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Translocator protein 18kDa (TSPO) is predominantly located in the mitochondrial outer membrane, playing an important role in steroidogenesis, inflammation, cell survival and proliferation. Its expression in central nervous system, mainly in glial cells, has been found to be upregulated in neuropathology, and brain injury. In this study, we investigated the anti-oxidative and anti-inflammatory effects of a group of TSPO ligands from the N,N-dialkyl-2-phenylindol-3-ylglyoxylamide class (PIGAs), highlighting the involvement of neurosteroids in their pharmacological effects. To this aim we used a well-known in vitro model of neurosteroidogenesis: the astrocytic C6 glioma cell line, where TSPO expression and localization, as well as cell response to TSPO ligand treatment, have been established. All PIGAs reduced l-buthionine-(S,R)-sulfoximine (BSO)-driven cell cytotoxicity and lipid peroxidation. Moreover, an anti-inflammatory effect was observed due to the reduction of inducible nitric oxide synthase and cyclooxygenase-2 induction in LPS/IFNγ challenged cells. Both effects were blunted by aminoglutethimide (AMG), an inhibitor of pregnenolone synthesis, suggesting neurosteroids' involvement in PIGA protective mechanism. Finally, pregnenolone evaluation in PIGA exposed cells revealed an increase in its synthesis, which was prevented by AMG pre-treatment. These findings indicate that these TSPO ligands reduce oxidative stress and pro-inflammatory enzymes in glial cells through the de novo synthesis of neurosteroids, suggesting that these compounds could be potential new therapeutic tools for the treatment of inflammatory-based neuropathologies with beneficial effects possibly comparable to steroids, but potentially avoiding the negative side effects of long-term therapies with steroid hormones.

The potential mechanisms of action of polyphenols in nonalcoholic fatty liver disease (NAFLD) are overlooked. Here, we evaluate the beneficial therapeutic effects of hydroxytyrosol (HT), the major metabolite of the oleuropein, in a nutritional model of insulin resistance (IR) and NAFLD by high-fat diet. Young male rats were divided into three groups receiving (1) standard diet (STD; 10.5% fat), (2) high-fat diet (HFD; 58.0% fat) and (3) HFD+HT (10 mg/kg/day by gavage). After 5 weeks, the oral glucose tolerance test was performed, and at 6th week, blood sample and tissues (liver and duodenum) were collected for following determinations. The HT-treated rats showed a marked reduction in serum AST, ALT and cholesterol and improved glucose tolerance and insulin sensitivity, reducing homeostasis model assessment index. HT significantly corrected the metabolic impairment induced by HFD, increasing hepatic peroxisome proliferator activated receptor PPAR-α and its downstream-regulated gene fibroblast growth factor 21, the phosphorylation of acetyl-CoA carboxylase and the mRNA carnitine palmitoyltransferase 1a. HT also reduced liver inflammation and nitrosative/oxidative stress decreasing the nitrosylation of proteins, reactive oxygen species production and lipid peroxidation. Moreover, HT restored intestinal barrier integrity and functions (fluorescein isothiocyanate-dextran permeability and mRNA zona occludens ZO-1). Our data demonstrate the beneficial effect of HT in the prevention of early inflammatory events responsible for the onset of IR and steatosis, reducing hepatic inflammation and nitrosative/oxidative stress and restoring glucose homeostasis and intestinal barrier integrity.

Background/Aims: Osteoarthritis (OA) is a joint degenerative biomechanical disorder involving immunity, metabolic alterations, inflammation, and cartilage degradation, where chondrocytes play a pivotal role. OA has not effective pharmacological treatments and new therapeutic targets are needed. Adipokines contribute to the low-grade systemic inflammation in OA. Here, we explored novel molecular mechanisms of sodium butyrate (BuNa) in modulating inflammation and chemotaxis in chondrocytes, demonstrating the direct involvement of its G protein-coupled receptor (GPR)-43. Methods: ATDC5 murine chondrocytes were stimulated with interleukin (IL)-1β, in the presence or not of BuNa, for 24 h. RT-PCR and Western blot analysis was performed to evaluate the expression of inflammatory mediators and structural proteins. Results: Butyrate reduced the expression of canonic pro-inflammatory mediators (Nos2, COX-2, IL-6), pro-inflammatory adipokines (lipocalin-2 and nesfatin-1) and adhesion molecule (VCAM-1 and ICAM-1) in IL-1β-stimulated chondrocytes, inhibiting several inflammatory signalling pathways (NFκB, MAPKinase, AMPK-α, PI3K/Akt). Butyrate also reduced metalloproteinase production and limited the loss of type II collagen in IL-1β-inflamed chondrocytes. The chemoattractant effect of butyrate, after different inflammatory challenges, was revealed by increased annexin (AnxA)1 levels and chemokines expression. The chemoattractant and anti-inflammatory activities of butyrate were completely blunted by GPR43 silencing using RNA interference. Conclusion: Taken together, our data suggest the potential application of sodium butyrate as a novel candidate in a multi-target approach for the treatment of chondrocyte inflammation and cartilage degenerative process.

Autism spectrum disorders (ASD) are a group of heterogeneous neurodevelopmental conditions characterized by impaired social interaction, and repetitive stereotyped behaviours. Interestingly, functional and inflammatory gastrointestinal diseases are often reported as a comorbidity in ASDs, indicating gut-brain axis as a novel emerging approach. Recently, a central role for peroxisome-proliferator activated receptor (PPAR)-α has been addressed in neurological functions, associated with the behaviour. Among endogenous lipids, palmitoylethanolamide (PEA), a PPAR-α agonist, has been extensively studied for its anti-inflammatory effects both at central and peripheral level. Based on this background, the aim of this study was to investigate the pharmacological effects of PEA on autistic-like behaviour of BTBR T+tf/J mice and to shed light on the contributing mechanisms. Our results showed that PEA reverted the altered behavioural phenotype of BTBR mice, and this effect was contingent to PPAR-α activation. Moreover, PEA was able to restore hippocampal BDNF signalling pathway, and improve mitochondrial dysfunction, both pathological aspects, known to be consistently associated with ASDs. Furthermore, PEA reduced the overall inflammatory state of BTBR mice, reducing the expression of pro-inflammatory cytokines at hippocampal, serum, and colonic level. The analysis of gut permeability and the expression of colonic tight junctions showed a reduction of leaky gut in PEA-treated BTBR mice. This finding together with PEA effect on gut microbiota composition suggests an involvement of microbiota-gut-brain axis. In conclusion, our results demonstrated a therapeutic potential of PEA in limiting ASD symptoms, through its pleiotropic mechanism of action, supporting neuroprotection, anti-inflammatory effects, and the modulation of gut-brain axis.

Non-dioxin-like (NDL) polychlorinated biphenyls (PCBs) are persistent organic pollutants, associated with a range of adverse health effects, including interference with the immune system. In this study, we investigate the capability of NDL-PCBs 101, 153, and 180, 3 of the 6 NDL-PCBs defined as indicators, to impair the immune response in lipopolysaccharide (LPS)-activated J774A.1 and primary murine macrophages. Our results clearly demonstrate that the exposure of J774A.1 and primary macrophages to NDL-PCB 153 or 180 or all NDL-PCBs mixtures causes a significant reduction in LPS-induced cytokine/chemokine synthesis, such as tumor necrosis factor-α and interleukin-6, together with monocyte chemoattractant protein-1, involved in cell recruitment. Moreover, PCBs were found to suppress LPS-stimulated NO production, and to reduce cyclooxygenase-2 and inducible nitric oxide synthase expression in J774A.1 and primary macrophages. At mechanistic level, PCBs significantly counteract the LPS-driven toll-like receptor (TLR) 4 and CD14 upregulation, therefore inhibiting downstream nuclear factor-κB (NF-κB) activation in J774A.1. Furthermore, PCBs determine a significant loss of macrophage endocytic capacity, a prerequisite for efficient antigen presentation. Taken together, these data indicate that NDL-PCBs reduce macrophage responsiveness, particularly when they are combined at concentrations per se inactive, impairing the capability to orchestrate a proper immune response to an infectious stimulus, disrupting TLR4/NF-κB pathway.

Alterations of microbiota-gut-brain axis have been invoked in the pathogenesis of autism spectrum disorders (ASD). Mouse models could represent an excellent tool to understand how gut dysbiosis and related alterations may contribute to autistic phenotype. In this study we paralleled gut microbiota (GM) profiles, behavioral characteristics, intestinal integrity and immunological features of colon tissues in BTBR T + tf/J (BTBR) inbred mice, a well established animal model of ASD. Sex differences, up to date poorly investigated in animal models, were specifically addressed. Results showed that BTBR mice of both sexes presented a marked intestinal dysbiosis, alterations of behavior, gut permeability and immunological state with respect to prosocial C57BL/6j (C57) strain. Noticeably, sex-related differences were clearly detected. We identified Bacteroides, Parabacteroides, Sutterella, Dehalobacterium and Oscillospira genera as key drivers of sex-specific gut microbiota profiles associated with selected pathological traits. Taken together, our findings indicate that alteration of GM in BTBR mice shows relevant sex-associated differences and supports the use of BTBR mouse model to dissect autism associated microbiota-gut-brain axis alteration.

Hypertension is an important risk factor for kidney failure and renal events in the general population. Palmitoylethanolamide (PEA) is a member of the fatty acid ethanolamine family with profound analgesic and anti-inflammatory effects, resulting from its ability to activate peroxisome proliferator activated receptor (PPAR)α. A role for this nuclear receptor has been addressed in cardiovascular system and PPARα ligands have been shown to protect against inflammatory damage especially resulting from angiotensin II hypertension. In this study, we demonstrated that PEA significantly reduced blood pressure in spontaneously hypertensive rats (SHR) and limited kidney damage secondary to high perfusion pressure. To investigate the mechanisms involved in PEA effect, we found that PEA reduced cytochrome P450 (CYP) hydroxylase CYP4A, epoxygenase CYP2C23 and soluble epoxide hydrolase enzyme expression in the kidney, accompanied by a reduction of 20-hydroxyeicosatetraenoic acid excretion in the urine. Moreover, it markedly reduced kidney oxidative and nitrosative stress accompanied by decreased expression of renal NAD(P)H oxidase and inducible nitric oxide synthase and increased expression of Cu/Zn superoxide dismutase, in the kidney of SHR. Moreover, angiotensin II receptor (AT) evaluation revealed a decrease in AT1 receptor expression and a restoration of AT2 receptor level in the kidney from PEA-treated SHR. Consistently, angiotensin converting enzyme expression was reduced, implying a decrease in angiotensin II synthesis. These results indicate that PEA treatment lowers blood pressure and can protect against hypertensive renal injury by increasing the antioxidant defense and anti-inflammatory response and modulating renin-angiotensin system.

The human gut is a composite anaerobic environment with a large, diverse and dynamic enteric microbiota, represented by more than 100 trillion microorganisms, including at least 1000 distinct species. The discovery that a different microbial composition can influence behavior and cognition, and in turn the nervous system can indirectly influence enteric microbiota composition, has significantly contributed to establish the well-accepted concept of gut-brain axis. This hypothesis is supported by several evidence showing mutual mechanisms, which involve the vague nerve, the immune system, the hypothalamic-pituitaryadrenal (HPA) axis modulation and the bacteria-derived metabolites. Many studies have focused on delineating a role for this axis in health and disease, ranging from stress-related disorders such as depression, anxiety and irritable bowel syndrome (IBS) to neurodevelopmental disorders, such as autism, and to neurodegenerative diseases, such as Parkinson Disease, Alzheimer’s Disease etc. Based on this background, and considering the relevance of alteration of the symbiotic state between host and microbiota, this review focuses on the role and the involvement of bioactive lipids, such as the N-acylethanolamine (NAE) family whose main members are N-arachidonoylethanolamine (AEA), palmitoylethanolamide (PEA) and oleoilethanolamide (OEA), and short chain fatty acids (SCFAs), such as butyrate, belonging to a large group of bioactive lipids able to modulate peripheral and central pathologic processes. Their effective role has been studied in inflammation, acute and chronic pain, obesity and central nervous system diseases. A possible correlation has been shown between these lipids and gut microbiota through different mechanisms. Indeed, systemic administration of specific bacteria can reduce abdominal pain through the involvement of cannabinoid receptor 1 in the rat; on the other hand, PEA reduces inflammation markers in a murine model of inflammatory bowel disease (IBD), and butyrate, producted by gut microbiota, is effective in reducing inflammation and pain in irritable bowel syndrome and IBD animal models. In this review, we underline the relationship among inflammation, pain, microbiota and the different lipids, focusing on a possible involvement of NAEs and SCFAs in the gut-brain axis and their role in the central nervous system diseases.

Fatty liver, oxidative stress, and mitochondrial dysfunction are key pathophysiological features of insulin resistance and obesity. Butyrate, produced by fermentation in the large intestine by gut microbiota, and its synthetic derivative, the N-(1-carbamoyl-2-phenyl-ethyl) butyramide, FBA, have been demonstrated to be protective against insulin resistance and fatty liver. Here, hepatic mitochondria were identified as the main target of the beneficial effect of both butyrate-based compounds in reverting insulin resistance and fat accumulation in diet-induced obese mice. In particular, butyrate and FBA improved respiratory capacity and fatty acid oxidation, activated the AMPK–acetyl-CoA carboxylase pathway, and promoted inefficient metabolism, as shown by the increase in proton leak. Both treatments consistently increased utilization of substrates, especially fatty acids, leading to the reduction of intracellular lipid accumulation and oxidative stress. Finally, the shift of the mitochondrial dynamic toward fusion by butyrate and FBA resulted in the improvement not only of mitochondrial cell energy metabolism but also of glucose homeostasis. In conclusion, butyrate and its more palatable synthetic derivative, FBA, modulating mitochondrial function, efficiency, and dynamics, can be considered a new therapeutic strategy to counteract obesity and insulin resistance.

Several pathogenetic factors have been involved in the onset and progression of Parkinson's disease (PD), including inflammation, oxidative stress, unfolded protein accumulation, and apoptosis. Palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, has been shown to be a neuroprotective and anti-inflammatory molecule, acting as a peroxisome proliferator activated receptor (PPAR)-α agonist. In this study we investigated the effects of PEA on behavioral alterations and the underlying pathogenic mechanisms in the 6-hydroxydopamine (6-OHDA)-induced model of PD in male mice. Additionally, we showed the involvement of PPAR-α in PEA protective effect on SH-SY5Y neuroblastoma against 6-OHDA damage. Here, we report that PEA (3-30mg/kg/days.c.) improved behavioral impairments induced by unilateral intrastriatal injection of 6-OHDA. This effect was accompanied by a significant increase in tyrosine hydroxylase expression at striatal level, indicating PEA preserving effect on dopaminergic neurons. Moreover, we found a reduction in the expression of pro-inflammatory enzymes, i.e. inducible nitric oxide synthase and cyclooxygenase-2, a modulation between pro- and anti-apoptotic markers, suggestive of PEA capability in controlling neuroinflammation and cell death. Interestingly, PEA also showed protective scavenging effect, through superoxide dismutase induction, and dampened unfolding protein response, interfering on glucose-regulated protein 78 expression and PERK-eIF2α pathway. Similar data were found in in vitro studies, where PEA treatment was found to rescue SH-SY5Y neuroblastoma cells from 6-OHDA-induced damage and death, partly by inhibiting endoplasmic reticulum stress detrimental response. Therefore, PEA, counteracting the pathogenetic aspects involved in the development of PD, showed its therapeutic potential, possibly integrating current treatments correcting dopaminergic deficits and motor dysfunction.

OBJECTIVES:Omega (ω)-3 polyunsaturated fatty acids (PUFA) are dietary compounds able to attenuate insulin resistance. Anyway, the precise actions of ω-3PUFAs in skeletal muscle are overlooked. We hypothesized that PUFAs, modulating mitochondrial function and efficiency, would ameliorate pro-inflammatory and pro-oxidant signs of nutritionally induced obesity. STUDY DESIGN:To this aim, rats were fed a control diet (CD) or isocaloric high fat diets containing either ω-3 PUFA (FD) or lard (LD) for 6 weeks. RESULTS:FD rats showed lower weight, lipid gain and energy efficiency compared to LD-fed animals, showing higher energy expenditure and O2 consumption/CO2 production. Serum lipid profile and pro-inflammatory parameters in FD-fed animals were reduced compared to LD. Accordingly, FD rats exhibited a higher glucose tolerance revealed by an improved glucose and insulin tolerance tests compared to LD, accompanied by a restoration of insulin signalling in skeletal muscle. PUFAs increased lipid oxidation and reduced energy efficiency in subsarcolemmal mitochondria, and increase AMPK activation, reducing both endoplasmic reticulum and oxidative stress. Increased mitochondrial respiration was related to an increased mitochondriogenesis in FD skeletal muscle, as shown by the increase in PGC1-α and -β. CONCLUSIONS:our data strengthened the association of high dietary ω3-PUFA intake with reduced mitochondrial energy efficiency in the skeletal muscle.

Ghrelin is a gastric peptide, discovered by Kojima et al. (1999) [55] as a result of the search for an endogenous ligand interacting with the “orphan receptor” GHS-R1a (growth hormone secretagogue receptor type 1a). Ghrelin is composed of 28 aminoacids and is produced mostly by specific cells of the stomach, by the hypothalamus and hypophysis, even if its presence, as well as that of its receptors, has been demonstrated in many other tissues, not least in gonads. Ghrelin potently stimulates GH release and participates in the regulation of energy homeostasis, increasing food intake, decreasing energy output and exerting a lipogenetic effect. Furthermore, ghrelin influences the secretion and motility of the gastrointestinal tract, especially of the stomach, and, above all, profoundly affects pancreatic functions. Despite of these previously envisaged activities, it has recently been hypothesized that ghrelin regulates several aspects of reproductive physiology and pathology. In conclusion, ghrelin not only cooperates with other neuroendocrine factors, such as leptin, in the modulation of energy homeostasis, but also has a crucial role in the regulation of the hypothalamic–pituitary gonadal axis. In the current review we summarize the main targets of this gastric peptide, especially focusing on the reproductive system.

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.

Ghrelin, an acylated 28 amino acid gastric peptide, was isolated from the stomach as an endogenous ligand for growth hormone (GH) secretagogue receptor in 1999. Circulating ghrelin is mainly produced by specific cells in the stomach's oxyntic glands. Ghrelin potently stimulates GH release and food intake and exhibits diverse effects, including ones on glucose metabolism and on secretion and motility of the gastrointestinal tract. Besides these effects on food intake and energy homeostasis, ghrelin is also involved in controlling reproductive functions, and a role for it as a novel regulator of the hypothalamic-pituitary gonadal axis is clearly emerging. We review recent ghrelin research with emphasis on its roles in the reproductive axis.

Leptin regulates glucose, lipid and energy homeostasis as well as feeding behavior, serving as a bridge between peripheral metabolically active tissues and the central nervous system (CNS). Indeed, this adipocyte-derived hormone, whose circulating levels mirror fat mass, not only exerts its anti-obesity effects mainly modulating the activity of specific hypothalamic neurons expressing the long form of the leptin receptor (Ob-Rb), but it also shows pleiotropic functions due to the activation of Ob-Rb in peripheral tissues. Nevertheless, several mechanisms have been suggested to mediate leptin resistance, including obesity-associated hyperleptinemia, impairment of leptin access to CNS and the reduction in Ob-Rb signal transduction effectiveness, among others. During the onset and progression of obesity, the dampening of leptin sensitivity often occurs, preventing the efficacy of leptin replacement therapy from overcoming obesity and/or its comorbidities. This review focuses on obesity-associated leptin resistance and the mechanisms underpinning this condition, to highlight the relevance of leptin sensitivity restoration as a useful therapeutic strategy to treat common obesity and its complications. Interestingly, although promising strategies to counteract leptin resistance have been proposed, these pharmacological approaches have shown limited efficacy or even relevant adverse effects in preclinical and clinical studies. Therefore, the numerous findings from this review clearly indicate a lack of a single and efficacious treatment for leptin resistance, highlighting the necessity to find new therapeutic tools to improve leptin sensitivity, especially in patients with most severe disease profiles.

Palmitoylethanolamide (PEA), a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR). Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF), and renin angiotensin system (RAS) modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day) for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the activation of angiotensin receptor 1 underlying pathways in mesenteric beds was shown in basal conditions in PEA-treated SHR. In conclusion, our data demonstrate the involvement of epoxyeicosatrienoic acids and renin angiotensin system in the blood pressure lowering effect of PEA.

Background: Although gut microbiota perturbation is recognized as a main contributing factor to the pathogenesis of inflammatory bowel disease, synbiotic therapies, as prevention or treatment, have remained overlooked. Objective: To verify whether Lactobacillus paracasei B21060‐based synbiotic therapy could prevent or repair colon damage in a mouse model of colitis, we performed treatments before and after colitis induction. Methods: The experimental study lasted 19 d. Experimental colitis was induced in BALB/c mice by dextran sodium sulfate (DSS, 2.5%) in drinking water (days 7‐12) followed by DSS-free water (days 13‐19) (DSS group). L. paracasei B21060 (2.5 3 10 7 bacteria/10g body weight) was orally administered 7db efore DSS synbiotic as preventive treatment (P-SYN) group] or 2 d after DSS [synbiotic as therapeutic treatment (T-SYN) group] until day 19. Another group was not treated with DSS or synbiotic and was given tap water (control group), for a total of 4 groups. Results: Compared with the DSS group, both synbiotic-treated groups had significantly less pronounced weight loss and colon damage. Consistently, mRNA levels of chemokine (C‐C motif) ligand 5 in the colon were reduced in both P-SYN and T-SYN mice compared with the DSS group (51%, P < 0.05 and 72%, P < 0.001, respectively). In the P-SYN and T-SYN groups, neutrophil elastase transcription was also reduced (51%, P < 0.01 and 59%, P < 0.001, respectively). Accordingly, oxidative/nitrosative stress was lower in P-SYN and T-SYN mice than inthe DSS group. In P-SYN and T-SYN mice, colonic gene expression of tumor necrosis factor (47%, P< 0.01 and 61%, P< 0.001, respectively) and prostaglandin-endoperoxide synthase 2 (45%, P < 0.01 and 35%, P < 0.05, respectively) was lower, whereas interleukin 10 mRNA was doubled compared with the DSS group (both P < 0.5). Remarkably, epithelial barrier integrity (zonulin and occludin) and gut protection (b-defensin and mucin expression) were completely restored in P-SYN and T-SYN mice. Conclusions: Our data highlight the beneficial effects of this synbiotic formulation in acutely colitic mice, suggesting that it may have therapeutic and possibly preventive efficacy in human colitis. J Nutr doi: 10.3945/jn.114.205989.

Environmental pollutants are known to have adverse effects on human health. However, the link between chemical exposure and osteoarthritis remains little investigated. This study sought to assess in vitro the effect of several non-dioxin-like polychlorinated biphenyls (NDL-PCBs) on chondrocytes viability and apoptosis induction. Murine chondrogenic ATDC-5 cell line and human T/C-28a2 immortalized chondrocytes were exposed to NDL-PCBs 101, 153 and 180. Cell viability was examined using MTT assay. Necrosis was evaluated by LDH assay. Expression of apoptotic related proteins, such as caspase-3, Bcl-2 and Bax was assessed by Western blot analysis. Finally, oxidative stress was evaluated by malondialdehyde (MDA) assay and the Oxidative Stress Index. In vitro exposure to NDL-PCBs caused strong reduction of cell viability in a concentration-dependent manner. Data from LDH assay showed cellular necrosis induction. Caspase-3 activation, as well as, altered Bcl2/Bax ratio and p38 MAP-kinase phosphorylation also suggested apoptosis induction. Finally, MDA levels and Oxidative Stress Index revealed that PCBs drive chondrocyte death via increase of oxidative stress. The viability of murine and human chondrocytes was reduced in presence of PCBs. The activity of PCBs on cell viability is likely to be mediated by complex alterations involving regulation mechanisms of apoptosis, necrosis and oxidative stress.

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.

During food deprivation, plasma T(4) and T(3) levels are decreased. Under this metabolic condition, hypothalamic deiodinase type 2 (D2) activity and mRNA levels are elevated, whereas TRH mRNA levels are suppressed. Systemic T(4) administration does not reverse these hypothalamic changes. The mechanism(s) that underlies this paradoxical regulation of D2 during fasting is unknown. We hypothesize that leptin and/or glucocorticoids play a role in these mechanisms, and their interactions may be an important regulator of the hypothalamic-pituitary-thyroid axis. Thus, we assessed the effects of these hormones on D2 activity levels of food-deprived as well as fed animals using enzyme activity measurements. In food-deprived animals, corticosterone replacement reversed the inhibitory effect of adrenalectomy (ADX) on D2 induction, whereas ADX and ADX plus corticosterone replacement did not significantly affect D2 activity levels in rats fed ad libitum. Leptin administration to fed animals did not change D2 activity, whereas in fasted rats, leptin decreased D2 activity by reducing corticosterone plasma levels. When leptin was administered to fasted animals that were either ADX or ADX plus corticosterone treated at a high dose, D2 activity did not increase. Our results show that during fasting, diminishing leptin levels play a permissive role to enable glucocorticoid-induced up-regulation of D2. Thus, our observations suggest that appropriate induction of D2 activity during negative energy balance is dependent upon both leptin and glucocorticoid signaling.

In the central nervous system, the active form of thyroid hormone, T3, derives from the cellular uptake and intracellular 5'-monodeiodination of T4 by type II 5'-monodeiodinase (DII). Here, we report that using an antiserum raised against the C-terminus of the full-length SeDII, immunolabeled cells were found in the rat hypothalamus in agreement with the DII mRNA distribution. Light and electron microscopy shows that DII is localized in astrocytes and tanycytes, supporting the hypothesis that these cells play an important role in the mediation peripheral signals, such as thyroid hormones, on hypothalamic functions.

Non-alcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and children. It is characterized by an aberrant lipid storage in hepatocytes, named hepatic steatosis. Simple steatosis remains a benign process in most affected patients, while some of them develop superimposed necroinflammatory activity with a non-specific inflammatory infiltrate and a progression to non-alcoholic steatohepatitis with or without fibrosis. Deep similarity and interconnections between innate immune cells and those of liver parenchyma have been highlighted and showed to play a key role in the development of chronic liver disease. The liver can be considered as an "immune organ" because it hosts non-lymphoid cells, such as macrophage Kupffer cells, stellate and dendritic cells, and lymphoid cells. Many of these cells are components of the classic innate immune system, enabling the liver to play a major role in response to pathogens. Although the liver provides a "tolerogenic" environment, aberrant activation of innate immune signaling may trigger "harmful" inflammation that contributes to tissue injury, fibrosis, and carcinogenesis. Pathogen recognition receptors, such as toll-like receptors and nucleotide oligomerization domain-like receptors, are responsible for the recognition of immunogenic signals, and represent the major conduit for sensing hepatic and non-hepatic noxious stimuli. A pivotal role in liver inflammation is also played by cytokines, which can initiate or have a part in immune response, triggering hepatic intracellular signaling pathways. The sum of inflammatory signals and deranged substrate handling induce most of the metabolic alteration traits: insulin resistance, obesity, diabetes, hyperlipidemia, and their compounded combined effects. In this review, we discuss the relevant role of innate immune cell activation in relation to NAFLD, the metabolic complications associated to this pathology, and the possible pharmacological tools.

Non-dioxin-like polychlorinated biphenyls (NDL-PCBs) are highly lipophilic environmental contaminants that accumulate in lipid-rich tissues, such as adipose tissue. Here, we reported the effects induced by PCBs 101, 153 and 180, three of the six NDL-PCBs defined as indicators, on mature 3T3-L1 adipocytes. We observed an increase in lipid content, in leptin gene expression and a reduction of leptin receptor expression and signaling, when cells were exposed to PCBs, alone or in combination. These modifications were consistent with the occurrence of "leptin-resistance" in adipose tissue, a typical metabolic alteration related to obesity. Therefore, we investigated how PCBs affect the expression of pivotal proteins involved in the signaling of leptin receptor. We evaluated the PCB effect on the intracellular pathway JAK/STAT, determining the phosphorylation of STAT3, a downstream activator of the transcription of leptin gene targets, and the expression of SOCS3 and PTP1B, two important regulators of leptin resistance. In particular, PCBs 153 and 180 or all PCB combinations induced a significant reduction in pSTAT3/STAT3 ratio and an increase in PTP1B and SOCS3, evidencing an additive effect. The impairment of leptin signaling was associated with the reduction of AMPK/ACC pathway activation, leading to the increase in lipid content. These pollutants were also able to increase the transcription of inflammatory cytokines (IL-6 and TNFα). It is worthy to note that the PCB concentrations used are comparable to levels detectable in human adipose tissue. Our data strongly support the hypothesis that NDL-PCBs may interfere with the lipid metabolism contributing to the development of obesity and related diseases.

The existence of acylethanolamides (AEs) in the mammalian brain has been known for decades. Among AEs, palmitoylethanolamide (PEA) is abundant in the central nervous system (CNS) and conspicuously produced by neurons and glial cells. Antihyperalgesic and neuroprotective properties of PEA have been mainly related to the reduction of neuronal firing and to control of inflammation. Growing evidence suggest that PEA may be neuroprotective during CNS neurodegenerative diseases. Advances in the understanding of the physiology and pharmacology of PEA have potentiated its interest as useful biological tool for disease management. Several rapid non-genomic and delayed genomic mechanisms of action have been identified for PEA as peroxisome proliferator-activated receptor (PPAR)-α dependent. First, an early molecular control, through Ca(+2)-activated intermediate- and/or big-conductance K(+) channels opening, drives to rapid neuronal hyperpolarization. This is reinforced by the increase of the inward Cl(-) currents due to the modulation of the gamma aminobutyric acid A receptor and by the desensitization of the transient receptor potential channel type V1. Moreover, the gene transcription-mediated mechanism sustains the long-term anti-inflammatory effects, by reducing pro-inflammatory enzyme expression and increasing neurosteroid synthesis. Overall, the integration of these different modes of action allows PEA to exert an immediate and prolonged efficacious control in neuron signaling either on inflammatory process or neuronal excitability, maintaining cellular homeostasis. In this review, we will discuss the effect of PEA on metabolism, behavior, inflammation and pain perception, related to the control of central functions and the emerging evidence demonstrating its therapeutic efficacy in several neurodegenerative diseases.

It has been suggested a role of fatty acid ethanolamides in control of feeding behavior. Among these, palmitoylethanolamide (PEA) has not been directly implicated in appetite regulation and weight gain. The aim of this study was to investigate the effect of PEA on food intake and body weight and the interaction between PEA and hypothalamic leptin signaling in ovariectomized rats. Ovariectomy produced hyperphagia and increased weight gain, making it an useful model of mild obesity. Ovariectomized rats were treated with PEA (30 mg/kg sc) for 5 weeks. Then, blood was collected, and hypothalamus and adipose tissue were removed for histological, cellular, and molecular measurements. We showed that PEA caused a reduction of food intake, body weight, and fat mass. The mechanisms underlying PEA effects involved an improvement in hypothalamic leptin signaling, through a raise in signal transducer and activator of transcription 3 phosphorylation. We also reported that PEA reduced AMP-activated protein kinase-α phosphorylation and modulated transcription of anorectic and orexigenic neuropeptides in the hypothalamus. Moreover, PEA increased AMP-activated protein kinase-α phosphorylation and carnitine palmitoyltransferase 1 transcription in adipose tissue, suggesting an increase in ATP-producing catabolic pathway. PEA also polarized adipose tissue macrophages to M2 lean phenotype, associated to a reduction of inflammatory cytokines/adipokines. To demonstrate the direct effect of PEA on leptin sensitivity without interference of adiposity loss, we obtained consistent data in PEA-treated sham-operated animals and in vitro in SH-SY5Y neuroblastoma cell line. Therefore, our data provide a rationale for the therapeutic use of PEA in obese postmenopausal woman.

Several polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) investigated in soft tissues of the frequently monitored Mytilus galloprovincialis were compared to those of Ensis siliqua, a highly dispersed and economically important bivalve species, though rarely investigated. Overall PCBs had higher concentrations than OCPs in both species with a prevalence of tri- tetra-and penta-chlorinated biphenyls in E. siliqua and a prevalence of hexa- hepta and octa-chlorinated biphenyls in M. galloprovincialis. E. siliqua emerges as a suitable complement to mussels for monitoring PCBs and OCPs pollution.

cope Virgin olive oil is an essential component of the Mediterranean diet. Its antioxidant and anti-inflammatory properties are mainly linked to phenolic contents. This study aims to evaluate the beneficial effects of a polyphenol-rich virgin olive oil (HPCOO) or olive oil without polyphenols (WPOO) in rats fed high-fat diet (HFD). Methods and results Male Sprague-Dawley rats were divided into four groups based on the different types of diet: (I) standard diet (STD); (II) HFD; (III) HFD containing WPOO, and (IV) HFD containing HPCOO. HPCOO and WPOO induced a significant improvement of HFD-induced impaired glucose homeostasis (by hyperglycemia, altered oral glucose tolerance, and HOMA-IR) and inflammatory status modulating pro- and anti-inflammatory cytokines (TNF-α, IL-1, and IL-10) and adipokines. Moreover, HPCOO and less extensively WPOO, limited HFD-induced liver oxidative and nitrosative stress and increased hepatic fatty acid oxidation. To study mitochondrial performance, oxidative capacity and energy efficiency were also evaluated in isolated liver mitochondria. HPCOO, but not WPOO, reduced H2O2 release and aconitase activity by decreasing degree of coupling, which plays a major role in the control of mitochondrial reactive oxygen species emission. Conclusion HPCOO limits HFD-induced insulin resistance, inflammation, and hepatic oxidative stress, preventing nonalcoholic fatty liver disease progression.

Fumonisin B-1 (FB1) is a water-soluble fungal metabolite that elicits a wide spectrum of toxicological effects. Cellular targets of FB1 include immune cells and in particular macrophages. In the present study the cytotoxic effect of FB1 (1-100 muM) was evaluated using a murine macrophage cell line (J774A.1) as model system. The effect of FB1 on nitric oxide (NO) and prostaglandin E-2 (PGE(2)) production induced by lipopolysaccharide (LPS, 10 and 100 ng/ml) was also investigated. Macrophages were pretreated with FB1 for 72 h and then stimulated with LPS for 24 h. The increase of LPS-induced production of these inflammatory mediators was observed at increasing concentrations of FB1 (0.1-10 10 muM) and was found to be concentration dependent. By western blot analysis we demonstrated that the observed increase of NO and PGE(2) production by FB1 was related to an enhancement of iNOS and COX-2 expression.

This paper is dedicated to the memory of our wonderful colleague Professor Alfredo Colonna, who passed away the same day of its acceptance. Fatty liver accumulation, inflammatory process and insulin resistance appear to be crucial in non-alcoholic fatty liver disease (NAFLD), nevertheless emerging findings pointed an important role also for iron overload. Here, we investigate the molecular mechanisms of hepatic iron metabolism in the onset of steatosis to understand whether its impairment could be an early event of liver inflammatory injury. Rats were fed with control diet or high fat diet (HFD) for 5 or 8 weeks, after which liver morphology, serum lipid profile, transaminases levels and hepatic iron content (HIC), were evaluated. In liver of HFD fed animals an increased time-dependent activity of iron regulatory protein 1 (IRP1) was evidenced, associated with the increase in transferrin receptor-1 (TfR1) expression and ferritin down-regulation. Moreover, ferroportin (FPN-1), the main protein involved in iron export, was down-regulated accordingly with hepcidin increase. These findings were indicative of an increased iron content into hepatocytes, which leads to an increase of harmful free-iron also related to the reduction of hepatic ferritin content. The progressive inflammatory damage was evidenced by the increase of hepatic TNF-α, IL-6 and leptin, in parallel to increased iron content and oxidative stress. The major finding that emerged of this study is the impairment of iron homeostasis in the ongoing and sustaining of liver steatosis, suggesting a strong link between iron metabolism unbalance, inflammatory damage and progression of disease.

Chemical investigation of two species of marine ascidians, Aplidium elegans and Ciona edwardsii, collected in Mediterranean area, led to isolation of a series of alkyl sulfates (compounds 1–5) including three new molecules 1–3. Structures of the new metabolites have been elucidated by spectroscopic analysis. Based on previously reported cytotoxic activity of these type of molecules, compounds 1–3 have been tested for their effects on the growth of two cell lines, J774A.1 (BALB/c murine macrophages) and C6 (rat glioma) in vitro. Compounds 1 and 2 induced selective concentration-dependent mortality on J774A.1 cells.

α-Melanocyte-stimulating hormone (α-MSH) is a critical regulator of energy metabolism. Prolyl carboxypeptidase (PRCP) is an enzyme responsible for its degradation and inactivation. PRCP-null mice ( PRCP gt/gt) showed elevated levels of brain α-MSH, reduced food intake, and a leaner phenotype compared with wild-type controls. In addition, they were protected against diet-induced obesity. Here, we show that PRCP gt/gt animals have improved metabolic parameters compared with wild-type controls under a standard chow diet (SD) as well as on a high-fat diet (HFD). Similarly to when they are exposed to SD, PRCP gt/gt mice exposed to HFD for 13 wk showed a leaner phenotype due to decreased fat mass, increased energy expenditure, and locomotor activity. They also showed improved insulin sensitivity and glucose tolerance compared with WT controls and a significant reduction in fasting glucose levels. These improvements occured before changes in body weight and composition were evident, suggesting that the beneficial effect of PRCP ablation is independent of the adiposity levels. In support of a reduced gluconeogenesis, liver PEPCK and G-6-Pase mRNA levels were reduced significantly in PRCPgt/gt compared with WT mice. A significant decrease in liver weight and hepatic triglycerides were also observed in PRCP gt/gt compared with WT mice. Altogether, our data suggest that PRCP is an important regulator of energy and glucose homeostasis since its deletion significantly improves metabolic parameters in mice exposed to both SD and HFD.

STUDY DESIGN Experimental study of spinal cord injury (SCI) using an organotypic slice culture. OBJECTIVE To clarify the protective mechanism of PPAR-δ agonist GW0742 in the injured spinal cord using an in vitro model. SUMMARY OF BACKGROUND DATA In vivo data suggest that ligands of the δ isoform have activity in a number of disease models that are partly driven by the inflammatory response. Moreover, reports from in vivo studies using models of ischemia reperfusion and Parkinson disease also have shown neuroprotection conferred by PPAR-δ. The biological role and function of PPAR-δ remains relatively unclear. METHODS Spinal cord from 6-week-old mice was cut into transverse slices of 400-μm thickness to generate the organotypic slice cultures. The slices were injured using a weight dropped onto the center of the slice. PPAR-δ agonist was applied at 10 μM at 1 hour before injury. RESULTS Our study shows that GW0742 incubation (10 μM) at 1 hour before transverse lesion significantly reduced (1) p38 mitogen-activated protein kinase (MAPK), (2) c-Jun N-terminal kinase (JNK/SAP kinase), (3) NF-κB activation, (4) loss of neurotrophic factors (BDNF, GDNF), (5) COX-2 expression, and (6) cell death. CONCLUSION GW0742 reduces the cellular and molecular changes occurring in SCI by targeting different downstream pathways modulating PPAR-δ receptors.

Objective: Various lines of evidence suggest that malfunctioning of the gut–liver axis contributes to hepatic damage of rodents and humans with nonalcoholic fatty liver disease. We evaluated the effects of short-term probiotic treatment in children with obesity-related liver disease who were noncompliant with lifestyle interventions. Patients and Methods: Twenty obese children (age 10.7 � 2.1 years) with persisting hypertransaminasemia and ultrasonographic (US) bright liver were enrolled in this double-blind, placebo-controlled pilot study. At baseline, patients underwent clinical and laboratory anthropometric evaluation, measurement of the US hepatorenal ratio, standard liver function tests, oral glucose tolerance test, serum tumor necrosis factoralpha, the glucose hydrogen breath test, and evaluation of serum antibodies to antipeptidoglycan-polysaccharide polymers. After exclusion of causes of liver disease other than obesity, patients received either probiotic Lactobacillus rhamnosus strain GG (12 billion CFU/day) or placebo for 8 weeks. Results: Multivariate analysis after probiotic treatment revealed a significant decrease in alanine aminotransferase (average variation vs placebo P ¼ 0.03) and in antipeptidoglycan-polysaccharide antibodies (average variation vs placebo P ¼ 0.03) irrespective of changes in BMI z score and visceral fat. Tumor necrosis factor-alpha, and US bright liver parameters remained fairly stable. Conclusions: Probiotic L rhamnosus strain GG warrants consideration as a therapeutic tool to treat hypertransaminasemia in hepatopathic obese children noncompliant with lifestyle interventions.

Non-dioxin-like polychlorinated biphenyls (PCBs) are stable and lipophilic chemicals that persist in the environment and tend to bioaccumulate in the food chains. In the present study, we have investigated the effect of PCBs 101, 153, and 180 on macrophage J774A.1 by assessing cell viability and apoptotic cell death. We have combined morphological techniques and biochemical ones to establish the relevance of apoptosis in macrophage cell death induced by PCBs, alone or in combination. Treatment with the examined PCBs caused the loss of cell viability and accelerated apoptosis in a concentration-dependent manner. Moreover, a synergistic effect on cell death and apoptosis was evidenced for all PCBs at concentrations which were inactive alone. The apoptosis induced by PCBs involved the increase of caspase-3 activity. Also, Bcl-2 and Bax proteins were assessed to elucidate the apoptosis machinery induced in macrophage cultures by PCBs. Our results indicate that the increase in PCB-induced apoptosis correlates with a reduction in the expression of antiapoptotic Bcl-2 and an increase in the expression of proapoptotic Bax. Interestingly, concentrations of PCBs inactive by themselves induce apoptosis when PCBs are combined. In conclusion, our findings suggest that, although less toxic than dioxin like congeners, the examined non-dioxin-like PCBs are equally dangerous as immunotoxic pollutants, also considering their presence as mixtures at higher levels than dioxin-like PCBs in biotic and abiotic matrices. © 2011 Elsevier Ireland Ltd.

The glycosylated phenylpropanoid verbascoside (VB), isolated from cultured cells of the medicinal plant Syringa vulgaris (Oleaceae), has previously been characterized as an effective scavenger of biologically active free radicals and an inhibitor of lipid peroxidation. The aim of the present study was to evaluate in a rat glioma cell line (C6) the effect of VB biotechnologically produced by S. vulgaris plant cell cultures in the regulation of the inflammatory response. We used a model of central nervous system inflammation induced by bacterial endotoxin/cytokine (lipopolysaccharide (LPS)/interferon (IFN)-gamma, 1 microg/ml and 100 U/ml, respectively). Our results show that the treatment with LPS/IFN-gamma for 24 h elicited the induction of inducible nitric oxide synthase (iNOS) activity as determined by NO(x) accumulation in the culture medium. Preincubation with VB (10-100 microg/ml) abrogated the mixed cytokine-mediated induction of iNOS. The effect was concentration-dependent. Our studies also showed an inhibitory effect of VB on neuronal nitric oxide synthase expression. Moreover, Western blot analysis showed that this glycoside prevents specifically the activation of the proinflammatory enzyme cyclooxygenase (COX)-2 in glioma cells without simultaneous inhibition of COX-1 enzyme. Moreover, we found that VB reduced the expression of proinflammatory enzymes in LPS/IFN-gamma through the inhibition of the activation of nuclear factor kappa B and mitogen-activated protein kinase signaling pathway. The mechanisms underlying in vitro the neuroprotective properties of VB involve modulation of transcription factors and consequent altered gene expression, resulting in downregulation of inflammation. These findings provide support that VB may provide a promising approach for the treatment of oxidative-stress-related neurodegenerative diseases.

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.

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.

Ochratoxin A (OTA) is a fungal metabolite with controversial immunomodulatory effects. A prolonged in vivo exposure to the mycotoxin may result in impaired immunity and decreased resistance to infections. In the present study, OTA modulation of lipopolysaccharide (LPS)-induced inflammatory process is described in the macrophagic cell line, J774A.1 in order to better understand the mechanisms underlying OTA immunotoxicity. OTA (30 nM–100 μM) induces a time and concentration dependent cytotoxic effect, increased when cells were co-stimulated with LPS (100 ng/ml), a concentration that alone did not modify the cellular viability. Moreover, OTA (3 μM) alone induces a significant increase in cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression, while at the highest concentration (10 μM) a reduced expression of both enzymes was shown, consistently with the mycotoxin cytotoxic profile. The role of nuclear factor-kB (NF-kB) in the mycotoxin effect was also demonstrated. Conversely, when cells were co-stimulated with LPS, OTA showed a concentration-dependent reduction of COX-2 and iNOS expression and their respective metabolites (PGE 2 and NO). These results confirm the pro-inflammatory role of OTA by itself, and demonstrate the impaired capability of OTA-treated macrophages to respond properly to noxious stimuli, such as LPS, mimicking the environmental co-exposure to both compounds.

Hypericum perforatum is a medicinal plant species containing many polyphenolic compounds, namely flavonoids and phenolic acids. Since polyphenolic compounds have high antioxidant potential, we have investigated the effects of H. perforatum extract on the development of multiple organ dysfunction syndrome caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in mice. Organ failure and systemic inflammation in rats was assessed 18 h after administration of zymosan and/or H. perforatum extract and monitored for 12 days (for loss of body weight and mortality). Treatment of mice with H. perforatum extract (30 mg/kg i.p., 1 and 6h after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells caused by zymosan, pulmonary, intestinal and pancreatic injury, and renal dysfunction as well as the increase in myeloperoxidase in the lung and intestine. Immunohistochemical analysis for inducible nitric oxide synthase (iNOS), nitrotyrosine, and poly(ADP-ribose) (PAR) revealed positive staining in lung and intestine tissues obtained from zymosan-injected mice. The degree of staining for nitrotyrosine, iNOS, and PAR was markedly reduced in tissue sections obtained from zymosan-treated mice, which received H. perforatum extract. In conclusion, this study provides evidence, for the first time, that H. perforatum extract attenuates the degree of zymosan-induced multiple organ dysfunction syndrome in mice.

Various evidences have documented that the pineal secretory product melatonin exerts an important anti-inflammatory effect in different experimental models including colitis. The aim of the present study was to evaluate whether melatonin regulates the inflammatory response of experimental colitis in rats at the level of signal transduction pathway. Colitis was induced by intracolonic instillation of dinitrobenzene sulfonic acid (DNBS). Four days after DNBS administration, a substantial increase of colon TNF-alpha production was associated with the colon damage. In DNBS-treated rats, the colon injury correlated with a significant rise of apoptosis (evaluated by TUNEL coloration) which was associated with a significant increased expression of proapoptotic Bax and decreased colon content of antiapoptotic Bcl-2. This inflammatory response was also related to activation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of c-Jun as well as FAS ligand expression in the colon. Treatment with melatonin (15 mg/kg daily i.p.) was associated with a remarkable amelioration of colonic disrupted architecture as well as a significant reduction of TNF-alpha. Melatonin also reduced the NF-kappaB activation and phosphorylation of c-Jun as well as the Fas ligand expression in the colon. Furthermore, melatonin reduced the expression of Bax and prevented the loss of Bcl-2 proteins as well as the presence of apoptotic cells caused by DNBS. The results of this study show that melatonin administration exerts beneficial effects in inflammatory bowel disease by modulating signal transduction pathways.

Multiple-organ failure (MOF) is defined as the progressive deterioration in function which occurs in several organs or systems in patients with septic shock, multiple trauma, severe burns, or pancreatitis. In the present study, we have investigated the effects of Green Tea extract (GTE) on the development of general inflammation caused by zymosan (500 mg/kg, administered i.p. as a suspension in saline) in mice. Organ failure and systemic inflammation in mice was assessed 18 hours after administration of zymosan and/or GTE and monitored for 12 days (for loss of body weight and mortality). Treatment of mice with GTE (25 mg/kg i.p., 1 and 6 hours after zymosan) attenuated the peritoneal exudation and the migration of polymorphonuclear cells (PMNs) caused by zymosan, GTE also attenuated the lung, liver and pancreatic injury and renal dysfunction caused by zymosan as well as the increase in myeloperoxidase (MPO) activity caused by zymosan in the lung and intestine. Immunohistochemical analysis for inducible nitric oxide synthase (iNOS), nitrotyrosine and poly(ADP-ribose) (PAR) revealed positive staining in lung and intestine tissues obtained from zymosan-treated mice. The degree of staining for nitrotyrosine, iNOS and PAR were markedly reduced in tissue sections obtained from zymosan-treated mice, which received GTE. In conclusion this study provides evidence, for the first time, that GTE attenuates the degree of zymosan induced generalized inflammation in mice.

Poly (ADP-ribose) polymerase (PARP), a nuclear enzyme activated by strand breaks in DNA, plays an important role in the colon injury associated with experimental colitis. The aim of the present study was to examine the effects of 3-aminobenzamide (3-AB), an inhibitor of PARP activity, in the development of acute pancreatitis caused by cerulein in mice. Intraperitoneal injection of cerulein in mice resulted in severe, acute pancreatitis characterized by oedema, neutrophil infiltration and necrosis and elevated serum levels of amylase and lipase. Infiltration of pancreatic and lung tissue with neutrophils (measured as increase in myeloperoxidase activity) was associated with enhanced expression of the intercellular adhesion molecule-1 (ICAM-1) and P-selectin. Immunohistochemical examination demonstrated a marked increase in the staining (immunoreactivity) for transforming growth factor-beta (TGF-beta) and vascular endothelial growth factor (VEGF) in the pancreas of cerulein-treated mice in comparison to sham-treated mice. Acute pancreatitis in vehicle-treated mice was also associated with a significant mortality (40% survival at 5 days after cerulein administration). In contrast, (1) the degree of pancreatic inflammation and tissue injury (histological score), (2) upregulation/formation of ICAM-1 and P-selectin, (4) neutrophils infiltration and (5) the expression of TGF-beta and VEGF was markedly reduced in pancreatic tissue obtained from cerulein-treated mice which have been treated with 3-AB. These findings provide the evidence that PARP inhibition reduce the degree of pancreas injury caused by acute pancreatitis induced by cerulein administration.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily of ligand-activated transcription factors that are related to retinoid, steroid, and thyroid hormone receptors. Three isotypes of PPARs have been identified: alpha, beta/delta, and gamma, encoded by different genes and distributed in various tissues. PPARs are implicated in the control of inflammatory responses and in energy homeostasis and, thus, can be defined as metabolic and anti-inflammatory transcription factors. They exert anti-inflammatory effects by inhibiting the induction of proinflammatory cytokines, adhesion molecules, and extracellular matrix proteins, or by stimulating the production of anti-inflammatory molecules. Moreover, PPARs modulate the proliferation, differentiation, and survival of immune cells. This review presents the current state of knowledge regarding the involvement of PPARs in the control of inflammatory response, and their potential therapeutic applications in several types of shock, as well as hemorrhagic, septic, and nonseptic shock.

In vitro and in vivo studies have demonstrated that methylguanidine, an inhibitor of nitric oxide synthase (NOS), is also able to reduce tumour necrosis factor-alpha (TNF-alpha) release. In the present study, we evaluated the anti-inflammatory potential of methylguanidine treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy) where oxyradical, nitric oxide (NO) and prostaglandins play a crucial role in the inflammatory processes. Our data show that methylguanidine, given intraperitoneally at the dose of 30 mg/kg, inhibits the inflammatory response reducing significantly (P

Obesity, from declining estrogen levels after menopause, increases the risk of heart disease, diabetes, and hypertension. Ovariectomy (OVX) in rats is a good model of estrogen insufficiency. The ensuing mild obesity is useful to study how hypoestrogenism alters adiposity. This study examines the hypothesis that in ovariectomized (OVX) rats modification of estrogen levels or treatment with a selective estrogen receptor modulator, raloxifene (RAL), alters leptinemia and modulates leptin receptor (Ob-R) abundance in hypothalamus and white adipose tissue, similar to the modification of adipose status induced by hypoestrogenism. Mid- and long-term studies (7 and 22 wk) were conducted to monitor the change in leptinemia in rats after estrogen loss by OVX and after estrogen replacement by 17-estradiol (OVXE2) or RAL treatment (OVXRAL). Leptin was significantly higher in OVX rats vs. controls, in a time-dependent manner. This effect was reversed by both E2 and RAL treatment at 7 wk (P < 0.05) and 22 wk (P < 0.001). Moreover, E2 or RAL treatment reversed the OVX-induced increases in food intake, body weight, and fat mass content; the modifications of serum parameters were examined to evaluate the different lipid profiles. We also evaluated Ob-R expression in hypothalamus and adipose tissue by Western blot analysis. The expression of the long functional isoform (Ob-Rb) increased at 7 wk only in adipose tissue and decreased at 22 wk in OVX rats in both tissues; these effects were reversed by E2 or RAL treatment. We provide evidence that central and peripheral Ob-Rb expression is related to modification of estrogen levels. (Endocrinology 145: 3115–3121, 2004)

Echinacea purpurea (L.) Moench and Hypericum perforatum (L.) were evaluated for their anti-inflammatory activity against carrageenan-induced paw oedema in mice. Each drug was administered orally to mice at 30 and 100 mg kg−1, twice daily. Only the higher dose significantly inhibited, time dependently, the formation of oedema, evaluated as area under the curve (echinacea P < 0.01; hypericum P < 0.05). Western blot analysis showed that in-vivo treatment with these extracts could modulate lipopolysaccharide (LPS) and interferon-γ induced cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) expression in peritoneal macrophages. In particular, treatment with 100 mg kg−1 hypericum inhibited both iNOS and COX-2 expression, whereas treatment with 100 mg kg−1 echinacea down-regulated only COX-2 expression. The present study suggests that the anti-inflammatory effect of these extracts could be in part related to their modulation of COX-2 expression.

The present study focuses on the effect of various naturally occurring flavonoids (apigenin, galangin, morin, naringenin, quercetin, and silymarin) on nitric oxide (NO) and prostaglandin E2 (PGE2) production induced by lipopolysaccharide (LPS) in the macrophage cell line J774A.1. Moreover, we evaluated flavonoid modulation of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) enzyme expression by western blot analysis. Apigenin and quercetin (0.5-50 microM) were the most potent inhibitors of NO production and this effect was concentration-dependent and significant at 5 and 50 microM. These data were consistent with the modulation of iNOS enzyme expression. A similar pattern was observed considering the inhibitory effect of flavonoids on LPS-induced PGE2 release and COX-2 expression. Quercetin, galangin, apigenin, and naringenin markedly decreased PGE2 release and COX-2 expression in a concentration-dependent manner. This study suggests that inhibition of iNOS and COX-2 expression by flavonoids may be one of the mechanisms responsible for their anti-inflammatory effects.

In the present study, we demonstrated that repeated treatment with recombinant human prolactin (rhPRL) protected mice against Salmonella typhimurium infection. The protective activity was statistically significant, dose-dependent and present only when rhPRL treatments were performed before the infection. This activity was probably related to the observed increases in phagocytosis and intracellular killing of peritoneal macrophages induced by the hormonal treatment. The number of peripheral leukocytes was not modified, excluding a mobilization of cells from other compartments. A decrease in the mortality rate after challenge was also observed in mice treated with the monoclonal antibody anti-PRL receptor U5, confirming that the protective activity was associated with receptor activation. Our studies also suggest that nitric oxide (NO) production was involved in the protective effect of rhPRL since pre-treatment of the animals with L-NAME, an inhibitor of NO-synthase, was able to completely revert the protective activity, whereas D-NAME, the inactive D-isomer, was without effect.