MECCANISMI FISIOPATOLOGICI ALLA BASE DELL'ENTEROPATIA INDOTTA DA FARMACI ANTI-INFIAMMATORI NON STEROIDEI:STUDIO DI CONFRONTO TRA CELECOXIB ED ETORICOXIB

I Farmaci anti-infiammatori non steroidei (FANS), rappresentano la categoria di medicinali maggiormente prescritti per la loro efficacia nel trattamento del dolore, febbre, infiammazione e malattie reumatiche. Tuttavia, è ampiamente riconosciuto che l’uso dei FANS si associa all’insorgenza di eventi avversi nel tratto gastrointestinale (GI) superiore, sebbene alcune evidenze sperimentali suggeriscano che questi farmaci siano anche in grado di esercitare effetti negativi sul tratto GI inferiore. Inoltre, i meccanismi patogenetici associati all’insorgenza di tossicità intestinale dei FANS risultano ancora poco conosciuti. Su tale base, l'obiettivo della presente tesi è stato quello di valutare gli effetti di celecoxib ed etoricoxib, come inibitori selettivi di COX-2, e di indometacina e diclofenac, come FANS non selettivi (ns-FANS), sull'integrità della mucosa intestinale in ratti anziani, allo scopo di identificare i meccanismi primari coinvolti nella fisiopatologia del danno intestinale. Gli animali sono stati sottoposti a somministrazione giornaliera con i farmaci in studio per via intra-gastrica, per 14 giorni. Al termine del trattamento gli animali sono stati sottoposti alle seguenti indagini: 1) valutazione dei livelli plasmatici di emoglobina come indice di sanguinamento del tratto digerente; 2) analisi istologica del danno intestinale nell’intestino tenue; 3) valutazione della funzionalità mitocondriale a livello epatico; 4) analisi western blot dell'espressione di ciclo-ossigenasi-1 e -2 (COX-1, COX-2) nella parete dell’intestino tenue; 5) misurazione dei livelli di malondialdeide (MDA), di mieloperossidasi (MPO) e di prostaglandina E2 (PGE2) nella parete dell’intestino tenue. I dati ottenuti hanno evidenziato che la somministrazione di indometacina e diclofenac ha determinato una riduzione dei livelli di emoglobina nel sangue, mentre nessun cambiamento significativo e stato rilevato negli animali trattati con celecoxib o etoricoxib. Inoltre, il danno istologico intestinale osservato in animali trattati con celecoxib è risultato di grado inferiore rispetto a quello osservato negli altri gruppi di trattamento. Negli animali trattati con indometacina e diclofenac è stata riscontrata una riduzione significativa di PGE2 nella mucosa del digiuno e dell'ileo, mentre etoricoxib e celecoxib non hanno determinato variazioni significative. L'analisi western blot, ha rivelato che la somministrazione dei farmaci non ha determinato nessun cambiamento nell'espressione di COX-1. Al contrario, è stato rilevato un aumento dell'espressione di COX-2 nei tessuti prelevati dai ratti trattati con indometacina, e in minore misura con diclofenac ed etoricoxib, mentre negli animali trattati con celecoxib l’espressione di COX-2 è risultata ridotta. Alcuni parametri di respirazione mitocondriale sono risultati alterati in seguito a trattamento con indometacina, diclofenac ed etoricoxib, mentre celecoxib si è dimostrato privo di effetti a questo livello. Indometacina e diclofenac hanno aumentato i livelli di MDA e MPO sia nel digiuno che nell' ileo. Etoricoxib e celecoxib non hanno modificato tali livelli nel digiuno, mentre nell'ileo etoricoxib, ma non celecoxib, ha determinato un aumento sia dei livelli di MDA che di MPO. I dati ottenuti dal presente studio suggeriscono che ns-FANS ed etoricoxib, caratterizzati da un basso valore di pKa, possono determinare l’insorgenza di enteropatia, attraverso effetti topici diretti sulla mucosa. Al contrario, celecoxib, che risulta sprovvisto di caratteristiche acide, si è dimostrato privo di azioni lesive significative sull’intestino tenue. Pertanto, il profilo di selettività nei confronti di COX-1 e/o COX-2 e i relativi effetti sulla produzione di PGE2, non sembrano sufficienti a giustificare gli effetti negativi sulla mucosa intestinale causati dai FANS, mentre sembra che la loro tossicità sia strettamente legata alla struttura chimica acida/lipofila

Alteration of colonic excitatory tachykininergic motility and enteric inflammation following dopaminergic nigrostriatal neurodegeneration

Background: Parkinson's disease (PD) is frequently associated with gastrointestinal (GI) symptoms, including constipation and defecatory dysfunctions. The mechanisms underlying such disorders are still largely unknown, although the occurrence of a bowel inflammatory condition has been hypothesized. This study examined the impact of central dopaminergic degeneration, induced by intranigral injection of 6-hydroxydopamine (6-OHDA), on distal colonic excitatory tachykininergic motility in rats. Methods: Animals were euthanized 4 and 8 weeks after 6-OHDA injection. Tachykininergic contractions, elicited by electrical stimulation or exogenous substance P (SP), were recorded in vitro from longitudinal muscle colonic preparations. SP, tachykininergic NK1 receptor, and glial fibrillary acidic protein (GFAP) expression, as well as the density of eosinophils and mast cells in the colonic wall, were examined by immunohistochemical analysis. Malondialdehyde (MDA, colorimetric assay), TNF, and IL-1 beta (ELISA assay) levels were also examined. The polarization of peritoneal macrophages was evaluated by real-time PCR. Results: In colonic preparations, electrically and SP-evoked tachykininergic contractions were increased in 6-OHDA rats. Immunohistochemistry displayed an increase in SP and GFAP levels in the myenteric plexus, as well as NK1 receptor expression in the colonic muscle layer of 6-OHDA rats. MDA, TNF, and IL-1 beta levels were increased also in colonic tissues from 6-OHDA rats. In 6-OHDA rats, the number of eosinophils and mast cells was increased as compared with control animals, and peritoneal macrophages polarized towards a pro-inflammatory phenotype. Conclusions: The results indicate that the induction of central nigrostriatal dopaminergic degeneration is followed by bowel inflammation associated with increased oxidative stress, increase in pro-inflammatory cytokine levels, activation of enteric glia and inflammatory cells, and enhancement of colonic excitatory tachykininergic motility

Fibrotic and Vascular Remodelling of Colonic Wall in Patients with Active Ulcerative Colitis

open16noIntestinal fibrosis is a complication of inflammatory bowel disease [IBD]. Although fibrostenosis is a rare event in ulcerative colitis [UC], there is evidence that a fibrotic rearrangement of the colon occurs in the later stages. This is a retrospective study aimed at examining the histopathological features of the colonic wall in both short-lasting [SL] and long-lasting [LL] UC. Surgical samples of left colon from non-stenotic SL [a parts per thousand currency sign 3 years, n = 9] and LL [a parts per thousand yen 10 years, n = 10] UC patients with active disease were compared with control colonic tissues from cancer patients without UC [n = 12] to assess: collagen and elastic fibres by histochemistry; vascular networks [CD31/CD105/nestin] by immunofluorescence; parameters of fibrosis [types I and III collagen, fibronectin, RhoA, alpha-smooth muscle actin [alpha-SMA], desmin, vimentin], and proliferation [proliferating nuclear antigen [PCNA]] by western blot and/or immunolabelling. Colonic tissue from both SL-UC and LL-UC showed tunica muscularis thickening and transmural activated neovessels [displaying both proliferating CD105-positive endothelial cells and activated nestin-positive pericytes], as compared with controls. In LL-UC, the increased collagen deposition was associated with an up-regulation of tissue fibrotic markers [collagen I and III, fibronectin, vimentin, RhoA], an enhancement of proliferation [PCNA] and, along with a loss of elastic fibres, a rearrangement of the tunica muscularis towards a fibrotic phenotype. A significant transmural fibrotic thickening occurs in colonic tissue from LL-UC, together with a cellular fibrotic switch in the tunica muscularis. A full-thickness angiogenesis is also evident in both SL- and LL-UC with active disease, as compared with controls.openIppolito, Chiara; Colucci, Rocchina; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, NunziaIppolito, Chiara; Colucci, ROCCHINA LUCIA; Segnani, Cristina; Errede, Mariella; Girolamo, Francesco; Virgintino, Daniela; Dolfi, Amelio; Tirotta, Erika; Buccianti, Piero; Di Candio, Giulio; Campani, Daniela; Castagna, Maura; Bassotti, Gabrio; Villanacci, Vincenzo; Blandizzi, Corrado; Bernardini, Nunzi

Protective effects of the combination Bifidobacterium longum plus lactoferrin against NSAID-induced enteropathy

Objectives Non-steroidal anti-inflammatory drugs can exert detrimental effects in the lower digestive tract. This study examined the protective effects of a combination of the probiotic Bifidobacterium longum BB536 (Bifidobacterium) with the prebiotic lactoferrin in a rat model of diclofenac-induced enteropathy. Methods Enteropathy was induced in 40-week-old male rats by intragastric diclofenac (4 mg/kg BID, 14 days). Lactoferrin (100 mg/kg BID), Bifidobacterium (2.5\u2022106 CFU/rat BID) or their combination were administered 1 hour before diclofenac. At the end of treatments, the ileum was processed for the evaluation of histological damage, myeloperoxidase (MPO) and malondialdehyde (MDA) levels, as well as the expression of toll-like receptors 2 and 4 (TLR-2/-4) and the activation of downstream signaling molecules (MyD88 and NF-kB p65). Blood hemoglobin and fecal calprotectin were also assessed. Results Diclofenac induced intestinal damage, along with increments of MPO and MDA, overexpression of TLR-2, TLR-4, MyD88 and NF-kB p65, increase in fecal calprotectin and decrease in blood hemoglobin levels. Lactoferrin or Bifidobacterium alone prevented diclofenac-induced enteric damage, and the changes in blood hemoglobin, MPO, MDA, fecal calprotectin and NF-kB p65. Bifidobacterium, but not lactoferrin, decreased TLR-4 expression, while none of them affected MyD88 overexpression. TLR-2 expression was slightly enhanced by all treatments. The combined administration of lactoferrin and Bifidobacterium reduced further the intestinal damage, and restored MPO and blood hemoglobin levels. Conclusions Diclofenac induced ileal mucosal lesions by activation of inflammatory and pro-oxidant mechanisms. These detrimental actions were prevented by the combination of lactoferrin with Bifidobacterium likely through the modulation of TLR-2/-4/NF-kB pro-inflammatory pathways

A Case Study of African American Parental Involvement in an Urban Middle School

Studying parental involvement offers the opportunity to develop new strategies and resources to increase involvement at the middle schools serving a similar demographic population. In a large economically disadvantaged urban middle school in the southeastern United States, very little parental involvement occurs from the African American population. The purpose of this qualitative single case study was to examine African American parents\u27 perception about their involvement in their middle school students\u27 education. Guided by Epstein, Simon, and Salinas\u27 parental involvement model, which describes 6 levels of parental involvement, the research questions guiding this project study examined African American parents\u27 perceptions about middle school children\u27s educational experiences, the level of parental involvement in middle school education, and parental beliefs about student success. A purposeful participant pool of 10 African American parents of Grade 7 and 8 students was used for data collection. Ten parents completed the preliminary paper questionnaire, 10 parents participated in 1-on-1 semi-structured interviews, and 7 parents participated in a focus group discussion. Thematic analysis of data followed the open coding process and identified categories and themes. The findings suggested the need for a parent education program involving the use of new strategies and resources for increasing African American parent involvement at the middle school level. Social change will occur by empowering African American parents to be involved in their middle school students\u27 education

The flavonoid compound apigenin prevents colonic inflammation and motor dysfunctions associated with high fat diet-induced obesity

When compared to SD mice, HFD animals displayed increased body weight, epididymal fat weight and metabolic indexes. HFD mice showed increments in colonic MDA, IL-1 beta and IL-6 levels, as well as a decrease in let-7f expression in both colonic and epididymal tissues. HFD mice displayed an increase in colonic eosinophil infiltration. Immunohistochemistry revealed an increase in SP and iNOS expression in myenteric ganglia of HFD mice. In preparations from HFD mice, electrically evoked contractions upon NOS blockade or mediated by tachykininergic stimulation were enhanced. In HFD mice, Apigenin counteracted the increase in body and epididymal fat weight, as well as the alterations of metabolic indexes. Apigenin reduced also MDA, IL-1 beta and IL-6 colonic levels as well as eosinophil infiltration, SP and iNOS expression, along with a normalization of electrically evoked tachykininergic and nitrergic contractions. In addition, apigenin normalized let-7f expression in epididymal fat tissues, but not in colonic specimens

Ghrelin restores nitric oxide availability in resistance circulation of essential hypertensive patients: role of NAD(P)H oxidase

We assessed whether acute intra-arterial infusion of exogenous ghrelin can improve endothelial dysfunction by restoring nitric oxide (NO) availability in the forearm microcirculation of essential hypertensive patients. The effect of ghrelin on endothelial dysfunction (pressurized myograph), vascular oxidative stress generation (fluorescent dihydroethidium), and phosphorylation of p47phox (western blot), an index of NAD(P)H oxidase activation, in isolated small arteries taken from essential hypertensive patients (subcutaneous biopsy) were also investigated

Effects of L-DOPA/Benserazide Co-Treatment on the Patterns of Colonic Neuromuscular Excitatory Cholinergic and Tachykininergic Pathways in the Presence of Experimental Parkinson's Disease

Introduction. Patients with Parkinson's disease (PD) can develop digestive motor dysfunctions associated with changes in enteric nervous system. The mainstay therapy for PD is represented by levodopa (L-DOPA) plus DOPA-decarboxylase inhibitors. However, the possible effects of these drugs on intestinal motility in PD are unclear. This study examines the effects of L-DOPA plus benserazide (BE) on colonic neuromuscular excitatory pathways in rats with experimental PD. Methods. PD was induced in male rats by intra-nigral injection of 6- hydroxydopamine (6-OHDA). Animals injected with 6-OHDA vehicle served as normal controls (sham-PD). PD or sham-PD animals were treated orally with L-DOPA/BE (6/15 mg/ Kg/day) or their vehicle for 28 days, starting 28 days after intra-nigral injection. At the end of drug treatment, colonic longitudinal muscle preparations were isolated, set up in organ baths, and connected to isometric transducers to record neurogenic contractions (g/g tissue) elicited by electrical stimulation (ES, 1-20 Hz) under the following conditions: 1) Krebs solution containing guanethidine (10 \u3bcM), N\u3c9-nitro-L-arginine methylester (L-NAME, 100 \u3bcM) and L-732,138 (NK1 receptor antagonist, 10 \u3bcM) to record cholinergic contractile responses; 2) Krebs solution containing L-NAME, guanethidine and atropine (1 \u3bcM) to obtain contractions driven primarily by tachykinins. Myogenic contractions elicited by carbachol (1 \u3bcM) or exogenous substance P (SP, 0.1 \u3bcM) in the presence of tetrodotoxin (1 \u3bcM) were also recorded. Results. In sham-PD animals, treatment with L-DOPA/BE did not modify all the patterns of stimulated motor activity, in comparison with drug vehicle treatment. When compared with sham-PD rats, PD animals displayed the following colonic motor alterations: 1) a reduction of atropine-sensitive neurogenic cholinergic contractions evoked by ES (10 Hz) (-24\ub12.3%); 2) an increase in myogenic contractions evoked by carbachol (+93\ub113%); 3) an enhancement of electrically evoked (10 Hz) neurogenic L-732,138-sensitive tachykininergic motor responses (+70\ub14%); 4) a potentiation of myogenic contractions elicited by exogenous SP (+113\ub17.6%). In PD animals, treatment with L-DOPA/BE was associated with a restoration of ES-evoked neurogenic cholinergic motor responses, while the changes in carbachol-evoked myogenic contractions were unaffected. On the other hand, treatment of PD animals with L-DOPA/BE did not produce any effect on the alterations of both ES- and SP-evoked tachykininergic motor responses. Conclusion. In experimental PD, treatment with L-DOPA/BE resulted in a recovery of colonic cholinergic excitatory neurogenic motility, suggesting a protective effect of L-DOPA/BE on the alterations of cholinergic neurotransmission associated with experimental PD