Anti-Inflammatory Properties of the Enaminone E121 in the Dextran Sulfate Sodium (DSS) Colitis Model.

Enaminones are synthetic compounds with an established role in the prevention of various forms of seizures. Recent evidence suggests potent anti-tussive, bronchodilation and anti-inflammatory properties. Pre-treatment with particularly E121 compound resulted in a decrease in leukocyte recruitment in the ovalbumin induced-model of asthma, immune cell proliferation and cytokine release in vitro. We hypothesize that E121 might serve as a therapeutic potential in intestinal inflammation through modulating immune cell functions.Colitis was induced by daily dextran sulfate sodium (DSS) administration for 5 days, and its severity was determined by gross and histological assessments. The plasma level of various cytokines was measured using flow cytometry-based assay. The colonic expression/ phosphorylation level of various molecules was determined by immunofluorescence and western blotting. The effects of E121 treatment on in vitro neutrophil chemotaxis (under-agarose assay), superoxide release (luminol oxidation assay) and apoptosis (annexin V/7AAD) were also determined.DSS-induced colitis in mice was significantly reduced by daily E121 treatment (30-100 mg/kg) at gross and histological levels. This effect was due to modulated plasma levels of interleukin (IL-2) and colonic expression levels of various signaling molecules and proteins involved in apoptosis. In vitro neutrophil survival, chemotaxis, and superoxide release were also reduced by E121 treatment.Our results indicate important anti-inflammatory actions of E121 in the pathogenesis of IBD

Effect of E121 treatment on the macroscopic score for colitis severity.

<p>Effect of E121 treatment on the macroscopic score for colitis severity.</p

Effect of E121 treatment on the histological score for colitis severity.

<p>Panels A and B represent histological assessment of colitis severity and the % of ulceration in the whole colon section respectively in the groups indicated. Histobars represent means ± SEM for 6 mice in each group. Asterisks denote significant difference from UT mice with p<0.05 (*) and, p<0.001 (**). Panel C illustrates a colon section taken from UT mouse showing normal regular mucosal architecture. Panel D illustrates a colon section taken from mouse treated by daily i.p vehicle plus DSS where there is significant mucosal destruction and immune cell recruitment. Panels E-H illustrate typical colon sections taken from mice treated with various doses of E121 plus DSS. Treatment with 60–100 mg/kg doses of E121 improved mucosal integrity and reduced the enhanced immune cell recruitment (panels G and H). Bars represent 150 μm. Left pictures were taken at 5x magnification, while right pictures were taken at 10x magnification.</p

Effect of E121 on total/phosphorylated levels of proteins involved in cell signaling and apoptosis.

<p>Colonic levels of various proteins involved in cell signaling and apoptosis were determined by western blotting in UT or mice treated with either vehicle or E121 (30 and 60 mg/kg) at day 5 post colitis induction. The blots represent one of 3 similar experiments.</p

Effect of E121 treatment on colitis severity.

<p>Panel A shows % body weight changes in DSS treated mice alone or receiving vehicle (solid circles and squares, black lines), or E121 treated mice (red lines) compared to untreated (UT) mice receiving tap water only (open circle, hatched green line). Panels B and C show % of circulating neutrophils and lymphocytes respectively determined at day 5 post colitis induction in DSS treated mice alone or receiving vehicle (solid bars) or E121 treated mice (hatched bars) compared to UT group (open bars). Colon length (panel D) and thickness (panel E) was determined in all groups. Histobars represent means ± SEM for 6 mice in each group. Asterisks denote significant difference from UT mice with p<0.05 (*), # denotes significant difference from DSS alone and DSS/i.p vehicle treated mice with p<0.05.</p

Effect of E121 on neutrophil recruitment <i>in vivo</i>.

<p>Colon sections taken from untreated (UT) mice or mice treated with DSS plus E121 (60 mg/kg) or vehicle were immunostained with antisera against the neutrophil marker Gr1 (panel A). Immunofluorescent (Alexa Fluor) signals shown in left side of panel A are overlaid with DAPI stain on right side to show tissue architecture for the conditions indicated. Panels B represent quantitative assessment of fluorescence intensity (arbitrary units). Histobars represent means ± SEM for 6 mice in each group. Asterisk denotes significant difference from UT mice, with p<0.05, and # denotes significant difference from DSS/ i.p vehicle treated mice, with p<0.05.</p

Effect of E121 on neutrophil apoptosis, chemotaxis, and superoxide release <i>in vitro</i>.

<p>Neutrophils were isolated from naïve mice and were treated with vehicle (solid bar) or various concentrations of E121 (open bars). Panels A and B represent % viable and % apoptotic cells. Panel C represents neutrophil chemotaxis, determined using the under agarose assay, towards WKYMVm (1μM) for vehicle treated (solid bar) or E121 treated (open bars) neutrophils. Panel D: superoxide released from neutrophils was determined by recording chemiluminescence emanating from oxidation of luminol substrate as described in Methods. Neutrophils were treated with vehicle only (solid circles), or 10 μM WKYMVm plus vehicle (solid squares) or various concentrations of E121 (gray lines). Histobars represent means ± SEMs for 3 independent experiments conducted with cells isolated from 3 mice in each group. Asterisks denote significant difference from vehicle group, with p<0.05.</p

Anti-Inflammatory Action of Angiotensin 1-7 in Experimental Colitis.

BACKGROUND:There is evidence to support a role for angiotensin (Ang) 1-7 in reducing the activity of inflammatory signaling molecules such as MAPK, PKC and SRC. Enhanced angiotensin converting enzyme 2 (ACE2) expression has been observed in patients with inflammatory bowel disease (IBD) suggesting a role in its pathogenesis, prompting this study. METHODS:The colonic expression/activity profile of ACE2, Ang 1-7, MAS1-receptor (MAS1-R), MAPK family and Akt were determined by western blot and immunofluorescence. The effect of either exogenous administration of Ang 1-7 or pharmacological inhibition of its function (by A779 treatment) was determined using the mouse dextran sulfate sodium model. RESULTS:Enhanced colonic expression of ACE2, Ang1-7 and MAS1-R was observed post-colitis induction. Daily Ang 1-7 treatment (0.01-0.06 mg/kg) resulted in significant amelioration of DSS-induced colitis. In contrast, daily administration of A779 significantly worsened features of colitis. Colitis-associated phosphorylation of p38, ERK1/2 and Akt was reduced by Ang 1-7 treatment. CONCLUSION:Our results indicate important anti-inflammatory actions of Ang 1-7 in the pathogenesis of IBD, which may provide a future therapeutic strategy to control the disease progression

Cocaine sensitization does not alter SP effects on locomotion or excitatory synaptic transmission in the NAc of rats

Substance P (SP) and cocaine employ similar mechanisms to modify excitatory synaptic transmission in the nucleus accumbens (NAc), a region implicated in substance abuse. Here we explored, using NAc slices, whether SP effects on these synaptic responses were altered in rats that have been sensitized to cocaine and whether SP could mimic cocaine in triggering increased locomotion in sensitized rats. Intraperitoneal (IP) injection of na\uefve rats with cocaine (15 mg/kg) caused increased locomotion by 408.5 \ub1 85.9% (n = 5) which further increased by 733.1 \ub1 157.8% (n = 5) following a week of cocaine sensitization. A similar challenge with 10 mg/kg of SP after cocaine sensitization did not produce significant changes in locomotion (170.6 \ub1 61.0%; n = 4). In contrast to cocaine, IP injection of rats with SP or SP5\u201311 (10\u2013100 mg/kg) with or without phosphoramidon did not elicit changes in locomotion. In electrophysiological studies, both cocaine and SP depressed evoked NMDA and non-NMDA receptor-mediated excitatory synaptic currents (EPSCs) in slices obtained from na\uefve rats. In slices derived from cocaine-sensitized rats, cocaine but not SP produced a more profound decrease in non-NMDA compared to NMDA responses. Similar to that in na\uefve rats, cocaine\u2019s effect on the EPSCs in these sensitized rats occluded those of SP. Thus, although SP and cocaine may employ similar mechanisms to depress EPSCs in the NAc, IP injection of SP does not mimic cocaine-induced hyperlocomotion indicating that not all of cocaine\u2019s effects are mimicked by SP.Peer reviewed: YesNRC publication: Ye