Endocannabinoid-related compounds in gastrointestinal diseases

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC-like compounds able to modulate ECS function without the typical central side effects of cannabinomimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases

Structural determinants in the group III truncated hemoglobin from Campylobacter jejuni.

Truncated hemoglobins (trHbs) constitute a distinct lineage in the globin superfamily, distantly related in size and fold to myoglobin and monomeric hemoglobins. Their phylogenetic analyses revealed that three groups (I, II, and III) compose the trHb family. Group I and II trHbs adopt a simplified globin fold, essentially composed of a 2-on-2 alpha-helical sandwich, wrapped around the heme group. So far no structural data have been reported for group III trHbs. Here we report the three-dimensional structure of the group III trHbP from the eubacterium Campylobacter jejuni. The 2.15-angstrom resolution crystal structure of C. jejuni trHbP (cyano-met form) shows that the 2-on-2 trHb fold is substantially conserved in the trHb group III, despite the absence of the Gly-based sequence motifs that were considered necessary for the attainment of the trHb specific fold. The heme crevice presents important structural modifications in the C-E region and in the FG helical hinge, with novel surface clefts at the proximal heme site. Contrary to what has been observed for group I and II trHbs, no protein matrix tunnel/cavity system is evident in C. jejuni trHbP. A gating movement of His(E7) side chain (found in two alternate conformations in the crystal structure) may be instrumental for ligand entry to the heme distal site. Sequence conservation allows extrapolating part of the structural results here reported to the whole trHb group III

Enteric glia: A new player in inflammatory bowel diseases

In addition to the well-known involvement of macrophages and neutrophils, other cell types have been recently reported to substantially contribute to the onset and progression of inflammatory bowel diseases (IBD). Enteric glial cells (EGC) are the equivalent cell type of astrocyte in the central nervous system (CNS) and share with them many neurotrophic and neuro-immunomodulatory properties. This short review highlights the role of EGC in IBD, describing the role played by these cells in the maintenance of gut homeostasis, and their modulation of enteric neuronal activities. In pathological conditions, EGC have been reported to trigger and support bowel inflammation through the specific over-secretion of S100B protein, a pivotal neurotrophic factor able to induce chronic inflammatory changes in gut mucosa. New pharmacological tools that may improve the current therapeutic strategies for inflammatory bowel diseases (IBD), lowering side effects (i.e. corticosteroids) and costs (i.e. anti-TNFα monoclonal antibodies) represent a very important challenge for gastroenterologists and pharmacologists. Novel drugs capable to modulate enteric glia reactivity, limiting the pro-inflammatory release of S100B, may thus represent a significant innovation in the field of pharmacological interventions for inflammatory bowel diseases

Ferrous Campylobacter jejuni truncated hemoglobin P displays an extremely high reactivity for cyanide - A comparative study

Campylobacter jejuni hosts two hemoglobins (Hbs). The Camplylobacter jejuni single-domain Hb (called Cgb) is homologous to the globin domain of flavohemoglobin, and it has been proposed to protect the bacterium against nitrosative stress. The second Hb is called Ctb (hereafter Cj-trHbP), belongs to truncated Hb group III, and has been hypothesized to be involved in O 2 chemistry. Here, the kinetics and thermodynamics of cyanide binding to ferric and ferrous Cj-trHbP [Cj-trHbP(III) and Cj-trHbP(II), respectively] are reported and analyzed in parallel with those of related heme proteins, with particular reference to those from Mycobacterium tuberculosis. The affinity of cyanide for Cj-trHbP(II) is higher than that reported for any known (in)vertebrate globin by more than three orders of magnitude (K = 1.2 × 10-6 m). This can be fully attributed to the highest (ever observed for a ferrous Hb) cyanide-binding association rate constant (kon = 3.3 × 103 m-1·s-1), even though the binding process displays a rate-limiting step (kmax = 9.1 s -1). Cj-trHbP(III) shows a very high affinity for cyanide (L = 5.8 × 10-9 m); however, cyanide association kinetics are independent of cyanide concentration, displaying a rate-limiting step (l max = 2.0 × 10-3 s-1). Values of the first-order rate constant for cyanide dissociation from Cj-trHbP(II)-cyanide and Cj-trHbP(III)-cyanide (koff =5.0 × 10-3 s -1 and loff ≥ 1 × 10-4 s-1, respectively) are similar to those reported for (in)vertebrate globins. The very high affinity of cyanide for Cj-trHbP(II), reminiscent of that of horseradish peroxidase(II), suggests that this globin may participate in cyanide detoxification. © 2008 The Authors

Rifaximin improves Clostridium difficile toxin A-induced toxicity in Caco-2 cells by the PXR-dependent TLR4/MyD88 /NF-?B pathway

Background: Clostridium difficile infections (CDIs) caused by Clostridium difficile toxin A (TcdA) lead to severe ulceration, inflammation and bleeding of the colon, and are difficult to treat. Aim: The study aimed to evaluate the effect of rifaximin on TcdA-induced apoptosis in intestinal epithelial cells and investigate the role of PXR in its mechanism of action. Methods: Caco‐2 cells were incubated with TcdA and treated with rifaximin (0.1−10 μM) with or without ketoconazole (10 μM). The transepithelial electrical resistance (TEER) and viability of the treated cells was determined. Also, the expression of zona occludens‐1 (ZO‐1), toll‐like receptor 4 (TLR4), Bcl‐2‐associated X protein (Bax), transforming growth factor‐β‐activated kinase‐1 (TAK1), myeloid differentiation factor 88 (MyD88) and nuclear factor‐kappaB (NF‐κB) was determined. Results Rifaximin treatment (0.1, 1.0 and 10 μM) caused a significant and concentration-dependent increase in the TEER of Caco-2 cells (360%, 480% and 680% vs TcdA treatment) 24 hours after the treatment and improved their viability (61%, 79% and 105%). Treatment also concentration-dependently decreased the expression of Bax protein (–29%, –65% and –77%) and increased the expression of ZO-1 (25%, 54% and 87%) and occludin (71%, 114% and 262%) versus TcdA treatment. The expression of TLR4 (–33%, –50% and –75%), MyD88 (–29%, –60% and –81%) and TAK1 (–37%, –63% and –79%) were also reduced with rifaximin versus TcdA treatment. Ketoconazole treatment inhibited these effects. Conclusions: Rifaximin improved TcdA‐induced toxicity in Caco‐2 cells by the PXR‐dependent TLR4/MyD88/NF‐κB pathway mechanism, and may be useful in the treatment of CDIs

Impaired duodenal Palmitoylethanolamide release underlies acid-induced mast cells activation in Functional Dyspepsia

Acid hypersensitivity is claimed to be a symptomatic trigger in functional dyspepsia (FD); however, the neuroimmune pathway(s) and the mediators involved in this process have not been systematically investigated. Palmitoylethanolamide (PEA) is an endogenous compound, able to modulate nociception and inflammation, but its role in FD has never been assessed

SPECIFIC DYSPEPTIC SYMPTOMS ARE ASSOCIATED WITH POOR RESPONSE TO THERAPY IN PATIENTS WITH GASTROESOPHAGEAL REFLUX DISEASE

Background: In gastroesophageal reflux disease (GORD) patients, coexistence of functional dyspepsia (FD) is known to be associated with poor response to proton pump inhibitors (PPIs), but the contribution of specific dyspepsia symptoms has not been systematically investigated yet. Objective: To characterize the impact of dyspepsia symptoms on PPIs response in GORD patients. Methods:. The enrolled subjects were 100 patients with diagnosis of GORD. All patients underwent a 24 hour pH-impedance test, while on PPIs-therapy. Patients were divided into two groups, refractory and responders, according to the persistence of GORD symptoms. A standardized questionnaire for FD was also administered to assess presence of dyspepsia symptoms. Results: In the subgroup of refractory patients FD was more prevalent than in responder ones, with postprandial fullness, nausea, vomiting, early satiation and epigastric pain being significantly prevalent in refractory GORD-patients. In the multivariate analysis only early satiation and vomiting were significantly associated with poor response to PPIs Conclusion: Coexistence of FD is associated with refractory-GORD. We showed that only early satiation and vomiting are risk factors for poor response to PPIs therapy. Our findings suggest that symptoms of early satiation and vomiting would help to identify the subset of PPIs-refractory GORD patients

HIV-1 Tat-induced diarrhea is improved by the PPARalpha agonist, palmitoylethanolamide, by suppressing the activation of enteric glia

Diarrhea is a severe complication in HIV-1-infected patients with Trans-activator of transcription (HIV-1 Tat) protein being recognized as a major underlying cause. Beside its direct enterotoxic effects, Tat protein has been recently shown to affect enteric glial cell (EGC) activity. EGCs regulate intestinal inflammatory responses by secreting pro-inflammatory molecules; nonetheless, they might also release immune-regulatory factors, as palmytoilethanolamide (PEA), which exerts anti-inflammatory effects by activating PPARα receptors. We aimed at clarifying whether EGCs are involved in HIV-1 Tat-induced diarrhea and if PEA exerts antidiarrheal activity

Rifaximin, a non-absorbable antibiotic, inhibits the release of pro-angiogenic mediators in colon cancer cells through a pregnane X receptor-dependent pathway

Activation of intestinal human pregnane X receptor (PXR) has recently been proposed as a promising strategy for the chemoprevention of inflammation-induced colon cancer. The present study was aimed at evaluating the effect of rifaximin, a non-absorbable antibiotic, in inhibiting angiogenesis in a model of human colorectal epithelium and investigating the role of PXR in its mechanism of action. Caco-2 cells were treated with rifaximin (0.1, 1.0 and 10.0 μM) in the presence or absence of ketoconazole (10 μM) and assessed for cell proliferation, migration and expression of proliferating cell nuclear antigen (PCNA). The release of vascular endothelial growth factor (VEGF) and nitric oxide (NO), expression of Akt, mechanistic target of rapamycin (mTOR), p38 mitogen activated protein kinases (MAPK), nuclear factor κB (NF-κB) and metalloproteinase-2 and -9 (MMP-2 and -9) were also evaluated. Treatment with rifaximin 0.1, 1.0 and 10.0 μM caused significant and concentration-dependent reduction of cell proliferation (-25, -40 and -68%), cell migration (-18, -30 and -46%) and PCNA expression (-29, -53 and -76%) in the Caco-2 cells vs. untreated cells. Treatment downregulated VEGF secretion (-32, -45 and -72%), NO release (-40, -69 and -87%), VEGFR-2 expression (-33, -58 and -65%) and MMP-2 (-25, -62 and -87%) and MMP-9 expression (-38, -56 and -78%) vs. untreated cells. Rifaximin treatment also resulted in a concentration-dependent decrease in the phosphorylation of Akt (-50, -75 and -86%), mTOR (-38, -56 and -78%), p38MAPK (-24, -62 and -71%) and inhibition of HIF-1α (-65, -82 and -92%), p70S6K (-27, -55 and -85%) and NF-κB (-29, -55 and -61%). Ketoconazole (PXR antagonist) treatment inhibited these effects. These findings demonstrated that rifaximin causes PXR-mediated inhibition of angiogenic factors in Caco-2 cell line and may be a promising anticancer tool

S100B‑p53 disengagement by pentamidine promotes apoptosis and inhibits cellular migration via aquaporin‑4 and metalloproteinase‑2 inhibition in C6 glioma cells

S100 calcium‑binding protein B (S100B) is highly expressed in glioma cells and promotes cancer cell survival via inhibition of the p53 protein. In melanoma cells, this S100B‑p53 interaction is known to be inhibited by pentamidine isethionate, an antiprotozoal agent. Thus, the aim of the present study was to evaluate the effect of pentamidine on rat C6 glioma cell proliferation, migration and apoptosis in vitro. The change in C6 cell proliferation following treatment with pentamidine was determined by performing a 3‑[4,5‑dimethylthiazol‑2‑yl]‑2,5 diphenyltetrazolium bromide‑formazan assay. Significant dose‑dependent decreases in proliferation were observed at pentamidine concentrations of 0.05 μM (58.5±5%; P<0.05), 0.5 μM (40.6±7%; P<0.01) and 5 μM (13±4%; P<0.001) compared with the control (100% viability). Furthermore, treatment with 0.05, 0.5 and 5 μM pentamidine was associated with a significant increase in apoptosis versus the untreated cells, as determined by DNA fragmentation assays, immunofluorescence analysis of C6 chromatin using Hoechst staining, and immunoblot analysis of B‑cell lymphoma‑2 (Bcl‑2)‑associated X protein (100%, P<0.05; 453%, P<0.01; and 1000%, P<0.001, respectively) and Bcl‑2 (‑60%, P<0.001; ‑80.13%, P<0.001; ‑95%, P<0.001, respectively). In addition, the administration of 0.05, 0.5 and 5 μM pentamidine significantly upregulated the protein expression levels of p53 (681±87.5%, P<0.05; 1244±94.3%, P<0.01; and 2244±111%, P<0.001, respectively), and significantly downregulated the expression levels of matrix metalloproteinase‑2 (42±2.3%, P<0.05; 71±2.5%, P<0.01; and 95.8±3.3%, P<0.001, respectively) and aquaporin 4 (38±2.5%, P<0.05; 69±2.6%, P<0.01; and 88±3.0%, P<0.001, respectively), compared with the untreated cells. The wound healing assay demonstrated that cell migration was significantly impaired by treatment with 0.05, 0.5 and 5 μM pentamidine compared with untreated cells (88±4.2%, P<0.05; 64±2%, P<0.01; and 42±3.1%, P<0.001, respectively). Although additional in vivo studies are required to clarify the current in vitro data, the present study indicates that pentamidine and S100B‑p53 inhibitors may represent a novel approach for the treatment of glioma