophysiological process in the development of atherosclerosis, which is the major cause of coronary artery disease (CAD). Polyphenol-rich foods, such as mulberry leaf, have been reported to reduce the risk of CAD. The effect of mulberry leaf extract (MLE) on cell growth was measured by a growth curve assay, on distribution of cells in the cell cycle by flow cytometry, and on cyclin-dependent kinase (CDK) activity and cell-cycle regulatory proteins by Western blot, immunoblotting, and immunoprecipitation analyses. The results showed that MLE induced phosphorylation of p53, promoted expression of p21 and p27, decreased CDK2/4 activity, inhibited phosphorylation of Rb, and thereby blocked the G1 to S transition in the cell cycle.

Mulberry water extracts (MWEs) ameliorated carbon tetrachloride-induced liver damages in rat.

Mulberry extracts are antidiabetic and antihyperlipidemic, as well as preventive of cardiovascular disease. The current study investigates the protective mechanisms of mulberry water extracts (MWEs) in carbon tetrachloride (CCl(4))-induced hepatic injury. Oral administration of MWEs significantly reduced the lipid peroxidation triggered by CCl(4), as shown by the reduced production of thiobarituric acid-reactive substance (TBARS). The levels of serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also reduced via cotreatment with MWEs compared with CCl(4) treatment alone. Cotreatment with MWE evidently reduced CCl(4)-induced liver weight and inhibited lipid deposition and fibrogenesis. In a similar manner, cotreatment with silymarin, a well-known liver protective agent, also reversed the CCl(4)-induced effects, such as reduced TBARS formation, decreased serum AST, ALT, and ALP levels, blocked lipid accumulation, and liver fibrosis. Furthermore, MWEs attenuated the proinflammatory genes such as cyclooxygenase 2, nuclear factor kappa B, and inducible nitric oxide synthase expression. The current findings suggest that MWEs such as silymarin exhibit protective and curative effects against CCl(4)-induced liver damage and fibrosis via decreased lipid peroxidation and inhibited proinflammatory gene expression.

Simvastatin inhibits cell cycle progression in glucose-stimulated proliferation of aortic vascular smooth muscle cells by up-regulating cyclin dependent kinase inhibitors and p53.

Simvastatin was reported to attenuate platelet-derived growth factor (PDGF)-induced vascular smooth muscle proliferation by up-regulation of cyclin dependent kinase (CDK) inhibitor p27, but had no effect on p16, p21, p53 expression. We investigate the mechanisms by which simvastatin inhibits vascular smooth muscle cell (VSMC) growth in high glucose conditions to mimic diabetes. Simvastatin was added to A7r5 cells cultured in high glucose (25 mM) medium, mimicking diabetes. We used an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate cell viability; flow cytometric analysis for cell counts distribution in the cell cycle; and Western blot, immunoblotting, and immunoprecipitation analyses to evaluate the effects of simvastatin on CDK activity and cell cycle regulatory proteins. Cell counts were significantly increased in G0/G1 phase and significantly decreased in S and G2/M phases. In our study, low dose of simvastatin had no significant inhibitory effect on VSMC growth in normal glucose condition. However, both low and high doses of simvastatin inhibited VSMC growth significantly in a dose-dependent manner in high glucose status. We also found that simvastatin inhibited phosphorylation of Rb, promoted expression of p53, p16, p21, p27 and decreased CDK2/4 activity. In conclusion, simvastatin inhibits VSMC proliferation in high glucose status, mimicking diabetes, inducing a G0/G1 phase cell cycle growth arrest by acting on multiple steps upstream of pRb, including inhibition of CDK2/4 expression and up-regulation of p53, p21, p16, and p27. We propose that statins may be used more extensively in diabetic patients regardless of lipid status for preventing atherosclerosis and restenosis after PCI.

Mulberry water extracts inhibit rabbit atherosclerosis through stimulation of vascular smooth muscle cell apoptosis via activating p53 and regulating both intrinsic and extrinsic pathways.

Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in rabbits. Apoptosis of vascular smooth muscle cells (VSMCs) is the key determinant of the number of VSMCs in remodeling. To improve the recovery from atherosclerosis pathology, it would be ideal to induce regression of atherosclerotic plaques and apoptosis of VSMCs. In this study, we treated high-cholesterol-diet-fed (HCD-fed) rabbits with MWEs, and we found that the MWEs effectively inhibited HCD-fed-induced intimal hyperplasia of vessel walls. We also found that MWEs initially activate JNK/p38 and p53, which in turn activate both Fas-ligand and mitochondria pathways, thereby causing mitochondria translocation of Bax and the reduction of Bcl-2 that trigger the cleavage of procaspases, finally resulting in apoptosis of VSMCs. In addition, 2.5-5.0 g/day of MWEs for humans may be enough to prevent atherosclerosis.

Mulberry Water Extracts Inhibit Atherosclerosis through Suppression of the Integrin-β3/Focal Adhesion Kinase Complex and Downregulation of Nuclear Factor κB Signaling in Vivo and in Vitro

Previous studies have shown that mulberry water extracts (MWEs), which contain polyphenolic compounds, have an antiatherosclerotic effect in vivo and in vitro through stimulating apoptosis of vascular smooth muscle cells (VSMCs). Histological analysis was performed on atherosclerotic lesions from high-cholesterol diet (HCD)-fed rabbits after treatment with 0.5-1% MWEs for 10 weeks. Immunohistochemistry showed that the expressions of SMA, Ras, and matrix metalloproteinase-2 in the VSMCs were dose-dependently inhibited after MWE treatment. The antimigratory effects of MWEs on A7r5 VSMCs were assessed by western blot analysis of migration-related proteins, visualization of F-actin cytoskeleton, and reverse transcription polymerase chain reaction. The results showed that MWEs inhibited VSMC migration through reducing interactions of the integrin-β3/focal adhesion kinase complex, alterations of the cytoskeleton, and downregulation of glycogen synthase kinase 3β/nuclear factor κB signaling. Taken together, MWEs inhibited HCD-induced rabbit atherogenesis through blocking VSMC migration via reducing interactions of integrin-β3 and focal adhesion kinase and downregulating migration-related proteins.

Impact of polyphenolic components from mulberry on apoptosis of vascular smooth muscle cells

BACKGROUND Previous studies have shown that mulberry polyphenolic compounds have an anti-atherosclerotic effect in rabbits. Apoptosis of vascular smooth muscle cells (VSMCs) is the key determinant of the number of VSMCs in remodeling. To examine the effect of mulberry polyphenol extracts (MPEs) on the apoptosis of VSMCs and thus the prevention of atherosclerosis, this study investigated the ability of MPEs to induce apoptosis in vitro and the underlying mechanism. RESULTS It was found that MPEs initially activated JNK/p38 and p53, which in turn activated both Fas-ligand and mitochondrial pathways, thereby causing mitochondrial translocation of Bax and a reduction in Bcl-2. This then triggered the cleavage of procaspases, finally resulting in apoptosis of VSMCs. CONCLUSION This study shows that MPEs may suppress atherosclerosis through stimulating apoptosis of VSMCs via activating JNK/p38 and p53 signaling.