Antihypertensive Effect of a Combination of Uracil and Glycerol Derived from Lactobacillus plantarum Strain TWK10-Fermented Soy Milk

We previously demonstrated that angiotensin-converting enzyme (ACE) could be inhibited by soy milk that had been fermented with the Lactobacillus plantarum strain TWK10, suggesting great potential for the development of antihypertensive products. In this work, the bioactive ACE inhibitors in TWK10-fermented soy milk water extracts were isolated, and a combination of uracil and glycerol (CUG) was identified as one of the ACE inhibitors. We then examined the physiological effects of CUG treatment in short-term and long-term studies using spontaneously hypertensive rats (SHRs) as an experimental model. The results revealed that the fermented soy milk extracts and CUG decreased blood pressure by 11.97 ± 3.71 to 19.54 ± 9.54 mmHg, 8 h after oral administration, and exhibited antihypertensive effects in SHRs in a long-term study. In addition, CUG was shown to decrease blood pressure by suppressing either the renin activity or the ACE activity and, thus, decreasing the downstream vasoconstricting peptide angiotensin II and the hormone aldosterone. CUG also promoted nitric oxide production, resulting in vasodilation and further improvement to hypertension. This important finding suggests that TWK10-fermented soy milk and its functional ingredients, uracil and glycerol, exhibit antihypertensive effects via multiple pathways and provide a healthier and more natural antihypertensive functional food

Graphical scheme of the anti-proliferative mechanism of viscolin in PDGF-treated HASMCs.

<p>Graphical scheme of the anti-proliferative mechanism of viscolin in PDGF-treated HASMCs.</p

Viscolin induces G0/G1 arrest and alters the expression of cell cycle regulatory proteins in PDGF-BB-treated HASMCs.

<p>(A) Cells were left untreated or were treated with 40 μM viscolin for 24 h and then with PDGF-BB for an additional 24 h. The proportions of cells in the G0/G1, S, or G2/M phases were determined by FACScan analysis. The mean±SEM of three independent experiments were shown. *<i>P</i><0.05 vs. the untreated group; ^†<i>P</i><0.05 vs. the PDGF-BB-treated group. (B-G) Serum-starved HASMCs were cultured in the presence of 10, 20, 30, or 40 μM viscolin for 24 h and then treated with 30 ng/mL of PDGF-BB for an additional 24 h. (B) The expression of Cyclin D1, (C) Cyclin E, (D) p27^Kip1, (E) CDK4, (F) CDK2, (G) or p21^Cip1 proteins was determined by Western blot analysis. GAPDH or α-tubulin was processed in parallel as an internal control for protein loading. The results are shown as the fold increase in expression relative to that in untreated controls. The data are the means ± SEM (n = 4). *<i>P</i><0.05 vs. the untreated controls; ^†<i>P</i><0.05 vs. the PDGF-BB-treated cells.</p

Effects of MAPK inhibitors on cell cycle regulator protein expression in PDGF-BB-treated HASMCs.

<p>Cells were treated with 30 μM MAPK inhibitors for 1 h, and then incubated with 30 ng/mL PDGF-BB for 24 h. The expression of (A) Cyclin D1, (B) Cyclin E, (C) p27^Kip1, (D) CDK4, (E) CDK2, (F) or p21^Cip1 proteins was determined by Western blot analysis. β-actin was processed in parallel as an internal control for protein loading. The histograms show the phosphorylated band/β-actin ratio (A-F). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the PDGF-BB-treated cells.</p

Effects of ERK, P38, and JNK-specific siRNA on cell cycle regulator protein expression in PDGF-BB-treated HASMCs.

<p>The expression of the (A) Cyclin D1, (B) CDK4, (C) Cyclin E, (D) CDK2, and (E) p21^Cip1 proteins was determined by Western blot analysis after ERK, P38, and JNK silencing, viscolin and PDGF-BB treatment. (F) ERK, P38, and JNK-specific siRNAs reduced the total levels of the ERK, P38, and JNK proteins compared with the non-treated cells. The values are the means±SEM. *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the PDGF-BB-treated cells.</p

Effects of viscolin expression on the phosphorylation of MAPKs and AKT in PDGF-BB-treated HASMCs.

<p>Cells were treated with 40 μM viscolin for 24 h and then incubated with or without 30 ng/mL PDGF-BB for 30 min. Phosphorylated (A) JNK, (B) P38, (C) ERK1/2, or (D) AKT levels were determined by Western blot analysis. GAPDH was processed in parallel as an internal control for protein loading. The histograms show the phosphorylated band/GAPDH ratio for (A-D). *<i>P</i><0.05 vs. the untreated control, ^†<i>P</i><0.05 vs. the PDGF-BB-treated cells.</p

Viscolin reduces neointimal hyperplasia <i>in vivo</i>.

<p>(A) Representative cross-sections of injured femoral arteries from ED/saline-injected mice (left) and ED/viscolin-injected mice (right) were stained with Resorcin–Fuchsin. Neointimal hyperplasia was shown between two arrows. Bar = 50 μm. (B) Quantification of the neointima/media (I/M) area ratio, intimal area, luminal area, and medial area. The values are means±SEM. *<i>P</i><0.05 vs. the ED/saline-injected mice. (C) Immunohistochemical staining with anti-PCNA antibodies. The positive reaction and internal elastic lamina are indicated by arrowheads and an arrow, respectively. Bar = 50 μm. (D) Immunohistochemical staining for the smooth muscle cell marker, α-actin. The internal elastic lamina is indicated by an arrow. Bar = 50 μm.</p

Viscolin Inhibits <i>In Vitro</i> Smooth Muscle Cell Proliferation and Migration and Neointimal Hyperplasia <i>In Vivo</i>

<div><p>Viscolin, an extract of <i>Viscum coloratum</i>, has anti-inflammatory and anti-proliferative properties against harmful stimuli. The aim of the study was to examine the anti-proliferative effects of viscolin on platelet derived growth factor-BB (PDGF)-treated human aortic smooth muscle cells (HASMCs) and identify the underlying mechanism responsible for these effects. Viscolin reduced the PDGF-BB-induced HASMC proliferation and migration <i>in vitro</i>; it also arrested HASMCs in the G0/G1 phase by decreasing the protein expression of Cyclin D1, CDK2, Cyclin E, CDK4, and p21^Cip1 as detected by Western blot analysis. These effects may be mediated by reduced PDGF-induced phosphorylation of ERK1/2, JNK, and P38, but not AKT as well as inhibition of PDGF-mediated nuclear factor (NF)-κB p65 and activator protein 1 (AP-1)/c-fos activation. Furthermore, viscolin pre-treatment significantly reduced neointimal hyperplasia of an endothelial-denuded femoral artery <i>in vivo</i>. Taken together, viscolin attenuated PDGF–BB-induced HASMC proliferation <i>in vitro</i> and reduced neointimal hyperplasia <i>in vivo</i>. Thus, viscolin may represent a therapeutic candidate for the prevention and treatment of vascular proliferative diseases.</p></div

Viscolin reduces the migration and proliferation of PDGF-BB-treated HASMCs.

<p>Serum-starved HASMCs were cultured with PDGF-BB (PDGF) alone or with viscolin (VIS). (A) Cell viability was measured using the MTT assay. (B) Cell death was assessed using the TUNEL assay. Cells treated with 1 mg/mL DNase I were the positive controls (PC). (C) BrdU incorporation was used to determine HASMC proliferation. Some cells were pretreated with 30 μM MAPK inhibitors for 30 min prior to treatment with viscolin or PDGF-BB. Representative photographs are shown in the left panel, and the quantitative data relative to the control value (without any treatment) were shown in the right panel. (D) Cell migration was examined in a wound healing assay. Serum-starved HASMCs were wounded by scratch injury (black lines on Fig). The wound closure area was determined by measuring the wound area/initial wound area. Before wound formation and PDGF-BB and or/ viscolin treatment, some cells were pretreated with an ERK1/2 inhibitor (PD98059; PD), P38 inhibitor (SB203580; SB) or JNK inhibitor (SP600125; SP) for 30 min. Representative inverted phase contrast light microscopy photographs are shown in the left panel and the quantified data are shown in the right panel. In A, C, and D, the data are means±SEM for four independent experiments. *<i>P</i><0.05 vs. the untreated group; ^†<i>P</i><0.05 vs. the PDGF-BB-treated group; ^‡<i>P</i><0.05 vs. the viscolin/PDGF-BB group. The scale bars in B- D = 100 μm.</p

Physalin F Induces Cell Apoptosis in Human Renal Carcinoma Cells by Targeting NF-kappaB and Generating Reactive Oxygen Species

<div><h3>Background</h3><p>The aim of this study was to determine the molecular mechanisms of physalin F, an effective purified extract of <em>Physalis angulata</em> L. (Solanacae), in renal carcinoma A498 cells.</p> <h3>Methodology/Principal Findings</h3><p>Physalin F was observed to significantly induce cytotoxicity of three human renal carcinoma A498, ACHN, and UO-31 cells in a concentration-dependent manner; this was especially potent in A498 cells. The physalin F-induced cell apoptosis of A498 cells was characterized by MTT assay, nuclear DNA fragmentation and chromatin condensation. Using flow cytometry analysis, physalin F induced A498 cell apoptosis as demonstrated by the accumulation of the sub-G1 phase in a concentration- and time-dependent manner. Moreover, physalin F-mediated accumulation of reactive oxygen species (ROS) caused Bcl-2 family proteins, Bcl-2, and Bcl-xL degradation, which led to disruption of mitochondrial membrane potential and release of cytochrome <em>c</em> from the mitochondria into the cytosol. These effects were associated with induction of caspase-3 and caspase-9 activity, which led to poly(ADP-ribose) polymerase cleavage. However, the antioxidant <em>N</em>-acetyl-_L-cysteine (NAC) and glutathione (GSH) resulted in the inhibition of these events and reversed physalin F-induced cell apoptosis. In addition, physalin F suppressed NF-κB activity and nuclear translocation of p65 and p50, which was reversed by NAC and GSH.</p> <h3>Conclusion</h3><p>Physalin F induced cell apoptosis through the ROS-mediated mitochondrial pathway and suppressed NF-κB activation in human renal cancer A498 cells. Thus, physalin F appears to be a promising anti-cancer agent worthy of further clinical development.</p> </div