Galactosylated Pro-Drug of Ursodeoxycholic Acid: Design, Synthesis, Characterization, and Pharmacological Effects in a Rat Model of Estrogen-Induced Cholestasis.

Ursodeoxycholic acid (UDCA) is considered the first-choice therapy for cholestatic disorders. To enhance solubility and exploit specific transporters in liver, we synthesized a new galactosyl pro-drug of UDCA (UDCAgal). Ethinylestradiol (EE)-induced cholestasis was used to study and compare the effects of UDCAgal with UDCA on bile flow, hepatic canalicular efflux transporter expression, and inflammation. UDCAgal resulted quite stable both at pH 7.4 and 1.2 and regenerated the parent drug after incubation in human plasma. Its solubility, higher than UDCA, was pH- and temperature-independent. UDCAgal displayed a higher cell permeation compared to UDCA in liver HepG2 cells. Moreover, in cholestatic rats, UDCAgal showed a higher potency compared to UDCA in reducing serum biomarkers (AST, ALT, and ALP) and cytokines (TNF-α and IL-1ß). The higher effect of UDCAgal on the increase in bile salt export pump and multidrug resistance-associated protein 2 transcription indicated an improved spillover of bile acids from the liver. UDCAgal showed a reduction in CCL2, as well as TNF-α, IL-1ß, and cyclooxygeanse-2 mRNAs, indicating a reduction in hepatic neutrophil accumulation and inflammation. Moreover, UDCAgal, similarly to UDCA, heightens bile flow and modulates biliary acids secretion. These results indicate that UDCAgal has a potential in the treatment of cholestatic disease.

Inflammatory Myopathy in Horses With Chronic Piroplasmosis.

Horses affected by chronic piroplasmosis may develop poor performance and muscle atrophy. Here we investigate the pathological and immunopathological aspects of myopathy occurring in chronic equine piroplasmosis. The study included 16 horses serologically positive for equine piroplasms presenting with clinical signs and supporting serum biochemical evidence of a myopathy. Skeletal muscle was evaluated by histopathology, immunohistochemistry, indirect immunofluorescence, and molecular detection of piroplasms and inflammatory cytokines in skeletal muscle. Histologic lesions included muscle fiber atrophy (100% of cases), degenerative changes (13/16, 81%), and perivascular perimysial and endomysial lymphocytic infiltrates (81% of cases). In 15 cases (94%), muscle fibers had strong immunostaining for major histocompatibility complex classes I and II. T lymphocyte populations were mainly CD3+, CD8+, and CD4+ in equal proportions, with a lower number of CD79α+ cells. The serum from affected horses was tested by indirect immunofluorescence for binding of IgG, IgM, or IgA to sections of normal equine muscle to detect circulating autoantibodies against muscle antigen(s). In all cases, distinct sarcolemmal staining was detected in sections incubated with serum from affected horses, in contrast to sections incubated with phosphate-buffered saline or equine control sera. Reverse transcription polymerase chain reaction (RT-PCR) testing of muscles from affected animals revealed a significant increase of interferon-γ, interleukin-12, and tumor necrosis factor-α gene expression compared to healthy controls. Theileria equi or Babesia caballi was not detected in samples of affected muscle by RT-PCR. Thus, inflammatory myopathy associated with equine piroplasmosis may involve an autoimmune pathogenesis with upregulation of inflammatory cytokines that may cause myofiber atrophy and degeneration.

Hydroxytyrosol prevents metabolic impairment reducing hepatic inflammation and restoring duodenal integrity in a rat model of NAFLD.

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.

Galactosyl prodrug of palmitoylethanolamide: synthesis, stability, cell permeation and cytoprotective activity.

N-Palmitoylethanolamide (PEA) is emerging as a novel therapeutic agent in the treatment of neuropathic pain and neurodegenerative diseases. Unfortunately, PEA poorly reaches the central nervous system (CNS), after peripheral administration, since it is inactivated through intracellular hydrolysis by lipid amidases. Since prodrug approach is one of the most popular methods used to increase cell permeability, the aim of this paper consists in the synthesis of a new galactosyl prodrug of PEA, the palmitoylethanolamide-succinamyl-D-galactos-6'-yl ester (PEAGAL). Biological experiments both in neuroblastoma and in C6 glioma cells, together with quantitative analyses performed through a LC-MS-MS technique, demonstrate the better efficacy of PEAGAL compared to PEA and its higher cell permeation. Our results encourage further experiments in animal models of neuropathic pain and of neurological disorders and/or neurodegenerative diseases, in order to promote a more effective peripherally administrated derivative of PEA.

New bioactive alkyl sulfates from Mediterranean tunicates.

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.

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.

Raloxifene, a selective estrogen receptor modulator, reduces carrageenan-induced acute inflammation in normal and ovariectomized rats.

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.

Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from ovariectomized rats.

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 17beta-estradiol (OVX+E(2)) or RAL treatment (OVX+RAL). Leptin was significantly higher in OVX rats vs. controls, in a time-dependent manner. This effect was reversed by both E(2) and RAL treatment at 7 wk (P < 0.05) and 22 wk (P < 0.001). Moreover, E(2) 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 E(2) or RAL treatment. We provide evidence that central and peripheral Ob-Rb expression is related to modification of estrogen levels.

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

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-gamma-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. 2. After 24 h of incubation, leptin (1-10 micro g ml(-1)) potently synergized with IFN-gamma (100 U ml(-1)) in nitric oxide (NO) release, evaluated as nitrite and nitrate (NO(x)), and prostaglandin E(2) (PGE(2)) production in culture medium. 3. The observed increase of NO and PGE(2) was related to enhanced expression of the respective inducible enzyme isoforms, measured in mRNA and protein by RT-PCR and Western blot analysis, respectively. 4. When cells were stimulated only with leptin, a weak induction of NO and PGE(2) release and of the expression of related inducible enzymes was observed. 5. Moreover IFN-gamma increased the expression of the functional form of leptin receptor (Ob-Rb) and this effect was potentiated by leptin in a concentration-dependent manner. 6. 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.