Giuseppina Autore

Inhibition of intestinal motility and secretion by flavonoids in mice and rats: Structure-activity relationships

Abstract— Intraperitoneal administration of some flavonoids (apigenin, flavone, kaempferol, morin, myricetin, naringin and rutin; 12·5–50 mg kg−1) significantly (P < 0·05–0·01) reduced small (28–69%) and large (83–134%) intestinal transit in mice. Other flavonoids (naringenin, silibinin, silymarin and taxifolin, 100–200 mg kg−1) reduced (23–41%; P < 0·5–0·01) intestinal transit at doses of 100–200 mg kg−1 while hesperitin, catechin and phloridzin (up to 200 mg kg−1) had no effect. This effect was antagonized by yohimbine (87–96%) and phentolamine (87–91%) but not by prazosin, propranolol, atropine, hexamethonium, mepyramine, cyproheptadine and naloxone. Yohimbine (92–96%) also antagonized the inhibitory effect of flavonols (12·5–50 mg kg−1) (P < 0·05–0·01) on intraluminal accumulation of fluid and diarrhoea induced by castor oil. By contrast, verapamil potentiated the flavonol effect. It is suggested that these effects, influenced by the structure of the molecules, are mediated by α2‐adrenergic receptors and calcium.

Probiotics Reduce the Inflammatory Response Induced by a High-Fat Diet in the Liver of Young Rats

Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease in the pediatric population. Preliminary evidence suggests a potential therapeutic utility of probiotics for this condition. Here, we tested the potential effect of the probiotic VSL#3 (a multistrain preparation composed of Streptococcus thermophilus and several species of Lactobacillus and Bifidobacteria) on oxidative and inflammatory damage induced by a high-fat diet in the liver of young rats. At weaning, young male Sprague-Dawley rats were randomly divided into 3 groups (n = 6) fed a standard, nonpurified diet (Std; 5.5% of energy from fat) or a high-fat liquid diet (HFD; 71% of energy from fat). One of the HFD groups received by gavage VSL#3 (13 x 10(9) bacteria x kg(-1) x d(-1)). After 4 wk, the HFD rats had greater body weight gain, fat mass, serum aminotransferase, and liver weight than rats fed the Std diet. The HFD induced liver lipid peroxidation, tumor necrosis factor (TNFalpha) production, protein S-nitrosylation, inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2 expression, and metalloproteinase (MMP) activity. Moreover, in the HFD group, PPARalpha expression was less than in rats fed the Std diet. In rats fed the HFD diet and treated with VSL#3, liver TNFalpha levels, MMP-2 and MMP-9 activities, and expression of iNOS and COX-2 were significantly lower than in the HFD group. In VSL#3-treated rats, PPARalpha expression was greater than in the HFD group. A modulation of the nuclear factor-kappaB pathway by VSL#3 was also demonstrated. Our data suggest that VSL#3 administration could limit oxidative and inflammatory liver damage in patients with NAFLD.

Laxatives and the production of autacoids by rat colon.

The effects of some laxatives were examined on the formation of histamine, 5‐hydroxytryptamine (5‐HT) and prostaglandin‐like material (PG‐LM) by rat intestine in‐vitro. Castor oil, senna, sulphosuccinate and bisacodyl, but not mannitol or lactulose, in doses that cause laxation, increased the formation of histamine, 5‐HT and PG‐LM. Indomethacin or hydrocortisone reduced the increase of PG‐LM formation. The data support the idea that the laxative effects of these intestinal secretagogues are due to increased intestinal production of PG‐LM, histamine and 5‐HT.

Antipyretic and antibacterial actions of Teucrium polium (L.)

The antipyretic and antibacterial activities of the ethanolic extract of the flowering tops of Teucrium polium (L.) were been studied. The extract was effective against both yeast and carrageenin pyrexia in rats. It also exhibited a marked antibacterial action against both gram positive and gram negative organisms and was found to be non toxic in acute studies.

Oxidative stress in patients with cardiovascular disease and chronic renal failure

Abstract Oxidative response regulates many physiological response in human health, but if not properly regulated it could also lead to a number of deleterious effects. The importance of oxidative stress injury depends on the molecular target, the severity of the stress, and the mechanism by which the oxidative stress is imposed: it has been implicated in several diseases including cancer, neurodegenerative diseases, malaria, rheumatoid arthritis and cardiovascular and kidney disease. Most of the common diseases, such as hypertension, atherosclerosis, heart failure, and renal dysfunction, are associated with vascular functional and structural alterations including endothelial dysfunction, altered contractility, and vascular remodeling. Common to these processes is increased bioavailability of reactive oxygen species (ROS), decreased nitric oxide (NO) levels, and reduced antioxidant capacity. Oxidative processes are up-regulated also in patients with chronic renal failure (CRF) and seem to be a cause of elevated risk of morbidity and mortality in these patients. In this review, we highlight the role of oxidative stress in cardiovascular and renal disease.

Very low protein diet reduces indoxyl sulfate levels in chronic kidney disease.

Background and Objectives: High levels of indoxyl sulfate (IS) are associated with chronic kidney disease (CKD) progression and increased mortality in CKD patients. The aim of this pilot study was to assess whether a very low protein diet (VLPD; 0.3 g/kg bw/day), with a consequent low phosphorus intake, would reduce IS serum levels compared to a low protein diet (LPD; 0.6 g/kg bw/day) in CKD patients not yet on dialysis. Material and Methods: This is a post hoc analysis of a preceding cross-over study aimed to analyze FGF23 during VLPD. Here we performed a prospective randomized controlled crossover study in which 32 patients were randomized to receive either a VLPD (0.3 g/kg bw/day) supplemented with ketoanalogues during the first week and an LPD during the second week (group A, n = 16), or an LPD during the first week and a VLPD during the second week (group B, n = 16 patients). IS serum levels were measured at baseline and at the end of each study period. We compared them to 24 hemodialysis patients (HD) and 14 healthy subjects (control). Results: IS serum concentration was significantly higher in the HD (43.4 ± 12.3 µM) and CKD (11.1 ± 6.6 µM) groups compared to the control group (2.9 ± 1.1 µM; p < 0.001). IS levels also correlated with creatinine values in CKD patients (R2 = 0.42; p < 0.0001). After only 1 week of a VLPD, even preceded by an LPD, CKD patients showed a significant reduction of IS serum levels (37%). Conclusions: VLPD supplemented with ketoanalogues reduced IS serum levels in CKD patients not yet on dialysis.

The Uremic Toxin Indoxyl Sulphate Enhances Macrophage Response to LPS

Indoxyl sulphate (IS) is a protein-bound uremic toxin that results from the metabolism of dietary tryptophan normally excreted by kidney through the proximal tubules. Thus the toxin accumulates in the blood of patients with impaired renal function such as in chronic kidney disease (CKD). High IS serum levels in patients with CKD suggest its involvement in CKD progression and in the onset of complications. Its presence in plasma is also a powerful predictor of overall and cardiovascular morbidity/mortality. IS is a well known nephrovascular toxin but very little is known regarding its effects on the immune system and in particular during inflammation. In this study we examined the effect of IS on macrophage activation in response to lipopolysaccharide from E. coli (LPS), a gram negative bacterial endotoxin associated with inflammation and septic shock. To simulate the uremic condition, J774A.1 macrophages were incubated with IS at concentrations observed in uremic patients (1000–62.5 µM) both alone and during LPS challenge. IS alone induced release of reactive oxygen species (ROS), through a mechanism involving pro- and anti-oxidant systems, and alteration in intracellular calcium homeostasis. When added to J774A.1 macrophages in presence of LPS, IS significantly increased the nitric oxide (NO) release, inducible nitric oxide synthase (iNOS) and cycloxygenase-2 (COX-2) expression. IS pre-treatment was also associated with an increase in tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) production by macrophages stimulated with LPS. Mechanistic studies revealed that IS increased LPS-induced NF-kB nuclear translocation, ROS release and altered calcium concentrations, mainly because of mitochondrial calcium overloading. Moreover also in primary mouse peritoneal macrophages IS enhances the inflammatory response to LPS increasing ROS, NO, iNOS, COX-2, TNF-α, IL-6 and NF-kB levels. This study provides evidences that IS stimulates macrophage function and enhances inflammatory reasponse associated with LPS, thus contributing to altered immune response dysfunctions observed in CKD.

Modulation of macrophage activity by aflatoxins B1 and B2 and their metabolites aflatoxins M1 and M2.

Aflatoxins are natural contaminants frequently found both in food and feed. Many of them exert immunomodulatory properties in mammals; therefore, the aim of the current study was to investigate immune-effects of AFB1, AFB2, AFM1 and AFM2, alone and differently combined, in J774A.1 murine macrophages. MTT assay showed that AFB1, alone and combined with AFB2, possess antiproliferative activity only at the highest concentration; such effect was not shown by their hydroxylated metabolites, AFM1 and AFM2, respectively. However, the immunotoxic effects of the aflatoxins evaluated in the current study may be due to the inhibition of production of active oxygen metabolites such as NO. Cytofluorimetric assay in macrophages exposed to aflatoxins (10-100 μM) revealed that their cytoxicity is not related to apoptotic pathways. Nevertheless, a significant increase of the S phase cell population accompanied by a decrease in G0/G1 phase cell population was observed after AFB1 treatment. In conclusion, the results of the current study suggest that aflatoxins could compromise the macrophages functions; in particular, co-exposure to AFB1, AFB2, AFM1 and AFM2 may exert interactions which can significantly affect immunoreactivity.

Involvement of ATP‐sensitive potassium channels in a model of a delayed vascular hyporeactivity induced by lipopolysaccharide in rats

We have investigated the role of ATP‐sensitive potassium (KATP) channels in an experimental model of a delayed phase of vascular hyporeactivity induced by lipopolysaccharide (LPS) in rats. After 24 h, from LPS treatment, in anaesthetized rats the bolus injection of phenylephrine (PE) produced an increase in mean arterial pressure (MAP) significantly (P<0.05) reduced in LPS‐treated rats compared to the vehicle‐treated rats. This reduction was prevented by pre‐treatment of rats with glibenclamide (GLB), a selective inhibitor of KATP channels. GLB administration did not affect the MAP in vehicle‐treated rats but produced an increase of MAP in rats treated with LPS. Cromakalim (CRK), a selective KATP channel opener, produced a reduction of MAP that was significantly (P<0.05) higher in LPS‐ than in vehicle‐treated rats. In contrast, the hypotension induced by glyceryl trinitrate (GTN) in LPS‐treated rats was not distinguishable from that produced in vehicle‐treated rats. Experiments in vitro were conducted on aorta rings collected from rats treated with vehicle or LPS 24 h before sacrifice. The concentration‐dependent curve to PE was statistically (P<0.005) reduced in aorta rings collected from LPS‐ compared to vehicle‐treated rats. This difference was totally abolished by tetraethylammonium (TEA), a non‐selective inhibitor of K+ channels. CRK produced a relaxation of PE precontracted aorta rings higher in rings from LPS‐ than in vehicle‐treated rats. GLB inhibited CRK‐induced relaxation in both tissues, abolishing the observed differences. In conclusion, our results indicate an involvement of KATP channels to the hyporesponsiveness of vascular tissue after 24 h from a single injection of LPS in rats. We can presume an increase in the activity of KATP channels on vascular smooth muscle cells but we cannot exclude an increase of KATP channel number probably due to the gene expression activation.

An extract of Apium graveolens var. dulce leaves: structure of the major constituent, apiin, and its anti-inflammatory properties

Flavonoids, natural compounds widely distributed in the plant kingdom, are reported to affect the inflammatory process and to possess anti‐inflammatory as well as immunomodulatory activity in‐vitro and in‐vivo. Since nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is one of the inflammatory mediators, the effects of the ethanol/water (1:1) extract of the leaves of Apium graveolens var. dulce (celery) on iNOS expression and NO production in the J774.A1 macrophage cell line stimulated for 24 h with Escherichia coli lipopolysaccharide (LPS) were evaluated. The extract of A. graveolens var. dulce contained apiin as the major constituent (1.12%, w/w, of the extract). The extract and apiin showed significant inhibitory activity on nitrite (NO) production in‐vitro (IC50 0.073 and 0.08 mg mL−1 for the extract and apiin, respectively) and iNOS expression (IC50 0.095 and 0.049 mg mL−1 for the extract and apiin, respectively) in LPS‐activated J774.A1 cells. The croton‐oil ear test on mice showed that the extract exerted anti‐inflammatory activity in‐vivo (ID50 730 μg cm−2), with a potency seven‐times lower than that of indometacin (ID50 93 μg cm−2), the non‐steroidal anti‐inflammatory drug used as reference. Our results clearly indicated the inhibitory activity of the extract and apiin in‐vitro on iNOS expression and nitrite production when added before LPS stimulation in the medium of J774.A1 cells. The anti‐inflammatory properties of the extract demonstrated in‐vivo might have been due to reduction of iNOS enzyme expression.