Measuring Myeloperoxidase Activity Research Articles

Evaluation of intestinal injury, inflammatory response and oxidative stress following intracerebral hemorrhage in mice.

Intestinal injury is a common complication following intracerebral hemorrhage (ICH), which leads to malnutrition, impaired immunity and unsatisfactory prognosis. Previous studies have revealed the pathogenesis of intestinal injury following traumatic brain injury using ischemic stroke models. However, the effects of ICH on intestinal injury remain unknown. The present study aimed to investigate the pathological alterations and molecular mechanism, as well as the time course of intestinal injury following ICH in mice. Male C57BL/6 mice were randomly divided into the following sevengroups (n=6mice/group): Control group, which underwent a sham operation, and six ICH groups (2, 6, 12 and 24h, and days3and7). The ICH model was induced by stereotactically injecting autologous blood in two stages into the brain. Subsequently, intestinal tissue was stained with hematoxylin and eosin for histopathological examination. Small intestinal motility was measured by charcoal meal test, and gut barrier dysfunction was evaluated by detecting the plasma levels of endotoxin. Quantitative polymerase chain reaction (qPCR), immunohistochemistry and ELISA analysis were performed to evaluate the mRNA and protein expression levels of inflammatory cytokines [interleukin (IL)‑1β, IL‑6, tumor necrosis factor‑α, intercellular adhesion molecule1, monocyte chemotactic protein1 and chemokine (C‑C motif) ligand‑5] in intestinal tissue and serum. Furthermore, intestinal leukocyte infiltration was detected by measuring myeloperoxidase activity. Oxidative stress was indirectly detected by measuring reactive oxygen species‑associated markers (malondialdehyde content and superoxide dismutase activity assays) and the mRNA and protein expression levels of antioxidant genes [nuclear factor (erythroid‑derived 2)‑like2, manganese superoxide dismutase and heme oxygenase1] by qPCR and western blot analysis. The results demonstrated that significant destruction of the gut mucosa, delayed small intestinal motility, intestinal barrier dysfunction, and increased inflammatory responses and oxidative stress occurred rapidly in response to ICH. These symptoms occurred as early as 2h after ICH and persisted for 7days. These findings suggested that ICH may induce immediate and persistent damage to gut structure and barrier function, which may be associated with upregulation of inflammation and oxidative stress markers.

Entire Peroxidation Reaction System of Myeloperoxidase Correlates with Progressive Low-Density Lipoprotein Modifications via Reactive Aldehydes in Atherosclerotic Patients with Hypertension

Background/Aims: Reactive oxygen species (ROS) contribute to the dysfunction of serum lipoproteins, which triggers lipid metabolism abnormalities in the development of atherosclerosis and hypertension. Myeloperoxidase (MPO) is involved in ROS modifications, triggering lipid peroxidation and aldehyde formation. However, the relationship between the entirety of the MPO reaction system and oxidative modification of serum lipoproteins in atherosclerotic patients with hypertension remains unclear. Methods: We measured MPO activity (peroxidation and chlorination), 4-hydroxynonenal-modified low-density lipoprotein (HNE-LDL), malondialdehyde-modified low-density lipoprotein (MDA-LDL), H₂O₂, reduced glutathione (GSH), and oxidized glutathione (GSSG) using a corresponding commercial kit in atherosclerotic patients with hypertension and healthy participants. We used Spearman’s correlation analysis to investigate the correlation between MPO activity and the levels of these oxidative and anti-oxidative stress-related indices and performed response surface regression to investigate the relationship between the MPO reaction system and the levels of HNE-LDL, MDA-LDL, and the GSH/GSSG ratio. Results: Our results showed no association between the levels of MPO peroxidation activity, MPO chlorination activity, H₂O₂, and Cl^- and those of HNE-LDL, MDA-LDL, GSH, and GSSG, and the GSH/GSSG ratio in healthy participants. In addition, no effects of the peroxidation reaction system of MPO (PRSM) and the chlorination reaction system of MPO (CRSM) on GSH/GSSG were found in this investigation. However, we found that the PRSM rather than the CRSM correlated with progressive low-density lipoprotein (LDL) modifications by HNE-LDL and MDA-LDL in atherosclerotic patients with hypertension. Conclusion: The PRSM rather than the CRSM correlated with progressive LDL modifications via reactive aldehydes in atherosclerotic patients with hypertension. Further investigation is warranted to evaluate whether the PRSM may serve as a potential index for monitoring LDL function in atherosclerosis and hypertension.

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.

Trans-chalcone added in topical formulation inhibits skin inflammation and oxidative stress in a model of ultraviolet B radiation skin damage in hairless mice

Trans-chalcone (TC) is a common precursor of flavonoids. However, the pharmacological properties of TC remain to be fully understood. The present study investigated whether topical formulation containing TC (TFcTC) presents therapeutic effect in UVB radiation-induced skin damage using disease, enzyme activity, antioxidant activity, protein and mRNA parameters. Control topical formulation (CTF) and TFcTC were applied in hairless mice before and after exposure to UVB radiation. Dorsal skin samples were collected after UVB exposure to evaluate: i) skin edema (weight) was measured by punch biopsy; ii) spectrophotometric assays were used to measure myeloperoxidase (MPO) and catalase activities, ferric (FRAP) and ABTS cation reducing antioxidant power, superoxide anion production and levels of reduced glutathione (GSH); iii) enzymography was used to measure matrix metalloproteinase-9 (MMP-9) activity; iv) chemiluminescence was used to measure the lipid peroxidation (LPO); v) enzyme-linked immunosorbent assay (ELISA) was used to measure tumor necrosis factor alpha (TNF-α) levels; vi) reverse transcription quantitative PCR (RT-qPCR) was used to measure cyclooxygenase-2 (COX-2), gp91phox (NADPH oxidase sub-unity), glutathione peroxidase-1 (Gpx1), glutathione reductase (Gr), nuclear factor erythroid 2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) mRNA expression. TFcTC inhibited UVB-induced skin edema, MPO activity, MMP-9 activity, TNF-α production, and COX-2 mRNA expression. TFcTC inhibited UVB-induced LPO, down-regulated superoxide anion levels and gp91phox mRNA expression, and improved antioxidant potential and GSH skin levels. The mRNA expression of detoxification systems such as Nrf2, HO-1, Gpx1 and Gr, and catalase activity were also enhanced by treatment with TFcTC. In conclusion, TFcTC protects mice skin from UVB radiation by inhibiting inflammation, and improving antioxidant and detoxification systems. Therefore, topical treatment with TC is a novel therapeutic approach for the treatment of UVB radiation skin damages, which merits further pre-clinical and clinical investigation.

The influence of lipoic acid on caveolin-1-regulated antioxidative enzymes in the mouse model of acute ulcerative colitis.

This study was undertaken to verify if two-weeks treatment of lipoic acid (LA) influence colon damage and pro-inflammatory cytokine synthesis during DSS-induced acute colitis. Moreover, as LA has anti-oxidative properties, we analyzed its influence on the level of antioxidative enzymes, HO-1 and eNOS, and their regulator- caveolin-1. LA was administrated to male C57/BALBc mice at a dose of 25 or 50mg/kg/day (i.p.) for 21days. Acute colitis was induced by administration of 4% DSS (w/v) in drinking water for 5days, followed by 2days of normal drinking water. Mice in LA+DSS groups were treated with LA (25 or 50mg/kg/day; i.p.) starting 14days prior to 4% DSS. Control group received saline for 21days. In the colon tissue we measured myeloperoxidase activity (MPO), IL-1β, IL-6, IL-17A, IL-23 (ELISA method), and tissue level of cav-1, phospho-eNOS, total eNOS and HO-1 (Western blot). Administration of DSS significantly increased total colon damage (p<0.001), myeloperoxidase (MPO) activity (p<0.05) and pro-inflammatory IL-6 (p<0.05). There was also a tendency towards higher IL-1β, IL-17A, and IL-23 in the colon. LA alone did not influence total colon damage, MPO activity, and pro-inflammatory cytokines concentration compared to control (p<0.05). Notably, mice treated with LA and DSS had significantly decreased total colon damage score (p<0.001), despite augmented colon MPO activity (p<0.01), but similar (IL-17A) or even significantly higher level (IL-1β, IL-23) as compared to the DSS group (p<0.05). IL-6 was insignificantly decreased after LA treatment at a dose of 50mg/kg. In acute colitis there was a tendency towards an increase in cav-1 and HO-1 and a decrease p-eNOS/total eNOS ratio. Moreover, the LA+DSS groups had higher expression of HO-1 and p-eNOS/total eNOS (p<0.05) compared to the DSS group, and a tendency towards higher cav-1 level. The changes did not depend on LA dose. Our study indicated that LA, at lower doses, may influence cav-1-regulated antioxidative enzyme levels (HO-1 and p-eNOS/total eNOS) despite an increase in colon pro-inflammatory cytokine levels during acute colitis. Hence, LA treatment may be - to some extent - beneficial in attenuation of acute colitis.

Curcumin alleviates ischemia reperfusion-induced acute kidney injury through NMDA receptor antagonism in rats

Objective: The present study investigated the role of N-methyl-d-aspartate (NMDA) receptors in curcumin-mediated renoprotection against ischemia reperfusion (I/R)-induced acute kidney injury (AKI) in rats.Methods: Rats were subjected to bilateral renal I/R (40 min I, 24 hours R) to induce AKI. Kidney injury was assessed by measuring creatinine clearance, blood urea nitrogen, plasma uric acid, potassium level, fractional excretion of sodium, and macroproteinuria. Oxidative stress in renal tissues was assessed by measuring myeloperoxidase activity, thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione content. Hematoxylin & eosin staining was done to assess histological changes in renal tissues. Curcumin (30 and 60 mg/kg) was administered one hour before subjecting rats to AKI. In separate groups, NMDA receptor agonists, glutamic acid (200 mg/kg), and spermidine (20 mg/kg) were administered prior to curcumin treatment in rats followed by AKI.Results: I/R-induced AKI was demonstrated by significant change in plasma and urine parameters along with marked increase in oxidative stress and histological changes in renal tissues that were aggravated with pretreatment of glutamic acid and spermidine in rats. Administration of curcumin resulted in significant protection against AKI. However, glutamic acid and spermidine pretreatments prevented curcumin-mediated renoprotection.Conclusion: It is concluded that NMDA receptor antagonism significantly contributes towards curcumin-mediated protection against I/R-induced AKI.

Inhibition of c-Jun N-terminal Kinase Signaling Pathway Alleviates Lipopolysaccharide-induced Acute Respiratory Distress Syndrome in Rats.

Background:An acute respiratory distress syndrome (ARDS) is still one of the major challenges in critically ill patients. This study aimed to investigate the effect of inhibiting c-Jun N-terminal kinase (JNK) on ARDS in a lipopolysaccharide (LPS)-induced ARDS rat model.Methods:Thirty-six rats were randomized into three groups: control, LPS, and LPS + JNK inhibitor. Rats were sacrificed 8 h after LPS treatment. The lung edema was observed by measuring the wet-to-dry weight (W/D) ratio of the lung. The severity of pulmonary inflammation was observed by measuring myeloperoxidase (MPO) activity of lung tissue. Moreover, the neutrophils in bronchoalveolar lavage fluid (BALF) were counted to observe the airway inflammation. In addition, lung collagen accumulation was quantified by Sircol Collagen Assay. At the same time, the pulmonary histologic examination was performed, and lung injury score was achieved in all three groups.Results:MPO activity in lung tissue was found increased in rats treated with LPS comparing with that in control (1.26 ± 0.15 U in LPS vs. 0.77 ± 0.27 U in control, P < 0.05). Inhibiting JNK attenuated LPS-induced MPO activity upregulation (0.52 ± 0.12 U in LPS + JNK inhibitor vs. 1.26 ± 0.15 U in LPS, P < 0.05). Neutrophils in BALF were also found to be increased with LPS treatment, and inhibiting JNK attenuated LPS-induced neutrophils increase in BALF (255.0 ± 164.4 in LPS vs. 53 (44.5-103) in control vs. 127.0 ± 44.3 in LPS + JNK inhibitor, P < 0.05). At the same time, the lung injury score showed a reduction in LPS + JNK inhibitor group comparing with that in LPS group (13.42 ± 4.82 vs. 7.00 ± 1.83, P = 0.001). However, the lung W/D ratio and the collagen in BALF did not show any differences between LPS and LPS + JNK inhibitor group.Conclusions:Inhibiting JNK alleviated LPS-induced acute lung inflammation and had no effects on pulmonary edema and fibrosis. JNK inhibitor might be a potential therapeutic medication in ARDS, in the context of reducing lung inflammatory.

Development and characterization of novel 1-(1-Naphthyl)piperazine-loaded lipid vesicles for prevention of UV-induced skin inflammation

1-(1-Naphthyl)piperazine (1-NPZ) has shown promising effects by inhibiting UV radiation-induced immunosuppression. Ultradeformable vesicles are recent advantageous systems capable of improving the (trans)dermal drug delivery. The aim of this study was to investigate 1-NPZ-loaded transethosomes (NPZ-TE) and 1-NPZ-loaded vesicles containing dimethyl sulfoxide (NPZ-DM) as novel delivery nanosystems, and to uncover their chemopreventive effect against UV-induced acute inflammation. Their physicochemical properties were evaluated as follows: vesicles size and zeta potential by dynamic and electrophoretic light scattering, respectively; vesicle deformability by pressure driven transport; rheological behavior by measuring viscosity and I-NPZ entrapment yield by HPLC. In vitro topical delivery studies were performed in order to evaluate the permeation profile of both formulations, whereas in vivo studies sought to assess the photoprotective effect of the selected formulation on irradiated hairless mice by measuring myeloperoxidase activity and the secretion of proinflammatory cytokines.Either NPZ-TE or NPZ-DM exhibited positive results in terms of physicochemical properties. In vitro data revealed an improved permeation of 1-NPZ across pig ear skin, especially by NPZ-DM. In vivo studies demonstrated that NPZ-DM exposure was capable of preventing UVB-induced inflammation and blocking mediators of inflammation in mouse skin. The successful results here obtained encourage us to continue these studies for the management of inflammatory skin conditions that may lead to the development of skin cancers.

Methods for measuring myeloperoxidase activity toward assessing inhibitor efficacy in living systems

Myeloperoxidase aids in clearance of microbes by generation of peroxidase-mediated oxidants that kill leukocyte-engulfed pathogens. In this review, we will examine 1) strategies for in vitro evaluation of myeloperoxidase function and its inhibition, 2) ways to monitor generation of certain oxidant species during inflammation, and 3) how these methods can be used to approximate the total polymorphonuclear neutrophil chemotaxis following insult. Several optical imaging probes are designed to target reactive oxygen and nitrogen species during polymorphonuclear neutrophil inflammatory burst following injury. Here, we review the following 1) the broad effect of myeloperoxidase on normal physiology, 2) the difference between myeloperoxidase and other peroxidases, 3) the current optical probes available for use as surrogates for direct measures of myeloperoxidase-derived oxidants, and 4) the range of preclinical options for imaging myeloperoxidase accumulation at sites of inflammation in mice. We also stress the advantages and drawbacks of each of these methods, the pharmacokinetic considerations that may limit probe use to strictly cell cultures for some reactive oxygen and nitrogen species, rather than in vivo utility as indicators of myeloperoxidase function. Taken together, our review should shed light on the fundamental rational behind these techniques for measuring myeloperoxidase activity and polymorphonuclear neutrophil response after injury toward developing safe myeloperoxidase inhibitors as potential therapy for chronic obstructive pulmonary disease and rheumatoid arthritis.

Investigation of the role of nitric oxide/soluble guanylyl cyclase pathway in ascorbic acid-mediated protection against acute kidney injury in rats.

The present study investigated the possible involvement of nitric oxide/soluble guanylyl cyclase (NO/sGC) pathway in ascorbic acid (AA)-mediated protection against acute kidney injury (AKI) in rats. The rats were subjected to bilateral renal ischemia by occluding renal pedicles for 40 min followed by reperfusion for 24 h. The AKI was assessed in terms of measuring creatinine clearance (CrCl), blood urea nitrogen (BUN), plasma uric acid, potassium level, fractional excretion of sodium (FeNa), and microproteinuria. The NO level and oxidative stress in renal tissues were assessed by measuring myeloperoxidase activity, thiobarbituric acid reactive substances, superoxide anion generation, and reduced glutathione level. AA (50 and 100 mg/kg, p.o.) was administered for 3 days before subjecting rats to AKI. In separate groups, the nitric oxide synthase inhibitor, L-NAME (20 mg/kg, i.p.) and sGC inhibitor, methylene blue (50 mg/kg, i.p.) was administered prior to AA treatment in rats. The significant decrease in CrCl and increase in BUN, plasma uric acid, potassium, FeNa, microproteinuria, and oxidative stress in renal tissues demonstrated ischemia-reperfusion-induced AKI in rats. The AA treatment ameliorated ischemia-reperfusion-induced AKI along with the increase in renal NO level. The pretreatment with L-NAME and methylene blue abolished protective effect of AA. It is concluded that AA protects against ischemia-reperfusion-induced AKI. Moreover, the NO/sGC pathway finds its definite involvement in AA-mediated reno-protective effect.

Involvement of stimulation of α7 nicotinic acetylcholine receptors in the suppressive effect of tropisetron on dextran sulfate sodium-induced colitis in mice

Ulcerative colitis (UC) involves chronic inflammation of the large intestine. Several agents are used to treat UC, but adverse side effects are remaining problems. We examined the effect of tropisetron as a new type of drug for UC using a dextran sulfate sodium (DSS)-induced model of colitis in mice. We developed a DSS-induced model of colitis and calculated the Disease Activity Index and colon length. We measured myeloperoxidase activity and determined the protein level and mRNA level of cytokines in the colon. DSS-induced colitis was ameliorated by administration of tropisetron and PNU282987. Pre-administration of methyllycaconitine diminished the suppressive effect of tropisetron upon DSS-induced colitis. These findings suggested that α7 nicotinic acetylcholine receptors (α7 nAChRs) were related to the suppressive effect of tropisetron on DSS-induced colitis. Additionally, stimulation of α7 nAChRs decreased the colon level of interleukin-6 and interferon-γ upon DSS administration. Furthermore, stimulation of α7 nAChRs decreased macrophage infiltration, with expression of α7 nAChR increased by DSS administration. These results suggest that the underlying mechanism of this suppressive effect on DSS-induced colitis is via stimulation of α7 nAChRs and involves suppression of expression of pro-inflammatory cytokines. Tropisetron could be a new type of therapeutic agent for UC.

A rapid bioluminescence assay for measuring myeloperoxidase activity in human plasma

Myeloperoxidase (MPO) is a circulating cardiovascular disease (CVD) biomarker used to estimate clinical risk and patient prognosis. Current enzyme-linked immunosorbent assays (ELISA) for MPO concentration are costly and time-intensive. Here we report a novel bioluminescence assay, designated MPO activity on a polymer surface (MAPS), for measuring MPO activity in human plasma samples using the bioluminescent substrate L-012. The method delivers a result in under an hour and is resistant to confounding effects from endogenous MPO inhibitors. In a pilot clinical study, we compared MAPS and two clinical ELISAs using 72 plasma samples from cardiac catheterization patients. Results from parallel MAPS and ELISAs were concordant within 2±11 μg l−1 MPO with similar uncertainty and reproducibility. Results between parallel MAPS and ELISA were in better agreement than those between independent ELISAs. MAPS may provide an inexpensive and rapid assay for determining MPO activity in plasma samples from patients with CVD or potentially other immune and inflammatory disorders.

Leukocyte function-associated antigen-1 deficiency impairs responses to polymicrobial sepsis.

To determine the role of leukocyte function-associated antigen-1 (LFA-1) in polymicrobial sepsis model in mice. Cecal ligation and puncture model was used to study polymicrobial sepsis in wild type and LFA-1 knockout (KO) (= CD11a KO) mice. Their survivals were examined. Neutrophil recruitment to the abdominal cavity, bacterial tissue load and bacterial killing by neutrophils, tissue cytokine profiles, and serum cytokines were examined. Apoptosis of tissues was assessed using cleaved-caspase 3 and TUNNEL staining. The recruitment of neutrophils to various tissues was assessed using myeloperoxidase staining or measuring myeloperoxidase activity. LFA-1 deficiency significantly decreased survival (P = 0.0024) with the reduction of neutrophil recruitment to the abdominal cavity and higher bacterial load in blood. It was also associated with increased apoptosis in spleen and more organ injuries probed by interleukin-6 mRNA level. However, the deficiency of LFA-1 did not prevent neutrophil recruitment to lung, liver, spleen or kidney, which suggested the existence of LFA-1 independent recruitment mechanism in these organs. LFA-1 deficiency did not attenuate neutrophil recruitment to various organs to adequately mitigate secondary tissue injury in sepsis. It was associated with decreased neutrophil recruitment to the abdominal cavity, higher bacterial load, leading to increased mortality in an abdominal, polymicrobial sepsis.

Berberine ameliorates intestinal mucosal barrier damage induced by peritoneal air exposure.

Berberine, an isoquinoline alkaloid derived from many medicinal plants, has been extensively used to treat various gastrointestinal diseases. In the present study, we investigated whether berberine could ameliorate intestinal mucosal barrier damage induced by peritoneal air exposure for 3 h. Peritoneal air-exposure rats received 100, 150, and 200 mg/kg berberine orally via gavage four times before and after surgery. Blood and terminal ileum samples were collected 24 h after surgery. The serum D-lactate levels were determined using an enzyme-linked immunosorbent assay (ELISA) kit. Intestinal permeability was determined by measuring the intestinal clearance of fluorescein isothiocyanate (FITC)-dextran (FD4). Intestinal inflammation was assessed by measuring myeloperoxidase activity. Intestinal histopathology was also assessed. The results revealed that berberine decreased the serum D-lactate level, intestinal FD4 clearance, and myeloperoxidase activity. Edema and inflammation were reduced by berberine in the intestinal mucosa and submucosa, and the Chiu's scores, indices of intestinal mucosal injury, also decreased in the berberine-treated group. In addition, berberine exerted these protective effects in a dose-dependent manner, with a significant difference from the control group at doses of 150 and 200 mg/kg. The results suggest that berberine could ameliorate intestinal mucosal barrier damage induced by peritoneal air exposure, which is linked to its anti-inflammatory activity. Berberine may be a promising treatment for intestinal mucosal barrier damage in open abdominal surgery.

Plecanatide and dolcanatide, novel guanylate cyclase-C agonists, ameliorate gastrointestinal inflammation in experimental models of murine colitis.

To evaluate the effect of orally administered plecanatide or dolcanatide, analogs of uroguanylin, on amelioration of colitis in murine models. The cyclic guanosine monophosphate (cGMP) stimulatory potency of plecanatide and dolcanatide was measured using a human colon carcinoma T84 cell-based assay. For animal studies all test agents were formulated in phosphate buffered saline. Sulfasalazine or 5-amino salicylic acid (5-ASA) served as positive controls. Effect of oral treatment with test agents on amelioration of acute colitis induced either by dextran sulfate sodium (DSS) in drinking water or by rectal instillation of trinitrobenzene sulfonic (TNBS) acid, was examined in BALB/c and/or BDF1 mice. Additionally, the effect of orally administered plecanatide on the spontaneous colitis in T-cell receptor alpha knockout (TCRα(-/-)) mice was also examined. Amelioration of colitis was assessed by monitoring severity of colitis, disease activity index and by histopathology. Frozen colon tissues were used to measure myeloperoxidase activity. Plecanatide and dolcanatide are structurally related analogs of uroguanylin, which is an endogenous ligand of guanylate cyclase-C (GC-C). As expected from the agonists of GC-C, both plecanatide and dolcanatide exhibited potent cGMP-stimulatory activity in T84 cells. Once-daily treatment by oral gavage with either of these analogs (0.05-0.5 mg/kg) ameliorated colitis in both DSS and TNBS-induced models of acute colitis, as assessed by body weight, reduction in colitis severity (P < 0.05) and disease activity index (P < 0.05). Amelioration of colitis by either of the drug candidates was comparable to that achieved by orally administered sulfasalazine or 5-ASA. Plecanatide also effectively ameliorated colitis in TCRα(-/-) mice, a model of spontaneous colitis. As dolcanatide exhibited higher resistance to proteolysis in simulated gastric and intestinal juices, it was selected for further studies. This is the first-ever study reporting the therapeutic utility of GC-C agonists as a new class of orally delivered and mucosally active drug candidates for the treatment of inflammatory bowel diseases.

Anti-inflammatory Effects of Flavonoids on TNBS-induced Colitis of Rats.

It has been shown that the extracts including eupatilin and quercetin-3-β-D-glucuronopyranoside had mucoprotective effects on the esophagus and stomach through their antioxidant activities. This study was designed to investigate the anti-inflammatory effect of these flavonoid compounds in an animal model of inflammatory bowel disease induced by 2,4,6-trinitrobenzene sulfonic acid. Experimental colitis was induced by intracolonic administration of 2,4,6-trinitrobenzene sulfonic acid. Extracts including eupatilin or quercetin-3-β-D-glucuronopyranoside were orally administered to animals 48, 24, and 1 h prior to the induction of colitis and then again 24 h later. The animals were sacrificed 48 h after by 2,4,6-trinitrobenzene sulfonic acid treatment and the macroscopic appearance of the colonic lesions was scored in a blinded manner on a scale of 1 to 10. The inflammatory response to colitis induction was assessed by measuring myeloperoxidase activity, nitric oxide production, tumor necrosis factor-α expression, total glutathione levels, and malondialdehyde concentrations in the colon. The results indicated that extracts including eupatilin and extracts including quercetin-3-β-D-glucuronopyranoside dose-dependently improved the morphology of the lesions induced by 2,4,6-trinitrobenzene sulfonic acid and reduced the ulcer index accordingly. In addition, rats receiving extracts including eupatilin and extracts including quercetin-3-β-D-glucuronopyranoside showed significantly decreased levels of mucosal myeloperoxidase activity, nitric oxide production, tumor necrosis factor-α expression, and malondialdehyde levels, and increased total glutathione levels. Extracts including eupatilin and extracts including quercetin-3-β-D-glucuronopyranoside ameliorated the inflammatory response and colonic injury in acute colitis by decreasing oxidative stress and neutrophil activation. Extracts including eupatilin and extracts including quercetin-3-β-D-glucuronopyranoside may inhibit acute colitis.

The role of macrophage 1 antigen in polymicrobial sepsis.

Macrophage 1 antigen (Mac-1, CD11bCD18) is a leukocyte adhesion molecule that is involved in many functions including leukocyte recruitment, phagocytosis, and neutrophil apoptosis. The previous report of mild polymicrobial, abdominal sepsis showed that the administration of anti-CD11b-blocking antibody administration attenuated lung injury without any survival benefit. Here we tested the impact of Mac-1 deficiency in severe polymicrobial abdominal sepsis model. Polymicrobial sepsis was studied using cecal ligation and puncture model in wild-type (WT) or Mac-1-deficient (CD11b knockout [KO]) mice, and their outcomes were examined. Bacterial tissue load and the recruitment of neutrophils to the abdominal cavity were assessed. In vitro bacterial killing assay was performed. Serum cytokine levels were measured using multiarray. Apoptosis of spleen tissues was assessed using Western blot analysis and immunohistochemistry (cleaved caspase 3 and TUNEL staining). In addition, in vitro apoptosis assay was performed using primary splenocytes from both WT and KO mice. The recruitment of neutrophils to lung was assessed by measuring myeloperoxidase activity. Macrophage 1 antigen deficiency significantly decreased survival (survival percentage WT 43.5% vs. KO 13.0%; P = 0.0038) with higher bacterial load in blood and more severe systemic inflammation. Knockout mice demonstrated higher apoptosis both in vivo and in vitro. The recruitment of neutrophils to lung was not different between WT and KO mice. Macrophage 1 antigen deficiency was associated with poorer outcomes, more bacterial load, systemic inflammation, and splenic apoptosis. However, Mac-1 deficiency did not attenuate neutrophil recruitment to lung.

High-altitude pulmonary edema can be prevented by heat shock protein 70-mediated hyperbaric oxygen preconditioning.

The primary goal of this study was to test whether high-altitude exposure (HAE of 9.7% O2 at 0.47 absolute atmosphere [ATA] for 3 days) was capable of increasing lung edema, neutrophil, and hemorrhage scores as well as decreasing lung levels of both aquaporin 1 (AQP1) and AQP5 proteins and messenger RNA (mRNA) expression in rats, with a secondary goal to test whether a preinduction of heat shock protein 70 (HSP70) by hyperbaric oxygen preconditioning (HBO2P of 100% O2 at 2.0 ATA for 1 hour per day for 5 consecutive days) attenuated the HAE-induced increased lung injury scores and decreased lung AQP1 and AQP5 protein and mRNA expressions. Rats were assigned to (1) non-HBO2P (21% O2 at 1.0 ATA) + non-HAE (21% O2 at 1.0 ATA) group; (2) non-HBO2P + HAE group; (3) HBO2P + HAE group; and HBO2P + HSP70 antibodies (Ab) + HAE group. For the HSP70 Ab group, a neutralizing HSP70 Ab was injected intravenously at 24 hours before HAE. All the physiologic and biochemical parameters were obtained at the end of HAE or the equivalent period of non-HAE. The cardiovascular and blood gas parameters were monitored for all experiments. Bronchoalveolar lavage (BAL) was performed to determine proinflammatory cytokines (interleukin 6, interleukin 1β, and tumor necrosis factor α). Parts of the lung were excised for myeloperoxidase activity measurement, whereas the rest was collected for lung damage score assessments. AQP1 and AQP5 protein and mRAN expressions were also determined in the lung tissues. In the non-HBO2P + HAE group, the animals displayed higher values of lung myeloperoxidase activity, BAL proinflammatory cytokines, lung water weight, and acute lung injury scores compared with those of the non-HBO2P + non-HAE controls. In contrast, the non-HBO2P + HAE group rats had lower values of lung AQP1 and AQP5 protein and mRNA expressions, mean arterial pressure, heart rate, SO2, Paco2, HCO3, and pH compared with those of non-HBO2P + non-HAE group rats. The increased acute lung edema, neutrophil, and hemorrhage scores; increased BAL levels of proinflammatory cytokines; decreased lung AQP1 and AQP5 protein and mRNA expressions; and hypotension, bradycardia, hypoxia, and acidosis caused by HAE were all significantly attenuated by HBO2P. Our data indicate that HBO2P may attenuate high-altitude acute lung injury by a preinduction of lung HSP70 in rats.

Guided bone regeneration produced by new mineralized and reticulated collagen membranes in critical-sized rat calvarial defects.

The aim of this study was to evaluate the bone regenerative effect of glutaraldehyde (GA) cross-linking on mineralized polyanionic collagen membranes in critical-sized defects on rat calvarias. Bone calvarial defects were induced in Wistar rats, which were then divided into five groups: a sham group; a control group, which received a commercial membrane; and GA, 25GA, and 75GA groups, which received one of three different polyanionic collagen membranes mineralized by 0, 25, or 75 hydroxyapatite cycles and then cross-linked by GA. Bone formation was evaluated based on digital radiography and computerized tomography. Histological analyses were performed 4 and 12 weeks after the surgical procedure to observe bone formation, membrane resorption, and fibrous tissue surrounding the membranes. Measurement of myeloperoxidase activity, tumor necrosis factor alpha, and interleukin 1beta production was performed 24 h after surgery. The percentage of new bone formation in the GA, 25GA, and 75GA groups was higher compared with the control and sham groups. In the GA and 25 GA groups, the membranes were still in place and were contained in a thick fibrous capsule after 12 weeks. No significant difference was found among the groups regarding myeloperoxidase activity and interleukin 1beta levels, although the GA, 25GA, and 75GA groups presented decreased levels of tumor necrosis factor alpha compared with the control group. These new GA cross-linked membranes accelerated bone healing of the calvarium defects and did not induce inflammation. In addition, unlike the control membrane, the experimental membranes were not absorbed during the analyzed period, so they may offer advantages in large bone defects where prolonged membrane barrier functions are desirable.

Bothrops jararacussu snake venom-induces a local inflammatory response in a prostanoid- and neutrophil-dependent manner

Local tissue reactions provoked by Bothrops venoms are characterized by edema, hemorrhage, pain, and inflammation; however, the mechanisms of tissue damage vary depending upon the species of snake. Here, we investigated the mechanisms involved in the local inflammatory response induced by the Bothrops jararacussu venom (BjcuV). Female Swiss mice were injected with either saline, BjcuV (0.125–8 μg/paw) or loratadine (an H1 receptor antagonist), compound 48/80 (for mast cell depletion), capsaicin (for C-fiber desensitization), infliximab (an anti-TNF-α antibody), indomethacin (a non-specific COX inhibitor), celecoxib (a selective COX-2 inhibitor) or fucoidan (a P- and L-selectins modulator) given before BjcuV injection. Paw edema was measured by plethysmography. In addition, paw tissues were collected for the measurement of myeloperoxidase activity, TNF-α and IL-1 levels, and COX-2 immunoexpression. The direct chemotactic effect of BjcuV and the in vitro calcium dynamic in neutrophils were also investigated. BjcuV caused an edematogenic response with increased local production of TNF-α and IL-1β as well as COX-2 expression. Both edema and neutrophil migration were prevented by pretreatment with indomethacin, celecoxib or fucoidan. Furthermore, BjcuV induced a direct in vitro neutrophil chemotaxis by increasing intracellular calcium. Therefore, BjcuV induces an early onset edema dependent upon prostanoid production and neutrophil migration.