A phloroglucinol from Melicope ptelefolia attenuates IgE-mediated mast cell degranulation via calcium-dependent signalling pathways

Mast cells are important effector cells of the innate immune system that participate in allergic reactions through activation of Lyn, Syk, mitogen activated protein kinase (MAPK), increase of intracellular calcium ion concentration ([Ca2+]i), degranulation and cytokine production. Our previous studies demonstrated that tHGA, a synthetic phloroglucinol originally found in Melicope ptelefolia, was able to inhibit IgE-mediated mast cell degranulation, evidenced by reduced amount of histamine, β-hexosaminidase, PGD2 and LTC4, IL-4 and TNF-α. However, the inhibitory mechanism remains unknown. This current study aims to understand the inhibitory mechanism of tHGA in IgE-mediated mast cell degranulation. IgE-sensitized RBL-2H3 cells were pre-treated with tHGA for 20 minutes and challenged with DNP-BSA for 30-60 min. The effect of tHGA on the influx of calcium ion into cells was studied by using Calcium Detection Kit. The inhibitory pathways of tHGA was determined by examining major signalling molecules involved in IgE-mediated mast cell degranulation using Western blot. The results from calcium influx assay showed that tHGA decreased the influx of calcium into cells. Western blot further confirmed that tHGA does not attenuate the phosphorylation of signalling molecules in calcium-independent NF- -κB signalling pathway but significantly attenuated the phosphorylation of signalling molecules in calcium-dependent pathways including MAPK, at 5 μM and 20 μM (P< 0.05). In a conclusion, tHGA attenuates IgE-mediated mast cell degranulation via calcium dependent signalling pathway. The molecular target of tHGA shall be further confirmed to provide an insight into the design of novel pharmacological agents which may be used to regulate the mast cell response

IL-8 as a potential in-vitro severity biomarker for dengue disease

Dengue is a common infection, caused by dengue virus. There are four different dengue serotypes, with different capacity to cause severe dengue infections. Besides, secondary infections with heterologous serotypes, concurrent infections of multiple dengue serotypes may alter the severity of dengue infection. This study aims to compare the severity of single infection and concurrent infections of different combinations of dengue serotypes in-vitro. Human mast cells (HMC)-1.1 were infected with single and concurrent infections of multiple dengue serotypes. The infected HMC-1.1 supernatant was then added to human umbilical cord vascular endothelial cells (HUVEC) and severity of dengue infections was measured by the percentage of transendothelial electrical resistance (TEER). Levels of IL- 10, CXCL10 and sTRAIL in HMC-1.1 and IL-8, IL-10 and CXCL10 in HUVEC culture supernatants were measured by the ELISA assays. The result showed that the percentage of TEER values were significantly lower in single infections (p0.4), indicating that IL-8 may be suitable as an in-vitro severity biomarker. In conclusion, this in-vitro model presented few similarities with regards to the conditions in dengue patients, suggesting that it could serve as a severity model to test for severity and levels of severity biomarkers upon different dengue virus infections

A phloroglucinol from Melicope ptelefolia preserves barrier integrity of lipopolysaccharide-induced endothelial cells: study on junctional proteins and cytoskeleton remodeling

Under normal condition, the endothelial cells form a compact monolayer to line the blood vessel. The integrity of this monolayer can be disrupted by lipopolysaccharide (LPS), where LPS could increase the permeability of the endothelium via dissociation of cell-to-cell junctions and rearrangement of cytoskeleton. Prolonged LPS-induced inflammation may lead to excessive plasma leakage and leukocyte migration, leading to fatal outcomes such as shock and multiple organ dysfunctions. Control on this hallmark of inflammation was suggested as a mean of therapeutic interference to improve inflammation. In this study, we investigated the capability of tHGA on maintaining barrier integrity of LPS-induced endothelial cells. tHGA is a phloroglucinol found in Melicope ptelefolia. The compound was previously proven to have strong therapeutic potential with anti-asthma, anti cancer and anti-inflammatory properties. In murine experimental asthma model, tHGA has been shown to inhibit cytokine secretion and leukocyte infiltration, both of which are hallmarks of LPS-induced inflammation. Therefore, it is highly probable that tHGA may have anti-inflammatory effects on LPS-induced inflammation, Human Umbilical Vein Endothelial Cells (HUVECs) were used as an in vitro representative of endothelial cells. HUVECs were seeded to confluence in cell culture insert, where the cells were pretreated with tHGA and induced with LPS. Tracer dyes or monocytes were then allowed to pass the endothelium to examine severity of vascular leakage. To examine F-actin rearrangement, pretreated HUVECs were stained with Alexa-Fluur 488 Phalloidin prior to microscopic examination. The effect of tHGA on preserving junctional protein expression were examined via Western Blot. tHGA reduced permeability and monocyte transendothelial migration at 5µM and 20µM. Furthermore, tHGA inhibited LPS-induced cytoskeletal rearrangement of HUVECs. Additionally, tHGA is able to preserve the protein expression of F-cadherin and Occludin. tHGA is able to preserve barrier integrity of LPS-induced endothelial cells via preservation of actin cytoskeleton and junctional proteins

2, 4, 6,-trihydroxy- 3-geranylacetophenone (tHGA) inhibits newly synthesized mediators release in IgE-mediated mast cell degranulation

Mast cells are important effector cells of the innate immune system and participate in allergy reaction. Upon FCERI aggregation, mast cells secrete preformed mediators such as histamine as well as newly synthesized mediators including prostaglandins, leukotrienes and proinflammatory cytokines. Our previous studies demonstrated that tHGA, an active compound isolated from Melicope ptelefolia, was able to dose-dependently inhibit the release of histamine and β-hexosaminidase in IgE-mediated mast cell degranulation. However, the inhibitory effects of tHGA on the release of newly synthesized mediators during mast cell degranulation still remain unknown. The current study aims to investigate the in vitro inhibitory effects of tHGA on newly synthesized mediators released in IgE-mediated mast cell degranulation by examining the level of arachidonic acid metabolites such as prostaglandins D2 and leukotriene C4, as well as cytokines such as interleukin 4 and tissue necrosis factor alpha. IgE-sensitized RBL-2H3 cells were pre-treated with tHGA for 20 minutes and later challenged with DNP-BSA for 6 hours. The level of all four mediators released by the degranulated cells was measured by using ELISA kits according to the manufacturer’s protocol. Real time-PCR was also performed to examine the effect of tHGA on the gene expression of IL-4 and TNF-α. The results showed that pre-treatment of IgE-sensitized RBL-2H3 cells with non-cytoxic concentrations of tHGA (1.25, 5 and 20 µM) significantly decreased the release and gene expression of all four mediators in a concentration-dependent manner (p<0.005). This preliminary study demonstrated that tHGA does not only inhibit the release of preformed mediators, but also attenuate the release of newly synthesized mediators during mast cell degranulation, via inhibition on their respective gene expression. Further study should look into the effect of tHGA on major signalling pathways to understand the mechanism of action of tHGA that contributes to its inhibitory effects in IgE-mediated mast cell degranulation

Barrier protective effects of 2,4,6-trihydroxy-3-geranyl acetophenone on lipopolysaccharides-stimulated inflammatory responses in human umbilical vein endothelial cells

Pharmocological relevance: 2,4,6-trihydroxy-3-geranyl acetophenone (tHGA), is a phloroglucinol compound found naturally in Melicope ptelefolia. Melicope ptelefolia has been used traditionally for centuries as natural remedy for wound infections and inflammatory diseases. Aim of the study: Endothelial barrier dysfunction is a pathological hallmark of many diseases and can be caused by lipopolysaccharides (LPS) stimulation. Therefore, this study aims to investigate the possible barrier protective effects of tHGA upon LPS-stimulated inflammatory responses in human umbilical vein endothelial cells (HUVECs). Materials and methods: HUVECs were pretreated with tHGA prior to LPS stimulation, where inflammatory parameters including permeability, monocyte adhesion and migration, and release of pro-inflammatory mediators were examined. Additionally, the effect of tHGA on F-actin rearrangement and adhesion protein expression of LPS-stimulated HUVECs was evaluated. Results: It was found that pretreatment with tHGA inhibited monocyte adhesion and transendothelial migration, reduced endothelial hyperpermeability and secretion of prostaglandin E2 (PGE2). Additionally, tHGA inhibited cytoskeletal rearrangement and adhesion protein expression on LPS-stimulated HUVECs. Conclusion: As the regulation of endothelial barrier dysfunction can be one of the therapeutic strategies to improve the outcome of inflammation, tHGA may be able to preserve vascular barrier integrity of endothelial cells following LPS-stimulated dysfunction, thereby endorsing its potential usefulness in vascular inflammatory diseases

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

Physicochemical characteristics, nutritional composition, and phytochemical profiles of nine Algerian date palm fruit (Phoenix dactylifera L.) varieties

Nine varieties of Algerian date palm fruits (Phoenix dactylifera L.) including Lahmira, Timjouhert, Adham Talmine, Deglet Talmine, Adam Boullah, Tinasser, Deglet, Deglet Nour, and Takerbouch were distinguished via Proton Nuclear Magnetic Resonance (1H‐NMR) metabolomics approach. The quality was evaluated based on the metabolite composition, proximate analysis, physicochemical characteristics, and biological activities which include nitric oxide (NO) inhibition via the cell‐based approach and NO scavenging abilities. A total of 49 metabolites were tentatively characterized including amino acids, organic acids, and phenolic compounds. The Principal Component Analysis and Hierarchical Clustering Analysis revealed a consistent grouping of Deglet, Takerbouch, and Deglet Noor from the rest of six varieties. Among the metabolites that contributed toward the observed segregation were serine, glycine, glucose, fructose, and sucrose. With regard to metabolite composition, NO scavenging activities, total soluble solids, and proximate analysis, Deglet dates displayed a better quality as compared to the other Algerian dates

Plasma and urine metabolite profiling reveals the protective effect of Clinacanthus nutans in an ovalbumin-induced anaphylaxis model: ¹H-NMR metabolomics approach

The present study sought to identify the key biomarkers and pathways involved in the induction of allergic sensitization to ovalbumin and to elucidate the potential anti-anaphylaxis property of Clinacanthus nutans (Burm. f.) Lindau water leaf extract, a Southeast Asia herb in an in vivo ovalbumin-induced active systemic anaphylaxis model evaluated by 1H-NMR metabolomics. The results revealed that carbohydrate metabolism (glucose, myo-inositol, galactarate) and lipid metabolism (glycerol, choline, sn-glycero-3-phosphocholine) are the key requisites for the induction of anaphylaxis reaction. Sensitized rats treated with 2000 mg/kg bw C. nutans extract before ovalbumin challenge showed a positive correlation with the normal group and was negatively related to the induced group. Further 1H-NMR analysis in complement with Kyoto Encyclopedia of Genes and Genomes (KEGG) reveals the protective effect of C. nutans extract against ovalbumin-induced anaphylaxis through the down-regulation of lipid metabolism (choline, sn-glycero-3-phosphocholine), carbohydrate and signal transduction system (glucose, myo-inositol, galactarate) and up-regulation of citrate cycle intermediates (citrate, 2-oxoglutarate, succinate), propanoate metabolism (1,2-propanediol), amino acid metabolism (betaine, N,N-dimethylglycine, methylguanidine, valine) and nucleotide metabolism (malonate, allantoin). In summary, this study reports for the first time, C. nutans water extract is a potential anti-anaphylactic agent and 1H-NMR metabolomics is a great alternative analytical tool to explicate the mechanism of action of anaphylaxis

Neuroprotective effects of biochanin A against glutamate-induced cytotoxicity in PC12 cells via apoptosis inhibition

l-Glutamate plays a crucial role in neuronal cell death, which is known to be associated with various neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s diseases. In this study, we investigated the protective effects of biochanin A, a phytoestrogen compound found mainly in Trifolium pratense, against l-glutamate-induced cytotoxicity in a PC12 cell line. Exposure of the cells to 10 mM l-glutamate was found to significantly increase cell viability loss and apoptosis, whereas pretreatment with various concentrations of biochanin A attenuated the cytotoxic effects of l-glutamate. Specifically, the pretreatment led to not only decreases in the release of lactate dehydrogenase, the number of apoptotic cells, and the activity of caspase-3 but also an increase in the total glutathione level in the l-glutamate-treated PC12 cells. These results indicate that biochanin A may be able to exert neuroprotective effects against l-glutamate-induced cytotoxicity. Furthermore, our findings also imply that biochanin A may act as an antiapoptotic agent in order to perform its protective function

The effects of a synthetic curcuminoid analogue,2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone on proinflammatorysignaling pathways and CLP-induced lethal sepsis in mice.

We previously showed that 2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC), suppressed the synthesis of various proinflammatory mediators. In this study we explain the mechanism of action of BHMC in lipopolysaccharide (LPS)-induced U937 monocytes and further show that BHMC prevents lethality of CLP-induced sepsis. BHMC showed dose-dependent inhibitory effects on p38, JNK and ERK 1/2 activity as determined by inhibition of phosphorylation of downstream transcription factors ATF-2, c-Jun and Elk-1 respectively. Inhibition of these transcription factors subsequently caused total abolishment of AP-1–DNA binding. BHMC inhibited p65 NF-κB nuclear translocation and DNA binding of p65 NF-κB only at the highest concentration used (12.5 μM) but failed to alter phosphorylation of JNK, ERK1/2 and STAT-1. Since the inhibition of p38 activity was more pronounced we evaluated the possibility that BHMC may bind to p38. Molecular docking experiments confirmed that BHMC fits well in the highly conserved hydrophobic pocket of p38 MAP kinase. We also show that BHMC was able to improve survival from lethal sepsis in a murine caecal-ligation and puncture (CLP) model