Inhibited Myeloperoxidase Activity Research Articles

Carnosine markedly ameliorates H9N2 swine influenza virus-induced acute lung injury

Oxidative stress injury is an important pathogenesis of influenza virus in critically ill patients. The present study investigated the efficacy of carnosine, an antioxidant and free radical scavenger, on a model of acute lung injury (ALI) induced by H9N2 swine influenza virus. Female specific-pathogen-free BALB/c mice were randomized into four groups and treated as follows: (1) H9N2 group, (2) mock control group, (3) H9N2+carnosine group and (4) carnosine control group. The H9N2 group mice were inoculated intranasally with A/Swine/Hebei/012/2008/ (H9N2) virus (100 μl) in allantoic fluid (AF), whilst mock-infected animals were intranasally inoculated with non-infectious AF. Carnosine [10 mg (kg body mass)- 1] was administered orally (100 μl) for 7 days consecutively. The survival rate, lung water content, TNF-α and IL-1β levels, lung histopathology, myeloperoxidase (MPO) activity, and Toll-like receptor (TLR)-4 levels were determined at 2, 4, 6, 8 and 14 days after inoculation. Carnosine treatment effectively decreased the mortality (43 versus 75 %, P < 0.05), significantly ameliorated pathological lesions in lungs and decreased the lung wet/dry mass ratio (P < 0.05). It also inhibited MPO activity, suppressed TNF-α and IL-1β release, decreased the H9N2 viral titre, and markedly inhibited levels of TLR-4 mRNA and protein in the lungs of infected mice (P < 0.05), which supported the use of carnosine for managing severe influenza cases.

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.

Myeloperoxidase Inhibitors as Potential Drugs.

Myeloperoxidase (MPO) is an important member of the haem peroxidase - cyclooxygenase superfamily. This enzyme is physiologically expressed in circulating neutrophils, monocytes and some tissue macrophages including microglia. MPO plays an essential role in the antimicrobial and antiviral system of humans. The microbicidal activity of MPO exists due to its capability to oxidize halide and pseudohalide ions (CI(-), Br(-), I(-) and SCN(-)) by H2O2, thereby producing respective hypohalous acids (HOX). During the phagocytosis of pathogens, azurophilic granules release their content together with MPO into phagolysosomes. On the other hand, MPO can be discharged outside the phagocytes. Due to this, tissue damage during inflammation is greatly promoted by MPO-derived oxidants. Regarding its activity, MPO is a key factor in a great number of conditions within the group of cardiovascular diseases, inflammatory diseases, neurodegenerative diseases, kidney diseases and immune-mediated diseases. Therefore, MPO and its downstream inflammatory pathways might be attractive targets for both prognostic and therapeutic intervention in the prophylaxis of all mentioned illnesses. Nowadays, structure and reaction mechanism of MPO are known, which enable rational strategy in the development of specific MPO inhibitors that still preserve MPO activity during host defense from bacteria, but hinder pathophysiologically persistent activation of MPO. Various methods for MPO activity inhibition and unfavorable effects of MPO-derived oxidants remodeling will be discussed. Emphasis will be put on various known inhibitors, as well as on newly investigated natural products, which can also inhibit MPO activity.

Auricularia auricular-judae polysaccharide attenuates lipopolysaccharide-induced acute lung injury by inhibiting oxidative stress and inflammation.

Auricularia auricular-judae polysaccharide (AAP) has shown a variety of pharmacological properties. In the present study, the role of AAP in acute lung injury (ALI) induced by lipopolysaccharide (LPS) was analyzed in rats to further explore the possible underlying mechanisms. Adult Sprague-Dawley rats were randomly assigned into the control, AAP, LPS and LPS plus AAP groups. Rats were injected with LPS (10 mg/kg, intraperitoneal) to induce ALI. Rats in the LPS plus AAP group were treated with AAP for 7 days before the induction of ALI. The protein concentration in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by the wet/dry (W/D) weight ratio. The myeloperoxidase (MPO) activity and malondialdehyde (MDA) level were assayed by MPO and MDA kits, respectively. The levels of inflammatory mediators, tumor necrosis factor-α (TNF-α) and interleukin (IL)-6, were assayed by the enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed by hematoxylin and eosin staining. The data showed that treatment with AAP significantly improved LPS-induced lung pathological changes, attenuated protein concentration in the BALF, inhibited MPO activity and reduced the MDA level and lung W/D weight ratio. AAP also inhibited the release of TNF-α and IL-6 in blood. The results indicated that AAP has a protective effect on LPS-induced ALI in rats.

Interplay between enterobactin, myeloperoxidase and lipocalin 2 regulates E. coli survival in the inflamed gut.

During an inflammatory response in the gut, some commensal bacteria such as E. coli can thrive and contribute to disease. Here we demonstrate that enterobactin (Ent), a catecholate siderophore released by E. coli, is a potent inhibitor of myeloperoxidase (MPO), a bactericidal enzyme of the host. Glycosylated Ent (salmochelin) and non-catecholate siderophores (yersiniabactin and ferrichrome) fail to inhibit MPO activity. An E. coli mutant (ΔfepA) that overproduces Ent, but not an Ent-deficient double mutant (ΔaroB/ΔfepA), inhibits MPO activity and exhibits enhanced survival in inflamed guts. This survival advantage is counter-regulated by lipocalin 2, a siderophore-binding host protein, which rescues MPO from Ent-mediated inhibition. Spectral analysis reveals that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage.

Survival advantage of E. coli in the inflamed gut is mechanistically linked to enterobactin-mediated inhibition of myeloperoxidase and regulation by lipocalin 2 (MPF4P.736)

Abstract During an inflammatory response in the gut, several commensal bacteria such as E.coli not only survive but also thrive and consequently contribute to the disease pathogenesis. The mechanisms by which such opportunistic pathogens bloom despite the hostile pro-inflammatory milieu of the inflamed gut remain largely unknown. We demonstrated that enterobactin (Ent), a siderophore released by E. coli, is a potent inhibitor of a key bactericidal enzyme of the host innate immune system, myeloperoxidase (MPO). The inhibitory activity is specific as glycosylated Ent (salmochelin) and non-catecholate siderophores such as yersiniabactin and ferrichrome failed to inhibit MPO activity. An E. coli ferrienterobactin permease mutant (ΔfepA) overexpressing Ent, but not 3-dehydroquinate synthase (ΔaroB)/ΔfepA double mutant inhibited MPO activity and exhibited enhanced survival in inflamed guts. This survival advantage was counter-regulated by the host siderophore binding protein, lipocalin 2, which rescued MPO from Ent-mediated inhibition. Spectral analysis revealed that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage.

Anti-inflammatory effects of water extract of Taraxacum mongolicum hand.-Mazz on lipopolysaccharide-induced inflammation in acute lung injury by suppressing PI3K/Akt/mTOR signaling pathway

Ethnopharmacological relevanceTaraxacum mongolicum Hand.-Mazz is a famous medicinal plant in China, has been listed in the Pharmacopoeia of the People׳s Republic of China and used to treat infection, fever, upper respiratory tract infection, pneumonia, and other infectious diseases. This study aims to evaluate the possible mechanisms responsible for the anti-inflammation effects of water extract of T. mongolicum Hand.-Mazz (WETMHM) on lipopolysaccharide (LPS)-induced inflammatory in acute lung injury. Materials and methodsFemale BALB/c mice were randomly divided into five groups with 10 mice in each group: (1) control group (saline), (2) LPS group, (3) LPS+dexamethasone (LPS+Dex, 2mg/kg, administered by gavage), (4) LPS+WETMHM (5g/kg, administered by gavage), (5) LPS+WETMHM (10g/kg, administered by gavage). The cell counting in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity were assayed by SOD and MPO kits, respectively. The levels of inflammation mediators including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6 were assayed by an enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed by hematoxylin and eosin (HE) staining. The levels of P-PI3K, PI3K, P-Akt, Akt, P-mTOR and mTOR were measured by Western blotting. ResultsThe data showed that treatment with the WETMHM inhibited LPS-induced inflammation: (1) WETMHM attenuated inflammation cell numbers in the BALF, (2) decreased protein levels of lung PI3K/Akt/mTOR, and (3) improved SOD activity and (4) inhibited MPO activity; (5) histological studies demonstrated that WETMHM substantially inhibited LPS-induced neutrophils in lung tissue. ConclusionThe results indicated that the WETMHM had a protective effect on LPS-induced ALI in mice.

PTEN–Foxo1 signaling triggers HMGB1-mediated innate immune responses in acute lung injury

PTEN is a multifunctional phosphatase that regulates immune responses through a PI3K/Akt signaling cascade. HMGB1 plays an important role in the initiation of innate immune responses to induce acute lung injury (ALI). This study was designed to investigate the role of PTEN/Foxo1 signaling in the regulation of in vivo and in vitro innate immune responses in ALI. Using a mouse model of ALI, wild-type (WT) and myeloid-specific PTEN knockout (PTENM-KO) mice were instilled with a recombinant HMGB1 (rHMGB1) or PBS. In some experiments, Foxo1 siRNA or non-specific siRNA was injected into mice 6 h prior to rHMGB1 instillation into lung. We found that rHMGB1 treatment in WT mice increased the expression of PTEN, Foxo1, TLR4, and NF-κB in alveolar macrophages from WT mice. However, macrophage-specific PTEN ablation resulted in reduced Foxo1 and TLR4 while increasing β-catenin (Ser552) and Akt (Ser473) phosphorylation in these cells. Knockdown of Foxo1 with siRNA administration in WT mice ameliorated lung injury and inhibited myeloperoxidase activity followed by rHMGB1 treatment, which was accompanied by decreased mRNA expression coding for TNF-α, IL-1β, MIP2, and IP-10. Moreover, Foxo1 knockdown inhibited the expression of TLR4-dependent IRF3 and IFN-β both in vitro and in vivo. These results demonstrate that PTEN/Foxo1 signaling is critical for triggering HMGB1-mediated innate TLR4 activation during ALI. By identifying the molecular signaling pathways within innate immune system, our studies provide the potential therapeutic targets for ALI.

Myeloperoxidase propagates damage and is a potential therapeutic target for subacute stroke.

Few effective treatment options exist for stroke beyond the hyperacute period. Radical generation and myeloperoxidase (MPO) have been implicated in stroke. We investigated whether pharmacologic reduction or gene deletion of this highly oxidative enzyme reduces infarct propagation and improves outcome in the transient middle cerebral artery occlusion mouse model (MCAO). Mice were treated with 4-aminobenzoic acid hydrazide (ABAH), a specific irreversible MPO inhibitor. Three treatment regimens were used: (1) daily throughout the 21-day observational period, (2) during the acute stage (first 24 hours), or (3) during the subacute stage (daily starting on day 2). We found elevated MPO activity in ipsilateral brain 3 to 21 days after ischemia. 4-Aminobenzoic acid hydrazide reduced enzyme activity by 30% to 40% and final lesion volume by 60% (P<0.01). The MPO-knockout (KO) mice subjected to MCAO also showed a similar reduction in the final lesion volume (P<0.01). The ABAH treatment or MPO-KO mice also improved neurobehavioral outcome (P<0.001) and survival (P=0.01), but ABAH had no additional beneficial effects in MPO-KO mice, confirming specificity of ABAH. Interestingly, inhibiting MPO activity during the subacute stage recapitulated most of the therapeutic benefit of continuous MPO inhibition, suggesting that MPO-targeted therapies could be useful when given after 24 hours of stroke onset.

Ginkgo biloba extracts attenuate lipopolysaccharide-induced inflammatory responses in acute lung injury by inhibiting the COX-2 and NF-κB pathways

In the present study, we analyzed the role of Ginkgo biloba extract in lipopolysaccharide(LPS)-induced acute lung injury (ALI). ALI was induced in mice by intratracheal instillation of LPS. G. biloba extract (12 and 24 mg·kg−1) and dexamethasone (2 mg·kg−1), as a positive control, were given by i.p. injection. The cells in the bronchoalveolar lavage fluid (BALF) were counted. The degree of animal lung edema was evaluated by measuring the wet/dry weight ratio. The superoxidase dismutase (SOD) and myeloperoxidase (MPO) activities were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators, tumor necrosis factor-a, interleukin-1b, and interleukin-6, were assayed by enzyme-linked immunosorbent assay. Pathological changes of lung tissues were observed by H&E staining. The levels of NF-κB p65 and COX-2 expression were detected by Western blotting. Compared to the LPS group, the treatment with the G. biloba extract at 12 and 24 mg·kg−1 markedly attenuated the inflammatory cell numbers in the BALF, decreased NF-κB p65 and COX-2 expression, and improved SOD activity, and inhibited MPO activity. The histological changes of the lungs were also significantly improved. The results indicated that G. biloba extract has a protective effect on LPS-induced acute lung injury in mice. The protective mechanism of G. biloba extract may be partly attributed to the inhibition of NF-κB p65 and COX-2 activation.

Effect of bifidobacterium on defensin-5 expression in intestinal injury of preweaning rats.

To investigate the protective effect of bifidobacterium in endotoxin-induced intestinal injury in preweaning rats. Preweaning rats were randomly divided into three groups (n = 40 for each): a control group (group C), a model group (group E) and a treatment group (group T). Both groups E and T were intraperitoneally injected with lipopolysaccharide (LPS) at a dose of 5 mg/kg (5 mg/L in normal saline), and group T was intragastrically administrated with bifidobacterium suspension (2.0 × 10(9) CFU/mL, 0.5 mL each time, twice a day, until the end of the experiment) 7 d before LPS administration. Group C was intraperitoneally injected with normal saline. After intraperitoneal injection and intragastric administration, the rats were placed back to the initial cage to receive breast feeding. The rats were killed at 2, 6, 12, 24 or 72 h, respectively, after endotoxin or physiological saline injection to collect serum and ileal tissue samples. Myeloperoxidase (MPO) contents in serum and ileum were detected at different times, and expression of ileal defensin-5 mRNA was evaluated by reverse transcription-polymerase chain reaction. Serum and ileal MPO contents in group E were significantly higher than those in group C (serum contents: 107.50 ± 17.70 vs 157.14 ± 24.67, P < 0.05; ileal contents: 1.03 ± 0.21 vs 1.57 ± 0.33, P < 0.05), which peaked at 12 h and 6 h, respectively. MPO contents in group T were significantly lower than those in group E (serum contents: 114.38 ± 24.56 vs 145.25 ± 23.62, P < 0.05; ileal contents: 1.25 ± 0.24 vs 1.57 ± 0.33, P < 0.05). The expression of defensin-5 mRNA in group E was significantly higher than that in group C (0.953 ± 0.238 vs 0.631 ± 0.146, P < 0.05), which peaked at 2 h, and then decreased gradually. The expression of defensin-5 mRNA in group T was significantly lower than that in group E (0.487 ± 0.149 vs 0.758 ± 0.160, P < 0.05) apparently in 24 h. The expression of defensin-5 mRNA at 2 h in group T was significantly higher than that in group C (0.824 ± 0.158 vs 0.631 ± 0.146, P < 0.05). MPO and defensin-5 mRNA increase in preweaning rats with LPS-induced intestinal injury. Bifidobacterium protects the gut by inhibiting MPO activity, not by increasing defensin-5 secretion.

Tectorigenin inhibits the inflammation of LPS-induced acute lung injury in mice

AimIn a previous study, the anti-inflammatory effects of tectorigenin were disclosed. In this study, the anti-inflammatory effects of tectorigenin on acute lung injury using a lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model were investigated MethodThe cell-count in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by the wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity was assayed using SOD and MPO kits, respectively. The levels of inflammatory mediators, including tumor necrosis factor-α (TNF-α), IL-1β, and IL-6 were assayed using an enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed through HE staining. The inflammatory signal pathway related protein nuclear factor NF-κB p65 mRNA expression was measured by real-time PCR, and the protein level of NF-κB p65 was measured using Western blotting analysis. ResultsThe data showed that treatment with the tectorigenin markedly attenuated the inflammatory cell numbers in the BALF, decreased nuclear factor NF-κB p65 mRNA level and protein level in the lungs, and improved SOD activity and inhibited MPO activity. Histological studies showed that tectorigenin substantially inhibited LPS-induced neutrophils in lung tissue compared with the model group. ConclusionThe results indicated that tectorigenin had a protective effect on LPS-induced ALI in mice.

Thein vitroeffects of divalent metal ions on the activities of immune-related enzymes in coelomic fluid from the sea cucumberApostichopus japonicus

The immune-related enzymes in marine animals are very sensitive to divalent metal ions. To investigate the roles divalent metal ions play in the influence on the immunity of sea cucumber Apostichopus japonicus, one of the most important commercial species in Asian countries, the effects of eight divalent metal ions at concentrations of 2.5, 5, 10, 15, 20, 25 and 30 mmol L−1 on the activities of superoxide dismutase (SOD), phenoloxidase (PO), acid phosphatase (ACP), alkaline phosphatase (AKP) and myeloperoxidase (MPO) in coelomic fluid were determined with the nitro blue tetrazolium chloride (NBT) method, dopachrome formation method, p-nitrophenyl phosphate (pNPP) method and 3,3′,5,5′-tetramethyl benzidine (TMB) method. The results indicated that Mg2+ enhanced the activities of SOD, PO, ACP and AKP significantly and showed no obvious effect on MPO activity; Zn2+ increased the activities of SOD, ACP and AKP, and showed no obvious effect on the activities of PO and MPO; Cu2+ enhanced the activities of ACP, AKP and MPO and activated SOD and PO at a certain concentration range; Ca2+ and Mn2+ inhibited the activities of ACP and AKP; Fe2+ had strong inhibitory effect on SOD activity; Pb2+ showed inhibitions on the activities of SOD, PO, ACP and AKP; and Cd2+ inhibited MPO activity greatly. The data obtained in this study collectively suggest that Mg2+, Zn2+ and Cu2+ have potential in promotion of A. japonicus immunity, while Ca2+, Fe2+, Pb2+, Cd2+ and Mn2+ might be limiting factors to the immune response of A. japonicus.

Hybrid molecules inhibiting myeloperoxidase activity and serotonin reuptake: a possible new approach of major depressive disorders with inflammatory syndrome

Major depressive disorder (MDD) is accompanied with an imbalance in the immune system and cardiovascular impairments, such as atherosclerosis. Several mechanisms have been pointed out to underlie this rather unexpected association, and among them the activity of myeloperoxidase (MPO). The aim of our study was to find compounds that inhibit both MPO and serotonin transporter (SERT) for treating MDD associated with cardiovascular diseases. SERT inhibition was assessed with measuring of [(3) H]-serotonin uptake using HEK-293 MSR cells. MPO inhibition was determined by taurine chloramine test on 3-(aminoalkyl)-5-fluoroindole derivatives and on clinically relevant antidepressants. All kinetic measurements were performed using a temperature-controlled stopped-flow apparatus (model SX-18 MV). Promising lead compounds were docked onto SERT 3D structure modelled using the LeuT structure complexed to tryptophan (PDB code 3F3A). Their toxicological profile was also assessed. 3-(aminoalkyl)-5-fluoroindole derivative with 5 carbons on the side chain and paroxetine showed the best activity on both MPO and SERT at the nanomolar range. Paroxetine was found to be the first irreversible MPO inhibitor at nanomolar concentrations. Our results put forward the first hybrid molecule (compound 25) and drug (paroxetine) that can be especially used in MDD associated with inflammatory syndrome.

Anti-inflammatory effects of Reduning Injection on lipopolysaccharide-induced acute lung injury of rats.

ObjectiveTo evaluate the protective effects of Reduning Injection (热毒宁注射液, RDN), a patent Chinese medicine, on lipopolysaccharide (LPS)-induced acute lung injury (ALI) in rats and its underlying mechanisms of action.MethodsSixty male Sprague-Dawley rats were randomly divided into 6 groups, including normal control, model, dexamethasone (DEX, 5 mg/kg), RDN-H (720 mg/kg), RDN-M (360 mg/kg) and RDN-L (180 mg/kg) groups, with 10 rats in each group. Rats were challenged with intravenous injection of LPS 1 h after intraperitoneal treatment with RDN or DEX. At 6 h after LPS challenge, lung tissues and bronchoalveolar lavage fluid (BALF) were collected, and the number of inflammatory cells was determined. The right lungs were collected for histopathologic examination, measurement of gene and protein expressions, superoxide dismutase (SOD) and myeloperoxidase (MPO) activities.Results In vivo pretreatment of RDN (360, 720 mg/kg) significantly reduced the weight of wet to dry (W/D) ratio of lung, protein content in BALF, and led to remarkable attenuation of LPS-induced histopathological changes in the lungs. Meanwhile, RDN enormously decreased BALF total inflammatory cells, especially neutrophil and macrophage cell numbers. Moreover, RDN increased SOD activity, inhibited MPO activity, alleviated LPS-induced tumor neurosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression in lung tissues. Furthermore, RDN (720 mg/kg) efficiently weakened nuclear factorkappa B (NF-κB) gene and protein expression.ConclusionAnti-inflammatory effects of RDN was demonstrated to be preventing pulmonary neutrophil infiltration, lowering MPO activity, TNF-α and iNOS gene expression by inhibiting NF-κB activity in LPS-induced ALI.

The effects of morin on lipopolysaccharide-induced acute lung injury by suppressing the lung NLRP3 inflammasome.

In previous study, the anti-inflammatory effect of morin had been found. In this study, we investigated anti-inflammatory effects of morin on acute lung injury using lipopolysaccharide (LPS)-induced acute lung injury (ALI) mouse model. The cell counting in the bronchoalveolar lavage fluid (BALF) was measured. The animal lung edema degree was evaluated by wet/dry weight (W/D) ratio. The superoxidase dismutase (SOD) activity and myeloperoxidase (MPO) activity were assayed by SOD and MPO kits, respectively. The levels of inflammatory mediators including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-18, and IL-6 were assayed by enzyme-linked immunosorbent assay method. Pathological changes of lung tissues were observed by hematoxylin and eosin (HE) staining. The protein level of lung NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome was measured by Western blotting. The data showed that treatment with the morin markedly attenuated inflammatory cell numbers in the BALF, decreased lung NLRP3 inflammasome protein level, and improved SOD activity and inhibited MPO activity. Histological studies demonstrated that morin substantially inhibited LPS-induced neutrophils in lung tissue compared with model group. The results indicated that the morin had a protective effect on LPS-induced ALI in mice.

Inhibition of myeloperoxidase activity by the alkaloids of Peganum harmala L. (Zygophyllaceae)

Ethnopharmacological relevanceSeeds and aerial parts of Peganum harmala L. are widely used in Algeria as anti-inflammatory remedies. Evaluation of Peganum harmala total alkaloids extracts and pure β-carboline compounds as an anti-inflammatory treatment by the inhibition of an enzyme key of inflammatory, myeloperoxidase (MPO) and HPLC quantification of the alkaloids from the different parts of plant. Materials and methodsMPO inhibition was tested using taurine chloramine test. The inhibition of LDL oxidation induced by MPO was carried out. The molecular docking analysis of Peganum harmala alkaloids on MPO was performed using the Glide XP docking protocol and scoring function and the redox potential of alkaloids was determined using an Epsilon potentiostat. The concentration of harmala alkaloids was determined using HPLC analysis. ResultsThe HPLC profiling of the active total alkaloids indicates that β-carboline e.g. harmine, harmaline, harmane, harmol and harmalol are major components. As β-carbolines resemble tryptamine, of which derivatives are efficient inhibitors of MPO, the harmala alkaloids were tested for their activity on this enzyme. Total alkaloids of the seeds and of the aerial parts strongly inhibited MPO at 20µg/mL (97±5% and 43±4%, respectively) whereas, at the same concentration, those of the roots showed very low inhibition (15±6%). Harmine, harmaline and harmane demonstrated a significant inhibition of MPO at IC50 of 0.26, 0.08 and 0.72µM respectively. These alkaloids exerted a similar inhibition effects on MPO-induced LDL oxidation. Molecular docking analysis of Peganum harmala alkaloids on MPO showed that all active Peganum harmala alkaloids have a high affinity on the active site of MPO (predicted free energies of binding up to −3.1kcal/mol). Measurement of redox potentials versus the normal hydrogen electrode clearly differentiated (i) the high MPO inhibitory activity of harmine, harmaline and harmane (+1014, 1014 and 1003mV, respectively); and (ii) the low activity of harmalol and harmol (+629/778 and 532/644mV, respectively). A reverse phase HPLC method has been developed to determine simultaneously five alkaloids of Peganum harmala. Seeds contained all five β-carboline derivatives with the main active alkaloids, harmaline and harmine, being up to 3.8% and 2.9%, respectively. Up to 3.2% of harmine was determined in the roots. The four β-carboline derivatives, harmine, harmaline, harmane and harmalol were identified in the aerial parts. The highest inhibitory effect observed in seeds and the moderate effect of aerial parts could be explained by their harmine and harmaline content. In contrast, the very weak inhibition of the root extract, despite the presence of harmine, may tentatively be explained by the high concentration of harmol which can reduce Compound II of MPO to the native form. ConclusionThe inhibition of MPO by Peganum harmala β-carboline alkaloids, herein reported for the first time, may explain the anti-inflammatory effect traditionally attributed to its herbal medicine.

Myeloperoxidase deficiency attenuates nitrogen mustard-induced skin injuries

The pathologic mechanisms of skin injuries, following the acute inflammatory response induced by vesicating agents sulfur mustard (SM) and nitrogen mustard (NM) exposure, are poorly understood. Neutrophils which accumulate at the site of injury, abundantly express myeloperoxidase (MPO), a heme protein that is implicated in oxidant-related antimicrobial and cytotoxic responses. Our previous studies have shown that exposure to SM analog 2-chloroethyl ethyl sulfide (CEES) or NM results in an inflammatory response including increased neutrophilic infiltration and MPO activity. To further define the role of neutrophil-derived MPO in NM-induced skin injury, here we used a genetic approach and examined the effect of NM exposure (12h and 24h) on previously established injury endpoints in C57BL/6J wild type (WT) and B6.129X1-MPOtm1Lus/J mice (MPO KO), homozygous null for MPO gene. NM exposure caused a significant increase in skin bi-fold thickness, epidermal thickness, microvesication, DNA damage and apoptosis in WT mice compared to MPO KO mice. MPO KO mice showed relatively insignificant effect. Similarly, NM induced increases in the expression of inflammatory and proteolytic mediators, including COX-2, iNOS and MMP-9 in WT mice, while having a significantly lower effect in MPO KO mice. Collectively, these results show that MPO, which generates microbicidal oxidants, plays an important role in NM-induced skin injuries. This suggests the development of mechanism-based treatments against NM- and SM-induced skin injuries that inhibit MPO activity and attenuate MPO-derived oxidants.

Carbon monoxide-releasing molecule 3 inhibits myeloperoxidase (MPO) and protects against MPO-induced vascular endothelial cell activation/dysfunction

Polymorphonuclear leukocyte (PMN)-derived myeloperoxidase (MPO) contributes to the pathophysiology of numerous systemic inflammatory disorders through: (1) direct peroxidation of targets and (2) production of strong oxidizing compounds, e.g., hypohalous acids, particularly hypochlorous acid, which furthers oxidant damage and contributes to the propagation of inflammation and tissue injury/dysfunction. Carbon monoxide-releasing molecules (CORMs) offer potent anti-inflammatory effects; however, the mechanism(s) of action is not fully understood. This study assessed the potential of MPO activity inhibition by a water-soluble CORM, CORM-3. To this end, we used in vitro assays to study CORM-3-dependent modulation of MPO activity with respect to: (1) the inhibition of MPO’s catalytic activity generally and (2) the specific inhibition of MPO’s peroxidation and halogenation (i.e., production of hypochlorous acid) reactions. Further, we employed primary human umbilical vein endothelial cells (HUVECs) to investigate MPO-dependent cellular activation and dysfunction by measuring intracellular oxidant stress (DHR-123 oxidation) and HUVEC permeability (flux of Texas red–dextran), respectively. The results indicate that CORM-3 significantly inhibits MPO activity as well as MPO’s peroxidation and hypohalous acid cycles specifically (p<0.05 vs uninhibited MPO). In addition, CORM-3 significantly decreases PMN homogenate- or rhMPO-induced intracellular DHR-123 oxidation in HUVECs and rhMPO-induced HUVEC monolayer permeability (p<0.05 vs untreated). In all assays the inactivated CORM-3 was significantly less effective than CORM-3 (p<0.05). Taken together our findings indicate that CORM-3 is a novel MPO inhibitor and mitigates inflammatory damage at least in part through a mechanism involving the inhibition of neutrophilic MPO activity.

Characterization and antioxidant properties of six Algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Because propolis contains many types of antioxidant compounds such as polyphenols and flavonoids, it can be useful in preventing oxidative damages. Ethyl acetate extracts of propolis from several Algerian regions show high activity by scavenging free radicals, preventing lipid peroxidation and inhibiting myeloperoxidase (MPO). By fractioning and assaying ethyl acetate extracts, it was observed that both polyphenols and flavonoids contribute to these activities. A correlation was observed between the polyphenol content and the MPO inhibition. However, it seems that kaempferol, a flavonoid, contributes mainly to the MPO inhibition. This molecule is in a high amount in the ethyl acetate extract and demonstrates the best efficiency towards the enzyme with an inhibiting concentration at 50% of 4 ± 2 μM.