Rosaria Meli

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.

Rapid broad-spectrum analgesia through activation of peroxisome proliferator-activated receptor-α

Severe pain remains a major area of unmet medical need. Here we report that agonists of the nuclear receptor PPAR-α (peroxisome proliferator-activated receptor-α) suppress pain behaviors induced in mice by chemical tissue injury, nerve damage, or inflammation. The PPAR-α agonists GW7647 [2-(4-(2-(1-cyclohexanebutyl)-3-cyclohexylureido)ethyl)phenylthio)-2-methylpropionic acid], Wy-14643 [4-chloro-6-(2,3-xylidino)-2-pyrimidinylthioacetic acid], and palmitoylethanolamide (PEA) reduced nocifensive behaviors elicited in mice by intraplantar (i.pl.) injection of formalin or i.p. injection of magnesium sulfate. These effects were absent in PPAR-α-null mice yet occurred within minutes of agonist administration in wild-type mice, suggesting that they were mediated through a transcription-independent mechanism. Consistent with this hypothesis, blockade of calcium-operated IKca (KCa3.1) and BKca (KCa1.1) potassium channels prevented the effects of GW7647 and PEA in the formalin test. Three observations suggest that PPAR-α agonists may inhibit nocifensive responses by acting on peripheral PPAR-α. (i) PEA reduced formalin-induced pain at i.pl. doses that produced no increase in systemic PEA levels; (ii) PPAR-α was expressed in dorsal root ganglia neurons of wild-type but not PPAR-α-null mice; and (ii) GW7647 and PEA prevented formalin-induced firing of spinal cord nociceptive neurons in rats. In addition to modulating nociception, GW7647 and PEA reduced hyperalgesic responses in the chronic constriction injury model of neuropathic pain; these effects were also contingent on PPAR-α expression and were observed following either acute or subchronic PPAR-α agonist administration. Finally, acute administration of GW7647 and PEA reduced hyperalgesic responses in the complete Freunds adjuvant and carrageenan models of inflammatory pain. Our results suggest that PPAR-α agonists may represent a novel class of analgesics.

Dual inhibitors of cyclooxygenase and 5-lipoxygenase. A new avenue in anti-inflammatory therapy?

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a mainstay in the treatment of inflammatory disease and are among the most widely used drugs worldwide. They are anti-inflammatory, antipyretic, and analgesic and are prescribed as first choice for the treatment of rheumatic disorders and, in general, inflammation. The main limitation in using NSAIDs consists in their side-effects, including gastrointestinal ulcerogenic activity and bronchospasm. The mechanism of action of these drugs is attributed to the inhibition of cyclooxygenase (COX), and, consequently, the conversion of arachidonic acid into prostaglandins. It is hypothesized that the undesirable side-effects of NSAIDs are due to the inhibition of COX-1 (constitutive isoform), whereas the beneficial effects are related to the inhibition of COX-2 (inducible isoform). Arachidonic acid can also be converted to leukotrienes (LTs) by the action of 5-lipoxygenase (5-LOX). LTC(4,) LTD(4,) and LTE(4) are potent bronchoconstrictors, whereas LTB(4) is chemotactic for leukocytes and plays an important role in the development of gastrointestinal ulcers by contributing to the inflammatory process. Thus, developing dual inhibitor compounds that will simultaneously inhibit COX and 5-LOX could enhance their individual anti-inflammatory effects and reduce the undesirable side-effects associated with NSAIDs, especially of the gastrointestinal tract. The most promising COX/5-LOX inhibitor is ML3000 ([2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-pyrrolizine-5-yl]-acetic acid), now in Phase III clinical trials. This new approach will certainly help to unravel the mechanisms at the root of the undesirable effects of NSAIDs and to develop safer NSAIDs.

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.

Effects of Lactobacillus rhamnosus Strain GG in Pediatric Obesity-related Liver Disease

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 factor-alpha, 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 = 0.03) and in antipeptidoglycan-polysaccharide antibodies (average variation vs placebo P = 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.

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.