Analysis of Free Radicals, Radical Modifications and Redox Signalling Analysis of oxidized and chlorinated lipids by mass spectrometry and relevance to signalling

Abstract Oxidized and chlorinated phospholipids are generated under inflammatory conditions and are increasingly understood to play important roles in diseases involving oxidative stress. MS is a sensitive and informative technique for monitoring phospholipid oxidation that can provide structural information and simultaneously detect a wide variety of oxidation products, including chain-shortened and -chlorinated phospholipids. MS n technologies involve fragmentation of the compounds to yield diagnostic fragment ions and thus assist in identification. Advanced methods such as neutral loss and precursor ion scanning can facilitate the analysis of specific oxidation products in complex biological samples. This is essential for determining the contributions of different phospholipid oxidation products in disease. While many pro-inflammatory signalling effects of oxPLs (oxidized phospholipids) have been reported, it has more recently become clear that they can also have anti-inflammatory effects in conditions such as infection and endotoxaemia. In contrast with free radical-generated oxPLs, the signalling effects of chlorinated lipids are much less well understood, but they appear to demonstrate mainly pro-inflammatory effects. Specific analysis of oxidized and chlorinated lipids and the determination of their molecular effects are crucial to understanding their role in disease pathology

Interaction between green tea and perindopril reduces inhibition of angiotensin-converting enzyme activity

Purpose: To investigate the blood pressure-lowering effect of green tea (GT) extract alone and in combination with an angiotensin converting enzyme (ACE) inhibitor, perindopril, on rats. Methods: The study consisted of four groups of five spontaneously hypertensive rats (SHR): negative control (2 % tragacanth mucilage), positive control group (perindopril, 0.36 mg/kg/day) and two treatment groups (green tea, 25 mg/kg/day; and combined green tea/perindopril). The treatments were given orally for 14 days. Systolic blood pressure was measured before and after treatment using the tail cuff technique. Angiotensin converting enzyme activity in the lung homogenate of the hypertensive rats was determined spectrophotometrically. Results: Green tea extract significantly reduced the rats’ systolic blood pressure (p < 0.05) but did not inhibit the angiotensin-converting enzyme. The combination of green tea extract with perindopril also caused a significant decline in blood pressure (p < 0.001). However, the green tea extract attenuated the inhibition of the angiotensin-converting enzyme activity by perindopril. Conclusion: Green tea extract produces anti-hypertensive activity in rats, but its mechanism of action is not via inhibition of angiotensin-converting enzyme. The interaction of GT extract with perindopril results in a reduction of ACE inhibitory activity

Synthetic chalcone derivatives inhibit cytokine secretion via inhibition of ERK and JNK pathways in human U937 macrophage

Purpose: To investigate the inhibitory effects of a chalcone derivative on lipopolysaccharide (LPS)- induced interleukin (IL)-6 and IL-8 secretions and on LPS-induced mitogen-activated protein kinases (MAPK) and nuclear factor kappa B (NF-κB) activation in human U937 macrophage-like cell line. Methods: The effects of chalcone derivative on LPS-induced secretion of IL-6 and IL-8 in endothelial cells were determined by enzyme-linked immunosorbent assay while the effects of chalcone on the activation of MAPK and NF-kB pathway were determined by Western blotting. Results: The results showed that 3-(5-methyl-furan-2-yl)-naphthalen-1-yl-propenone (compound 1) significantly inhibited the secretion of LPS-induced IL-6 and IL-8 in U937 macrophages. This compound also demonstrated significant suppression of c-Jun N-terminal kinases (JNK) and extracellular signalregulated kinases (ERK) phosphorylation. However, the compound did not reverse the degradation of inhibitor kappa B alpha (IκBα) and did not inhibit the phosphorylation of NF-κB subunit and P-38 MAPK. Conclusion: Compound 1 inhibits the secretion of cytokines via the inhibition of ERK and JNK pathways. These results suggest that chalcone derivative could act as an antiinflammatory agent by altering cytokine secretion and inflammatory pathways

Roselle is cardioprotective in diet-induced obesity rat model with myocardial infarction

Aims: Obesity increase the risks of hypertension and myocardial infarction (MI) mediated by oxidative stress. This study was undertaken to investigate the actions of roselle aqueous extract (R) on cardiotoxicity in obese (OB) rats and thereon OB rats subjected to MI. Main methods: Male Sprague-Dawley rats were fed with either normal diet or high-fat diet for 8 weeks. Firstly, OB rats were divided into (1) OB and (2) OB+ R (100 mg/kg, p.o, 28 days). Then, OB rats were subjected to MI (ISO, 85 mg/kg, s.c, 2 days) and divided into three groups: (1) OB +MI, (2) OB +MI+R and (3) OB +MI + enalapril for another 4 weeks. Key findings: Roselle ameliorated OB and OB +MI's cardiac systolic dysfunction and reduced cardiac hypertrophy and fibrosis. The increased oxidative markers and decreased antioxidant enzymes in OB and OB +MI groups were all attenuated by roselle. Significance: These observations indicate the protective effect of roselle on cardiac dysfunction in OB and OB + MI rats, which suggest its potential to be developed as a nutraceutical product for obese and obese patients with MI in the future

Camellia sinensis and Phyllanthus amarus ethanol extracts induced apoptosis and cell cycle arrest on human leukemic cell lines

Leukaemia is a heterogeneous hematologic malignancy characterized by unregulated proliferation of the early blood-forming cells which starts in bone marrow. The basic strategy of leukaemia therapy involves the induction of leukemic cells apoptosis. Research on natural products have shown that some plant derivatives have anticancer properties by inducing apoptosis of leukemic cells. Plants such as Camellia sinensis and Phyllanthus amarus are those that had gained a wide interest due to their anti-cancer effect. The aim of this study was to investigate the anti-cancer effects of C. sinensis and P. amarus extracts on human leukemic cell lines by analysing the cell cycle and determining the apoptotic state. The cell lines were treated with ethanolic plant extracts at the concentrations of 31.25 - 500 µg/mL for 24 h followed by MTT assay to determine the IC50. The IC50 of C. sinensis and P. amarus on the U937 cells were 170±10.39 and 210±6.78 µg/mL, respectively. Flow cytometric analysis of apoptosis using Annexin V/propidium iodide (PI) staining was also performed. C. sinensis extract at 170 µg/mL significantly increase apoptosis in U-937 (p<0.001), Jurkat (p<0.05) and K-562 cells (p<0.01) when compared to untreated cells. Meanwhile, P. amarus extract at 210 µg/mL significantly induced apoptosis in both U937 and K562 cells (p<0.05) but not Jurkat cells and caused cell cycle arrest at S phase in U-937 cells (p<0.001) and at G0/G1 in K652 cells (p<0.05) when compared to control. Based on the findings, both C. sinensis and P. amarus extracts showed potential in inducing apoptosis in human leukemic cell lines. In addition, P. amarus has the capability to disrupt cell cycle

Inhibitory effects of Gynura procumbens ethanolic extract on nitric oxide production and inducible nitric oxide synthase (iNOS) protein expression in macrophages

Nitric oxide (NO) overproduction by inducible nitric oxide synthase (iNOS) may be associated with acute and chronic inflammations. Macrophages as important cells in the innate immune system are able to be stimulated and can lead to iNOS activation and excessive NO production. Gynura procumbens is a medicinal plant traditionally used in treating various ailments including inflammation but the mechanism of anti-inflammatory activity of this plant is still elusive. This study was carried out to investigate the anti-inflammatory therapeutic effects of Gynura procumbens ethanolic extract on NO production and iNOS protein expression in RAW 264.7 macrophages stimulated with lipopolysaccharide (LPS). Cell viability of RAW 264.7 macrophages treated with Gynura procumbens ethanolic extract was determined by MTT assay. NO production was determined by Griess assay following Gynura procumbens ethanolic extract treatment alone or in combination with LPS stimulation. Protein expression of iNOS was determined by western blot. RAW 264.7 macrophages viability of more than 90% was observed after 24 h treatment with Gynura procumbens ethanolic extract concentration range of 3.9 μg/mL to 500 μg/mL. Significant inhibition of NO production level has been identified in LPS-stimulated RAW 264.7 cells pre-treated with 250 μg/mL Gynura procumbens ethanolic extract (p<0.05) while all selected concentrations of Gynura procumbens ethanolic extract showed no significant alteration of NO production in the absence of LPS stimulation. Pre-treatment of 250 μg/mL Gynura procumbens ethanolic extract also demonstrated significant suppression of iNOS protein expression in LPS-stimulated RAW 264.7 cells (p<0.05). In conclusion, this study demonstrates that Gynura procumbens ethanolic extract exhibits anti-inflammatory potential through inhibition of NO production and iNOS protein expression in LPS-stimulated macrophages, suggesting that this plant could be further researched for its beneficial use in inflammatory disorders

Synthesis of unsymmetrical monocarbonyl curcumin analogues with potent inhibition on prostaglandin E2 production in LPS-induced murine and human macrophages cell lines

The syntheses and bioactivities of symmetrical curcumin and its analogues have been the subject of interest by many medicinal chemists and pharmacologists over the years. To improve our understanding, we have synthesized a series of unsymmetrical monocarbonyl curcumin analogues and evaluated their effects on prostaglandin E2 production in lipopolysaccharide-induced RAW264.7 and U937 cells. Initially, compounds 8b and 8c exhibited strong inhibition on the production of PGE2 in both LPS-stimulated RAW264.7 (8b, IC50 = 12.01 μM and 8c, IC50 = 4.86 μM) and U937 (8b, IC50 = 3.44 μM and 8c, IC50 = 1.65 μM) cells. Placing vanillin at position Ar2 further improved the potency when both compounds 15a and 15b significantly lowered the PGE2 secretion level (RAW264.7: 15a, IC50 = 0.78 μM and 15b, IC50 = 1.9 μM while U937: 15a, IC50 = 0.95 μM and 15b, IC50 = 0.92 μM). Further experiment showed that compounds 8b, 8c, 15a and 15b did not target the activity of downstream inflammatory COX-2 mediator. Finally, docking simulation on protein targets COX-2, IKK-β, ERK, JNK2, p38α and p38β were performed using the conformation of 15a determined by single-crystal XRD

A comparison of five lipid extraction solvent systems for lipidomic studies of human LDL

Lipidome profile of fluids and tissues is a growing field as the role of lipids as signaling molecules is increasingly understood, relying on an effective and representative extraction of the lipids present. A number of solvent systems suitable for lipid extraction are commonly in use, though no comprehensive investigation of their effectiveness across multiple lipid classes has been carried out. To address this, human LDL from normolipidemic volunteers was used to evaluate five different solvent extraction protocols [Folch, Bligh and Dyer, acidified Bligh and Dyer, methanol (MeOH)-tert-butyl methyl ether (TBME), and hexane-isopropanol] and the extracted lipids were analyzed by LC-MS in a high-resolution instrument equipped with polarity switching. Overall, more than 350 different lipid species from 19 lipid subclasses were identified. Solvent composition had a small effect on the extraction of predominant lipid classes (triacylglycerides, cholesterol esters, and phosphatidylcholines). In contrast, extraction of less abundant lipids (phosphatidylinositols, lyso-lipids, ceramides, and cholesterol sulfates) was greatly influenced by the solvent system used. Overall, the Folch method was most effective for the extraction of a broad range of lipid classes in LDL, although the hexane-isopropanol method was best for apolar lipids and the MeOH-TBME method was suitable for lactosyl ceramides

The pro- or anti-inflammatory effects of oxidized or chlorinated lipids and its signalling mechanisms

Strathclyde theses - ask staff. Thesis no. : T13371It is currently well accepted that atherosclerosis is an inflammatory disease and accumulation of oxidized low density lipoprotein (OxLDL) is a principal risk factor for this disease. Since the finding that oxidized phospholipids (OxPLs) is active components of OxLDL, many studies have demonstrated their pro- and antiinflammatory effects as well as signalling mechanisms. In comparison, less is known about chlorinated lipids, although several reports show that they are found in atherosclerotic lesions and inflammatory loci, and induce mainly pro-inflammatory effects. This study investigated the role of chlorinated lipids on pro-inflammatory cytokine production and the signalling mechanisms induced by these modified lipids, in comparison with OxPLs. Treatment of myeloid (U937) cells with 1-stearoyl-2- oleoyl-sn-3-glycerophosphocholine chlorohydrin (SOPC ClOH) but not its native lipid enhanced the effect of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) and treatment with 2-chlorohexadecanal (2-ClHDA) inhibited LPS-induced tumor necrosis-alpha (TNF-α) production. However, treatment of these compounds alone did not increase the level of IL-8 and TNF-α)pression. Using mouse fibrosarcoma cells (L929sA) that were stably transfected with nuclear factor-kappaB (NF-kB) dependent promoter, it was demonstrated that SOPC ClOH and 2-ClHDA did not stimulate NF-kB-driven genes activity and did not inhibit TNFα-induced NF-kB driven genes activity. SOPC ClOH treatment of human embryonic kidney 293 (HEK 293) cells overexpressing PPARα-driven gene expression. However, treatment of these cells with native SOPC and 2-ClHDA did not induce similar effects. In addition, treatment of human umbilical vein endothelial cells (HUVECs) with SOPC ClOH and 2-ClHDA did not induce degradation of Ikappa B alpha (IkB-α) or expression of mitogen activated protein kinases (MAPKs), and pretreatment with either SOPC ClOH or 2-ClHDA did not inhibit LPS-mediated activation of NF-kB and MAPK pathways. In conclusion, phospholipid induced pro-inflammatory effects and stimulated PPRE-driven gene activity in cells overexpressing PPARα whereas 2-ClHDA induced an anti-inflammatory effect. The effects were different to those observed with short-chain OxPLs, showing that the nature of the oxidative modification is important in determining cellular responseIt is currently well accepted that atherosclerosis is an inflammatory disease and accumulation of oxidized low density lipoprotein (OxLDL) is a principal risk factor for this disease. Since the finding that oxidized phospholipids (OxPLs) is active components of OxLDL, many studies have demonstrated their pro- and antiinflammatory effects as well as signalling mechanisms. In comparison, less is known about chlorinated lipids, although several reports show that they are found in atherosclerotic lesions and inflammatory loci, and induce mainly pro-inflammatory effects. This study investigated the role of chlorinated lipids on pro-inflammatory cytokine production and the signalling mechanisms induced by these modified lipids, in comparison with OxPLs. Treatment of myeloid (U937) cells with 1-stearoyl-2- oleoyl-sn-3-glycerophosphocholine chlorohydrin (SOPC ClOH) but not its native lipid enhanced the effect of lipopolysaccharide (LPS)-induced interleukin-8 (IL-8) and treatment with 2-chlorohexadecanal (2-ClHDA) inhibited LPS-induced tumor necrosis-alpha (TNF-α) production. However, treatment of these compounds alone did not increase the level of IL-8 and TNF-α)pression. Using mouse fibrosarcoma cells (L929sA) that were stably transfected with nuclear factor-kappaB (NF-kB) dependent promoter, it was demonstrated that SOPC ClOH and 2-ClHDA did not stimulate NF-kB-driven genes activity and did not inhibit TNFα-induced NF-kB driven genes activity. SOPC ClOH treatment of human embryonic kidney 293 (HEK 293) cells overexpressing PPARα-driven gene expression. However, treatment of these cells with native SOPC and 2-ClHDA did not induce similar effects. In addition, treatment of human umbilical vein endothelial cells (HUVECs) with SOPC ClOH and 2-ClHDA did not induce degradation of Ikappa B alpha (IkB-α) or expression of mitogen activated protein kinases (MAPKs), and pretreatment with either SOPC ClOH or 2-ClHDA did not inhibit LPS-mediated activation of NF-kB and MAPK pathways. In conclusion, phospholipid induced pro-inflammatory effects and stimulated PPRE-driven gene activity in cells overexpressing PPARα whereas 2-ClHDA induced an anti-inflammatory effect. The effects were different to those observed with short-chain OxPLs, showing that the nature of the oxidative modification is important in determining cellular respons