HPLC and GC Characterization of Dicliptera bupleuroides Aerial Parts and Evaluation of Its Anti-Inflammatory Potential in Vitro, in silico and in Vivo Using Carrageenan and Formalin Induced Inflammation in Rat Models.

The current study aimed to evaluate the anti-inflammatory activity of Dicliptera bupleuroides Nees aerial parts methanol extract and its different fractions namely hexane, chloroform, ethyl acetate and butanol in vitro using cyclooxygenase inhibitory assay (COX-2). In vivo anti-inflammatory evaluation was performed using carrageenan and formalin induced inflammation in rat models followed by molecular docking. High performance liquid chromatography (HPLC) and gas chromatography coupled with mass chromatography (GC/MS) analyses were used for chemical analyses of the tested samples. The tested samples showed significant inhibition in COX-2 inhibitory assay where methanol extract (DBM) showed the highest inhibitory potential at 100 µg/mL estimated by 67.86 %. At a dose of 400 mg/kg, all of the examined samples showed pronounced results in carrageenan induced acute inflammation in rat model at 4th h interval with DBM showed the highest efficiency displaying 65.32 % inhibition as compared to the untreated rats. Formalin model was employed for seven days and DBM exhibited 65.33 % and 69.39 % inhibition at 200 and 400 mg/kg, respectively approaching that of the standard on the 7th day. HPLC revealed the presence of caffeic acid, gallic acid and sinapic acid, quercetin and myricetin in DBM. GC/MS analysis of its hexane fraction revealed the presence of 16 compounds belonging mainly to fatty acids and sterols that account for 85.26 % of the total detected compounds. Molecular docking showed that hexadecanoic acid followed by decanedioic acid and isopropyl myristate showed the best fitting within cyclooxygenase-II (COX-II) while nonacosane followed by hexatriacontane and isopropyl myristate revealed the most pronounced fitting within the 5-lipoxygenase (5-LOX) active sites. Absorption, metabolism, distribution and excretion and toxicity prediction (ADMET/ TOPKAT) concluded that most of the detected compounds showed reasonable pharmacokinetic, pharmacodynamic and toxicity properties that could be further modified to be more suitable for incorporation in pharmaceutical dosage forms combating inflammation and its undesirable consequences.

Towards PET imaging of the dynamic phenotypes of microglia.

There is increasing evidence showing the heterogeneity of microglia activation in neuroinflammatory and neurodegenerative diseases. It has been hypothesized that pro-inflammatory microglia are detrimental and contribute to disease progression, while anti-inflammatory microglia play a role in damage repair and remission. The development of therapeutics targeting the deleterious glial activity and modulating it into a regenerative phenotype relies heavily upon a clearer understanding of the microglia dynamics during disease progression and the ability to monitor therapeutic outcome in vivo. To that end, molecular imaging techniques are required to assess microglia dynamics and study their role in disease progression as well as to evaluate the outcome of therapeutic interventions. Positron emission tomography (PET) is such a molecular imaging technique, and provides unique capabilities for non-invasive quantification of neuroinflammation and has the potential to discriminate between microglia phenotypes and define their role in the disease process. However, several obstacles limit the possibility for selective in vivo imaging of microglia phenotypes mainly related to the poor characterization of specific targets that distinguish the two ends of the microglia activation spectrum and lack of suitable tracers. PET tracers targeting translocator protein 18 kDa (TSPO) have been extensively explored, but despite the success in evaluating neuroinflammation they failed to discriminate between microglia activation statuses. In this review, we highlight the current knowledge on the microglia phenotypes in the major neuroinflammatory and neurodegenerative diseases. We also discuss the current and emerging PET imaging targets, the tracers and their potential in discriminating between the pro- and anti-inflammatory microglia activation states.

Inhibition of Opisthorchis felineus glutathione-dependent prostaglandin synthase by resveratrol correlates with attenuation of cholangiocyte neoplasia in a hamster model of opisthorchiasis.

Food-borne trematodiases represent major neglected parasitic diseases. Trematodes of the family Opisthorchiidae including Opisthorchis felineus, Opisthorchis viverrini and Clonorchis sinensis are ranked eight on the global list of the 24 most prevalent food-borne parasites. Chronic O. felineus infection symptoms include precancerous lesions with the potential for malignancy. In recent decades, liver flukes of the family Opisthorchiidae have been extensively scientifically explored, however despite this the molecular mechanisms of O. felineus pathogenicity and its carcinogenic potential have not been studied. Opisthorchis felineus glutathione-dependent prostaglandin synthase (GST σ) is the major component of the excretory-secretory product of this liver fluke. We hypothesised that the activity of this enzyme is involved in the infection pathogenesis, including the formation of precancerous lesions. To test this hypothesis and to gain insights into the mechanisms of precancerous lesion formation, we (i) investigated whether excretory parasitic GST σ retains its enzymatic activity, (ii) tested resveratrol (RSV) as a possible inhibitor of this enzyme, and (iii) assessed biliary neoplasia and oxidative DNA damage as well as the expression of neoplasia and fibrogenesis marker genes after prolonged administration of RSV in a hamster model. RSV was found to inhibit GST σ enzymatic activity in a dose-dependent manner (R = 0.85, P < 0.001; half-maximal effective dose (ED50) = 48.6 µM). Prolonged administration of RSV significantly suppressed high-grade biliary neoplasia (P = 0.008), attenuated upregulation of hyperplasia and fibrogenesis-related genes (Tgfb, α-SMA and CK7), and decreased the elevated oxidative DNA damage. Taking into account that RSV can influence a wide range of pathways, further research is needed to confirm the role of GST σ in O. felineus pathogenicity. Nevertheless, the chemopreventive effect of RSV targeting biliary neoplasia formation might be useful for improving the outcomes in infected populations and represents a compelling rationale for RSV testing in combination chemotherapy of opisthorchiasis.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage.

OBJECTIVES: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. METHODOLOGY: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). RESULTS: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. CONCLUSIONS: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Periapical bone response to bacterial lipopolysaccharide is shifted upon cyclooxygenase blockage

Abstract Objectives: Infection, inflammation and bone resorption are closely related events in apical periodontitis development. Therefore, we sought to investigate the role of cyclooxygenase (COX) in osteoclastogenesis and bone metabolism signaling in periapical bone tissue after bacterial lipopolysaccharide (LPS) inoculation into root canals. Methodology: Seventy two C57BL/6 mice had the root canals of the first molars inoculated with a solution containing LPS from E. coli (1.0 mg/mL) and received selective (celecoxib) or non-selective (indomethacin) COX-2 inhibitor. After 7, 14, 21 and 28 days the animals were euthanized and the tissues removed for total RNA extraction. Evaluation of gene expression was performed by qRT-PCR. Statistical analysis was performed using analysis of variance (ANOVA) followed by post-tests (α=0.05). Results: LPS induced expression of mRNA for COX-2 (Ptgs2) and PGE2 receptors (Ptger1, Ptger3 and Ptger4), indicating that cyclooxygenase is involved in periapical response to LPS. A signaling that favours bone resorption was observed because Tnfsf11 (RANKL), Vegfa, Ctsk, Mmp9, Cd36, Icam, Vcam1, Nfkb1 and Sox9 were upregulated in response to LPS. Indomethacin and celecoxib differentially modulated expression of osteoclastogenic and other bone metabolism genes: celecoxib downregulated Igf1r, Ctsk, Mmp9, Cd36, Icam1, Nfkb1, Smad3, Sox9, Csf3, Vcam1 and Itga3 whereas indomethacin inhibited Tgfbr1, Igf1r, Ctsk, Mmp9, Sox9, Cd36 and Icam1. Conclusions: We demonstrated that gene expression for COX-2 and PGE2 receptors was upregulated after LPS inoculation into the root canals. Additionally, early administration of indomethacin and celecoxib (NSAIDs) inhibited osteoclastogenic signaling. The relevance of the cyclooxygenase pathway in apical periodontitis was shown by a wide modulation in the expression of genes involved in both bone catabolism and anabolism.

Expression and localization of cyclooxygenases in the oviduct of laying hens during the ovulatory cycle.

Prostaglandins (PGs) play important roles in regulation of the functions of the hen oviduct. However, little is known about the expression and localization of the rate-limiting cyclooxygenases (COX-1 and COX-2) in the oviduct. The aim of this study was to determine the COXs expression and localization in the different segments of the oviduct and to investigate changes in their expression levels during the ovulatory cycle of laying hens. White Leghorn laying hens were killed at 0, 4, 7, 16 and 24 h after oviposition, and samples from the infundibulum, magnum, isthmus, uterus, and vagina were collected. Gene and protein expressions were examined by real-time PCR and western blot, respectively, for both COX-1 and COX-2. Localization of COX-1 and COX-2 in the hen oviduct was determined by immunohistochemistry and PCR analysis of samples collected by laser capture microdissection (LCM). The expression level of COX-1 was highest in the infundibulum, while that of COX-2 was significantly higher in the uterus than in the other segments. The expression levels of COX-1 in the infundibulum and COX-2 in the uterus were higher at 0 and 24 h after oviposition, just prior to subsequent ovulation and oviposition. Western blot analysis confirmed the presence of COX-1 and COX-2 in all oviductal segments. The density of COX-2 was the highest in the uterus, and did not change during the ovulatory cycle. COX-1 and COX-2 were localized in the surface epithelium of all oviductal segments besides the uterine tubular glands. We conclude that both COXs are differentially expressed in the different oviductal segments with a temporal association to ovulation and oviposition. COX-1 and COX-2 may play an important role in the infundibulum and uterus, respectively, and COX-2 may be one of the factors regulating the induction of oviposition.

Evaluation of the role of the cyclooxygenase signaling pathway during inflammation in skin and muscle tissues of ball pythons (Python regius).

OBJECTIVE To determine degrees of production of cyclooxygenase (COX)-1 and -2 and other mediators of inflammation in noninflamed and inflamed skin and muscle tissues in ball pythons (Python regius). ANIMALS 6 healthy adult male ball pythons. PROCEDURES Biopsy specimens of noninflamed skin and muscle tissue were collected from anesthetized snakes on day 0. A 2-cm skin and muscle incision was then made 5 cm distal to the biopsy sites with a CO2 laser to induce inflammation. On day 7, biopsy specimens of skin and muscle tissues were collected from the incision sites. Inflamed and noninflamed tissue specimens were evaluated for production of COX-1, COX-2, phosphorylated protein kinase B (AKT), total AKT, nuclear factor κ-light-chain-enhancer of activated B cells, phosphorylated extracellular receptor kinases (ERKs) 1 and 2, and total ERK proteins by western blot analysis. Histologic evaluation was performed on H&E-stained tissue sections. RESULTS All biopsy specimens of inflamed skin and muscle tissues had higher histologic inflammation scores than did specimens of noninflamed tissue. Inflamed skin specimens had significantly greater production of COX-1 and phosphorylated ERK than did noninflamed skin specimens. Inflamed muscle specimens had significantly greater production of phosphorylated ERK and phosphorylated AKT, significantly lower production of COX-1, and no difference in production of COX-2, compared with production in noninflamed muscle specimens. CONCLUSIONS AND CLINICAL RELEVANCE Production of COX-1, but not COX-2, was significantly greater in inflamed versus noninflamed skin specimens from ball pythons. Additional research into the reptilian COX signaling pathway is warranted.

Induction of omega 6 inflammatory pathway by sodium metabisulfite in rat liver and its attenuation by ghrelin.

BACKGROUND: Sodium metabisulfite is commonly used as preservative in foods but can oxidize to sulfite radicals initiating molecular oxidation. Ghrelin is a peptide hormone primarily produced in the stomach and has anti-inflammatory effects in many organs. This study aimed to assess endogenous omega-3 (n-3) and omega-6 (n-6) polyunsaturated fatty acids (PUFAs) in rat peripheral organs following sodium metabisulfite treatment and determine the possible effect of ghrelin on changes in n-6 inflammatory pathway. METHODS: Male Wistar rats included in the study were allowed free access to standard rat chow. Sodium metabisulfite was given by gastric gavage and ghrelin was administered intraperitoneally for 5 weeks. Levels of arachidonic acid (AA, C20:4n-6), dihomo-gamma-linolenic acid (DGLA, C20:3n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) in liver, heart and kidney tissues were determined by an optimized multiple reaction monitoring (MRM) method using ultra fast-liquid chromatography (UFLC) coupled with tandem mass spectrometry (MS/MS). Cyclooxygenase (COX) and prostaglandin E2 (PGE2) were measured in tissue samples to evaluate changes in n-6 inflammatory pathway. RESULTS: Omega-6 PUFA levels, AA/DHA and AA/EPA ratio were significantly increased in liver tissue following sodium metabisulfite treatment compared to controls. No significant change was observed in heart and kidney PUFA levels. Tissue activity of COX and PGE2 levels were also significantly increased in liver tissue of sodium metabisulfite treated rats compared to controls. Ghrelin treatment decreased n-6 PUFA levels and reduced COX and PGE2 levels in liver tissue of sodium metabisulfite treated rats. CONCLUSION: Current results suggest that ghrelin exerts anti-inflammatory action through modulation of n-6 PUFA levels in hepatic tissue.

[Extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process].

OBJECTIVE: To investigate extracellular splitting pattern of mitochondria and the depressant effects of CsA on the process and explore the mechanism of post-traumatic SIRS and its therapeutic strategy. METHODS: Ten male SD rats with 60 to 70 days age and 240 to 280 g weight were used for mitochondrial isolation. Freshly isolated mitochondria were randomly divided into two groups, which were cultured in blood plasma with or without CsA respectively for 8 h. COX and MDH were assayed by ELISA every 30 min. Meanwhile, Rat macrophage cell line NR8383 were treated as follows, control (group A): cultivation with normal medium; NR8383+CsA co-culture group (group B): culture medium was supplemented with CsA of 10 mmol/L; NR8383+intact mitochondria co-culture group (group C): culture medium was supplemented with intact mitochondria (mtDNA=5 g/ml); NR8383+intact mitochondria+CsA co-culture group (group D): culture medium was supplemented with intact mitochondria (mtDNA=5 µg/ml)and CsA of 10 mmol/L; NR8383+disrupted mitochondria co-culture group (group E): culture medium was supplemented with disrupted mitochondria (mtDNA=5 µg/ml); NR8383+disrupted mitochondria+CsA co-culture group (group F): culture medium was supplemented with disrupted mitochondria (mtDNA=5 µg/ml)and CsA of 10 mmol/L. TNF-α and IL-6 concentrations in supernatant were assessed at 1, 3, 5 h after culture. RESULTS: In the mitochondria plasma cultures, MDH and COX levels were increased with the time and peaked at about 3 h and 3.5 h; CsA can delay the appearance of peak to 4.5 h. Among different treated groups,there was no significant difference in TNF-α and IL-6 between group A and group B; there was significant difference in TNF-α and IL-6 other groups. After 1 h culture, compared with group C, no significant difference of TNF-α and IL-6 was observed in group D, while TNF-α and IL-6 were significant higher in group E; after 3 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while TNF-α and IL-6 were significantly higher in group E; after 5 h culture, compared with group C, TNF-α and IL-6 were significantly lower in group D, while no significant difference of TNF-α and IL-6 were observed in group E. At each time point, there was no significant difference in TNF-α and IL-6 between group F and group E. CONCLUSION: Mitochondria can split in serum with time, which will further activate macrophages. CsA has depressant effect to mitochondrial splitting on the process and will therefore inhibit the activation of macrophages.

2-Acetoxyverecynarmin C, a new briarane COX inhibitory diterpenoid from Pennatula aculeata.

A new briarane-type diterpenoid, named 2-acetoxyverecynarmin C, was isolated from the methanolic extract of an octocoral, Pennatula aculeata, that exhibited cyclooxygenase (COX) inhibitory activity. The structure of the compound was elucidated by ESI-HRMS, 1D and 2D NMR spectroscopy and comparison of the measured spectral data with those reported in the literature. The relative stereochemistry at chiral carbons was established from 2D NOESY experiments. 2-Acetoxyverecynarmin C is a tricyclic compound containing a furan ring at C-7,8 of a briarane skeleton. 2-Acetoxyverecynarmin C showed moderate inhibitory activity in in vitro COX-1 and COX-2 assays.

Effects of copper on CHO cells: insights from gene expression analyses.

Copper concentration can impact lactate metabolism in Chinese Hamster ovary (CHO) cells. In our previous study, a 20-fold increase in initial copper concentration enabled CHO cultures to shift from net lactate production to net lactate consumption, and achieve higher cell growth and productivity. In this follow-up study, we used transcriptomics to investigate the mechanism of action (MOA) of copper that mediates this beneficial metabolism shift. From microarray profiling (days 0-7), the number of differentially expressed genes increased considerably after the lactate shift (>day 3). To uncouple the effects of copper at early time points (days 0-3) from that of lactate per se (>day 3), and to validate microarray hits, we analyzed samples before the lactate shift by RNA-Seq. Out of 6,398 overlapping genes analyzed by both transcriptomic methods, only the early growth response 1 gene-coding for a transcription factor that activates signaling pathways in response to environmental stimuli-satisfied the differential expression criteria (fold change ≥ 1.5; P < 0.05). Gene expression correlation and biological pathway analyses further confirmed that copper differences exerted minimal transcriptional impact on the CHO cultures before the lactate shift. By contrast, genes associated with hypoxia network and oxidative stress response were upregulated after the lactate shift. These upregulations should boost cell proliferation and survival, but do not account for the preceding shift in lactate metabolism. The findings here indicate that the primary MOA of copper that enabled the shift in lactate metabolism is not at the transcriptional level.

Nocebo and placebo modulation of hypobaric hypoxia headache involves the cyclooxygenase-prostaglandins pathway.

Nocebo and placebo effects have been found to modulate several neurochemical systems, such as cholecystokinin, endogenous opioids, and endocannabinoids. Here we show that also the cyclooxygenase-prostaglandins pathway can be modulated by both nocebos and placebos. In fact, we found that negative expectation, the crucial element of the nocebo effect, about headache pain led to the enhancement of the cyclooxygenase-prostaglandins pathway, which, in turn, induced pain worsening. As an experimental model, we studied hypobaric hypoxia headache at high altitude in 2 populations of subjects. Whereas the experimental nocebo group received negative information by a single individual who was informed about the risk of headache, the control group did not know about the possible occurrence of headache. We found a significant increase in headache and salivary prostaglandins and thromboxane in the nocebo group compared to the control group, suggesting that negative expectations enhance cyclooxygenase activity. In addition, placebo administration to headache sufferers at high altitude inhibited the nocebo-related component of pain and prostaglandins synthesis, which indicates that the cyclooxygenase pathway can be modulated by both nocebos and placebos. Our results show for the first time how nocebos and placebos affect the synthesis of prostaglandins, which represent an important target of analgesic drugs, thus emphasizing once again the notion that placebos and drugs may use common biochemical pathways.

In vitro inhibition of the transcription factor NF-κB and cyclooxygenase by bamboo extracts.

Several bamboo species have been used in traditional medicine for the treatment of inflammatory conditions. The present study evaluates the in vitro anti-inflammatory properties of the traditionally used bamboo species Phyllostachys nigra (Lodd.) Munro and Sasa veitchii (Carr.) Rehder to explore their future research opportunities and therapeutic potential as anti-inflammatory agents. The extracts were evaluated for their potential inhibitory activity at the level of NF-κB-induced gene expression and suppression of cyclooxygenase (COX)-1 and COX-2 enzyme activities, representative pharmacological targets for the anti-inflammatory action of glucocorticoids and non-steroidal anti-inflammatory drugs, respectively. The activity of P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder was compared with bamboo species without traditional anti-inflammatory indications. High-performance liquid chromatography with diode-array detection and liquid chromatography-tandem mass spectrometry analyses were performed to phytochemically characterize the extracts. P. nigra (Lodd.) Munro leaf extract potently inhibited NF-κB-induced gene expression, while S. veitchii (Carr.) Rehder leaf extract exerted a selective COX-2 inhibition. The crude extracts consistently showed a more potent bioactivity than the solid phase extraction fractions. P. nigra (Lodd.) Munro and S. veitchii (Carr.) Rehder both exert anti-inflammatory properties, but act via a different molecular mechanism.

Phenolics content and antioxidant and anti-inflammatory activities of legume fractions.

Two faba bean (Vicia faba L.) subspecies major and minor and lentil seeds grown in Algeria were separated into cotyledons and hulls. These fractions, together with their corresponding whole seeds, were extracted with two solvents, aqueous (70%) acetone and (80%) ethanol, and evaluated for antioxidant activity in relation to their phenolic contents. Acetone selectively extracted tannins from faba beans. The hulls always exhibited high antioxidant activity, measured using the reducing power (RP), antiradical activity (DPPH) or oxygen radical absorbance capacity (ORAC) assays. Aqueous ethanol (80%) extract of lentil hulls exhibited high antioxidant and anti-inflammatory activities preferentially inhibiting 15-LOX (IC(50), 55 µg/ml), with moderate COX-1 (IC(50), 66 µg/ml) and COX-2 (IC(50), 119 µg/ml) inhibitory effects on the COX pathway, whereas faba bean hull extracts exerted relatively mild LOX inhibitory activity.

Immunolocalization, gene expression, and enzymatic activity of cyclooxygenases, prostaglandin e2-9-ketoreductase, and nitric oxide synthases in Mediterranean buffalo (Bubalus bubalis) corpora lutea during diestrus.

Immunopresence, gene expression, and enzymatic activity of cyclooxygenase 1 (COX1), COX2, PGE2-9-ketoreductase (PGE2-9-K), endothelial (eNOS), and inducible nitric oxide synthases (iNOS), and hormone in vitro production were examined in early, mid, late, and regressive buffalo corpora lutea (CL). COX1 immunosignals were detected in the cytoplasm of small luteal cells, COX2 in large luteal cells, and PGE2-9-K in all luteal cells. COX2 and PGE2-9-K immunosignals were greater in late CL. Immunopresence of both NOS types were evidenced in the nuclei and cytoplasm of all luteal cells, as well as in the nuclei of endothelial cells, during all stages studied. The eNOS and iNOS immunosignals increased during the early stage. COX1 transcripts were lower in late and regressive CL, COX2 in late, PGE2-9-K higher in regressive, and iNOS higher in early and lower in regressive CL. COX1 enzymatic activity was lower in regressive CL, COX2 increased in mid and late stages, and PGE2-9-K was higher in late CL. Endothelial NOS activity was higher during mid and late stages and lower in regressive, whereas iNOS was greater in late and lower in early. Progesterone in vitro release was higher in mid and lower in late phase, while PGF2α synthesis was higher in late CL and lower in regressive, and PGE2 was higher during regressive stage. These results support the idea that COX, NOS, and PGE2-9-K regulate buffalo CL life span. In particular, regressive CL seems involved in the development of the contralateral early CL, through the production of the luteotrophic PGE2.

Ovarian steroids modulate tumor necrosis factor-α and nitric oxide-regulated prostaglandin secretion by cultured bovine oviductal epithelial cells.

Ovarian steroids assure an optimum environment for the final maturation of oocytes, gamete transport, fertilization, and early embryonic development. The aim of experiment 1 was to examine the influence of ovarian steroids on tumor necrosis factor-α (TNF-α)- or nitric oxide (NO)-regulated prostaglandin (PG), and nitrite/nitrate (NO2/NO3) secretion by cultured bovine oviductal epithelial cells (BOECs). BOECs were pretreated with 17ß-estradiol (E2; 10⁻9 M) and/or progesterone (P4; 10⁻7 M) for 24 h. For the next 24 h, BOECs were treated with TNF-α (10 ng/mL) or spermine nitric oxide complex (NONOate; 10⁻5 M). Prostaglandin F(2α) and PGE2 secretion was measured in medium by ELISA. The pretreatment of cells with P4 (progesterone), E2 (17 ß-estradiol), or E2/P4 augmented TNF-α-induced PGF(2α) and PGE2 secretion (P < 0.01). The pretreatment of cells with E2 or E2/P4 increased NONOate-induced PGF(2α) and PGE2 secretion (P < 0.01). TNF-α induced NO2/NO3 production by BOECs. The pretreatment of cells with E2 augmented only TNF-α-induced NO2/NO3 production (P < 0.05). The aim of experiment 2 was to examine the influence of TNF-α, NO, and ovarian steroids on the protein content of enzymes specifically involved in PG and NO production, PG synthases, and NO synthases (NOSs). BOECs were treated with TNF-α (10 ng/mL) or NONOate (10⁻5 M). TNF-α increased the protein content of PGG/H synthase, PGF synthase, and PGE synthase (P < 0.05) and endothelial and inducible NOSs (P < 0.05). Nitric oxide increased the protein content of PGF synthase, PGE synthase, endothelial NOS, and inducible NOS (P < 0.05). These results show possible linkage between TNF-α and NO, modulated by ovarian steroids, in the regulation of PG synthesis by BOECs that may be important for triggering the process of oviductal contractions.

In vitro effects of gonadotropin-releasing hormone (GnRH) on Leydig cells of adult alpaca (Lama pacos) testis: GnRH receptor immunolocalization, testosterone and prostaglandin synthesis, and cyclooxygenase activities.

The main objective of this study was to examine the modulatory in vitro effects of gonadotropin-releasing hormone (GnRH) on isolated Leydig cells of adult alpaca (Lama pacos) testis. We first evaluated the presence of GnRH receptor (GnRHR) and cyclooxygenase (COX) 1 and COX2 in alpaca testis. We then studied the in vitro effects of buserelin (GnRH analogue), antide (GnRH antagonist), and buserelin plus antide or inhibitor of phospholipase C (compound 48/80) and COXs (acetylsalicylic acid) on the production of testosterone, PGE(2), and PGF(2α) and on the enzymatic activities of COX1 and COX2. Immunoreactivity for GnRHR was detected in the cytoplasm of Leydig cells and in the acrosomal region of spermatids. COX1 and COX2 immunosignals were noted in the cytoplasm of spermatogonia, spermatocytes, spermatids, Leydig cells, and Sertoli cells. Western blot analysis confirmed the GnRHR and COX1 presence in alpaca testis. The in vitro experiments showed that buserelin alone increased (P < 0.01) and antide and buserelin plus acetylsalicylic acid decreased (P < 0.01) testosterone and PGF(2α) production and COX1 activity, whereas antide and compound 48/80 counteracted buserelin effects. Prostaglandin E(2) production and COX2 activity were not affected by buserelin or antide. These data suggest that GnRH directly up-regulates testosterone production in Leydig cells of adult alpaca testis with a postreceptorial mechanism that involves PLC, COX1, and PGF(2α).

Antioxidant and anti-inflammatory activities of bean ( Phaseolus vulgaris L.) hulls.

Hulls obtained by mechanical abrasive dehulling from four bean cultivars were extracted with two solvents, aqueous (70%) acetone and water, and the extracts evaluated for antioxidant and anti-inflammatory activities in relation to their phenolic contents. Total phenolic content and antioxidant activity of bean hulls, measured using oxygen radical absorbance capacity (ORAC) values, were 6-8-fold those of corresponding whole beans. Aqueous acetone (70%) extracted over twice the amount of total phenolics from hulls that exhibited significantly higher antioxidant and stronger inhibitory effect on both cyclooxygenases, COX-1 and COX-2, than water. Acetone extract of black bean hull exhibited strong COX-1 (IC(50) = 1.2 microg/mL) and COX-2 (IC(50) = 38 microg/mL) inhibitory effects, even outperforming aspirin. Bean hull water extracts were stronger inhibitors of lipoxygenase, 15-LOX, than corresponding acetone extracts. Anti-inflammatory activity of bean hulls was dependent on their phenolic content and antioxidant activity that were significantly affected by cultivar and extracting solvent.

Dexamethasone inhibits tumor necrosis factor-alpha-stimulated gastric epithelial cell migration.

BACKGROUND: Cell migration (restitution) occurs in the early phase of gastric ulcer healing. Tumor necrosis factor (TNF)-alpha is overexpressed at the ulcer margin and plays a physiologic role in gastric ulcer healing. Dexamethasone, which is a potent corticosteroid, delays rat gastric ulcer healing. We evaluated whether dexamethasone inhibited TNF-alpha-stimulated gastric epithelial cell migration using a rat normal gastric epithelial cell line (RGM-1). METHODS: An artificial wound model was employed to measure cell migration. Western blot was performed to evaluate the possible mechanisms. Intracellular prostaglandin E2 level was measured using an enzyme-linked immunosorbent assay. RESULTS: TNF-alpha treatment (10 ng/mL) for 12-48 hours significantly increased RGM-1 cell migration, and TNF-alpha treatment increased cyclooxygenase (COX)-2 protein expression 8 hours later and prostaglandin E2 (PGE2) synthesis 12 hours later compared with control (p < 0.05). Dexamethasone (10(-6) M) significantly inhibited the stimulatory effect of TNF-alpha on RGM-1 cell migration, which was associated with a significant decrease in COX-2 expression and PGE2 level in cells (p < 0.05). CONCLUSION: TNF-alpha plays a regulatory role in rat gastric epithelial cell migration and dexamethasone inhibited TNF-alpha-stimulated cell migration, which was associated with a decrease in COX-2 expression and PGE2 formation.

Effect of toluene exposure on the antioxidant status and apoptotic pathway in organs of the rat.

The chronic abuse of the solvent toluene results in structural and functional impairment of various organs. However, the pathophysiological mechanisms that cause these impairments of function are not clearly understood. This study aims to assess the effect of chronic toluene exposure (15, 30 and 45 days) on the oxidative stress and antioxidant status of different organs in the rat. Also, cyclooxygenase-2 and caspase-3 activities (a marker of apoptosis) are studied. Forty male albino rats were used and divided into four groups: controls (group I) and three other groups receiving a single daily dose of toluene (650 mg/kg) for 15 days (group II), 30 days (group III) and 45 days (group IV). The animals were then sacrificed and the brain cortex, cerebellum, liver, kidney and testis were separated for the determination of thiobarbituric acid reactive substance (TBARS), GSH, glutathione disulphide (GSSG) and glutathione reductase (GR), glutathione peroxidase (GPx), glutathione-S-transferase (GST), superoxide dismutase (SOD), cyclooxygenase-2 (COX-2) and caspase-3 activity. Results showed a significant and time-dependent increase in the levels of TBARS, GSSG and in GST, SOD, COX-2 and caspase-3 activity, while GSH, GR and GPx showed a marked decline in most tissues. The brain (cortex and cerebellum) was the most affected organ, showing the greatest increase in one apoptotic marker (caspase-3), while the testis and kidneys were least affected. In conclusion, oxidative stress and derangement of the GSH:GSSG ratio, induced chronic inflammatory change and apoptosis may play an essential role in toluene toxicity