Effect of dietary β-glucan on growth performance,fecal microbial shedding and immunological responses after lipopolysaccharide challenge in weaned pigs

Two experiments (Exp.) were conducted to evaluate the effects of β-glucan inclusion in the diet on growth performance and immune function after lipopolysaccharide (LPS) challenge. In Exp. 1, a total of 40 weaned pigs (progeny of Landrace×Yorkshire sows by Duroc) with an initial body weight (BW) of 7.89 ± 0.84 kg (21 ± 2 d) of age) were used in a 28-day (d) experiment to determine the effects of dietary β-glucan on growth performance. Pigs were allotted randomly to two treatments consisting of addition of 0 or 0.1 g β-glucan/kg diet with four replicate pens per treatment and five pigs per pen. Growth performance was not affected by β-glucan supplementation throughout the experiment. However, dietary β-glucan reduced (P<0.05) the number of fecal Escherichia coli. In Exp. 2, a total of 20 weaned barrows (6.22 ± 0.25 kg of BW and 21 ± 2 d of age) individually raised in metabolic cages were used to evaluate immunological responses following LPS challenge. Pigs were fed 0 or 0.1 g β-glucan/kg diet for 42 d. At the end of the trial, half of the pigs (n = 5) from each treatment were injected intraperitoneal with E. coli LPS at a concentration of 100 μg/kg BW and the other half were injected with sterile saline solution. Treatments were arranged as a 2×2 factorial, with the main effect of LPS challenge (saline vs. LPS) and β-glucan supplementation (0 g/kg vs. 0.1 g/kg). After LPS injection, blood was taken at 0, 2, 4, 6, 8 and 12 hours (h) for the blood cell counts and blood inflammatory response. Dietary β-glucan increased (P<0.05) leukocytes counts at 4, 6 and 8 h, and blood lymphocyte concentrations at 2, 4 and 6 h and LPS challenge increased (P<0.05) counts of leukocytes at 2, 4, 6 and 8 h and blood lymphocyte at 2 and 4 h post-challenge. The rectal temperature was increased (P<0.05) at 2, 4, 6 and 8 h after LPS challenge. Dietary β-glucan reduced (P<0.05) and LPS challenge increased (P<0.05) blood plasma tumor necrosis factor-α (TNF-α) concentration at 2 and 4 h post-challenge. Dietary β-glucan increased (P<0.05) the concentration of the cluster of differentiation antigens 4 cells (CD4⁺) at 2, 4 and 6 h, and of 8 (CD8⁺) at 4 and 6 h post-challenge, respectively. The LPS challenge increased (P<0.05) CD4⁺ and CD8⁺ cell concentrations at 2, 4 and 6 h post-challenge. The CD4⁺:CD8⁺ ratio was reduced (P<0.05) by LPS challenge but was increased (P<0.05) by dietary β-glucan at 2, 4, 6 and 8 h post-challenge. In conclusion, dietary β-glucan decreased E. coli numbers but did not affect growth performance in weaned pigs and may offer benefits on immune function in weaned pigs challenged with LPS.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.     

In vitro and in vivo interaction of macrophages from vaccinated and non-vaccinated channel catfish (Ictalurus punctatus) to Edwardsiella ictaluri

Macrophages from catfish vaccinated with an Edwardsiella ictaluri vaccine and macrophages from non-vaccinated catfish were used in in vitro and in vivo studies with red-fluorescent E. ictaluri to assess phagocytic ability, reactive oxygen and nitric oxide production and bactericidal activity. In the in vitro experiment, macrophages were harvested from vaccinated and non-vaccinated fish and then exposed to red-fluorescent E. ictaluri. Results of this study showed that E. ictaluri can survive and replicate in macrophages from non-vaccinated catfish (relative percent killing, RPK, from 0.011 to 0.620 and from ?0.904 to 0.042 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) even in the presence of reactive oxygen and nitrogen products. Macrophages from vaccinated fish were significantly (p < 0.05) more efficient in killing E. ictaluri (RPK from 0.656 to 0.978 and from 0.011 to 0.620 with macrophage:bacteria ratios of 1:20 and 1:100, respectively) and produced significantly (p < 0.05) higher amounts of ROS (10-fold increase) and nitrogen oxide (about 10-fold increase) than macrophages from non-vaccinated fish. In the in vivo experiment, vaccinated and non-vaccinated catfish were injected with red-fluorescent E. ictaluri to allow the interaction between macrophages and other components of the immune system. After 6 h, macrophages were harvested from the fish and seeded in glass chamber slides and bactericidal activity was measured in vitro. Results showed in vivo interaction of other components of the immune system enhanced bactericidal activity of macrophages from vaccinated fish. In another set of experiments, catfish were intraperitoneally injected with fluorescent bacteria opsonized with immune serum or non-opsonized and necropsied in the first 48 h after bacterial challenge to observe localization of E. ictaluri between vaccinated and non-vaccinated catfish. Vaccinated fish were able to control the dispersion of E. ictaluri in the body and red-fluorescent bacteria were observed only in the spleen, anterior and trunk kidney. In non-vaccinated fish E. ictaluri was able to replicate and invade all organs with the exception of the brain. We further determined that macrophages seeded with E. ictaluri could cause infection in non-vaccinated fish upon reinoculation with in vitro infected-macrophages. Overall, the results indicated that macrophages from vaccinated fish are activated and responsible for rapid clearance of infection upon re-exposure to virulent E. ictaluri.     

Effects of dietary vitamin D3 administration on innate immune parameters of seabream (Sparus aurata L.)

The present study assesses the in vivo effect of vitamin D₃ or cholecalciferol on some innate immune parameters of the gilthead seabream (Sparus aurata L.). Cholecalciferol was orally administered to seabream specimens in a commercial pellet food supplemented with 0 (control); 3750; 18,750 or 37,500 U kg^?1 and fish were sampled after 1, 2 and 4 weeks of treatment. Serum and head– kidney leucocytes were obtained and humoral (peroxidase and complement activity) and cellular (leucocyte peroxidase content, phagocytic, respiratory burst and natural cytotoxic activities) innate immune parameters were measured. Diet supplementation with 37,500 U kg^?1 cholecalciferol for 2 or 4 weeks resulted in a significant increase in phagocytic ability or serum peroxidase content, respectively, whereas the 3750 and 18,750 U kg^?1 supplemented diets led to significant increases in the phagocytic capacity of leucocytes at week 2 compared with the values found in control fish. Natural cytotoxic activity was increased in leucocytes from fish fed for 1 week with 3750 U kg^?1 cholecalciferol. No significant differences were observed in complement activity or in respiratory burst activity in the assayed conditions. These results suggested that dietary vitamin D₃ administration has an effect on the innate immune parameters of gilthead seabream. The immunostimulant effect was greater on the cellular innate immune parameters assayed, suggesting that similar receptors to those present in mammals are involved in the action of this vitamin in the fish immune system.     

Growth performance and starch utilization in common carp (Cyprinus carpio L.) in response to dietary chromium chloride supplementation

A nutrition trial was conducted on juvenile common carp (Cyprinus carpio), initial mean body weight 15 ± 0.4 g within a controlled facility at 25 ± 0.5 °C. Six diets containing various levels of supplementary Cr (0, 0.2, 0.5, 1.0, 1.5, and 2.0) mg Cr/kg of diet as Cr chloride hexahydrate were fed to carp for a period of 10 weeks. Lower growth performance was observed in fish fed on the control diet and the diet supplemented with the highest level of Cr (2.0 mg Cr/kg). Although fish fed 0.5 mg Cr/kg showed the best growth performance, this was not significantly different (P > 0.05) from fish fed 1.0 mg Cr/kg. The regression of plasma glucose concentration was linear (R² = 0.97 and P value = 0.001) as the Cr content of the diet increased (up to 1.5 mg Cr/kg).Cr carcass content was elevated with an increasing level of dietary Cr supplementation up to 1.5 mg Cr/kg; but fish fed on the diet supplemented with the highest level of Cr (2.0 mg Cr/kg) showed a decrease in Cr carcass content.Histological examination to evaluate the impact of different Cr supplementation on liver and gut tissues showed notable changes. The higher level of Cr (2.0 mg Cr/kg) in the diet gave rise to elevated hepatocyte vacuolization and changes in gut tissue morphology.It appeared that Cr chloride significantly improved growth within a defined range (0.2–1.5) mg Cr/kg without any negative impact, while 2.0 mg Cr/kg in carp diet seems to be the threshold for the initiation of toxicity.     

Potential use of green macroalgae Ulva lactuca as a feed supplement in diets on growth performance,feed utilization and body composition of the African catfish,Clarias gariepinus

This study aimed to evaluate the effects of diet containing the green macroalgae, Ulva lactuca, on the growth performance, feed utilization and body composition of African catfish Clarias gariepinus. Four experimental diets were formulated: D1 as a control group and D2, D3 and D4 which included 10%, 20% and 30% U. lactuca meal, respectively. 180 African catfish, weighing 9.59 ± 0.43 g, and with an average length of 11.26 ± 0.21, (mean ± SE) were divided into four groups corresponding to the different feeding regimes. The final body weight of the fish showed insignificant differences (P > 0.05) between the control and fish fed D2, whereas, there was a significant difference (P < 0.05) between these two diets compared with D3 and D4, with weights of 70.52, 60.92, 40.57 and 35.66 g recorded for D1, D2, D3 and D4, respectively. In the same trend significant differences were also evident in weight gain, specific growth rate and feed utilization. Fish fed with a diet containing 20% or 30% U. lactuca meal had poorer growth performance and feed utilization. Protein productive value, protein efficiency ratio, daily dry feed intake and total feed intake were also significantly lower in fish fed with D3 and D4 than in the control D1 and D2.Overall, the results of the experiment revealed that African catfish fed a diet with U. lactuca included at 20% and 30% levels showed poorer growth and feed utilization than the control group and fish fed diets containing 10% of U. lactuca.     

Experimental antibacterial therapy with puroindolines,lactoferrin and lysozyme in Listeria monocytogenes-infected mice

Puroindoline A and puroindoline B from plant seeds, bovine lactoferrin and chicken eggs lysozyme are antimicrobial proteins of innate immune system that lyse invading organisms. We investigate their potential antibacterial activity against Listeria monocytogenes in a mouse model. Bacteria were isolated from various organs for 7 days after challenge. Livers displayed consistently higher bacterial count (up to 10⁷ cfu/g) than spleens, kidneys and brains. The efficacy of the AMPs was therefore established by measuring the infection level (cfu number) of these organs. Puroindoline A and puroindoline B (5 mg/mouse), lactoferrin and lysozyme (1.25 mg/mouse), intravenously injected individually, inhibited bacterial growth completely. Puroindoline A, puroindoline B and lactoferrin were effective when administered 24 h before infection; lysozyme was effective at the time of infection or 5 days after. Their combined use resulted in the enhancement of individual antibacterial activities. Complete inhibition of bacterial growth was observed using concurrently 0.059 mg/mouse of puroindoline A and 0.019 mg/mouse of puroindoline B, lactoferrin and lysozyme. Individual antimicrobial proteins reduced significantly the expression level of pro-inflammatory cytokines (IL-6, IL-8, INF-γ and TNF-α), acute phase proteins (C-reactive protein and fibrinogen) and the T lymphocyte antigens CD4, CD8a, CD8b and CD25. These results suggest their potential use for the control of L. monocytogenes infections.     

Elements of margin of safety,toxicity and action of sodium selenite in a lipopolysaccharide rat model

ProjectBoth septic shock and sodium selenite (Na₂SeO₃) lead to multiple organ failure through oxidation. Na₂SeO₃ has direct oxidant effects above the nutritional level and indirect anti-oxidant properties.In a lipopolysaccharide (LPS) rat model we assessed margin of safety, toxicity and beneficial effect of pentahydrate Na₂SeO₃ (5H₂O·Na₂SeO₃) at oxidant doses.ProcedureIn a three-step study on 204 rats we: (i) observed toxic effects of Na₂SeO₃ injected intraperitoneously (IP) and determined its Minimum Dose Without Toxic effect (MDWT) 0.25–0.35 mg/kg selenium (Se) content; (ii) injected IP LPS at 70% lethal dose (LD) followed, or not, one hour later by IP Na₂SeO₃ at MDWT and (iii) by doses > MDWT. At 48 h, in survivors, we measured plasma creatinine, lactate, aspartate and alanine aminotransferase (AST, ALT), nitric oxide (NO) and Se concentrations.Results(i) Na₂SeO₃ alone did not increase NO and lactate. Encephalopathy appeared at 1 mg Se/kg. Creatinine increased at 1–1.75 mg Se/kg, AST, ALT at 3–4.5 mg Se/kg, and the minimum LD was 3 mg Se/kg. (ii) Mortality after LPS was 37/50 (74%, [62–86%]) vs. 20/30 (67%, [50–84%]) when followed by Na₂SeO₃ at MDWT (p = 0.483) with a decreased in NO (−31%, p = 0.038) a trend for lactate decrease (−19%, p = 0.068) and an increased Se in plasma of survivals. (iii) All rats died at doses ≥0.6 mg/kg (p < 0.001).ConclusionMechanisms of LPS and Na₂SeO₃ toxicity differ (i.e. NO, lactate). In septic shock 5H₂O·Na₂SeO₃ toxicity increased, margin of safety decrease, but IP administration of dose considered as oxidant of 5H₂O·Na₂SeO₃ showed beneficial effects.     

Exploring LPS-induced sepsis in rats and mice as a model to study potential protective effects of the nociceptin/orphanin FQ system

The nociceptin receptor (NOP) and its ligand nociceptin/orphanin FQ (N/OFQ) have been shown to exert a modulatory effect on immune cells during sepsis. We evaluated the suitability of an experimental lipopolysaccharide (LPS)-induced sepsis model for studying changes in the nociceptin system. C57BL/6 mice BALB/c mice and Wistar rats were inoculated with different doses of LPS with or without a nociceptin receptor antagonist (UFP-101 or SB-612111). In C57BL/6 mice LPS 0.85 mg/kg injection produced no septic response, whereas 1.2 mg/kg produced a profound response within 5 h. In BALB/c mice, LPS 4 mg/kg produced no response, whereas 7 mg/kg resulted in a profound response within 24 h. In Wistar rats LPS 15 mg/kg caused no septic response in 6/10 animals, whereas 25 mg/kg resulted in marked lethargy before 24 h. Splenic interleukin-1β mRNA in BALB/c mice, and serum TNF-α concentrations in Wistar rats increased after LPS injection in a dose-dependent manner, but were undetectable in control animals, indicating that LPS had stimulated an inflammatory reaction. IL-1β and TNF-α concentrations in LPS-treated animals were unaffected by administration of a NOP antagonist. Similarly NOP antagonists had no effect on survival or expression of mRNA for NOP or ppN/OFQ (the N/OFQ precursor) in a variety of tissues. In these animal models, the dose–response curve for LPS was too steep to allow use in survival studies and no changes in the N/OFQ system occurred within 24 h. We conclude that LPS-inoculation in rodents is an unsuitable model for studying possible changes in the NOP-N/OFQ system in sepsis.     

Effects of PEGylated porcine glucagon-like peptide-2 therapy in weaning piglets challenged with lipopolysaccharide

This study aims to evaluate the therapeutic effect of polyethylene glycosylated porcine glucagon-like peptide-2 (pGLP-2), a long-acting form of pGLP-2, in lipopolysaccharide (LPS)-challenged piglets. Eighteen 21-day-old weaning piglets were randomly assigned into three groups: control (saline solution), LPS (100 μg/kg LPS), and PEG–pGLP-2 (10 nmol/kg PEG–pGLP-2 + 100 μg/kg LPS). All treatments were administered intraperitoneally. Compared with the control treatment, LPS treatment significantly decreased (P < 0.05) the villus heights of the duodenum and jejunum, as well as the villus height/crypt depth ratio of the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P > 0.05). Specifically, PEG–pGLP-2 infusion significantly increased the villus height/crypt depth ratio of the duodenum (P < 0.05) compared with LPS treatment. Compared with the control treatment, LPS treatment significantly increased (P < 0.05) the mRNA expression levels of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) in the jejunum. However, PEG–pGLP-2 therapy reduced these effects (P < 0.05). Specifically, PEG–pGLP-2 infusion significantly decreased (P < 0.05) the mRNA expression levels of interleukin (IL)-8 and TNF-α in the duodenum and jejunum, IL-10 in the duodenum, and IFN-γ in the jejunum compared with the LPS treatment. LPS treatment increased the caspase-3 activity of the ileum mucosal (P < 0.05), and this effect was significantly reduced by PEG–pGLP-2 treatment. These results indicate that PEG–pGLP-2 infusion alleviates the severity of intestinal injury in weaning piglets by reducing the secretion of inflammatory cytokines and the caspase-3 activity, and increasing the villus height/crypt depth ratio.     

Heterologous production of caffeic acid from tyrosine in Escherichia coli

Caffeic acid is a plant secondary metabolite and its biological synthesis has attracted increased attention due to its beneficial effects on human health. In this study, Escherichia coli was engineered for the production of caffeic acid using tyrosine as the initial precursor of the pathway. The pathway design included tyrosine ammonia lyase (TAL) from Rhodotorula glutinis to convert tyrosine to p-coumaric acid and 4-coumarate 3-hydroxylase (C3H) from Saccharothrix espanaensis or cytochrome P450 CYP199A2 from Rhodopseudomonas palustris to convert p-coumaric acid to caffeic acid. The genes were codon-optimized and different combinations of plasmids were used to improve the titer of caffeic acid. TAL was able to efficiently convert 3 mM of tyrosine to p-coumaric acid with the highest production obtained being 2.62 mM (472 mg/L). CYP199A2 exhibited higher catalytic activity towards p-coumaric acid than C3H. The highest caffeic acid production obtained using TAL and CYP199A2 and TAL and C3H was 1.56 mM (280 mg/L) and 1 mM (180 mg/L), respectively. This is the first study that shows caffeic acid production using CYP199A2 and tyrosine as the initial precursor. This study suggests the possibility of further producing more complex plant secondary metabolites like flavonoids and curcuminoids.     

Evaluation of red seaweed Gracilaria arcuata as dietary ingredient in African catfish,Clarias gariepinus

The aim of this study was to evaluate the use of dried marine seaweed, Gracilaria arcuata for the first time as dietary ingredient in partial substitution of fishmeal on the growth performance, feed utilization and body composition of African catfish, Clarias gariepinus. Four experimental diets were formulated: D1 as a control group; D2; D3 and D4 which included 10%, 20% and 30% G. arcuata meal respectively. One hundred and eighty African catfish weighing 9.62 ± 0.42 g, (mean ± SE) was divided into four groups corresponding to the different feeding regimes. The final body weight of the fishes showed significant differences (P < 0.05) between the control (D1); D2 and other treated groups D3 and D4, with weights of 66.98, 59.60, 47.34 and 30.73 g recorded for D1, D2, D3 and D4, respectively. Significant differences (P < 0.05) were also evident in weight gain, specific growth rate, and feed utilization between treatment and control groups. However, no significant differences (P > 0.05) were observed between the control group and fishes fed D2 for all previous parameters. Protein productive value, protein efficiency ratio, daily dry feed intake and total feed intake were also significantly lower in fish fed with a diet containing G. arcuata than in the control group and D2 which contains 10% of G. arcuata. Overall, the results of the experiment revealed that African catfish fed a diet with G. arcuata included in 20% and 30% levels showed poorer growth and feed utilization than the control group and D2. However, the study recommended that C. gariepinus can accept this ingredient up to 10% in their diets. More defined experiments therefore seem to be necessary in order to determine the maximum level of this marine seaweed in diets with amino acid supplementation for African catfish.     

Chronic treatment with cyclosporine affects systemic purinergic parameters,homocysteine levels and vascular disturbances in rats

Vascular disease is a major cause of morbidity and mortality among transplanted recipients and cyclosporine (CsA) treatment has been consistently implicated in this event. In this study we assessed total blood homocysteine levels (tHcy), ecto-nucleotidase activities and adenine nucleotide/nucleoside levels searching for parameters related to the mechanisms of vascular damage induced by chronic CsA treatment in non-transplanted rats. Thirty male Wistar rats were divided in three groups: control group treated with corn oil, CsA 5 mg/kg and CsA 15 mg/kg, administered by daily gastric gavage during 8 weeks. CsA 15 mg/kg treatment increased blood levels of tHcy. Both CsA treatments (5 mg/kg and 15 mg/kg) decreased adenine nucleotides hydrolysis by ecto-nucleotidases in serum, which negatively correlated with tHcy levels (r: ?0.74, r: ?0.63 and r: ?0.63, p < 0.004, for ATP, ADP and AMP, respectively). CsA 15 mg/kg induced a statistically significant increase in ADP and decrease in adenosine (ADO) plasma levels compared to control group. THcy levels were positively correlated with plasma ADP levels and negatively correlated with ADO levels (r: 0.84, p < 0.0001 and r: ?0.68, p < 0.0001, respectively). Rats under CsA 15 mg/kg treatment presented cell injury and inflammatory responses in the endothelium and intima layer of the aorta artery. In conclusion, blood ecto-nucleotidases activity, tHcy, and ADP and ADO levels may be implicated in vascular injury induced by CsA treatment.     

The effects of feeding on the swimming performance and metabolic response of juvenile southern catfish,Silurus meridionalis,acclimated at different temperatures

To test whether the effects of feeding on swimming performance vary with acclimation temperature in juvenile southern catfish (Silurus meridionalis), we investigated the specific dynamic action (SDA) and swimming performance of fasting and feeding fish at acclimation temperatures of 15, 21, 27, and 33 °C. Feeding had no effect on the critical swimming speeding (U_crit) of fish acclimated at 15 °C (p = 0.66), whereas it elicited a 12.04, 18.70, and 20.98% decrease in U_crit for fish acclimated at 21, 27 and 33 °C, respectively (p < 0.05). Both the maximal postprandial oxygen consumption rate (VO_2peak) and the active metabolic rate (VO_2active, maximal aerobic sustainable metabolic rate of fasting fish) increased significantly with temperature (p < 0.05). The postprandial maximum oxygen consumption rates during swimming (VO_2max) were higher than the VO_2active of fasting fish at all temperature groups (p < 0.05). The VO_2max increased with increasing temperature, but the relative residual metabolic scope (VO_2max? VO_2peak) during swimming decreased with increasing in temperature. The present study showed that the impairment of postprandial swimming performance increased with increasing temperature due to the unparalleled changes in the catfish's central cardio-respiratory, peripheral digestive and locomotory capacities. The different metabolic strategies of juvenile southern catfish at different temperatures may relate to changes in oxygen demand, imbalances in ion fluxes and dissolved oxygen levels with changes in temperature.     

Anti-apoptotic activity of gentiopicroside in d-galactosamine/lipopolysaccharide-induced murine fulminant hepatic failure

This study investigated the hepatoprotective effects of gentiopicroside on d-galactosamine (d-GalN) and lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were administrated orally with gentiopicroside (40 or 80 mg/kg body weight) at 12 h and 1 h before d-GalN (700 mg/kg)/LPS (10 μg/kg) injection. Gentiopicroside markedly reduced the increases in serum aminotransferase activities and lipid peroxidation. The glutathione content decreased in d-GalN/LPS alone group, and this decrease was attenuated by gentiopicroside. Increases in serum tumor necrosis factor-α (TNF-α), which were observed in d-GalN/LPS alone group, were significantly reduced by gentiopicroside. Importantly, gentiopicroside attenuated d-GalN/LPS-induced apoptosis of hepatocytes, as estimated by the caspase-3 cleavage, poly(ADP-ribose) polymerase (PARP) cleavage, and DNA fragmentation. d-GalN/LPS-induced caspase-8 and -9 activation was significantly suppressed by gentiopicroside. Moreover, increased cytosolic cytochrome c protein was reduced by gentiopicroside. Also, the increased ratio of Bax and Bcl-2 protein was significantly attenuated by gentiopicroside. After 6 h of d-GalN/LPS injection, phosphorylated c-jun N-terminal kinase (JNK) and extracellular signal regulated kinase (ERK) was significantly increased, whereas phosphorylation JNK and ERK were attenuated by gentiopicroside. Our results suggest that gentiopicroside offers remarkable hepatoprotection against damage induced by d-GalN/LPS related with its anti-apoptotic activities.     

The protective efficacy of magnolol in hind limb ischemia-reperfusion injury

We investigated the protective effects of magnolol, an active antioxidant and free radical scavenger extracted from Magnolia officinalis, in a hind limb ischemic-reperfusion animal model. Adult male Spraque-Dawley rats were subjected to hind limb ischemic insult for 2 hours and were intravenously treated with magnolol at 0.01 mg/kg (n=8), 0.3 mg/kg (n=8) mg/kg or 1 mg/kg (n=8) mg/kg, or vehicle (n=8). At 24 h post-insult, the levels of nitrite/nitrate (NOX), malondialdehyde (MDA) and myeloperoxidase (MPO), as well as the degree of muscle damage, were assessed. Relative to controls, animals treated with magnolol (0.3 and 1 mg/kg) had attenuated muscular inflammation, edema and damage. Magnolol (0.3–1 mg/kg) also effectively reduced postischemic rises in the MDA, NOx and MPO levels (p<0.05, respectively). Magnolol administrated at 0.01 mg/kg, however, failed to protect against the ischemic-perfusion limb injury. In addition, magnolol (0.01–1 mg/kg) did not affect local muscular blood reperfusion or other physiological parameters, including hematocrit, glucose, arterial blood gases and mean arterial blood pressure. Thus, intravenous administration with magnolol at 0.3–1 mg/kg protects against ischemic limb damage in rats. This cytoprotection may be attributed to its antioxidant, anti-nitrosative and anti-inflammatory actions.     

Seasonal variations in the body composition and bioaccumulation of heavy metals in Nile tilapia collected from drainage canals in Al-Ahsa,Saudi Arabia

The body composition of Nile tilapia (Oreochromis niloticus) collected from drainage canals in Al-Ahsa, Saudi Arabia and the concentration of four heavy metals; zinc (Zn), cadmium (Cd), cobalt (Co) and lead (Pb) in both fish muscles and the water collected from this environment were assessed across the four seasons. The body composition was found to change with the seasons, with the best body composition being recorded in autumn and winter, where higher levels of protein (17.24, 17.65%), and fat (0.58, 0.71%) and lower water content (80.15, 79.86%) respectively were noted. The concentration of heavy metals in both fish muscles and the water body also varied significantly with the seasons. In the fish muscles, the highest content of Zn (0.409 mg/kg dry weight) and Cd (4.140 mg/kg dry weight) was recorded in winter, however, the highest concentration of Co (0.318 mg/kg dry weight) and Pb (1.96 mg/kg dry weight) was observed in spring and summer respectively. On the other hand, the water samples collected in autumn showed the maximum concentration of Cd (1.385 mg/L), Co (0.762 mg/L) and Pb (0.18 mg/L) however, the maximum concentration of Zn (0.0041 mg/L) was recorded in winter. With the exception of Cd, the accumulation of the studied heavy metals in fish muscles was within the safe limits for seafood recommended by various organizations.     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 1

We investigated proinflammatory cytokine TNFα production inhibitors in order to develop novel anti-inflammatory agents. According to the results, we found that 17, a pyrrole derivative possessing a tetrahydropyridine group at the β-position, showed potent inhibitory activity in vitro (inhibition of lipopolysaccharide (LPS) induced TNFα production in human whole blood, IC₅₀ = 1.86 μM) and in vivo (inhibition of LPS induced TNFα production in mice, ID₅₀ = 5.98 mg/kg).     

Tetrahydropyridine derivatives with inhibitory activity on the production of proinflammatory cytokines: Part 3

In order to develop a new class of anti-rheumatic drug which inhibits production of proinflammatory cytokines such as TNFα, IL-1β, IL-6, and IL-8, a series of 3-pyridylpyrrole derivatives possessing a bicyclic tetrahydropyridine moiety at the 4-position of the pyrrole ring were synthesized and their pharmacological activities were evaluated. The derivatives were found to have potent inhibitory activities on the production of the cytokines both in vitro and in vivo. Among them, compound 4a, (S)-2-(4-fluorophenyl)-4-(1,2,3,5,6,8a-hexahydroindolizin-7-yl)-3-(pyridin-4-yl)-1H-pyrrole (R-132811), achieved the most promising results in various in vitro and in vivo tests including several rheumatoid arthritis models ((i) inhibition of p38α, p38β, p38γ, and p38δ MAP kinases: IC₅₀ = 0.034, 0.572, >10, and >10 μM, respectively; (ii) inhibition of TNFα, IL-1β, IL-6, and IL-8 production in human whole blood: IC₅₀ = 0.026, 0.020, 0.88, and 0.016 μM, respectively; (iii) inhibition of LPS induced TNFα, IL-1β and IL-6 production in mice: ID₅₀ = 0.93, 8.63, and 0.11 mg/kg, po, respectively; (iv) inhibition of anti-collagen antibody-induced arthritis in mice: ID₅₀ = 2.22 mg/kg, po; (v) inhibition of collagen-induced arthritis in mice: ID₅₀ = 2.38 mg/kg, po; (vi) prophylactic effect on adjuvant-induced arthritis in rats: ID₅₀ = 3.1 mg/kg, po; (vii) therapeutic effect on adjuvant-induced arthritis in rats: ID₅₀ = 4.9 mg/kg, po; (viii) analgesic effect on adjuvant-induced arthritic pain in rats: ID₅₀ = 2.9 mg/kg, po). As a result, compound 4a was chosen as a candidate for further pre-clinical studies.