Salvianolic acid B attenuates cyclooxygenase-2 expression in vitro in LPS-treated human aortic smooth muscle cells and in vivo in the apolipoprotein-E-deficient mouse aorta

Inflammation plays an essential role in atherosclerosis and post-angioplasty restenosis and the synthesis and release of inflammatory cytokines from vascular smooth muscle cells is an important contributor to these pathologies. It is assumed that drugs that prevent the overproduction of inflammatory cytokines may inhibit cardiovascular disorders. In the present study, the effects of a water-soluble antioxidant, salvianolic acid B (Sal B), derived from a Chinese herb, on the expression of cyclooxygenase (COX) in lipopolysaccharide (LPS)-treated human aortic smooth muscle cells (HASMCs) and in the aortas of cholesterol-fed apoE deficient mice were investigated. In unstimulated HASMCs, COX-2 mRNA and protein were almost undetectable, but were strongly upregulated in response to LPS. In contrast, HASMCs with or without LPS treatment showed constitutive expression of COX-1 mRNA and protein. The activation of COX-2 protein synthesis in LPS-stimulated HASMCs was shown to involve the activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2), c-Jun NH(2)-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathway. Incubation of HASMCs with Sal B before LPS stimulation resulted in pronounced downregulation of COX-2 expression. Sal B treatment suppressed ERK1/2 and JNK phosphorylation and attenuated the increase in prostaglandin E(2) production and NADPH oxidase activity in LPS-treated HASMCs. When apoE-deficient mice were fed a 0.15% cholesterol diet with or without supplementation with 0.3% Sal B for 12 weeks, the intima/media area ratio in the thoracic aortas was significantly reduced in the Sal B group (0.010 +/- 0.009%) compared to the apoE-deficient group (0.114 +/- 0.043%) and there was a significant reduction in COX-2 protein expression in the thickened intima. These results demonstrate that Sal B has anti-inflammatory properties and may explain its anti-atherosclerotic properties. This new mechanism of action of Sal B, in addition to its previously reported inhibition of LDL oxidation, may help explain its efficacy in the treatment of atherosclerosis.     

MRI measured epicardial adipose tissue thickness at the right AV groove differentiates inflammatory status in obese men with metabolic syndrome

Epicardial adipose tissue (EAT) is a metabolically active visceral fat, which secretes inflammatory cytokines and adipokines. In this study, our aim was to examine which measurements of EAT thickness by magnetic resonance imaging (MRI) could best help differentiate inflammatory status, classified by levels of high-sensitivity C-reactive protein (hs-CRP), in obese men with metabolic syndrome (MetS). We prospectively enrolled 32 men with central obesity (waist circumference ≥90 cm) and at least two other MetS criteria. MRI examinations for measurements of EAT, subcutaneous fat, and abdominal visceral fat as well as recordings of anthropometric parameters and tests for serum inflammatory cytokines and adipokines were conducted. Subjects with MetS (N = 32) were divided into three subgroups: (i) low inflammatory status (hs-CRP < 0.1 mg/dl, N = 8), [corrected] (ii) intermediate inflammatory status (hs-CRP 0.1-0.3 mg/dl, N = 15), and (iii) high inflammatory status (hs-CRP >0.3 mg/dl, N = 9). EAT thickness at the right atrioventricular (AV) groove showed a significant linear trend among the three subgroups of MetS (P for trend = 0.004). High inflammatory status MetS subgroup had a significantly thicker right AV groove EAT than did the low inflammatory status MetS subgroup (19.3 ± 3.1 vs. 14.4 ± 3.3 mm, P = 0.015). In binary logistic regression analysis, right AV groove EAT thickness was an independent predictor for differentiating inflammatory status in MetS while abdominal visceral fat area and insulin-resistance index were not. In conclusion, MRI measured EAT thickness at the right AV groove could be a useful marker for differentiating the inflammatory status in obese men with MetS.     

Inhibition of NADPH oxidase-related oxidative stress-triggered signaling by honokiol suppresses high glucose-induced human endothelial cell apoptosis

Angiopathy is a major complication of diabetes. Abnormally high blood glucose is a crucial risk factor for endothelial cell damage. Nuclear factor-kappaB (NF-kappaB) has been demonstrated as a mediated signaling in hyperglycemia or oxidative stress-triggered apoptosis of endothelial cells. Here we explored the efficacy of honokiol, a small molecular weight natural product, on NADPH oxidase-related oxidative stress-mediated NF-kappaB-regulated signaling and apoptosis in human umbilical vein endothelial cells (HUVECs) under hyperglycemic conditions. The methods of morphological Hoechst staining and annexin V/propidium iodide staining were used to detect apoptosis. Submicromolar concentrations of honokiol suppressed the increases of NADPH oxidase activity, Rac-1 phosphorylation, p22(phox) protein expression, and reactive oxygen species production in high glucose (HG)-stimulated HUVECs. The degradation of IkappaBalpha and increase of NF-kappaB activity were inhibited by honokiol in HG-treated HUVECs. Moreover, honokiol (0.125-1 microM) also suppressed HG-induced cyclooxygenase (COX)-2 upregulation and prostaglandin E(2) production in HUVECs. Honokiol could reduce increased caspase-3 activity and the subsequent apoptosis and cell death triggered by HG. These results imply that inhibition of NADPH oxidase-related oxidative stress by honokiol suppresses the HG-induced NF-kappaB-regulated COX-2 upregulation, apoptosis, and cell death in HUVECs, which has the potential to be developed as a therapeutic agent to prevent hyperglycemia-induced endothelial damage.     

A possibility of detection of the non-charge based analytes using ultra-thin body field-effect transistors

Ultra-thin body of p-type field-effect transistors were developed as transducer for biosensors. Changes of conductance resulted from the changes of the surface potentials of ultra-thin body field-effect transistors (UTB-FETs) due to surface chemical modifications were demonstrated. The channel surface of UTB-FETs were modified with N-[3-(trimethoxysilyl)propyl]ethylenediamine (AEAPTMS) and then gold nanoparticles (AuNPs) to immobilize the bio-component, the genetically engineered Delta(5)-3-ketosteroid isomerase (Art_KSI) or the Art_KSI conjugated with charged reporter (Art_KSI_mA51). The binding of charge-based molecules or nanoparticles has been demonstrated to strongly affect the conductivity of UTB-FETs; the increase or decrease of the conductance depends on the polarity of the immobilized molecules or nanoparticles. A new protocol involving the detection of a non-charged analyte relied on the competitive binding of analyte (19-norandrostendione) and a charged reporter (mA51) with KSI. When exposed to a 19-norandrostendione solution (10 microM), the conductance of Art_KSI_mA51-modified UTB-FET increased by 265 nS ( approximately 12%). On the other hand, conductance of Art_KSI-modified UTB-FET showed no distinct change under the same detection conditions.     

Trpm2 enhances physiological bioenergetics and protects against pathological oxidative cardiac injury: Role of Pyk2 phosphorylation

The mechanisms by which Trpm2 channels enhance mitochondrial bioenergetics and protect against oxidative stress-induced cardiac injury remain unclear. Here, the role of proline-rich tyrosine kinase 2 (Pyk2) in Trpm2 signaling is explored. Activation of Trpm2 in adult myocytes with H₂O₂ resulted in 10- to 21-fold increases in Pyk2 phosphorylation in wild-type (WT) myocytes which was significantly lower (~40%) in Trpm2 knockout (KO) myocytes. Pyk2 phosphorylation was inhibited (~54%) by the Trpm2 blocker clotrimazole. Buffering Trpm2-mediated Ca²⁺ increase with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) resulted in significantly reduced pPyk2 in WT but not in KO myocytes, indicating Ca²⁺ influx through activated Trpm2 channels phosphorylated Pyk2. Part of phosphorylated Pyk2 translocated from cytosol to mitochondria which has been previously shown to augment mitochondrial Ca²⁺ uptake and enhance adenosine triphosphate generation. Although Trpm2-mediated Ca²⁺ influx phosphorylated Ca²⁺-calmodulin kinase II (CaMKII), the CaMKII inhibitor KN93 did not significantly affect Pyk2 phosphorylation in H₂O₂-treated WT myocytes. After ischemia/reperfusion (I/R), Pyk2 phosphorylation and its downstream prosurvival signaling molecules (pERK1/2 and pAkt) were significantly lower in KO-I/R when compared with WT-I/R hearts. After hypoxia/reoxygenation, mitochondrial membrane potential was lower and superoxide level was higher in KO myocytes, and were restored to WT values by the mitochondria-targeted superoxide scavenger MitoTempo. Our results suggested that Ca²⁺ influx via tonically activated Trpm2 phosphorylated Pyk2, part of which translocated to mitochondria, resulting in better mitochondrial bioenergetics to maintain cardiac health. After I/R, Pyk2 activated prosurvival signaling molecules and prevented excessive increases in reactive oxygen species, thereby affording protection from I/R injury.     

The Pyruvate Dehydrogenase Complex Is Partially Inactivated During Early Recirculation Following Short-Term Forebrain Ischemia in Rats

Abstract: The mechanisms of selective neuronal loss after short-term global ischemia remain undefined, but processes including increased proteolytic activity, impaired protein synthesis, and oxidative damage have been proposed to contribute. A decrease in activity of the pyruvate dehydrogenase complex in the dorsolateral striatum, an ischemia-susceptible region, is one change apparently differentiating this region from ischemia-resistant areas during early recirculation. To provide an insight into processes contributing to postischemic cell damage, the changes in the pyruvate dehydrogenase complex during early recirculation have been further characterized. These studies provide clear confirmation that the activity of the pyruvate dehydrogenase complex is reduced in mitochondria from the dorsolateral striatum by 3 h of recirculation. The decrease in activity was not accompanied by a loss of antigenic sites or by changes in electrophoretic mobility of the components of the complex. A reduction in activity of the E1 component of the complex (39–42% decrease), but not the E2 and E3 components, was observed that was apparently sufficient to explain the decrease in activity of the whole complex. These results indicate that the changes in activity of the pyruvate dehydrogenase complex in the dorsolateral striatum are not due to loss or gross disruption of the constituent proteins but rather most likely reflect a selective inactivation of a specific component of the complex.     

Activation of phosphoinositide 3-kinase in response to inflammation and nitric oxide leads to the up-regulation of cyclooxygenase-2 expression and subsequent cell proliferation in mesangial cells

In this study, we showed that nitric oxide (NO) donors induced the mesangial cell proliferation and cyclooxygenase-2 (COX-2) protein expression in murine mesangial cells. An inflammatory condition [lipopolysaccharide (LPS) plus interferon-gamma (IFN-gamma)] could also induce cell proliferation and significantly enhance inducible nitric oxide synthase (iNOS) and COX-2 expression. Phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, inhibited these responses. LPS/IFN-gamma-induced COX-2 expression in mesangial cells could be inhibited by iNOS inhibitor, aminoguanidine. Selective COX-2 inhibitor, NS398, was capable of inhibiting NO donor- or LPS/IFN-gamma-induced mesangial cell proliferation. Both NO donor and LPS/IFN-gamma markedly activated the PI3K activity and the phosphorylation of Akt and nuclear factor (NF)-kappaB DNA binding activity in mesangial cells, which could be inhibited by LY294002 and transfection of dominant-negative vectors of PI3K/p85 and Akt. These results indicate that a PI3K/Akt-dependent pathway involved in the NO-regulated COX-2 expression and cell proliferation in mesangial cells under inflammatory condition.     

Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice

We present evidence to show that the alpha-amylase gene family in rice is under two different modes of regulation: 1) hormonal regulation in germinating seeds, and 2) metabolic repression in cultured cells by available carbohydrate nutrients. Expression of alpha-amylase genes in deembryoed rice seeds is known to be induced by exogenous gibberellic acid. On the other hand, expression of alpha-amylase genes in suspension-cultured cells is induced by the deprivation of carbohydrate nutrient. A lag period of 2-4 h is required for the induction of alpha-amylase mRNA in sucrose-depleted medium. The induction of alpha-amylase expression is extraordinarily high and levels of alpha-amylase mRNA can be increased 8-20-folds after 24 h of sucrose starvation. The synthesis and secretion of alpha-amylase is also dependent upon the level of carbon source. The derepression or repression of alpha-amylase synthesis can be readily reversed by the deprivation or replenishment of sucrose in the medium, respectively. Glucose and fructose exert a repression on the alpha-amylase synthesis similar to that of sucrose. A hypothesis that explains the induction of alpha-amylase synthesis by carbohydrate starvation is proposed. Our data have suggested a hitherto undiscovered, potentially important control mechanism of carbohydrate metabolism in higher plants.