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Palmitoylethanolamide (PEA) regulates many pathophysiological processes in the central nervous system, including pain perception, convulsions and neurotoxicity, and increasing evidence points to its neuroprotective action. In the present study, we report that PEA, acting as a ligand of peroxisome-proliferator activated receptor (PPAR)-α, might regulate neurosteroidogenesis in astrocytes, which, similar to other glial cells and neurones, have the enzymatic machinery for neurosteroid de novo synthesis. Accordingly, we used the C6 glioma cell line and primary murine astrocytes. In the mitochondrial fraction from cells stimulated with PEA, we demonstrated an increase in steroidogenic acute regulatory protein (StAR) and cytochrome P450 enzyme (P450scc) expression, both comprising proteins considered to be involved in crucial steps of neurosteroid formation. The effects of PEA were completely blunted by GW6471, a selective PPAR-α antagonist, or by PPAR-α silencing by RNA interference. Accordingly, allopregnanolone (ALLO) levels were increased in supernatant of PEA-treated astrocytes, as revealed by gas chromatography-mass spectrometry, and this effect was inhibited by GW6471. Moreover, PEA showed a protective effect, reducing malondialdehyde formation in cells treated with l-buthionine-(S,R)-sulfoximine, a glutathione depletor and, interestingly, the effect of PEA was partially inhibited by finasteride, a 5α-reductase inhibitor. A similar profile of activity was demonstrated by ALLO and the lack of an additive effect with PEA suggests that the reduction of oxidative stress by PEA is mediated through ALLO synthesis. The present study provides evidence indicating the involvement of the saturated acylethanolamide PEA in ALLO synthesis through PPAR-α in astrocytes and explores the antioxidative activity of this molecule, confirming its homeostatic and protective role both under physiological and pathological conditions.

Palmitoylethanolamde (PEA) is an endogenous lipid neuromodulator that mediates a broad spectrum of pharmacological effects by activation of peroxisome proliferator-activated receptor alpha (PPAR-alpha). Detectable or high levels of PEA in the CNS have been found, but the specific function of this lipid remains to be clarified. Here we report evidence that PEA, activating PPAR-alpha receptor and involving neurosteroids de novo synthesis, modulates pentobarbital-evoked hypnotic effect. A single i.c.v. administration of PEA (1-5microg) increases pentobarbital induced loss of righting reflex (LORR) duration in mice. This effect is mimicked by GW7647 (3microg), a synthetic PPAR-alpha agonist, and disappears in PPAR-alpha knockout mice. Antagonism experiments strongly support the engaging of neurosteroidogenic pathway in the increase of LORR duration induced by PEA. This effect disappeared using two inhibitors blocking the key steps of neurosteroids synthesis, aminogluthetimide and finasteride. Moreover, we demonstrated that in brainstem PEA increased the expression of steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc), both involved in neurosteroidogenesis. Accordingly, allopregnanolone (ALLO) levels were in turn higher in brainstem of PEA and pentobarbital treated mice vs pentobarbital alone, as revealed by quantitative analysis using gas chromatography-mass spectrometry. A Our results demonstrate that exogenous administration of PEA, through a PPAR-alpha-dependent mechanism, modulates neurosteroids formation increasing ALLO levels and leading to a positive modulation of GABA(A) receptor. These data further strengthen our previous data on the role of PPAR-alpha in PEA's actions and could provide a new framework to understand its role in the CNS.

Despite the clear roles played by peroxisome proliferators-activated receptor alpha (PPAR-alpha) in lipid metabolism, inflammation and feeding, the effects of its activation in the central nervous system (CNS) are largely unknown. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-alpha agonist, exerting analgesic and anti-inflammatory effects. Both PPAR-alpha and PEA are present in the CNS, but the specific functions of this lipid and its receptor remain to be clarified. Using the carrageenan-induced paw model of hyperalgesia in mice, we report here that intracerebroventricular administration of PEA (0.1-1 microg) 30 min before carrageenan injection markedly reduced mechanical hyperalgesia up to 24 h following inflammatory insult. This effect was mimicked by GW7647 (1 microg), a synthetic PPAR-alpha agonist. The obligatory role of PPAR-alpha in mediating PEA's actions was confirmed by the lack of anti-hyperalgesic effects in mutant mice lacking PPAR-alpha. PEA significantly reduced the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) in sciatic nerves and restored carrageenan-induced reductions of PPAR-alpha in the L4-L6 dorsal root ganglia (DRG). To investigate the mechanism by which PEA attenuated hyperalgesia, we evaluated inhibitory kB-alpha (IkB-alpha) degradation and p65 nuclear factor kB (NF-kappaB) activation in DRG. PEA prevented IkB-alpha degradation and p65 NF-kappaB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral hyperalgesia. These results add further support to the broad-spectrum of biological and pharmacological effects induced by PPAR-alpha agonists, suggesting a centrally mediated component for these drugs in controlling inflammatory pain.

Leptin and/or ghrelin, initially thought to be considered messengers of energy metabolism, are now considered to play a role in normal and complicated pregnancy. In this study, pregnant, spontaneously hypertensive rats (SHR) have been used to evaluate, for the first time, the modification of leptin and ghrelin both at serum and tissue levels. In SHR, we evaluate plasma leptin level and tissue protein expression in both placenta and adipose tissue at the end of gestation (day 20) versus normotensive Wistar–Kyoto (WKY) animals. The expression of functional leptin receptor (Ob-Rb) in peripheral tissues and in the hypothalamus was evaluated. Moreover, we measured plasma ghrelin level and its mRNA expression in the stomach and placenta. SHR strain presented significantly lower plasma leptin levels when compared with those found in pregnant or not WKY controls. Interestingly, in the placenta, leptin gene expression was higher in SHR than normotensive WKY. Moreover, we demonstrated a resistance to the effects of leptin via ‘downregulation’ of hypothalamic receptors in pregnant SHR. Conversely, SHR presented significantly higher ghrelin plasma levels when compared with those found in pregnant or not WKY. However, we observed that ghrelin level in the stomach of SHR did not change during pregnancy, and on the opposite, mRNA ghrelin in the placenta of SHR was lower than that of normotensive rats, suggesting a different production of this hormone in the fetal–placental unit. These data gain further insight into metabolic hormone modifications observed in a model of pre-existing hypertension associated with pregnancy.

BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) is the most common form of chronic liver disease. Insulin resistance (IR) appears to be critical in its pathogenesis. We evaluated the effects of sodium butyrate (butyrate) and its synthetic derivative N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA) in a rat model of insulin resistance and steatosis induced by high-fat diet (HFD). METHODS: After weaning, young male Sprague-Dawley rats were divided into 4 groups receiving different diets for 6 weeks: 1. control group (standard diet); 2. HFD; 3. HFD plus butyrate (20 mg/kg/die) and 4. HFD plus FBA (42.5 mg/Kg/die, the equimolecular dose of butyrate). Liver tissues of the rats were analyzed by Western blot and real-time PCR. Insulin resistance, liver inflammation and Toll-like pattern modifications were determined. RESULTS: Evaluation of these two preparations of butyrate showed a reduction of liver steatosis and inflammation in HFD fed animals. The compounds showed a similar potency in the normalisation of several variables, such as transaminases, homeostasis model assessment for insulin resistance index, and glucose tolerance. Both treatments significantly reduced hepatic TNF-α expression and restored GLUTs and PPARs, either in liver or adipose tissue. Finally, FBA showed a higher potency in reducing pro-inflammatory parameters in the liver, via suppression of Toll-like receptors and NF-κB activation. CONCLUSIONS: Our results demonstrated a protective effect of butyrate in limiting molecular events underlying the onset of IR and NAFLD, suggesting a potential clinical relevance for this substance. In particular, its derivative, FBA, could represent an alternative therapeutic option to sodium butyrate, sharing a comparable efficacy, but a better palatability and compliance.

Astrocytes in the CNS produce inflammatory mediators in response to several stimuli and cytokines. Here we investigated the in vitro effect of leptin on inducible nitric oxide synthase (iNOS) expression in a glioma cell line (C6). After hormone stimulation, culture media were analysed for accumulated stable oxidation products of NO (NO2(-) and NO3(-), designated as NO(x)), cellular RNA was extracted to determine iNOS mRNA level by RT-PCR and cellular lysates were prepared for protein expression. Leptin induced a concentration-dependent increase of NO release, related to iNOS induction. This effect was potentiated by IFN-gamma, or TNF-alpha, or IFN-gamma plus IL-1beta. Pyrrolidine dithiocarbamate (PDTC) and N-alpha-tosyl-L-lysine chloromethyl ketone (TLCK), two inhibitors of NF-kappaB activation, as well as the specific proteasome inhibitor MG132, blocked leptin-induced iNOS. The role of NF-kappaB was also confirmed by time course studies on degradation of IkappaB-alpha, which began to degrade 5 min after treatment with leptin and returned to basal level after 30-60 min. Pre-incubation of cells with MG132 inhibited leptin-induced IkappaB-alpha degradation. These results confirm the pro-inflammatory role of leptin and identify it as a potential up-regulator of cytokine-induced inflammatory response in the CNS.

Raloxifene (RAL) is a selective estrogen receptor modulator presenting tissue-specific agonist activity. The aim of this study was to examine whether RAL has an estrogenic effect on carrageenan-induced acute inflammation. Adult female rats were ovariectomized (OVX) 7 wk before edema or pleurisy to deplete circulating estrogens. Edema formation and selected inflammatory markers in inflamed paw tissue were measured in intact (sham-operated) and OVX rats. Groups of OVX rats were treated with RAL (1, 3, or 10 mg/kg) or 17beta-estradiol (E2, 25 microg/kg), and these treatments began 2 d after surgery and continued until carrageenan paw edema or pleurisy. Ovariectomy amplifies the inflammation, and we found that RAL, as well as E2, attenuates inflammation and tissue damage associated with paw edema and pleurisy. In treated rats, there is a decrease in edema development and formation, and in polymorphonuclear cell infiltration and migration, as shown by myeloperoxidase measurement and cell counting. RAL and E2 treatments decrease cyclooxygenase-2 and inducible nitric oxide synthase expression in inflamed areas and counteract the inhibition of peroxisome proliferators-activated receptor-gamma expression caused by ovariectomy, restoring this receptor protein expression to sham-operated levels and identifying a possible peroxisome proliferators-activated receptor-dependent antiinflammatory effect of these drugs. Moreover, RAL and E2 increase cytoprotective heat shock protein 72 expression, which seems to be closely associated with the remission of the inflammatory reaction. In addition, we confirm the antiinflammatory effect of RAL in male rats, using a single administration of RAL or E2.

Insulin resistance (IR) has been identified as crucial pathophysiological factor in the development and progression of non-alcoholic fatty liver disease (NAFLD). Although mounting evidence suggests that perturbation of gut microflora exacerbates the severity of chronic liver diseases, therapeutic approaches using synbiotic has remained overlooked. Here, we show that a synbiotic composed by Lactobacillus paracasei B21060 plus arabinogalactan and fructo-oligosaccharides lessens NAFLD progression in a rat model of high fat feeding. IR and steatosis were induced by administration of high fat diet (HFD) for 6 weeks. Steatosis and hepatic inflammation, Toll-like receptor (TLR) pattern, glucose tolerance, insulin signaling and gut permeability were studied. Liver inflammatory markers were down-regulated in rats receiving the synbiotic, along with an increased expression of nuclear peroxisome proliferator-activated receptors and expression of downstream target genes. The synbiotic improved many aspects of IR, such as fasting response, hormonal homeostasis and glycemic control. Indeed it prevented the impairment of hepatic insulin signaling, reducing the phosphorylation of insulin receptor substrate-1 in Ser 307 and down-regulating suppressor of cytokine signaling 3. Gene expression analysis revealed that in the liver the synbiotic reduced cytokines synthesis and restored the HFD-dysregulated TLR 2, 4 and 9 mRNAs toward a physiological level of expression. The synbiotic preserved gut barrier integrity and reduced the relative amount of Gram–negative Enterobacteriales and Escherichia coli in colonic mucosa. Overall, our data indicate that the L. paracasei B21060 based synbiotic is effective in reducing the severity of liver injury and IR associated with high fat intake, suggesting its possible therapeutic/preventive clinical utilization.

Fulminant hepatic failure is defined by the presence of severe impairment of liver function, with or without encephalopathy, in patients with no underlying chronic liver disease. We report the case of a 4-month-old infant who developed fulminant hepatitis B infection and recovered concomitant with tenofovir therapy without liver transplantation.

We investigated the involvement of de novo neurosteroid synthesis in the mechanisms underlying the analgesic and antihyperalgesic effects of N-palmitoylethanolamine (PEA) in two models of acute and persistent pain, the formalin test and carrageenan-induced paw edema. The pivotal role of peroxisome proliferator-activated receptor (PPAR)-α in the antinocifensive effect of PEA was confirmed by the lack of this effect in PPAR-α-null mice. PEA antinociceptive activity was partially reduced when the animals were treated with aminoglutethimide or finasteride, implying that de novo neurosteroid synthesis is involved in the effect of PEA. Accordingly, in the spinal cord, the allopregnanolone (ALLO) levels were increased by PEA treatment both in formalin- and carrageenan-exposed mice, as revealed by gas chromatography-mass spectrometry. In agreement with those data, in both pain models, PEA administration in challenged mice specifically restored the expression of two proteins involved in neurosteroidogenensis, the steroidogenic acute regulatory protein (StAR) and cytochrome P450 side-chain cleavage (P450scc) in the ipsilateral horns of spinal cord, without affecting their expression in the contralateral side. These results provide new information about the involvement of de novo neurosteroid synthesis in the modulation of pain behavior by PEA.

Iron is essential for many biological processes and its deficiency or excess is involved in pathological conditions. At cellular level, the maintenance of iron homeostasis is largely accomplished by the transferrin receptor (TfR-1) and by ferritin, whose expression is mainly regulated post-transcriptionally by iron regulatory proteins (IRPs). This study examines the hypothesis that modification of serum estrogen levels by ovariectomy and 17beta-estradiol (E(2)) treatment in rats modulate serum iron-status parameters and iron metabolism in adipose tissue. In particular, we evaluated the RNA binding of IRP1 by electrophoretic mobility-shift assay and IRP1, ferritin, and TfR-1 expression in adipose tissue by Western blot analysis. Ovariectomy, besides a lowered serum iron and transferrin iron binding capacity, remarkably decreased the binding activity of IRP1 in peritoneal and subcutaneous adipose tissues, and these effects were reversed by E(2) treatment. Moreover, ovariectomy determined a decrease of IRP1 expression, which was significant in subcutaneous adipose tissue. Consistent with IRP1 regulation, an increase of ferritin and a decrease of TfR-1 expression were observed in peritoneal adipose tissue from ovariectomized animals, while the treatment with E(2) reconstituted TfR-1 level. A similar expression profile of TfR-1 was observed in subcutaneous adipose tissue, where ferritin level did not change in ovariectomized animals, and was increased after E(2) treatment. Our results indicate that estrogen level changes can regulate the binding activity of the IRP1, and consequently ferritin and TfR-1 expression in adipose tissue, suggesting a relationship among serum and tissue iron parameters, estrogen status and adiposity.

Only in the last few years has non-alcoholic steatohepatitis been recognized as an important and relatively common liver disease. To date, the therapeutic options are limited, vitamin E and metformin have been proposed for the treatment of this condition, although their mechanisms are not completely clarified as yet. The aim of this study was to investigate the anti-inflammatory and anti-oxidative mechanisms of these drugs in an experimental model of non-alcoholic steatohepatitis in the young rat. Male rats, just after weaning, were divided into four groups: a control group that received a standard diet; a high fat diet group; two high fat diet fed groups treated with vitamin E or metformin, respectively. After 4 weeks, we evaluated in the liver the modification of lipid peroxidation, assessed by malondialdehyde, TNF-alpha levels, S-nitrosylated protein, inducible nitric oxide sinthase (iNOS), and peroxisome proliferators-activated receptors (PPAR) expression and metalloproteinase activity. High fat diet increased malondialdehyde, nitrotyrosilated proteins, and TNF-alpha tissue content. Moreover, a decrease of PPAR-alpha and an increase of PPAR-gamma expression were observed. An increase of metalloproteinase activity was also shown. Among drug treatments, metformin reduced body weight gain and fat mass, metalloproteinase activity, and TNF-alpha tissue content, while it restored PPAR-alpha expression and downregulated PPAR-gamma expression. Vitamin E reduced the oxidative damage, protein nitrotyrosilation, and tissue TNF-alpha levels. Moreover a decrease of PPAR-gamma expression was also shown. These findings confirm the efficacy of both drugs as therapeutic tools in preventing the early onset of liver damage and non-alcoholic fatty liver disease progression.

BACKGROUND Mechanisms of normalization of blood pressure in spontaneously hypertensive rats (SHR) during pregnancy were investigated. We hypothesized that at the end of pregnancy (20th day), the modified renal renin-angiotensin system (RAS) plays a pivotal role in this effect associated with reduced inflammation and oxidative damage. METHODS We measured blood pressure and heart rate (HR) using a noninvasive tail-cuff method in conscious SHR and Wistar Kyoto rats (WKY). Nonpregnant (-NP) or pregnant (-P) SHR and WKY were used to compare the changes of angiotensin II (ANG II) type 1 (AT1) and type 2 (AT2) receptor expression in the kidney. Renal modification of proinflammatory enzyme expression, cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) and their transcription factor nuclear factor-kappaB (NF-kappaB) were also evaluated. Renal malonyldialdehyde (MDA) content and protein nitrotyrosylation, as indicators of oxidative stress, were assessed. Moreover monocyte chemotactic protein-1 (MCP-1) mRNA was determined. RESULTS Our findings indicate that the significant reduction of blood pressure induced by pregnancy in the SHR strain could be related to reduced AT1 and increased AT2 expression. We also saw a significant decline in renal NF-kappaB, COX-2, iNOS, and macrophage infiltration, as well as the fall in oxidative stress indicators. CONCLUSIONS The increased proinflammatory and oxidative variables, seen in SHR, are strongly ameliorated by pregnancy. In pregnant SHR animals, the adaptive and compensative changes of RAS and inflammation in the kidney seem to contribute to the reduction of blood pressure near term.

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.

and seeds (5.1%), fruits and vegetables (3.1%), fish (1.8%), peanuts (1.3%), crustaceans (0.7%), other specified foods in 6.2%. 31.3% of cases received a final diagnosis of food-induced anaphylaxis but the particular food was not indicated. Conclusions: Significant increase in admission for food-induced anaphylaxis occurred in Italian children between the period 2001–2005. Similar data have been recently reported in other Western countries. Our data suggest the importance of more research to investigate the causative factors and the necessity to improve the healthcare service for this condition.

Melatonin (N-acetyl-5-methoxytryptamine), an indole hormone, is the chief secretory product of the pineal gland and is an efficient free radical scavenger and antioxidant, both in vitro and in vivo. The role of melatonin as an immunomodulator is, in some cases, contradictory. Although melatonin is reported to influence a variety of inflammatory and immune responses, evidence supporting its effects on important glioma cells-derived mediators is incomplete. We studied, in rat glioma cell line (C6), the role of melatonin (100 microm-1 mm) in the regulation of the expression of nitric oxide synthase (NOS) caused by incubation with lipopolysaccharide (LPS)/interferon (IFN)-gamma (1 microg/mL and 100 U/mL, respectively) and defined the mode of melatonin's action. Treatment with LPS/IFN-gamma for 24 hr elicited the induction of inducible (iNOS) activity as determined by nitrite and nitrate (NO(x)) accumulation in the culture medium. Preincubation with melatonin abrogated the mixed cytokines-mediated induction of iNOS. The effect of melatonin was concentration-dependent. Moreover, Western blot analysis showed that melatonin inhibited LPS/IFN-gamma-induced expression of COX-2 protein, but not that of constitutive cyclooxygenase. Inhibition of iNOS and COX-2 expression was associated with inhibition of activation of the transcription factor nuclear factor kappa B (NF-kappaB). The ability of melatonin to inhibit NF-kappaB activation was further confirmed by studies on the degradation of the inhibitor of NF-kappaB, IkappaB-alpha. Increased production of lipid peroxidation products using thiobarbituric acid assay were found in cellular contents from activated cultures. Lipid peroxidation was decreased by melatonin treatment in a concentration-dependent manner. Moreover, several genes having roles in heat-shock response were downregulated in melatonin-treated cells, such as 70 proteins, reflecting the reduced oxidative stress in these cells. The mechanisms underlying in vitro the neuroprotective properties of melatonin involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation.