Capsaicin inhibits aortic valvular interstitial cell calcification via the redox-sensitive NFκB/AKT/ERK1/2 pathway.

Calcific aortic valve stenosis (CAVS), the third most prevalent cardiovascular disorder is known to impose a huge social and economic burden on patients. However, no pharmacotherapy has yet been established. Aortic valve replacement is the only treatment option, although its lifelong efficacy is not guaranteed and involves inevitable complications. So, there is a crucial need to find novel pharmacological targets to delay or prevent CAVS progression. Capsaicin is well known for its anti-inflammatory and antioxidant properties and has recently been revealed to inhibit arterial calcification. We thus investigated the effect of capsaicin in attenuating aortic valve interstitial cells (VICs) calcification induced by pro-calcifying medium (PCM). Capsaicin reduced the level of calcium deposition in calcified VICs, along with reductions in gene and protein expression of the calcification markers Runx2, osteopontin, and BMP2. Based on Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway analysis oxidative stress, AKT and AGE-RAGE signaling pathways were selected. The AGE-RAGE signaling pathway activates oxidative stress and inflammation-mediated pathways including ERK and NFκB signaling pathways. Capsaicin successfully inhibited oxidative stress- and reactive oxygen species-related markers NOX2 and p22phox. The markers of the AKT, ERK1/2, and NFκB signaling pathways, namely, phosphorylated AKT, ERK1/2, NFκB, and IκBα were upregulated in calcified cells, while being significantly downregulated upon capsaicin treatment. Capsaicin attenuates VICs calcification in vitro by inhibition of redox-sensitive NFκB/AKT/ERK1/2 signaling pathway, indicating its potential as a candidate to alleviate CAVS.

Taxifolin as a Major Bioactive Compound in the Vasorelaxant Effect of Different Pigmented Rice Bran Extracts.

Cardiovascular disease is one of the leading causes of morbidity and mortality in recent years. The intake of polyphenol rich diets has been associated with improved cardiovascular function and reduced cardiovascular risks. Oryza sativa L. is one of the most common cereals worldwide. Rice bran, a byproduct of the rice milling process, contains many bioactive ingredients, including polyphenols, polysaccharides, proteins, and micronutrients. It is also consumed as a healthy diet in the form of rice bran oil and powder in many Asian countries like Japan, South Korea, and India for its several health benefits as a natural antioxidant. Thus, this study evaluated the vasorelaxant effect of ethanolic extracts of brown, green, red, and black rice bran and investigated its underlying vasorelaxant mechanism. Among the four rice bran extracts (RBEs) examined, the red rice bran extract (RRBE) had a strong endothelium-dependent vasorelaxant effect, which was markedly prevented by N-ω-nitro-L-arginine [endothelial nitric oxide synthase (eNOS) inhibitor], wortmannin [phosphoinositide-3 kinase (PI3K) inhibitor], and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (inhibitor of guanylate cyclase). Likewise, RRBE induced the phosphorylation of eNOS and Src in cultured endothelial cells, thereby stimulating NO formation. Altogether, these findings propose that RRBE induces endothelium-dependent relaxation, involving at least in part, NO-mediated signaling through the PI3K/eNOS pathway. Further, LC-PDA analysis conducted on the four RBEs also revealed that RRBE highly contained taxifolin, which is an active flavanonol that induces endothelium-dependent vasorelaxation, compared to other RBEs. Subsequently, the underlying mechanism of taxifolin was assessed through vascular reactivity studies with pharmacological inhibitors similar to that of RRBE. These findings deciphered a distinct difference in vasorelaxant effects between RRBE and the other RBEs. We also observed that RRBE induced a potent endothelium-dependent NO-mediated relaxation in coronary artery rings, which involved the Src/PI3K pathway that activates eNOS. Additionally, taxifolin exhibited, at least in part, similar vasoprotective effects of RRBE. Therefore, we propose that RRBE may serve as natural sources of functional phytochemicals that improve cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

Prevention of Fine Dust-Induced Vascular Senescence by Humulus lupulus Extract and Its Major Bioactive Compounds.

Both short- and long-term exposure to fine dust (FD) from air pollution has been linked to various cardiovascular diseases (CVDs). Endothelial cell (EC) senescence is an important risk factor for CVDs, and recent evidence suggests that FD-induced premature EC senescence increases oxidative stress levels. Hop plant (Humulus lupulus) is a very rich source of polyphenols known to have nutritional and therapeutic properties, including antioxidant behavior. The aims of this study were to evaluate whether Humulus lupulus extract prevents FD-induced vascular senescence and dysfunction and, if so, to characterize the underlying mechanisms and active components. Porcine coronary arteries and endothelial cells were treated with FD in the presence or absence of hop extract (HOP), and the senescence-associated-beta galactosidase (SA-ß-gal) activity, cell-cycle progression, expression of senescence markers, oxidative stress level, and vascular function were evaluated. Results indicated that HOP inhibited FD-induced SA-ß-gal activity, cell-cycle arrest, and oxidative stress, suggesting that HOP prevents premature induction of senescence by FD. HOP also ameliorated FD-induced vascular dysfunction. Additionally, xanthohumol (XN) and isoxanthohumol (IX) were found to produce the protective effects of HOP. Treatment with HOP and its primary active components XN and IX downregulated the expression of p22phox, p53, and angiotensin type 1 receptor, which all are known FD-induced redox-sensitive EC senescence inducers. Taken together, HOP and its active components protect against FD-induced endothelial senescence most likely via antioxidant activity and may be a potential therapeutic agent for preventing and/or treating air-pollution-associated CVDs.

Endothelium-Dependent Relaxation Effects of Actinidia arguta Extracts in Coronary Artery: Involvement of eNOS/Akt Pathway.

Cardiovascular diseases (CVD) are the major cause of death globally. Bioavailability of nitric oxide, antioxidative activity, and regulation of ionic homeostasis are the key targets for prevention of CVD. Actinidia arguta (AA) has shown promising effect for anticancer, anti-hypercholesterolemia, and antioxidant agents. However, the vascular effect of AA remains unclear. Therefore, we investigated the vascular relaxation of AA extract as well as the underlying mechanisms. Vascular reactivity was assessed in organ baths using porcine coronary arteries and antioxidant properties were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Methanol extract of AA stem (AASE) induced significantly vasorelaxation of porcine coronary artery and its effects is endothelium-dependent without cytotoxicity effects. In addition, ASSE scavenged reactive oxygen species (ROS) in vitro and strongly inhibited NADPH-oxidase activity, which is major source of ROS in vasculature. AASE strongly and dose-dependently activate endothelial nitric oxide synthase (eNOS), the major vascular protective enzyme, and Akt, the upstream signaling protein of eNOS, in porcine coronary artery endothelial cell. Altogether, these results have demonstrated that AASE is a potent endotheliumdependent vasodilator and this effect was involved in, at least in part, Akt/eNOS/NO pathway with strong anti-oxidant properties. The present findings indicate that AA stem could be a valuable candidate of herbal medicine for cardiovascular diseases associated with endothelial dysfunction and atherosclerosis.

Smooth Muscle Cell Derived Microparticles Acts as Autocrine Activation of Smooth Muscle Cell Proliferation by Mitogen Associated Protein Kinase Upregulation.

Microparticles (MP); also know as microvesicles are extracellular vesicles (0.1 to 1.0 µm) released by cells in response to cell activation or apoptosis. The high level of circulating MP is one of the important indicators of altered vascular function. Vascular smooth muscle cell (VSMC) derived MP could mediate proliferation and migration of VSMC leading to vascular inflammation. Proliferation of VSMC is mediated by mitogen associated protein kinase (MAPK) and proliferation cell nuclei antigen (PCNA) signaling pathway whereas migration is mediated by metalloproteinase and cytoskeletal remodeling pathway. In this study, VSMC-MP were isolated from confluent VSMC stimulated with tumor necrosis factor (TNF)-α. VSMC-MP were treated to VSMC to investigate the VSMC proliferation marker using in vitro assay. In comparison to normal (VSMC-MP untreated group), VSMC-MP treatment results in proliferation of VSMC as revealed by MTT assay. VSMC-MP and TNF-α induce proliferation by 34% and 67% respectively. VSMC-MP also induce over expression of PCNA in both immuno-fluorescence and western blot experiment. VSMC-MP and TNF-α increase the PCNA expression by 1.86-fold and 1.95-fold respectively. Similarly, VSMC-MP treatment results in over expression of MAPK pathway protein expression in VSMC. As compared to normal, the MAPK protein (pERK1/2, pP38 and pJNK) were increased by 1.41-fold, 1.42-fold and 1.48-fold, respectively in VSMC-MP treated VSMC. Our results provide the evidence of VSMC-MP involvement in proliferation of VSMC. Thus, VSMC-MP could be considered as a potential marker of vascular inflammatory disorder such as atherosclerosis.

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery.

Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect the cardiovascular system, in part, by stimulating the endothelial formation of nitric oxide (NO). EPA:DHA 6:1 has been identified as a potent omega 3 PUFA formulation to induce endothelium-dependent vasorelaxation and activation of endothelial NO synthase (eNOS). This study examined whether intake of EPA:DHA 6:1 (500 mg/kg/day) for 2 weeks improves an established endothelial dysfunction in old rats (20 months old), and, if so, the underlying mechanism was subsequently determined. In the main mesenteric artery rings, an endothelial dysfunction characterized by a blunted NO component, an abolished endothelium-dependent hyperpolarization component, and increased endothelium-dependent contractile responses (EDCFs) are observed in old rats compared to young rats. Age-related endothelial dysfunction was associated with increased vascular formation of reactive oxygen species (ROS) and expression of eNOS, components of the local angiotensin system, senescence markers, and cyclooxygenase-2 (COX-2), and the downregulation of COX-1. The EPA:DHA 6:1 treatment improved the NO-mediated relaxation, reduced the EDCF-dependent contractile response and the vascular formation of ROS, and normalized the expression level of all target proteins in the old arterial wall. Thus, the present findings indicate that a 2-week intake of EPA:DHA 6:1 by old rats restored endothelium-dependent NO-mediated relaxations, most likely, by preventing the upregulation of the local angiotensin system and the subsequent formation of ROS.

Fine air pollution particles induce endothelial senescence via redox-sensitive activation of local angiotensin system.

Fine dust (FD) is a form of air pollution and is responsible for a wide range of diseases. Specially, FD is associated with several cardiovascular diseases (CVDs); long-term exposure to FD was shown to decrease endothelial function, but the underlying mechanism remains unclear. We investigated whether exposure to FD causes premature senescence-associated endothelial dysfunction in endothelial cells (ECs) isolated from porcine coronary arteries. The cells were treated with different concentrations of FD and senescence associated-beta galactosidase (SA-ß-gal) activity, cell cycle progression, expression of endothelial nitric oxide synthase (eNOS), oxidative stress level, and vascular function were evaluated. We found that FD increased SA-ß-gal activity, caused cell cycle arrest, and increased oxidative stress, suggesting the premature induction of senescence; on the other hand, eNOS expression was downregulated and platelet aggregation was enhanced. FD exposure impaired vasorelaxation in response to bradykinin and activated the local angiotensin system (LAS), which was inhibited by treatment with the antioxidant N-acetyl cysteine (NAC) and angiotensin II receptor type 1 (AT1) antagonist losartan (LOS). NAC and LOS also suppressed FD-induced SA-ß-gal activity, increased EC proliferation and eNOS expression, and improved endothelial function. These results demonstrate that FD induces premature senescence of ECs and is associated with increased oxidative stress and activation of LAS. This study can serve as a pharmacological target for prevention and/or treatment of air pollution-associated CVD.

Combination of Garcinia cambogia Extract and Pear Pomace Extract Additively Suppresses Adipogenesis and Enhances Lipolysis in 3T3-L1 Cells.

BACKGROUND: Inhibition of adipogenesis has been a therapeutic target for reducing obesity and obesity-related disorders such as diabetes, hypertension, atherosclerosis, and cancer. For decades, anti-adipogenic potential of many herbal extracts has been investigated. One example is Garcinia cambogia extract (GE) containing (-)-hydroxycitric acid as an active ingredient. GE is currently marketed as a weight loss supplement, used alone or with other ingredients. Pear pomace extract (PE), another natural product, has been also shown to have anti-adipogenic activity in a recent report. OBJECTIVE: It was tested if the mixture of PE and GE (MIX) would produce more effective anti-adipogenic activity than PE or GE alone. MATERIALS AND METHODS: Differentiation of 3T3-L1 preadipocyte was induced by adding insulin, dexamethasone, and isobutylmethylxanthine and lipid accumulation was measured by Oil Red O staining. Cellular markers for adipogenesis and lipolysis such as CCAAT/enhancer binding protein (C/EBP-α), peroxisome proliferator-activated receptor gamma (PPAR-γ), fatty acid synthase (FAS), and hormone-sensitive lipase (HSL) was measured using immunocytochemistry. RESULTS: MIX, compared to PE or GE alone, showed greater inhibition of lipid accumulation. Furthermore, MIX reduced the expression of adipogenesis-related factors C/EBP-α, PPAR-γ, and FAS more than PE or GE alone did. In contrast, the expression of HSL the enzyme required for lipolysis was further enhanced in MIX-treated adipocytes compared to the PE or GE alone treated groups. CONCLUSIONS: Anti-adipogenic effect of PE and GE appears synergistic, and the MIX may be a useful therapeutic combination for the treatment of obesity and obesity-related diseases. SUMMARY: PE and GE efficiently inhibited adipocyte differentiation by suppressing the expression of adipogenic transcription factor CEBP-α and PPAR-γ.PE and GE significantly decreased the expression of adipogenic enzyme FAS.PE and GE increased the expression of lipid degrading enzyme HSL.Mixture of PE and GE exhibited additive or moderately synergistic effect on adipocyte differentiation and lipid accumulation. Abbreviations used: CEBP-a: CCAT/enhancer binding protein alpha, CI: Combination Index, FAS: Fatty acid synthase, GE: Garcinia cambogia extract, HSL: Hormone sensitive lipase, PE: Pear pomace extract, PPAR-γ: Peroxisome proliferator-activated receptor gamma.

Potential mechanisms underlying cardiovascular protection by polyphenols: Role of the endothelium.

Epidemiological studies have indicated that regular intake of polyphenol-rich diets such as red wine and tea, are associated with a reduced risk of cardiovascular diseases. The beneficial effect of polyphenol-rich products has been attributable, at least in part, to their direct action on the endothelial function. Indeed, polyphenols from tea, grapes, cacao, berries, and plants have been shown to activate endothelial cells to increase the formation of potent vasoprotective factors including nitric oxide (NO) and to delay endothelial ageing. Moreover, intake of such polyphenol-rich products has been associated with the prevention and/or the improvement of an established endothelial dysfunction in several experimental models of cardiovascular diseases and in Humans with cardiovascular diseases. This review will discuss both experimental and clinical evidences indicating that polyphenols are able to promote endothelial and vascular health, as well as the underlying mechanisms.

Preparation and In Vitro Evaluation of Elastic Nanoliposomes for Topical Delivery of Highly Skin-Permeable Growth Factors.

Percutaneous delivery of growth factors is often used to treat wounds, and for cosmetic purposes, as a way of accelerating healing and skin regeneration, respectively. However, the therapeutic effects of growth factors are diminished by their poor absorption when delivered percutaneously, in addition to their rapid degradation by proteinases. To overcome these obstacles, we constructed two skin-permeable compounds. Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) were both genetically paired with low-molecular-weight protamine (LMWP), to yield the compounds LMWP-bFGF and LMWP-VEGF-A, respectively. The molecular weights and N-terminal amino acid sequences of LMWP-bFGF and LMWP-VEGF-A confirmed that the N-terminus-specific conjugation of LMWP with bFGF and VEGF-A had been successful. The biological abilities of the native factors to stimulate human fibroblast (CCD-986sk) and endothelial cell proliferation were preserved. Both compounds significantly promoted wound (scratch) recovery and enhanced procollagen type I C-peptide synthesis in CCD-986sk cells (to levels 184 and 133% those of the native compounds, respectively). The LMWP-conjugated growth factors were significantly more permeable than the native forms (by 7.29- and 29.22-fold, respectively). Finally, encapsulation of the compounds in positively charged elastic nanoliposomes (115 ± 1.54 nm in diameter with a zeta potential of 57.2 ± 3.05 mV) further improved both permeation and stability. Thus, nanoliposomes loaded with LMWP-conjugated growth factors are expected to enhance skin regeneration; the materials will find applications in wound-healing therapies and anti-wrinkle cosmetics.

Rice Bran Extract Inhibits TMEM16A-Involved Activity in the Neonatal Rat Cochlea.

TMEM16A is a Ca²âº-activated Cl⁻ channel found in secretory glands, GI and respiratory tracts, and sensory organs, playing a major physiological role in fluid secretion, autonomous GI motility, and sensory transduction. In addition, overexpression of TMEM16A has been associated with cancer cell proliferation and invasion. Suppression of upregulated TMEM16A has been proposed as an effective anti-cancer strategy. While searching for a potential TMEM16A inhibitor, components of rice bran attracted our attention due to their anti-cancer potential in colon cancer cells, a type of cells known to overexpressing TMEM16A. Here, it was tested whether rice bran extract exhibits anti-TMEM16A activity. Rice bran extract was tested in the neonatal rat cochlear tissues where TMEM16A-involved spontaneous activity is generated as a part of normal development of the auditory pathway. Rice bran extract readily inhibited the TMEM16A-involved activity in the cochlear tissues and the effect was reversible upon washout. Taken together, rice bran extract appears to contain a putative TMEM16A inhibitor and the rice byproduct might serve as a source of a new anti-cancer agent.

Cacao Polyphenols Potentiate Anti-Platelet Effect of Endothelial Cells and Ameliorate Hypercoagulatory States Associated with Hypercholesterolemia.

Platelets are related to the formation of blood clots that play a crucial role in thrombosis and other cardiovascular diseases. Cocoa, derived from Theobroma cocoa, has been widely used as functional food for improving cardiovascular health. In the present study, the direct and indirect effects of cacao polyphenols (CPs) were investigated on human platelet aggregation associated with endothelial cells (ECs) senescence. In addition, the effect of CPs on high-fat diet- (HFD-) induced hypercoagulatory states in rats was evaluated. CPs directly inhibited the human platelet aggregation induced by thromboxane analogue, U46619, and treatment of CPs on senescent endothelial cells markedly restored inhibitory effect of ECs on platelet aggregation. Nitric oxide (NO) from ECs is known as modulator of platelet aggregation and CPs increased eNOS activity in ECs and coronary artery. In animal model, increased TG level induced by high fat diet (HFD) was significantly decreased by CPs administration. In addition, the HFD animal had shorter bleeding time, and CPs administration attenuated the HFD-induced changes. Taken together, the present study indicates that CPs have potent anti-platelet effects most likely by direct and indirect effect via ECs and have the potential for lowering the risk of cardiovascular disease-related hypercoagulation due to hypercholesterolemia.

The Effect of Quercus salicina Leaf Extracts on Vascular Endothelial Function: Role of Nitric Oxide.

Dysfunction of the vascular endothelium is reported as a hallmark of cardiovascular diseases. Many evidences suggest that polyphenols are associated with a decreased global mortality and might be involved in protection against cardiovascular risk. This beneficial effect of polyphenol may be due to many actions as antioxidant that increases bioavailability of nitric oxide, vasodilation or anti-hypertensive properties. To identify new natural medicine candidate for cardiovascular protection, plant extracts used in traditional medicine were evaluated by vascular reactivity system. Porcine coronary artery rings were suspended in organ chambers for the measurement of changes in isometric tension. Screening results indicated that the ethanolic extract of leaf from Quercus salicina (QSE) has been found to exhibit potent vasorelaxant activity. QSE dose-dependently induced endothelium-dependent relaxations, which were abolished by inhibitors of nitric oxide synthase (Nomega-nitro-L-arginine). In addition, QSE strongly and dose-dependently activate endothelial nitric oxide synthase (eNOS) in porcine coronary artery endothelial cell. Taken together, the present study has demonstrated that QSE is a powerful endothelium-dependentvasodilator and that this effect involves increased nitric oxide bioavailability. In conclusion, QSE could be a cardiovascular protective herbal medicine candidate associated with cardiovascular diseases and endothelial dysfunction.

Anthocyanidins, novel FAK inhibitors, attenuate PDGF-BB-induced aortic smooth muscle cell migration and neointima formation.

AIMS: Abnormal migration of human aortic smooth muscle cells (HASMCs) causes intimal thickening of the aorta, a pivotal step in atherosclerotic development. Although many studies have demonstrated that high anthocynidins intake confers protective effects against atherosclerosis, the direct molecular targets, and mechanisms of action responsible remain unclear. Here, we investigated the preventive effect of anthocyanidins on atherosclerosis and the underlying mechanisms involved. METHODS AND RESULTS: We analysed six major anthocyanidins, and found that petunidin exhibited the most potent inhibitory effects against platelet-derived growth factor (PDGF)-BB-induced HASMC migration in the Boyden chamber and wound healing assays. Petunidin also suppressed PDGF-BB-induced ex vivo rat aortic sprouting and in vivo rat neointima formation. Western blot analysis showed that petunidin inhibited PDGF-BB-induced phosphorylation of focal adhesion kinase (FAK) at the low concentration of 5 µM, whereas phosphorylation of Src, mitogen-activated protein kinases, and Akt was only slightly inhibited at 20 µM. In vitro and ex vivo FAK activity assays demonstrated that petunidin directly suppresses FAK activity by binding in an ATP non-competitive manner. Moreover, anthocyanidins that reduced HASMC migration also inhibited PDGF-BB-induced FAK phosphorylation, F-actin reduction, and FAK activity, and directly bound with FAK. PDGF-BB-induced migration, F-actin reduction by HASMCs, and ex vivo aortic sprouting were all inhibited by treatment with a commercial FAK inhibitor, PF-228. CONCLUSION: The results of the present study demonstrate that anthocyanidins can directly bind with and suppress the activity of FAK with atherosclerosis-preventive effects.

The limited intestinal absorption via paracellular pathway is responsible for the low oral bioavailability of doxorubicin.

1. Doxorubicin exhibited dose-independent pharmacokinetics after intravenous (5-20 mg/kg) and oral (20-100 mg/kg) administration to rats. Nearly all (82.1-99.7%) of the orally administered doxorubicin remained unabsorbed, and the hepatic first-pass extraction ratio and oral bioavailability of doxorubicin were approximately 0.5% and 1%, respectively. Based on these results, it is likely that the primary factor responsible for the low oral bioavailability of doxorubicin is the limited intestinal absorption, rather than the CYP3A4-mediated first-pass metabolism. 2. Moreover, the in vitro transport and cellular uptake studies using Caco-2 cell monolayers have revealed that doxorubicin crosses the intestinal epithelium primarily via the paracellular pathway (accounting for 85.6% of the overall absorptive transport) probably due to its physicochemical properties (hydrophilic cation; pKa = 9.67, log P = -0.5). These results suggest that P-glycoprotein (P-gp)-mediated efflux activity does not play a significant role in limiting the intestinal absorption of doxorubicin, attenuating the absorptive transport by only 5.56-13.2%. 3. Taken together, the present study demonstrated that the limited and paracellular intestinal absorption of doxorubicin was a major factor responsible for its low oral bioavailability, restricting the role of CYP3A4-mediated first-pass metabolism and P-gp-mediated efflux.

Enhanced IL-12p40 production in LPS-stimulated macrophages by inhibiting JNK activation by artemisinin.

Artemisinin can be isolated from Artemisia annua L. In addition to its well-known anti-malarial activity, artemisinin has antitumor and anti-microbial effects. In this study, we investigated the effect of artemisinin on the production of IL-12p40, which is important in the generation of T helper 1 responses. Artemisinin significantly induced IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. To elucidate the signaling molecules regulated by artemisinin in induced IL-12p40 production, the DNA-binding activity of several transcription factors and activation of mitogen-activated protein kinase (MAPK)s were investigated. The band intensities of NF-κB, AP-1, and SP1, and the activation of p38 MAPK and ERK were not changed by artemisinin. However, the induced phosphorylation of JNK was significantly decreased by artemisinin, and inhibition of the JNK signaling pathway further increased IL-12p40 production in LPS-stimulated RAW264.7 macrophage cells. Taken together, these data suggest that artemisinin induces the production of IL-12p40 in LPS-stimulated macrophage cells by inhibiting JNK activity.

Decaffeinated green tea extract improves hypertension and insulin resistance in a rat model of metabolic syndrome.

BACKGROUND: Oxidative stress and endothelial dysfunction are closely associated with hypertension and insulin resistance (IR) in metabolic syndrome (MetS). It is still controversial whether green tea extract (GTE) may have blood pressure (BP) lowering effect. Decaffeinated GTE might be presumed to have strong antioxidative effect and BP-lowering effect as compared with catechins. Thus we investigated whether decaffeinated-GTE could attenuate hypertension and IR by improving endothelial dysfunction and reducing oxidative stress in a rat model of MetS. METHODS AND RESULTS: 20 Otsuka Long-Evans Tokushima Fatty (OLETF) rats at 13 weeks old, MetS rats, were randomized into a saline treated group (OLETF; n = 10) and a group treated with decaffeinated-GTE (25 mg/kg/day) (GTE-OLETF; n = 10). Intraperitoneal glucose tolerance tests and BP measurements were performed at 13 and 25 weeks. Decaffeinated-GTE significantly reduced BP (OLETF vs. GTE-OLETF; 130 ± 7 vs. 121 ± 3 mmHg, p = 0.01), fasting/postprandial 2 h glucose (141 ± 18/159 ± 13 vs. 115 ± 7/132 ± 16 mg/dL, p = 0.009/0.002) and insulin levels (4.8 ± 2.3 vs. 2.4 ± 1.3 ng/mL, p < 0.001). Decaffeinated-GTE significantly reduced vascular reactive oxygen species (ROS) formation and NADPH oxidase activity, and improved endothelium dependent relaxation in the thoracic aorta of OLETF rats. Decaffeinated-GTE also suppressed the expression of p47 and p22phox (NADPH oxidase subunits) in the immunohistochemical staining, and stimulated phosphorylation of endothelial nitric oxide synthase (eNOS) and Akt in the immunoblotting of aortas. CONCLUSIONS: Decaffeinated-GTE reduced the formation of ROS and NADPH oxidase activity and stimulated phosphorylation of eNOS and Akt in the aorta of a rat model of MetS, which resulted in improved endothelial dysfunction and IR, and eventually lowered BP.

Kaempferol attenuates 4-hydroxynonenal-induced apoptosis in PC12 cells by directly inhibiting NADPH oxidase.

Kaempferol, a natural flavonoid isolated from various plant sources, has been identified as a potential neuroprotectant. In this study, we investigated the protective effect of kaempferol against 4-hydroxynonenal (HNE)-induced apoptosis in PC12 rat pheochromocytoma cells. Kaempferol inhibited 4-HNE-mediated apoptosis, characterized by nuclear condensation, down-regulation of antiapoptotic protein Bcl-2, and activation of proapoptotic caspase-3. Kaempferol inhibited 4-HNE-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). More importantly, kaempferol directly bound p47(phox), a cytosolic subunit of NADPH oxidase (NOX), and significantly inhibited 4-HNE-induced activation of NOX. The antiapoptotic effects of kaempferol were replicated by the NOX inhibitor apocynin, suggesting that NOX is an important enzyme in its effects. Our results suggest that kaempferol attenuates 4-HNE-induced activation of JNK and apoptosis by binding p47(phox) of NOX and potently inhibiting activation of the NOX-JNK signaling pathway in neuron-like cells. Altogether, these results suggest that kaempferol may be a potent prophylactic against NOX-mediated neurodegeneration.

Lysimachia clethroides extract promote vascular relaxation via endothelium-dependent mechanism.

Lysimachia clethroides is widely used in traditional herbal medicine for many purposes, especially for blood vessel-related diseases in Korea and East Asia. Experiments were undertaken to determine whether hydro-alcoholic extract obtained from L. clethroides (LCE) has vasorelaxant activity in the rat aorta rings and, if so, to elucidate the underlying mechanism. Rat aorta rings were suspended in organ chambers for the measurement of changes in isometric tension in the presence or absence of several inhibitors. LCE induced endothelium-dependent vasodilation (ED50 = 6.1 mug/mL) and that was abolished by nitric oxide synthase inhibitor, N-nitro-L-arginine, and guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one, PI3-kinase inhibitor, wortmannin, and cell permeable superoxide dismutase. In addition, LCE decreased vessels contraction by phenylephrine. Prostaglandin synthesis inhibitor, indometacin, and inhibitors of endothelium-derived hyperpolarizing factor, charybdotoxin plus apamin, did not affect vasodilatory effect of LCE. In cultured endothelial cells, LCE-induced phosphorylation of serine 1177-endothelial nitric oxide synthase and serine 473-Akt. LCE inhibited strongly nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in smooth muscle cells and angiotensin II-induced contraction of rat aorta. Finally, increased oxidative stress in rat aorta-induced by angiotensin II is ameliorated by LCE. Taken together, LCE induces an endothelium-dependent vasodilation and might be involved, at least in part, the activation of the nitric oxide-cyclic guanosine monophosphate pathway. In addition, LCE decreases oxidative stress in aorta by inhibition of NADPH oxidase activity. The present findings indicate that LCE could be a candidate of herbal medicine for cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

Cocoa procyanidins inhibit expression and activation of MMP-2 in vascular smooth muscle cells by direct inhibition of MEK and MT1-MMP activities.

AIMS: Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. METHODS AND RESULTS: Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 microg/mL and 5 microM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P < 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P < 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of pro-MMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). CONCLUSION: Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.