Insulin promotes vascular smooth muscle cell proliferation and apoptosis via differential regulation of tumor necrosis factor‐related apoptosis‐inducing ligand

Background: Insulin regulates glucose homeostasis but can also promote vascular smooth muscle (VSMC) proliferation, important in atherogenesis. Recently, we showed that tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) stimulates intimal thickening via accelerated growth of VSMCs. The aim of the present study was to determine whether insulin‐induced effects on VSMCs occur via TRAIL. Methods: Expression of TRAIL and TRAIL receptor in response to insulin and glucose was determined by polymerase chain reaction. Transcriptional activity was assessed using wild‐type and site‐specific mutations of the TRAIL promoter. Chromatin immunoprecipitation studies were performed. VSMC proliferation and apoptosis was measured. Results: Insulin and glucose exposure to VSMC for 24 h stimulated TRAIL mRNA expression. This was also evident at the transcriptional level. Both insulin‐ and glucose‐inducible TRAIL transcriptional activity was blocked by dominant‐negative specificity protein‐1 (Sp1) overexpression. There are five functional Sp1‐binding elements (Sp1‐1, Sp1‐2, Sp‐5/6 and Sp1‐7) on the TRAIL promoter. Insulin required the Sp1‐1 and Sp1‐2 sites, but glucose needed all Sp1‐binding sites to induce transcription. Furthermore, insulin (but not glucose) was able to promote VSMC proliferation over time, associated with increased decoy receptor‐2 (DcR2) expression. In contrast, chronic 5‐day exposure of VSMC to 1 µg/mL insulin repressed TRAIL and DcR2 expression, and reduced Sp1 enrichment on the TRAIL promoter. This was associated with increased cell death. Conclusions: The findings of the present study provide a new mechanistic insight into how TRAIL is regulated by insulin. This may have significant implications at different stages of diabetes‐associated cardiovascular disease. Thus, TRAIL may offer a novel therapeutic solution to combat insulin‐induced vascular pathologies

Growth and lovastatin production by Aspergillus terreus under different carbohyrates as carbon sources

Carbon source is a key component of metabolites synthesis in microorganisms. This work examined the effects of selected carbon sources in the form of carbohydrates, on the growth of Aspergillus terreus ATCC 20542 and the production of lovastatin. Slowly metabolised carbohydrates, such as D-galactose (consumption rate, r=3.11), produced a high microbial biomass, XFINAL (9.44 g/L) compared to other carbohydrates, but with a low biomass yield coefficient (YLOV/X=1.68). In contrast, D-ribose (YLOV/X=) which showed moderate biomass growth (XFINAL=8.78 g/L) and consumption rate (r=5.44 g/day), produced the highest lovastatin amount (51.81 mg/L, day 6). These indicate little correlation between biomass growth and lovastatin production. Notably, culture consisting of pellets with short hairy surface feature is associated with enhanced lovastatin production. Our findings suggest that the production of lovastatin by Aspergillus terreus is highly influenced by the choice of carbohydrates that will shape the pellet morphology rather than the rate of carbohydrates metabolism

Crosstalk between signaling pathways involved in the regulation of airway smooth muscle cell hyperplasia

Increased ASM mass, primarily due to ASM hyperplasia, has been recognized as a hallmark of airway remodeling in asthma. Increased ASM mass is the major contributor to the airway narrowing, thus worsening the bronchoconstriction in response to stimuli. Inflammatory mediators and growth factors released during inflammation induce increased ASM mass surrounding airway wall via increased ASM proliferation, diminished ASM apoptosis and increased ASM migration. Several major pathways, such as MAPKs, PI3K/AKT, JAK2/STAT3 and Rho kinase, have been reported to regulate these cellular activities in ASM and were reported to be interrelated at certain points. This article aims to provide an overview of the signaling pathways/molecules involved in ASM hyperplasia as well as the mapping of the interplay/crosstalk between these major pathways in mediating ASM hyperplasia. A more comprehensive understanding of the complexity of cellular signaling in ASM cells will enable more specific and safer drug development in the control of asthma

2,4,6,-trihydroxy-3-geranylacetophenone (tHGA) suppresses chronic allergic airway inflammation in ovalbumin-sensitized mice via intraperitoneal route

Introduction: Asthma is a condition characterized by eosinophilic airway inflammation and remodelling that involves several pathological changes, including subepithelial fibrosis, mucus hypersecretion, smooth muscle growth, and vascular changes. The present study aimed to determine the effect of tHGA administered intraperitoneally in a chronic asthma mouse model that closely mimics the human asthma. Methods: Ovalbumin-sensitized and challenged BALB/c mice were i.p. administered with tHGA at different doses (20 and 2 mg/kg). Respiratory function was measured, and brochoalveolar lavage, blood and lung samples were then obtained and analyzed. Results: The airways of OVA-induced mice developed increased pulmonary inflammation with increased levels of cytokines, chemokines, and changes in vascular permeability. Intraperitoneal administration of tHGA in OVA-induced mice significantly and dose-dependently inhibited the airway inflammation, production of immunoglobulin E, Th2-type cytokines and chemokines, and inflammatory mediators. Treatment with tHGA also significantly reduced the airway hyperresposiveness in response to increased methacholine doses. Conclusion: This study demonstrates that the efficacy of tHGA in alleviating chronic asthmatic symptoms in mouse model improved significantly when administered intraperitoneally compared to oral route. Furthermore, this study also supports that tHGA has a therapeutic potential in chronic asthma management by acting as a cysteinyl leukotrienes (CysLT) inhibitor

The geranyl acetophenone tHGA attenuates human bronchial smooth muscle proliferation via inhibition of AKT phosphorylation

Increased airway smooth muscle (ASM) mass is a prominent hallmark of airway remodeling in asthma. Inhaled corticosteroids and long-acting beta2-agonists remain the mainstay of asthma therapy, however are not curative and ineffective in attenuating airway remodeling. The geranyl acetophenone 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), an in-house synthetic non-steroidal compound, attenuates airway hyperresponsiveness and remodeling in murine models of asthma. The effect of tHGA upon human ASM proliferation, migration and survival in response to growth factors was assessed and its molecular target was determined. Following serum starvation and induction with growth factors, proliferation and migration of human bronchial smooth muscle cells (hBSMCs) treated with tHGA were significantly inhibited without any significant effects upon cell survival. tHGA caused arrest of hBSMC proliferation at the G1 phase of the cell cycle with downregulation of cell cycle proteins, cyclin D1 and diminished degradation of cyclin-dependent kinase inhibitor (CKI), p27Kip1. The inhibitory effect of tHGA was demonstrated to be related to its direct inhibition of AKT phosphorylation, as well as inhibition of JNK and STAT3 signal transduction. Our findings highlight the anti-remodeling potential of this drug lead in chronic airway disease

The potential use of honey as a remedy for allergic diseases: a mini review

Honey has been conventionally consumed as food. However, its therapeutic properties have also gained much attention due to its application as a traditional medicine. Therapeutic properties of honey such as anti-microbial, anti-inflammatory, anti-cancer and wound healing have been widely reported. A number of interesting studies have reported the potential use of honey in the management of allergic diseases. Allergic diseases including anaphylaxis, asthma and atopic dermatitis (AD) are threatening around 20% of the world population. Although allergic reactions are somehow controllable with different drugs such as antihistamines, corticosteroids and mast cell stabilizers, modern dietary changes linked with allergic diseases have prompted studies to assess the preventive and therapeutic merits of dietary nutrients including honey. Many scientific evidences have shown that honey is able to relieve the pathological status and regulate the recruitment of inflammatory cells in cellular and animal models of allergic diseases. Clinically, a few studies demonstrated alleviation of allergic symptoms in patients after application or consumption of honey. Therefore, the objective of this mini review is to discuss the effectiveness of honey as a treatment or preventive approach for various allergic diseases. This mini review will provide insights into the potential use of honey in the management of allergic diseases in clinical settings

Level of chemical and microbiological contaminations in chili bo (Paste).

The objective of this study was to determine the level of preservatives and microbiological loads in various brands of commercially available chili bo (paste). Fifteen different brands of chili bo obtained from the local market and hypermarkets were analyzed for pH, moisture and benzoic acid content, microbiological loads (aerobic, anaerobic, aerobic spores, and fungi), and thermophilic microorganisms. Results showed that both moisture content and pH vary among samples. The concentrations of benzoic acid detected in chili bo were found to be in the range of 537 to 5,435 mg/kg. Nine of fifteen brands were found to exceed the maximum level permitted by the Malaysian Food Law in accordance with the Codex Alimentarius (1,000 mg/kg for benzoic acid). An apparent correlation between benzoic acid concentration and microbiological loads present in the chili bo was observed. The microbiological loads were found to be relatively low in the end products containing high amounts of benzoic acid. The heat-resistant (70 to 80 degrees C) microorganisms present in chili bo were identified as Ochrobacterum tritici, Stenotrophomonas rhizophila, Microbacterium maritypicum, Roseomonas spp., CDC group II-E subgroup A, Flavimonas oryzihabitans, and Pseudomonas aeruginosa, with M. maritypicum being the most frequently found (in 9 of 15 samples) microorganism. Most of these identified microorganisms were not known to cause foodborne illnesses

Effects of 3-(2-Hydroxyphenyl)-1-(5-methyl-furan-2-y-l) propenone (HMP) upon signalling pathways of lipopolysaccharide-induced iNOS synthesis in RAW 264.7 cells.

NO synthesis in the RAW 264.7 murine macrophage line. The inhibition of NO synthesis was related to inhibition of p38 phosphorylation and kinase activity that led to significant inhibition of phosphorylation of ATF-2. This effect in turn caused inhibition of AP-1-DNA binding which partially explains the inhibitory effect upon the synthesis of iNOS. HMP had no effect upon phosphorylation of JNK, ERK1/2 and STAT-1. Kinase activity of JNK and ERK1/2 was also not affected by HMP as determined by levels of phosphorylated c-jun and phosphorylated elk-1. Furthermore HMP failed to block phosphorylation of IκBα, and subsequent nuclear translocation and DNA-binding activity of p65 NF-κB in IFN-γ/LPS-induced RAW 264.7 cells. Molecular docking experiments confirmed that HMP fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We conclude that the synthetic HMP is a chalcone analogue that selectively inhibits the p38/ATF-2 and AP-1 signaling pathways in the NO synthesis by the macrophage RAW 264.7

LAT is essential for the mast cell stabilising effect of tHGA in IgE-mediated mast cell activation

Mast cells play a central role in the pathogenesis of allergic reaction. Activation of mast cells by antigens is strictly dependent on the influx of extracellular calcium that involves a complex interaction between signalling molecules located within the cells. We have previously reported that tHGA, an active compound originally isolated from a local shrub known as Melicope ptelefolia, prevented IgE-mediated mast cell activation and passive systemic anaphylaxis by suppressing the release of interleukin-4 (IL-4) and tumour necrosis factor (TNF)-α from activated rat basophilic leukaemia (RBL)-2H3 cells. However, the mechanism of action (MOA) as well as the molecular target underlying the mast cell stabilising effect of tHGA has not been previously investigated. In this study, DNP-IgE-sensitised RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA. To dissect the MOA of tHGA in IgE-mediated mast cell activation, the effect of tHGA on the transcription of IL-4 and TNF-α mRNA was determined using Real Time-Polymerase Chain Reaction (qPCR) followed by Calcium Influx Assay to confirm the involvement of calcium in the activation of mast cells. The protein lysates were analysed by using Western Blot to determine the effect of tHGA on various important signalling molecules in the LAT-PLCγ-MAPK and PI3K-NFκB pathways. In order to identify the molecular target of tHGA in IgE-mediated mast cell activation, the LAT and LAT2 genes in RBL-2H3 cells were knocked-down by using RNA interference to establish a LAT/LAT2 competition model. The results showed that tHGA inhibited the transcription of IL-4 and TNF-α as a result of the suppression of calcium influx in activated RBL-2H3 cells. The results from Western Blot revealed that tHGA primarily inhibited the LAT-PLCγ-MAPK pathway with partial inhibition on the PI3K-p65 pathway without affecting Syk. The results from RNAi further demonstrated that tHGA failed to inhibit the release of mediators associated with mast cell degranulation under the LAT/LAT2 competition model in the absence of LAT. Collectively, this study concluded that the molecular target of tHGA could be LAT and may provide a basis for the development of a mast cell stabiliser which targets LAT

Anti-allergic activity of 2,4,6-trihydroxy-3-geranylacetophenone (tHGA) via attenuation of IgE-mediated mast cell activation and inhibition of passive systemic anaphylaxis

tHGA, a geranyl acetophenone compound originally isolated from a local shrub called Melicope ptelefolia, has been previously reported to prevent ovalbumin-induced allergic airway inflammation in a murine model of allergic asthma by targeting cysteinyl leukotriene synthesis. Mast cells are immune effector cells involved in the pathogenesis of allergic diseases including asthma by releasing cysteinyl leukotrienes. The anti-asthmatic properties of tHGA could be attributed to its inhibitory effect on mast cell degranulation. As mast cell degranulation is an important event in allergic responses, this study aimed to investigate the anti-allergic effects of tHGA in cellular and animal models of IgE-mediated mast cell degranulation. For in vitro model of IgE-mediated mast cell degranulation, DNP-IgE-sensitized RBL-2H3 cells were pre-treated with tHGA before challenged with DNP-BSA to induce degranulation. For IgE-mediated passive systemic anaphylaxis, Sprague Dawley rats were sensitized by intraperitoneal injection of DNP-IgE before challenged with DNP-BSA. Both in vitro and in vivo models showed that tHGA significantly inhibited the release of preformed mediators (β-hexosaminidase and histamine) as well as de novo mediators (interleukin-4, tumour necrosis factor-α, prostaglandin D2 and leukotriene C4). Pre-treatment of tHGA also prevented IgE-challenged RBL-2H3 cells and peritoneal mast cells from undergoing morphological changes associated with mast cell degranulation. These findings indicate that tHGA possesses potent anti-allergic activity via attenuation of IgE-mediated mast cell degranulation and inhibition of IgE-mediated passive systemic anaphylaxis. Thus, tHGA may have the potential to be developed as a mast cell stabilizer for the treatment of allergic diseases in the future