Giuseppina Mattace Raso

Inhibition of inducible nitric oxide synthase and cyclooxygenase-2 expression by flavonoids in macrophage J774A.1.

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.

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

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

Probiotics as an emerging therapeutic strategy to treat NAFLD: focus on molecular and biochemical mechanisms

Nonalcoholic fatty liver disease (NAFLD) is currently the most common liver disease worldwide, both in adults and in children. NAFLD is characterized by aberrant lipid storage in hepatocytes (hepatic steatosis) and inflammatory progression to nonalcoholic steatohepatitis. Evidences so far suggest that intrahepatic lipid accumulation does not always derive from obesity. Gut microbiota has been considered as a regulator of energy homeostasis and ectopic fat deposition, suggesting its implications in metabolic diseases. Probiotics are live microbial that alter the enteric microflora and have beneficial effects on human health. Although the molecular mechanisms of probiotics have not been completely elucidated yet, many of their effects have proved to be beneficial in NAFLD, including the modulation of the intestinal microbiota, an antibacterial substance production, an improved epithelial barrier function and a reduced intestinal inflammation. Given the close anatomical and functional correlation between the bowel and the liver, and the immunoregulatory effects elicited by probiotics, the aim of this review is to summarize todays knowledge about probiotics in NAFLD, focusing in particular on their molecular and biochemical mechanisms, as well as highlighting their efficacy as an emerging therapeutic strategy to treat this condition.

Acute Intracerebroventricular Administration of Palmitoylethanolamide, an Endogenous Peroxisome Proliferator-Activated Receptor-α Agonist, Modulates Carrageenan-Induced Paw Edema in Mice

Peroxisome proliferator-activated receptor (PPAR)-α is a nuclear transcription factor. Although the presence of this receptor in different areas of central nervous system (CNS) has been reported, its role remains unclear. Palmitoylethanolamide (PEA), a member of the fatty-acid ethanolamide family, acts peripherally as an endogenous PPAR-α ligand, exerting analgesic and anti-inflammatory effects. High levels of PEA in the CNS have been found, but the specific function of this lipid remains to be clarified. Using carrageenan-induced paw edema in mice, we show that i.c.v. administration of PEA may control peripheral inflammation through central PPAR-α activation. A single i.c.v. administration of 0.01 to 1 μg of PEA, 30 min before carrageenan injection, reduced edema formation in the mouse carrageenan test. This effect was mimicked by 0.01 to 1 μg of GW7647 [2-[[4-[2-[[(cyclohexylamino)carbonyl](4-cyclohexylbutyl)amino]ethyl]phenyl]thio]-2-methylpropanoic acid], a synthetic PPAR-α agonist. Moreover, central PEA administration significantly reduced the expression of the proinflammatory enzymes cyclooxygenase-2 and inducible nitric-oxide synthase, and it significantly restored carrageenan-induced PPAR-α reduction in the spinal cord. To investigate the mechanism by which i.c.v. PEA attenuated the development of carrageenan-induced paw edema, we evaluated inhibitor κB-α (IκB-α) degradation and nuclear factor-κB (NF-κB) p65 activation in the cytosolic or nuclear extracts from spinal cord tissue. PEA prevented IkB-α degradation and NF-κB nuclear translocation, confirming the involvement of this transcriptional factor in the control of peripheral inflammation. The obligatory role of PPAR-α in mediating the effects of PEA was confirmed by the lack of the compounds anti-inflammatory effects in mutant mice lacking PPAR-α. In conclusion, our data show for the first time that PPAR-α activation in the CNS can control peripheral inflammation.

Leptin potentiates IFN-γ-induced expression of nitric oxide synthase and cyclo-oxygenase-2 in murine macrophage J774A.1

Leptin, a pleiotropic hormone believed to regulate body weight, has recently been associated with inflammatory states and immune activity. Here we have studied the effect of leptin on expression of IFN‐γ‐induced nitric oxide synthase (iNOS) and cyclo‐oxygenase‐2 (COX‐2), both prominent markers of macrophage activation, using the murine macrophage J774A.1 cell line. After 24 h of incubation, leptin (1–10 μg ml−1) potently synergized with IFN‐γ (100 U ml−1) in nitric oxide (NO) release, evaluated as nitrite and nitrate (NOx), and prostaglandin E2 (PGE2) production in culture medium. The observed increase of NO and PGE2 was related to enhanced expression of the respective inducible enzyme isoforms, measured in mRNA and protein by RT–PCR and Western blot analysis, respectively. When cells were stimulated only with leptin, a weak induction of NO and PGE2 release and of the expression of related inducible enzymes was observed. Moreover IFN‐γ increased the expression of the functional form of leptin receptor (Ob‐Rb) and this effect was potentiated by leptin in a concentration‐dependent manner. These data suggest that macrophages, among the peripheral immune cells, represent a target for leptin and confirm the relevance of this hormone in the pathophysiology of inflammation.

Central administration of palmitoylethanolamide reduces hyperalgesia in mice via inhibition of NF-κB nuclear signalling in dorsal root ganglia

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 PEAs 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.

In-vivo and in-vitro anti-inflammatory effect of echinacea purpurea and Hypericum perforatum

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.

Signal transduction pathways involved in protective effects of melatonin in C6 glioma cells

Abstract:  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 μm‐1 mm) in the regulation of the expression of nitric oxide synthase (NOS) caused by incubation with lipopolysaccharide (LPS)/interferon (IFN)‐γ (1 μg/mL and 100 U/mL, respectively) and defined the mode of melatonin’s action. Treatment with LPS/IFN‐γ for 24 hr elicited the induction of inducible (iNOS) activity as determined by nitrite and nitrate (NOx) 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‐γ‐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‐κB). The ability of melatonin to inhibit NF‐κB activation was further confirmed by studies on the degradation of the inhibitor of NF‐κB, IκB‐α. 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.

Role of innate immune response in non-alcoholic fatty liver disease: metabolic complications and therapeutic tools

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.

Prolactin Induction of Nitric Oxide Synthase in Rat C6 Glioma Cells

Abstract : We have examined the neuroimmunoregulatory function of prolactin (PRL) on astrocytic inducible nitric oxide synthase (iNOS) expression in the C6 glioma cell line. After 24 h of PRL (5‐100 nM) stimulation, a concentration‐dependent increase of NO release, evaluated as nitrite, was observed in C6 culture medium. Moreover, both NO release and iNOS expression induced by interferon‐γ (250 U/ml) were enhanced by PRL (18‐100 nM). PRL‐induced NO release was inhibited by dexamethasone, an inhibitor of de novo iNOS synthesis. We used erbstatin (5 μg/ml), a potent inhibitor of protein tyrosine kinases, to test whether these proteins were required for signaling events evoked by PRL in these cells. This inhibitor was able to inhibit completely the PRL‐induced NO production and iNOS expression. In conclusion, we provide evidence that NO production in glial cells can be regulated not only by cytokines but also by neuroimmunoregulatory hormones such as PRL, which is present in normal brain but may be elevated in several pathological states.