Luminol-amplified Chemiluminescence Research Articles

Eugenol Inhibits Neutrophils Myeloperoxidase In Vitro and Attenuates LPS-Induced Lung Inflammation in Mice.

Eugenol (Eug) is a polyphenol extracted from the essential oil of Syzygium aromaticum (L.) Merr. and Perry (Myrtaceae). The health benefits of eugenol in human diseases were proved in several studies. This work aims to evaluate the effect of eugenol on lung inflammatory disorders. For this, using human neutrophils, the antioxidant activity of eugenol was investigated in vitro. Furthermore, a model of LPS-induced lung injury in mice was used to study the anti-inflammatory effect of eugenol in vivo. Results showed that eugenol inhibits luminol-amplified chemiluminescence of resting neutrophils and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLF) peptide or phorbol myristate acetate (PMA). This effect was dose dependent and was significant from a low concentration of 0.1 µg/mL. Furthermore, eugenol inhibited myeloperoxidase (MPO) activity without affecting its degranulation. Eugenol has no scavenging effect on hydrogen peroxide (H2O2) and superoxide anion (O2-). Pretreatment of mice with eugenol prior to the administration of intra-tracheal LPS significantly reduced neutrophil accumulation in the bronchoalveolar lavage fluid (BALF) and decreased total proteins concentration. Moreover, eugenol clearly inhibited the activity of matrix metalloproteinases MMP-2 (21%) and MMP-9 (28%), stimulated by LPS administration. These results suggest that the anti-inflammatory effect of eugenol against the LPS-induced lung inflammation could be exerted via inhibiting myeloperoxidase and metalloproteinases activity. Thus, eugenol could be a promising molecule for the treatment of lung inflammatory diseases.

Translational suppression of SARS-COV-2 ORF8 protein mRNA as a Viable therapeutic target against COVID-19: Computational studies on potential roles of isolated compounds from Clerodendrum volubile leaves

The open reading frame 8 (ORF8) protein of SARS-CoV-2 has been implicated in the onset of cytokine storms, which are responsible for the pathophysiology of COVID-19 infection. The present study investigated the potential of isolated compounds from Clerodendrum volubile leaves to stall oxidative bursts in vitro and interact with ORF8 mRNA segments of the SARS-CoV-2 whole genome using computational tools. Five compounds, namely, harpagide, 1-(3-methyl-2-butenoxy)-4-(1-propenyl)benzene, ajugoside, iridoid glycoside and erucic acid, were isolated from C. volubile leaves, and their structures were elucidated using conventional spectroscopy tools. Iridoid glycoside is being reported for the first time and is thus regarded as a new compound. The ORF8 mRNA sequences of the translation initiation sites (TIS) and translation termination sites (TTSs) encoding ORF8 amino acids were retrieved from the full genome of SARS-CoV-2. Molecular docking studies revealed strong molecular interactions of the isolated compounds with the TIS and TTS of ORF8 mRNA. Harpagide showed the strongest binding affinity for TIS, while erucic acid was the strongest for TTS. The immunomodulatory potentials of the isolated compounds were investigated on neutrophil phagocytic respiratory bursts using luminol-amplified chemiluminescence technique. The compounds significantly inhibited oxidative burst, with 1-(3-methyl-2-butenoxy)-4-(1-propenyl)benzene having the best activity. Ajugoside and erucic acid showed significant inhibitory activity on T-cell proliferation. These results indicate the potential of C. volubile compounds as immunomodulators and can be utilized to curb cytokine storms implicated in COVID-19 infection. These potentials are further corroborated by the strong interactions of the compounds with the TIS and TTS of ORF8 mRNA from the SARS-CoV-2 whole genome.

One night of sleep fragmentation does not affect exercise-induced leukocyte trafficking or mitogen-stimulated leukocyte oxidative burst in healthy men

PurposeThis study examined whether one night of sleep fragmentation alters circulating leukocyte counts and mitogen-stimulated oxidative burst by leukocytes at rest and in response to an acute bout of vigorous exercise. MethodsIn a randomised crossover design, nine healthy men (mean ± SD: age 22 ± 2 years; BMI 24.9 ± 1.9 kg/m2) were exposed to one night of fragmented or uninterrupted sleep before cycling for 45 min at 71% ± 4% V̇O2peak. Finger-tip blood samples were collected at rest, immediately post-exercise and one-hour post-exercise. Total leukocytes, lymphocytes, monocytes and neutrophils were counted. Leukocyte oxidative burst was assessed in whole blood by measuring Reactive Oxygen Species (ROS) production with luminol-amplified chemiluminescence after stimulation with phorbol 12-myristate 13-acetate (PMA). ResultsExercise elicited the expected trafficking pattern of leukocytes, lymphocytes, monocytes and neutrophils. Compared to rest, PMA-stimulated ROS production was increased one-hour post-exercise (+73% ± 65%; p = 0.019; data combined for fragmented and uninterrupted sleep). There were no statistically significant effects of fragmented sleep on leukocyte, lymphocyte, monocyte, and neutrophil counts or on ROS production at rest, immediately post-exercise or one-hour post-exercise (p > 0.05). However, with fragmented sleep, there was a +10% greater lymphocytosis immediately post-exercise (fragmented +40% ± 37%; uninterrupted +30% ± 35%; p = 0.51) and a −19% smaller neutrophilia by one-hour post-exercise (fragmented +103% ± 88%; uninterrupted +122% ± 131%; p = 0.72). ConclusionFragmented sleep did not substantially alter the magnitude or pattern of exercise-induced leukocyte trafficking or mitogen-stimulated oxidative burst by leukocytes.

Membranotropic and Antiradical Properties of 2-Nitroxysuccinate 3-Hydroxy-6-Methyl-2-Ethylpyridine.

We studied membranotropic properties of NO donor 2-nitroxysuccinate 3-hydroxy-6-methyl-2-ethylpyridine and its structural analog succinate 3-hydroxy-6-methyl-2-ethylpyridine (Mexidol). It was shown that the compounds under study are incorporated into modeled membranes and form long-living complexes with pyrene in the region of fatty acid tails of phospholipids. Luminol-amplified chemiluminescence analysis showed that both compounds exhibited antiradical activity and in a concentration of 0.1 mM reduced chemiluminescence intensity by more than 70%. 2-Nitroxysuccinate 3-hydroxy-6-methyl-2-ethylpyridine inhibited catalytic activity of monoamine oxidase A more efficiently than its structural analogue Mexidol.

Syzygium aromaticum aqueous extract inhibits human neutrophils myeloperoxidase and protects mice from LPS-induced lung inflammation

Context: Syzygium aromaticum (L.) Merr. & Perry (Myrtaceae), commonly known as clove, originally found in the Muluku Islands in East Indonesia, is widely used as a spice and has numerous medicinal properties.Objective: This study investigated the antioxidant potential of S. aromaticum aqueous extract (SAAE) in vitro and its protective effects on lipopolysaccharide (LPS)-induced lung inflammation in mice.Material and methods: Neutrophils were isolated from healthy donors and reactive oxygen species (ROS) generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by cytochrome c reduction assay. H2O2 was detected by DCFH fluorescence assay. Myeloperoxidase (MPO) activity was mesured by tetramethyl benzidine oxidation method. To study the anti-inflammatory activity of SAAE, lung inflammation was induced in mice (BALB/c) by intra-tracheal instillation of lypopolysaccharide (5 µg/mouse), and SAAE (200 mg/kg body weight) was injected intraperitoneally prior to LPS administration. Bronchoalveolar lavage and lung tissue were collected to assess inflammatory cells count and total protein content. Metalloproteinases activity was detected by zymography technique.Results: SAAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine- or phorbol myristate acetate-stimulated neutrophils, with an inhibitory effect starting at a concentration as low as 0.5 µg/mL. Moreover, SAAE reduced significantly MPO activity and it exhibits a dose-dependent action (IC50 = 0.5 µg/mL). In vivo results showed that SAAE decreased markedly neutrophil count (From 61% to 15%) and proteins leakage into bronchoalveolar lavage fluid. Gelatin zymography assay showed that S. aromaticum inhibited MMP-2 (15%) and MMP-9 (18%) activity in lung homogenates.Discussion and conclusion: Our results suggest that the anti-inflammatory activity of SAAE, in vivo, is due to the inhibition of ROS production and metalloproteinases activity via its action on MPO. According to these findings, SAAE could be a potential source of new compounds with anti-inflammatory activity.

Influence of heat shock proteins in individual sensitivity of human neutrophils to heat stress

We have developed a simple and reliable method to measure the sensitivity of individuals to oxidative stress. This method utilizes luminol-amplified chemiluminescence to quantify production of reactive oxygen species (ROS) by opsonized zymosan-stimulated neutrophils that have been subjected to short-term stress via heat shock. In this study, the chemiluminescence reaction was used to monitor the dynamics of ROS production in neutrophils derived from 17 patients of different ages and genders before and after these neutrophils were subjected to heat shock. In addition, we determined expression of Toll-like receptors using fluorescent-labeled antibody. The effects of adrenaline, dexamethasone, aspirin, and indomethacin, as well as different doses of exogenous heat shock protein 70 (Hsp70), on the production of ROS by stimulated neutrophils was also investigated. Our data showed that adrenaline and exogenous Hsp70 both suppressed ROS production by stimulated neutrophils. Furthermore, TLR4 expression was upregulated upon heat stress. Thus, adrenaline, HSPs, and TLRs may all play a role in regulating stress responses in phagocytes.

Flavonoids and triterpenes from Combretum fragrans with anti-inflammatory, antioxidant and antidiabetic potential.

Despite the well-documented benefits of Combretum fragrans in Cameroon, only few scientific works have been done on it. In this study we isolated eight compounds from the leaves extract of C. fragrans: velutin (1), belamcanidin (2), cirsilineol (3), cirsimaritin (4), 3β-acetoxy-20,24-epoxy-11,25-hydroxy-dammarane (5), combretin A (6), combretin B (7) and a mixture of arjunolic acid (8a) and asiatic acid (8b). Compounds 6 and 7 presented potent anti-inflammatory, antioxidant and antidiabetic activities. Compounds 1, 3, 5 and the mixture of 8a and 8b were significantly active, and compounds 2 and 4 presented moderate activity for reactive oxygen species inhibitory and free-radical scavenging. All compounds were isolated using chromatographic techniques; their structures were elucidated by spectroscopic techniques and their spectroscopic data compared with those of the literature. Anti-inflammatory activity was evaluated via the oxidative burst assay using a luminol-amplified chemiluminescence technique, antioxidant activity by free-radical scavenging activity (DPPH) and antidiabetic activity via α-glucosidase inhibition. All of the isolated compounds (1-8) were reported to exhibit significant antioxidant activity. Compounds 1, 3, and 5-8 exhibited potent chemiluminescence inhibition effect, and only compounds 6 and 7 inhibited α-glucosidase. Thus, C. fragrans can be used as an effective natural source of anti-inflammatory, antioxidant and antidiabetic compounds.

Oleuropein and hydroxytyrosol inhibit the N-formyl-methionyl-leucyl-phenylalanine-induced neutrophil degranulation and chemotaxis via AKT, p38, and ERK1/2 MAP-Kinase inhibition.

Oleuropein and hydroxytyrosol are polyphenols that are extracted from olives and are major biological active components of olives and olive oil. Oleuropein and hydroxytyrosol exhibit interesting pharmacological effects on cells, and have been shown to have many health benefits such as anti-inflammatory effects. These effects were mainly attributed to their ability to scavenge the reactive oxygen species (ROS) produced by phagocytes such as neutrophils. The aim of this study was to investigate the effect of oleuropein and hydroxytyrosol on other neutrophil functions. Human neutrophils were isolated from healthy donors. ROS production was measured by luminol-amplified chemiluminescence. Degranulation was assessed by measuring myeloperoxidase activity and Western blots. Chemotaxis was assessed by the under-agarose chemotaxis assay. Phosphorylated proteins were assessed by gel electrophoresis and Western blots. We show that in addition to their ROS scavenging effect, oleuropein and hydroxytyrosol significantly inhibited the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF)-induced degranulation of azurophilic and specific granules as measured by myeloperoxidase and lactoferrin release, respectively. We also show that oleuropein and hydroxytyrosol reduced fMLF-induced neutrophil chemotaxis. Interestingly, both agents impaired the fMLF-induced AKT, p38MAPKinase, and ERK1/2 phosphorylation, signaling molecules that are involved in pathways regulating neutrophil functions. Our data suggest that the anti-inflammatory properties of oleuropein and hydroxytyrosol are not only restricted to their ROS scavenging effect, but also involve the inhibition of two other major pro-inflammatory neutrophil functions.

Pectolinarigenin from the leaves of Clerodendrum volubile shows potent immunomodulatory activity by inhibiting T − cell proliferation and modulating respiratory oxidative burst in phagocytes

There have been increasing interest in the use of plant-derived substance as immunomodulators for the treatment and management of inflammatory ailments. Clerodendrum volubile, a leafy vegetable is known for its folkloric applications in the treatments of several inflammatory related ailments, but with little scientific evidence. This study reports the isolation, structure elucidation and in vitro immunomodulatory potentials of pectolinarigenin from C. volubile leaves. The immunomodulatory potentials of the crude methanolic extract and fractions [n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n − butanol (BuOH)] were investigated on whole blood, neutrophil and macrophage phagocytic respiratory burst using luminol-amplified chemiluminescence technique. DCM fraction showed higher inhibitory activity on respiratory burst, indicating high suppressive immunomodulatory potency. The DCM fraction was further fractionated using a gravity column chromatography loaded with silica gel. The column was eluted with mixtures of Hex and DCM (92.5:7.5) in increasing order of polarity up to Hex: DCM (88:12) to afford 5,7-Dihydroxy-6,4′-dimethoxyflavone (pectolinarigenin). The structure of the compound was established using data obtained from 1H- and 13C NMR spectroscopies and mass spectrometry. The isolated flavone was investigated for its inhibitory activity of neutrophil phagocytes respiratory burst as well as T − Cell proliferation. The compound exhibited significant activities (at p <0.05) indicating high suppressive immunomodulatory potency. The potent suppressive effect of pectolinarigenin on polymorphonuclear neutrophils (PMNs) respiratory oxidative burst and T − cell proliferation suggests an immunomodulatory potential and pathway of the flavonoid.

Suppression of phagocytic oxidative burst, cytotoxic effect, and computational prediction of oral toxicity of dietary fatty acids of Clerodendrum volubile stem

Oil and fatty acids from the flowers and stems of Clerodendrum volubile were investigated for their antioxidative burst activities. The oils were methylated to obtain fatty acid methyl esters (FAMEs). The oils and fatty acids were then subjected to GC-MS analysis to identify the compounds present. Their inhibitory potentials on oxidative burst were investigated on blood phagocytes using the luminol-amplified chemiluminescence technique after activation with serum opsonized zymosan. Succinate derivatives were observed to be the most predominant compounds. C. volubile stem fatty acids showed potent inhibitory activity (IC50 = 18.52 ± 0.12) on blood ROS production. No cytotoxic effect was observed on treatment of CC-1 normal cell lines with the fatty acids. Oral toxicity prediction of the identified fatty acids of C. volubile stem via ProTox revealed their safety on swallowing. These results suggest the antioxidative burst activity of fatty acids from C. volubile stem, indicating a phagocytic action which may be attributed to the synergetic effect of the identified fatty acids.

Исследование функциональной активности полиморфно-ядерных лейкоцитов крови у пациентов с гнойно-воспалительными осложнениями при сахарном диабете II типа

In the article we present the results of estimating the phagocytic activity and priming effect in the polymorphonuclear leukocytes of blood under the effect of Streptococcus pyogenes complex antigen in patients having acute purulent inflammatory diseases of skin and soft tissues against the background of type 2diabetes (with diabetic foot syndrome) and without it. The examination of patient's blood was performed by the method of barium sulfate-stimulated luminol-amplified chemiluminescence. On the basic of the obtained results it has been found out that the patients with diabetic foot syndrome had the reduction in phagocytic activity and priming effect of polymorphonuclear leukocytes as compared to the patients with no carbohydrate metabolism disorders. The significant negative correlation between phagocytic activity, priming effect in the polymorphonuclear leukocytes and blood hyperglycemia in diabetic foot syndrome was detected.

Flowers of Clerodendrum volubile exacerbate immunomodulation by suppressing phagocytic oxidative burst and modulation of COX-2 activity.

The immunomodulatory potentials of the crude methanolic extract and fractions [n-hexane (Hex), n-dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH)] of Clerodendrum volubile flowers were investigated on whole blood phagocytic oxidative burst using luminol-amplified chemiluminescence technique. They were also investigated for their free radicals scavenging activities. The DCM fraction showed significant (p<0.05) anti-oxidative burst and free radical scavenging activities indicating high immunomodulatory and antioxidant potencies respectively. Cytotoxicity assay of the DCM fraction revealed a cytotoxic effect on CC-1 normal cell line. GCMS analysis revealed the presence of triacetin; 3,6-dimethyl-3-octanol; 2R - Acetoxymethyl-1,3,3-trimethtyl - 4t - (3-methyl-2-buten-1-yl) - 1c - cyclohexanol and Stigmastan - 3,5-diene in DCM fraction. These compounds were docked with the active sites of cyclooxygenase-2 (COX-2). Triacetin, 3,6-dimethyl-3-Octanol, and 2R-Acetoxymethyl-1,3,3-trimethtyl-4t-(3-methyl-2-buten-1-yl)-1c-cyclohexanol docked comfortably with COX-2 with good scoring function (-CDocker energy) indicating their inhibitory potency against COX-2. 3,6-dimethyl-3-Octanol, displayed the lowest predicted free energy of binding (-21.4kcalmol-1) suggesting its stronger interaction with COX-2, this was followed by 2R - Acetoxymethyl-1, 3, 3-trimethtyl-4t-(3-methyl-2-buten-1-yl)-1c-cyclhexanol (BE=-20.5kcalmol-1), and triacetin (BE=-10.9kcalmol-1). Stigmastan - 3,5-diene failed to dock with COX-2. The observed suppressive effect of the DCM fraction of C. volubile flower methanolic extract on phagocytic oxidative burst indicates an immunomodulatory potential. This is further reflected in its free scavenging activities and synergetic modulation of COX-2 activities by its identified compounds in silico.

Cytotoxic, Anti-inflammatory, and Leishmanicidal Activities of Diterpenes Isolated from the Roots of Caesalpinia pulcherrima.

A phytochemical investigation on the chloroform extract of Caesalpinia pulcherrima roots led to the isolation of ten known furanocassane diterpenoids, vouacapen-5α-ol (1), 8,9,11,14-didehydrovouacapen-5α-ol (2), 6β-cinnamoyl-7β-hydroxyvouacapen-5α-ol (3), pulcherrin A (4), pulcherrin B (5), pulcherrin J (6), pulcherrimin A (7), pulcherrimin B (8), pulcherrimin C (9), and pulcherrimin E (10). Chemical transformation of 3 and 7 gave compounds 6β-hydroxyisovouacapenol C (11), 6β-cinnamoyl-7β-acetoxyvouacapen-5α-ol (12), and pulcherrimin D (13). Cytotoxicity of compounds 1-13 was evaluated against three cancer cell lines (MCF-7, HeLa, and PC-3). Anti-inflammatory potential of the compounds was evaluated via the oxidative burst assay using a luminol-amplified chemiluminescence technique. Leishmanicidal activity was tested against promastigotes of Leishmania major in vitro. Compounds 3, 4, 8, 9, and 10 were found active against all three cancer cell lines with IC50s ranging from 7.02 ± 0.31 to 36.49 ± 1.39 µM. Compounds 8 and 13 exhibited a potent inhibitory effect on reactive oxygen species generated from human whole blood phagocytes (IC50 = 15.30 ± 1.10 µM and 8.00 ± 0.80 µM, respectively). Compounds 3, 9, and 13 showed significant activity against promastigotes of L.major (IC50 = 65.30 ± 3.20, 58.70 ± 2.80, and 55.90 ± 2.40 µM, respectively).

Chamomile decoction extract inhibits human neutrophils ROS production and attenuates alcohol-induced haematological parameters changes and erythrocytes oxidative stress in rat.

BackgroundThe aim of this study was to evaluate the protective effects of subacute pre-treatment with chamomile (Matricaria recutita L.) decoction extract (CDE) against stimulated neutrophils ROS production as well as ethanol (EtOH)-induced haematological changes and erythrocytes oxidative stress in rat.MethodsNeutrophils were isolated and ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. Adult male wistar rats were used and divided into six groups of ten each: control, EtOH, EtOH + various doses of CDE (25, 50, and 100 mg/kg, b.w.), and EtOH+ ascorbic acid (AA). Animals were pre-treated with CDE extract during 10 days.ResultsWe found that CDE inhibited (P ≤ 0.0003) luminol-amplified chemiluminescence of resting neutrophils and N-formyl methionylleucyl-phenylalanine (fMLF) or phorbolmyristate acetate (PMA) stimulated neutrophils, in a dose-dependent manner. CDE had no effect on superoxide anion, but it inhibited (P ≤ 0.0004) H2O2 production in cell free system. In vivo, CDE counteracted (P ≤ 0.0034) the effect of single EtOH administration which induced (P < 0.0001) an increase of white blood cells (WBC) and platelets (PLT) counts. Our results also demonstrated that alcohol administration significantly (P < 0.0001) induced erythrocytes lipoperoxidation increase and depletion of sulfhydryl groups (−SH) content as well as antioxidant enzyme activities as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). More importantly, we found that acute alcohol administration increased (P < 0.0001) erythrocytes and plasma hydrogen peroxide (H2O2), free iron, and calcium levels while the CDE pre-treatment reversed increased (P ≤ 0.0051) all these intracellular disturbances.ConclusionsThese findings suggest that CDE inhibits neutrophil ROS production and protects against EtOH-induced haematologiacal parameters changes and erythrocytes oxidative stress. The haematoprotection offered by chamomile might involve in part its antioxidant properties as well as its opposite effect on some intracellular mediators such as H2O2, free iron, and calcium.

Characterization and antioxidant activity of semi-purified extracts and pure delphinidin-glycosides from eggplant peel (Solanum melongena L.)

Eggplant peels containing delphinidin-3-rutinoside or delphinidin-3-(p-coumaroylrutinoside)-5-glucoside (nasunin) were compared and characterized for antioxidant properties, using three cell-free assays with electron paramagnetic resonance (EPR) and two biological assays on human neutrophils with luminol amplified chemiluminescence (LACL). Antioxidant solutions were made with semi-purified extracts depleted in chlorogenic acid or with purified anthocyanin crystals. The semi-purified nasunin extract was higher in scavenging capacity than delphinidin-3-rutinoside in four assays (superoxide anion, hydroxyl radical, LACL, and Fremy's salt). Nasunin crystals showed higher antioxidant indices in Fremy's salt and LACL assays, with no detectable activity against the hydroxyl radical. The EC50 against free radicals was 0.4–20.6 nmol/mL in semi-purified extracts, and 1.7–73.6 nmol/mL for anthocyanin crystals. The higher activity of semi-purified extracts than crystals was attributed to other antioxidants, such as ascorbic acid, glutathione and biogenic amines, thus representing a percentage close to 30% of the total antioxidant content of eggplant peels.

English

Citrillus colocynthis peel aqueous extract (CCPAE) is widely used to treat disorders such as inflammation, ulcers and infections, but its pharmacological target is not known. The objectives of this work were to study the effect of C. colocynthis peel aqueous extract, on human neutrophil reactive oxygen species (ROS) production in vitro, and to evaluate its protective effect on lipopolysaccharide (LPS)-induced lung inflammation in vivo in mice. Neutrophils were isolated from blood of healthy volunteers. ROS generation was measured by luminol-amplified chemiluminescence. Superoxide anion generation was detected by the cytochrome c reduction assay. H2O2 was detected by horseradish peroxidase (HRP)-amplified chemiluminescence assay. Myeloperoxidase (MPO) activity was measured by the tetramethylbenzidine oxidation method. Lung inflammation was induced in mice by LPS instillation. CCPAE inhibited luminol-amplified chemiluminescence of resting neutrophils and N-formyl-methionyl-leucyl-phenylalanine (fMLF)- or phorbolmyristate acetate (PMA)-stimulated neutrophils, in a concentration-dependent manner. CCPAE also inhibited superoxide anion generation; and did not scavenge H2O2 and superoxide anions nor inhibited MPO activity in vitro suggesting that it inhibits nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation. In vivo studies showed that CCPAE attenuated LPS-induced lung inflammation in mice. This study shows that CCPAE inhibits neutrophil ROS production and attenuates LPS-induced lung inflammation in mice. Inhibition of NADPH oxidase activation by CCPAE could explain its anti-inflammatory action. Key words: Citrullus colocynthis, colocynth, inflammation, neutrophils, reactive oxygen species (ROS), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

Escherichia coli LF82 differentially regulates ROS production and mucin expression in intestinal epithelial T84 cells: implication of NOX1.

Increased reactive oxygen species (ROS) production is associated with inflamed ileal lesions in Crohn's disease colonized by pathogenic adherent-invasive Escherichia coli LF82. We investigated whether such ileal bacteria can modulate ROS production by epithelial cells, thus impacting on inflammation and mucin expression. Ileal bacteria from patients with Crohn's disease were incubated with cultured epithelial T84 cells, and ROS production was assayed using the luminol-amplified chemiluminescence method. The gentamicin protection assay was used for bacterial invasion of T84 cell. The expression of NADPH oxidase (NOX) subunits, mucin, and IL-8 was analyzed by quantitative real-time PCR and Western blots. Involvement of NOX and ROS was analyzed using diphenyleneiodonium (DPI) and N-acetylcysteine (NAC). Among different bacteria tested, only LF82 induced an increase of ROS production by T84 cells in a dose-dependent manner. This response was inhibited by DPI and NAC. Heat- or ethanol-attenuated LF82 bacteria and the mutant LF82ΔFimA, which does not express pili type 1 and poorly adheres to epithelial cells, did not induce the oxidative response. The LF82-induced oxidative response coincides with its invasion in T84 cells, and both processes were inhibited by DPI. Also, we observed an increased expression of NOX1 and NOXO1 in response to LF82 bacteria versus the mutant LF82ΔFimA. Furthermore, LF82 inhibited mucin gene expression (MUC2 and MUC5AC) in T84 cells while increasing the chemotactic IL-8 expression, both in a DPI-sensitive manner. Adherent-invasive E. coli LF82 induced ROS production by intestinal NADPH oxidase and altered mucin and IL-8 expression, leading to perpetuation of inflammatory lesions in Crohn's disease.

FMLP-Induced IL-8 Release Is Dependent on NADPH Oxidase in Human Neutrophils.

N-Formyl-methionyl-leucyl-phenylalanine (fMLP) and platelet-activating factor (PAF) induce similar intracellular signalling profiles; but only fMLP induces interleukin-8 (IL-8) release and nicotinamide adenine dinucleotide phosphate reduced (NADPH) oxidase activity in neutrophils. Because the role of ROS on IL-8 release in neutrophils is until now controversial, we assessed if NADPH oxidase is involved in the IL-8 secretions and PI3K/Akt, MAPK, and NF-κB pathways activity induced by fMLP. Neutrophils were obtained from healthy volunteers. IL-8 was measured by ELISA, IL-8 mRNA by qPCR, and ROS production by luminol-amplified chemiluminescence, reduction of ferricytochrome c, and FACS. Intracellular pH changes were detected by spectrofluorescence. ERK1/2, p38 MAPK, and Akt phosphorylation were analysed by immunoblotting and NF-κB was analysed by immunocytochemistry. Hydroxy-3-methoxyaceto-phenone (HMAP), diphenyleneiodonium (DPI), and siRNA Nox2 reduced the ROS and IL-8 release in neutrophils treated with fMLP. HMAP, DPI, and amiloride (a Na+/H+ exchanger inhibitor) inhibited the Akt phosphorylation and did not affect the p38 MAPK and ERK1/2 activity. DPI and HMAP reduced NF-κB translocation induced by fMLP. We showed that IL-8 release induced by fMLP is dependent on NADPH oxidase, and ROS could play a redundant role in cell signalling, ultimately activating the PI3K/Akt and NF-κB pathways in neutrophils.

Scavenging effect of Trolox released from brushite cements

In this study a brushite cement was doped with the chain-breaking antioxidant Trolox. The effect of the antioxidant on the physical properties of the cement was evaluated and the release of Trolox was monitored by UV spectroscopy. The ability of the Trolox set free to scavenge reactive oxygen species (ROS) released by macrophages was determined in vitro using a luminol-amplified chemiluminescence assay. Trolox did not modify the crystalline phases of the set cement, which mainly formed crystalline brushite after 7days in humid conditions. The setting time, compressive strength and morphology of the cement also remained unaltered after the addition of the antioxidant. Trolox was slowly released from the cement following a non-Fickian transport mechanism and nearly 64% of the total amount was released after 3days. Moreover, the capacity of Trolox to scavenge the ROS released by macrophages increased in a dose-dependent manner. Trolox-loaded cements are expected to reduce some of the first harmful effects of acute inflammation and can thus potentially protect the surrounding tissue during implantation of these as well as other materials used in conjunction.

Antioxidant activity of hyaluronic acid investigated by means of chemiluminescence of equine neutrophil bursts and electron paramagnetic resonance spectroscopy.

Activated neutrophils (PMNs), the ROS/RNS released by PMNs and the derived inflammatory processes are involved in the pathogenesis and progression of human inflammatory airway diseases. Similar diseases are also present in horses which suffer from recurrent airway obstruction (RAO), exercise-induced pulmonary haemorrhage (EIPH) and inflammatory airway diseases (IAD). Hyaluronic acid (HA) plays numerous roles in modulating inflammatory processes. The aim of this study was to examine whether a preparation of HA (MW 900 000 Da) interferes with ROS/RNS during the course of equine PMN respiratory bursts, and to establish the lowest concentration at which it still has antioxidant activity by means of luminol-amplified chemiluminescence (LACL). Electron paramagnetic resonance (EPR) spectroscopy was also used to investigate the direct antiradical activity of HA. The hydroxyl radical was significantly scavenged in a concentration-dependent manner at HA concentrations ranging from 2.5 to 0.16 mg/mL. Superoxide anion, Tempol radical and the ABTS(•+) were significantly inhibited at concentrations ranging from 2.5 to 0.62 mg/mL. The LACL of stimulated equine neutrophils showed that HA induced a statistically significant concentration-effect reduction from 5 mg/mL to 1.25 mg/mL. These findings were confirmed also when l-Arg was added to investigate the inhibition of the resulting peroxynitrite anion. Our findings indicate that, in addition to the human use, HA can also be used to antagonize the oxidative stress generated by free radicals in horses peripheral blood mononuclear cells (PBMCs). In order to achieve therapeutic concentrations, a direct aerosol administration to horses with horse respiratory diseases can be considered, as this route of application is also recommended in human medicine.