Pregnane X receptor (PXR) deficiency promotes hepatocarcinogenesis via induction of Akr1c18 expression and prostaglandin F2α (PGF2α) levels.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Pregnane X receptor (PXR), a xenobiotic-sensing nuclear receptor, plays a critical role in the metabolism of endogenous and exogenous substances in the liver. Here, we investigate whether PXR plays a role in pathogenesis of HCC. We show that liver tumors were developed in diethylnitrosamine (DEN)-treated in PXR knockout (KO) mice. Hepatic levels of prostaglandin F2α (PGF2α) and aldo-keto reductase family 1 member C18 (Akr1c18), a prostaglandin synthase of catalyzing reduction of PGH2 to PGF2α, were significantly elevated in DEN-treated PXR KO mice. Hepatic mRNA levels of alpha fetoprotein (AFP), cyclin D1 (Ccnd1), fibroblast growth factor 21 (FGF21), and inflammatory cytokine interleukin 6 (IL-6) were significantly increased in DEN-treated PXR KO mice. Other members of Akr1c family, liver metabolizing enzymes including Cyp1a2, Cyp2b10 and Cyp3a11, and bile acid synthesis enzyme Cyp7a1 mRNA levels were significantly decreased in DEN-treated PXR KO mice. Our findings revealed that PXR deficiency promoted DEN-induced HCC in mice via induction of Akr1c18 expression and PGF2α levels and the increased PGF2α levels synthetized by Akr1c18 enhanced hepatocytes proliferation and induced inflammatory cytokine production, which accelerated liver tumor development after DEN treatment, suggesting that PXR deficiency may create a microenvironment that is more prone to DEN-induced liver tumors and targeting PXR and Akr1c18 to reduce PGF2α biosynthesis may be a potential and novel therapeutic strategy for HCC.

Acetaminophen reduces osteoprotegerin synthesis stimulated by PGE2 and PGF2α in osteoblasts: attenuation of SAPK/JNK but not p38 MAPK or p44/p42 MAPK.

Acetaminophen is one of the most widely used analgesic and antipyretic medicines, whose long-period use has reportedly been associated with an increased risk of bone fracture. However, the mechanism underlying this undesired effect remains to be investigated. The homeostatic control of bone tissue depends on the interaction between osteoblasts and osteoclasts. Osteoprotegerin produced by osteoblasts is known to play an essential role in suppressing osteoclast induction. We have previously reported that prostaglandin (PG) E2 and PGF2α induce osteoprotegerin synthesis through p38 mitogen-activated protein kinase (MAPK), p44/p42 MAPK and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the effects of acetaminophen on the osteoprotegerin synthesis induced by PGE2 and PGF2α in MC3T3-E1 cells. Acetaminophen significantly suppressed the osteoprotegerin release stimulated by PGE2 and PGF2α. The PGE2-induced expression of osteoprotegerin mRNA was also reduced by acetaminophen. Acetaminophen markedly downregulated the phosphorylation of SAPK/JNK stimulated by PGE2 and PGF2α, but not those of p38 MAPK or p44/p42 MAPK. SP600125, an inhibitor of SAPK/JNK, suppressed the levels of PGE2- and PGF2α-upregulated osteoprotegerin mRNA expression. Taken together, these results strongly suggest that acetaminophen reduces the PGE2- and PGF2α-stimulated synthesis of osteoprotegerin in osteoblasts, and that the suppressive effect is exerted via attenuation of SAPK/JNK. These findings provide a molecular basis for the possible effect of acetaminophen on bone tissue metabolism.

Protection of 6-OHDA neurotoxicity by PGF2α through FP-ERK-Nrf2 signaling in SH-SY5Y cells.

6-Hydroxydopamine (6-OHDA) is a neurotoxin that destroy dopaminergic neurons and widely used to establish animal models of Parkinson's disease. Prostaglandins (PGs) are involved in various cellular processes, including the damage and repair of neuronal cells. However, the function of PGF2α in neuronal cells remains unclear. In this study, we investigated the effects of PGF2α against 6-OHDA-mediated toxicity in human neuroblastoma SH-SY5Y cells and elucidated its underlying molecular mechanism. When the cells were treated with 6-OHDA (50 µM) for 6 h, the expression levels of PGF2α synthetic enzymes; cyclooxygenase-2 and aldo-keto reductase 1C3 as PGF2α synthase were enhanced in an incubation-time-dependent manner. In addition, the production of PGF2α was increased in 6-OHDA-treated cells. Fluprostenol, a PGF2α receptor (FP) agonist (500 nM), suppressed 6-OHDA-induced cell death by decreasing the production of reactive oxygen species (ROS) and increasing the expression of the anti-oxidant genes. These fluprostenol-mediated effects were inhibited by co-treatment with AL8810, an FP receptor antagonist (1 µM) or transfection with FP siRNA (20 nM). Moreover, 6-OHDA-induced phosphorylation of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase family, was inhibited by co-incubation with AL8810. Furthermore, fluprostenol itself enhanced ERK phosphorylation and further elevated the 6-OHDA-induced phosphorylation of ERK. In addition, 6-OHDA induced nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2), activating anti-oxidant gene expression, was repressed by co-culturing with AL8810. These results indicate that PGF2α suppressed 6-OHDA-induced neuronal cell death by enhancing anti-oxidant gene expression via the FP receptor-ERK-Nrf2 signaling. Thus, FP receptor is a potential target for inhibition of ROS-mediated neuronal cell death.

Selective inhibition of prostaglandin D2 biosynthesis in human mast cells to overcome need for multiple receptor antagonists: Biochemical consequences.

BACKGROUND: The major mast cell prostanoid PGD2 is targeted for therapy of asthma and other diseases, because the biological actions include bronchoconstriction, vasodilation and regulation of immune cells mediated by three different receptors. It is not known if the alternative to selectively inhibit the biosynthesis of PGD2 affects release of other prostanoids in human mast cells. OBJECTIVES: To determine the biochemical consequences of inhibition of the hematopoietic prostaglandin D synthase (hPGDS) PGD2 in human mast cells. METHODS: Four human mast cell models, LAD2, cord blood derived mast cells (CBMC), peripheral blood derived mast cells (PBMC) and human lung mast cells (HLMC), were activated by anti-IgE or ionophore A23187. Prostanoids were measured by UPLC-MS/MS. RESULTS: All mast cells almost exclusively released PGD2 when activated by anti-IgE or A23187. The biosynthesis was in all four cell types entirely initiated by COX-1. When pharmacologic inhibition of hPGDS abolished formation of PGD2 , PGE2 was detected and release of TXA2 increased. Conversely, when the thromboxane synthase was inhibited, levels of PGD2 increased. Adding exogenous PGH2 confirmed predominant conversion to PGD2 under control conditions, and increased levels of TXB2 and PGE2 when hPGDS was inhibited. However, PGE2 was formed by non-enzymatic degradation. CONCLUSIONS: Inhibition of hPGDS effectively blocks mast cell dependent PGD2 formation. The inhibition was associated with redirected use of the intermediate PGH2 and shunting into biosynthesis of TXA2 . However, the levels of TXA2 did not reach those of PGD2 in naïve cells. It remains to determine if this diversion occurs in vivo and has clinical relevance.

Oleic acid and derivatives affect human endothelial cell mitochondrial function and vasoactive mediator production.

BACKGROUND: Inhalation of common air pollutants such as diesel and biodiesel combustion products can induce vascular changes in humans which may contribute to increased mortality and morbidity associated with fine particulate matter exposures. Diesel, biodiesel, and other combustion byproducts contain fatty acid components capable of entering the body through particulate matter inhalation. Fatty acids can also be endogenously released into circulation following a systemic stress response to some inhaled pollutants such as ozone. When in the circulation, bioactive fatty acids may interact with cells lining the blood vessels, potentially inducing endothelial dysfunction. To examine whether fatty acids could potentially be involved in human vascular responses to air pollutants, we determined the effects of fatty acids and derivatives on important vascular cell functions. METHODS: Human umbilical vein endothelial cells (HUVEC) were exposed in vitro to oleic acid (OA) or OA metabolites for 4-48 h. Cytotoxicity, vasodilator production (by ELISA measurement), mitochondrial function (using Sea Horse assays), and iron metabolism (inferred by ICP-OES measurements) were examined, with standard statistical testing (ANOVA, t-tests) employed. RESULTS: Dose-dependent cytotoxicity was noted at 24 h, with 12-hydroxy OA more potent than OA. Mitochondrial stress testing showed that 12-hydroxy OA and OA induce mitochondrial dysfunction. Analysis of soluble mediator release from HUVEC showed a dose-dependent increase in prostaglandin F2α, a lipid involved in control of vascular tone, at 24 h (85% above controls) after OA-BSA exposure. RT-PCR analysis revealed OA did not induce changes in gene expression at noncytotoxic concentrations in exposed HUVEC, but 12-OH OA did alter ICAM and COX2 gene expression. CONCLUSIONS: Together, these data demonstrate that FA may be capable of inducing cytotoxic effects and altering expression of mediators of vascular function following inhalation exposure, and may be implicated in air pollutant-induced deaths and hospitalizations. (267 of max 350 words).

Oxytocin-induced prostaglandin F2-alpha release is low in early bovine pregnancy but increases during the second month of pregnancy†.

Circulating prostaglandin F2α metabolite (PGFM) after an oxytocin challenge was evaluated throughout the first 2 months of pregnancy in lactating Holstein cows. On day 11, 18, and 25 after artificial insemination (AI), and on days 32, 39, 46, 53, and 60 of pregnancy, cows were challenged with 50 IU oxytocin, i.m. Blood was collected before (0 min), 30, 60, 90, and 120 min after oxytocin for plasma PGFM concentrations. Ultrasound evaluations were performed for pregnancy diagnosis on day 32-60 post-AI. Nonpregnant (NP) cows on day 18 were designated by a lack of interferon-stimulated genes in peripheral blood leukocytes and Pregnant (P) based on day 32 ultrasound. On day 11, P and NP were similar with low PGFM and no effect of oxytocin on PGFM. On day 18, oxytocin increased PGFM (3-fold) in NP with little change in P cows. Comparing only P cows from day 11 to 60, basal circulating PGFM increased as pregnancy progressed, with day 11 and 18, lower than all days from day 25 to 60 of pregnancy. Oxytocin-induced PGFM in P cows on day 25 was greater than P cows on day 18 (2.9-fold). However, oxytocin-induced PGFM was lower on day 25 compared to day 53 and 60, with intermediate values on day 32, 39, and 46 of pregnancy. Thus, the corpus luteum (CL) of early pregnancy (day 11, 18) is maintained by suppression of PGF, as reflected by suppressed PGFM in this study. However, during the second month of pregnancy, uterine PGF secretion was not suppressed since basal PGFM and oxytocin-induced PGFM secretion were elevated. Apparently, mechanisms other than suppression of oxytocin receptors maintain CL after day 25 of pregnancy.

The processes associated with lipid peroxidation in human embryonic lung fibroblasts, treated with polycyclic aromatic hydrocarbons and organic extract from particulate matter.

Polycyclic aromatic hydrocarbons (PAHs) may cause lipid peroxidation via reactive oxygen species generation. 15-F2t-isoprostane (IsoP), an oxidative stress marker, is formed from arachidonic acid (AA) by a free-radical induced oxidation. AA may also be converted to prostaglandins (PG) by prostaglandin-endoperoxide synthase (PTGS) induced by NF-κB. We treated human embryonic lung fibroblasts (HEL12469) with benzo[a]pyrene (B[a]P), 3-nitrobenzanthrone (3-NBA) and extractable organic matter (EOM) from ambient air particulate matter <2.5 µm for 4 and 24 h. B[a]P and 3-NBA induced expression of PAH metabolising, but not antioxidant enzymes. The concentrations of IsoP decreased, whereas the levels of AA tended to increase. Although the activity of NF-κB was not detected, the tested compounds affected the expression of prostaglandin-endoperoxide synthase 2 (PTGS2). The levels of prostaglandin E2 (PGE2) decreased following exposure to B[a]P, whereas 3-NBA exposure tended to increase PGE2 concentration. A distinct response was observed after EOM exposure: expression of PAH-metabolising enzymes was induced, IsoP levels increased after 24-h treatment but AA concentration was not affected. The activity of NF-κB increased after both exposure periods, and a significant induction of PTGS2 expression was found following 4-h treatment. Similarly to PAHs, the EOM exposure was associated with a decrease of PGE2 levels. In summary, exposure to PAHs with low pro-oxidant potential results in a decrease of IsoP levels implying 'antioxidant' properties. For such compounds, IsoP may not be a suitable marker of lipid peroxidation.

The correlation between oxidative stress level and intra-abdominal fat in obese males.

This study aims to investigate the correlation between oxidative stress and intra-abdominal fat (IAF) in obese young and middle-aged males.The present study included 136 male examinees in the Examination Center of the First Hospital of Qinhuangdao from October 10, 2015 to December 10, 2015. Then, clinical data, oxidative stress indices (8-iso-prostaglandin F2α [8-iso-PGF2α], malondialdehyde [MDA], and superoxide dismutase [SOD]), and IAF area were recorded. All subjects were assigned into 3 groups according to body mass index (BMI): obese group (BMI ≥ 28 kg/m, 43 subjects), overweight group (24 ≤ BMI < 28 kg/m, 46 subjects), and control group (BMI < 24 kg/m, 47 subjects). Then, statistical analysis was performed.There were significant differences in IAF area, leptin, adiponectin, 8-iso-PGF2α, MDA, SOD, fasting insulin (FINS), fasting blood glucose (FBG), and homeostasis model assessment-insulin resistance (HOMA-IR) among these 3 groups (P < .05). Male subjects in the obese group had higher leptin, 8-iso-PGF2α, MDA, FINS, and HOMA-IR levels, compared to subjects in the overweight and control groups. Furthermore, subjects in the overweight group had a larger IAF area and higher 8-iso-PGF2α, MDA, and FBG levels, when compared to controls. In addition, SOD was significantly lower in the obese and overweight groups than in the control group. However, there were no statistical differences in age, systolic and diastolic blood pressure, lipids, and islet ß-cell secretion function (homeostasis model assessment-ß) among these 3 groups (P ≥ .05). Moreover, the IAF area was positively correlated to 8-iso-PGF2α and MDA, and negatively correlated to SOD.Oxidative stress is significantly associated with the IAF area in obese males, and abdominal obesity could increase oxidative stress level and insulin resistance.

Interleukin-1ß and prostaglandin-synthesizing enzymes as modulators of human omental and subcutaneous adipose tissue function.

IL-1ß stimulates expression of prostaglandin (PG)-synthesizing enzymes cyclooxygenase (COX)-2 and aldo-keto reductase (AKR)1B1 in human preadipocytes. We aimed to examine the impact of IL-1ß, COX-2 and AKR1B1 on markers of human visceral and subcutaneous adipose tissue function, and to assess whether PG synthesis by these enzymes mediates IL-1ß effects. Omental and subcutaneous fat samples were obtained from bariatric surgery patients. PG release and expression of inflammatory and adipogenic markers were assessed in explants treated with COX-2 inhibitor NS-398 or AKR1B1 inhibitor Statil, with or without IL-1ß. Preadipocyte differentiation experiments were also performed. IL-1ß decreased expression of PPARγ in both fat depots compared to control and increased expression of NF-κB1, IL-6, CCL-5, ICAM-1 and VEGFA, especially in visceral fat for IL-6, CCL-5 and VEGFA. Adding Statil or NS-398 to IL-1ß blunted PGF2α and PGE2 release, but did not alter IL-1ß effects on adipose tissue function markers. IL-1ß down-regulated adipocyte differentiation whereas NS-398 alone increased this process. However, NS-398 did not prevent IL-1ß inhibition of adipogenesis. We conclude that IL-1ß induces a pro-inflammatory response in human adipose tissues, particularly in visceral fat, and acts independently of concomitant PG release. IL-1ß and COX-2 appear to be critical determinants of adipose tissue pathophysiologic remodeling in obesity.

Production of prostaglandin F2α by molecular breeding of an oleaginous fungus Mortierella alpina.

Cyclooxygenases are responsible for the production of prostaglandin H2 (PGH2) from arachidonic acid. PGH2 can be converted into some bioactive prostaglandins, including prostaglandin F2α (PGF2α), a potent chemical messenger used as a biological regulator in the fields of obstetrics and gynecology. The chemical messenger PGF2α has been industrially produced by chemical synthesis. To develop a biotechnological process, in which PGF2α can be produced by a microorganism, we transformed an oleaginous fungus, Mortierella alpina 1S-4, rich in triacylglycerol consisting of arachidonic acid using a cyclooxygenase gene from a red alga, Gracilaria vermiculophylla. PGF2α was accumulated not only in the mycelia of the transformants but also in the extracellular medium. After 12 days of cultivation approximately 860 ng/g and 6421 µg/L of PGF2α were accumulated in mycelia and the extracellular medium, respectively. The results could facilitate the development of novel fermentative methods for the production of prostanoids using an oleaginous fungus.

Changes in the expression of prostaglandin family members in bovine corpus luteum during the estrous cycle and pregnancy.

The aim of this study was to characterize certain prostaglandin family members in the bovine corpus luteum (CL) during the estrous cycle and pregnancy. The CL tissue was assigned to the stages 1-2, 3-4, 5-7, 8-12, 13-16 and >18 days (after regression) of the estrous cycle and 1-2, 3-4, 6-7, and >8 months of pregnancy. In these samples, we investigated prostaglandin F2alpha (PTGF), prostaglandin E2 (PTGE), their receptors (PTGFR, PTGER2, and PTGER4), cyclooxygenase 2 (COX-2), PTGF synthase (PTGFS), and PTGE synthase (PTGES). The expression of messenger RNA (mRNA) was measured by reverse transcription quantitative polymerase chain reaction, hormones by enzyme immunoassay, and localization by immunohistochemistry. The mRNA expression of COX-2, PTGFS, and PTGES in CL during the early-luteal phase was high followed by a continuous and significant downregulation afterward, as well as during all phases of pregnancy. The concentration of PTGF in CL tissue was high during the early-luteal phase, decreased significantly in the mid-luteal phase, and increased again afterward. In contrast, the concentration of PTGE increased significantly during the late-luteal phase followed by a decrease during regression. The PTGE level increased again during late pregnancy. Immunohistochemically, the large granulose-luteal cells show strong staining for COX-2 and PTGES during the early-luteal stage followed by lower activity afterward. During pregnancy, most of the luteal cells were only weakly positive or negative. In conclusion, our results indicate that the examined prostaglandin family members are involved in the local mechanisms that regulate luteal function, specifically during CL formation, function, and regression and during pregnancy in the cow.

Heat stress affects prostaglandin synthesis in bovine endometrial cells.

Heat stress (HS) negatively affects reproduction in cattle; however, its effect on endocrine function in bovine endometrial cells remains unclear. In this study, we examined the effects of HS on the production of prostaglandin (PG) E2 and PGF2α in the cultured bovine endometrial epithelial and stromal cells separately. To evaluate the effect of HS on endocrine function, the cells were cultured at 38.5°C (control) or 40.5°C (HS). After treatment, PGE2 and PGF2α levels were measured via enzyme immunoassay (EIA) and mRNA expressions of enzymes involved in PG synthesis were examined via quantitative reverse transcription polymerase chain reaction (RT-PCR). HS did not influence the production of PGE2 or PGF2α in the epithelial cells; however, HS significantly enhanced the production of both PGE2 and PGF2α in the stromal cells (P < 0.05). In addition, HS significantly increased phospholipase A2 (PLA2), cyclooxygenase 2 (COX2), prostaglandin F synthase (PGFS), prostaglandin E synthase (PGES), and carbonyl reductase 1 (CBR1) mRNA expression in the stromal cells (P < 0.05). The overall results suggest that HS induces mRNA expression of enzymes involved in PG synthesis, resulting in the upregulation of PGE2 and PGF2α production in the stromal cells, but not in the epithelial cells. The HS-induced increase of PGE2 and PGF2α secretion in bovine endometrial stromal cells may disrupt the normal estrous cycle and cause infertility in cows during summer.

Role of luteal biosynthesis of prostaglandin F2α on function and structure of the corpus luteum during luteolysis in heifers.

The role of endogenous prostaglandin F2α (PGF) in the induction of luteolysis by exogenous PGF was studied by simultaneous inhibition of endogenous PGF with flunixin meglumine (FM). Groups were controls (n = 8), PGF treated (n = 8), and FM + PGF treated (n = 9). Treatments were given 10 d postovulation at hours 0, 8, and 16. The protocol was based on (1) the assumption that luteolytic characteristics of exogenous PGF would be altered if the synthesis of endogenous PGF is simultaneously inhibited and (2) the reports that luteolysis involves a direct effect of uterine PGF on large luteal cells followed by an effect of the large cells on the small cells. At hour 48, progesterone concentration was greater in the controls (7.6 ± 0.8 ng/mL) than that in the FM + PGF group (3.0 ± 0.5 ng/mL) and lower in the PGF group (0.7 ± 0.3 ng/mL) than in the FM + PGF group (interaction, P < 0.0001). The effects of each of the 3 groups on percentage change in CL volume were similar to the effects on progesterone. At hour 48, the percentage of CL tissue with color-Doppler signals of blood flow was similar between the controls (56.2% ± 3.8%) and FM + PGF group (50.0% ± 6.4%) and lowest in the PGF group (15.6% ± 7.2%) (interaction, P < 0.0001). A resurgence in progesterone concentration began at hours 24 or 48 in 6 of 9 heifers in the FM + PGF group compared to 0 of 8 heifers in each of the other groups (P < 0.007). The progesterone resurgence in the FM + PGF group was associated with the maintenance of percentage of CL tissue with blood-flow signals. The experimental hypothesis that an inhibitor of endogenous PGF reduces the luteolytic response to exogenous PGF was supported.

Cytobrush: A tool for sequential evaluation of gene expression in bovine endometrium.

Aims were to (i) compare specific transcript abundance between endometrial samples collected by transcervical biopsy and cytobrush and (ii) measure the abundance of endometrial transcripts involved in PGF2α synthesis in samples collected by cytobrush. In Experiment 1, endometrial samples were taken transcervically by cytobrush and biopsy 10 days after ovulation. Compared to biopsy samples, abundance of transcripts for MSTN, AKR1C4 and PGR was similar, VIM, FLT1 and PTGES was lower (p < .05) and KRT18 and CD3D was greater in cytobrush samples (p < .05). Thus, there was an enrichment of epithelial and immune cells in the cytobrush samples. In Experiment 2, endometrial samples were collected by cytobrush on days 10, 13, 16 and 19 after ovulation. Abundance of PGR2 mRNA was maximum on day 10 then decreased (p < .05). Abundance of ESR1 decreased gradually from day 10 to day 16 then increased again on day 19. The greatest abundance of OXTR was noted on day 19. The sequential alterations in abundance of these transcripts are consistent with the release of PGF2α associated with luteolysis. In summary, cytobrush sampling provides representative, physiologically relevant samples of the luminal epithelium in cattle.

Evidence for CB2 receptor involvement in LPS-induced reduction of cAMP intracellular levels in uterine explants from pregnant mice: pathophysiological implications.

STUDY QUESTION: What is the role of the endocannabinoid system (eCS) on the lipopolysaccharide (LPS) effects on uterine explants from 7-day pregnant mice in a murine model of endotoxin-induced miscarriage? SUMMARY ANSWER: We found evidence for cannabinoid receptor type2 (CB2) involvement in LPS-induced increased prostaglandin-F2α (PGF2α) synthesis and diminished cyclic adenosine monophosphate (cAMP) intracellular content in uterine explants from early pregnant mice. WHAT IS KNOWN ALREADY: Genital tract infections by Gram-negative bacteria are a common complication of human pregnancy that results in an increased risk of pregnancy loss. LPS, the main component of the Gram-negative bacterial wall, elicits a strong maternal inflammatory response that results in embryotoxicity and embryo resorption in a murine model endotoxin-induced early pregnancy loss. We have previously shown that the eCS mediates the embryotoxic effects of LPS, mainly via CB1 receptor activation. STUDY DESIGN, SIZE, DURATION: An in vitro study of mice uterine explants was performed to investigate the eCS in mediating the effects of LPS on PGF2α production and cAMP intracellular content. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eight to 12-week-old virgin female BALB/c or CD1 (wild-type [WT] or CB1-knockout [CB1-KO]) mice were paired with 8- to 12-week-old BALB/c or CD1 (WT or CB1-KO) males, respectively. On day 7 of pregnancy, BALB/c, CD1 WT or CD1 CB1-KO mice were euthanized, the uteri were excised, implantation sites were removed and the uterine tissues were separated from decidual and embryo tissues. Uterine explants were cultured and exposed for an appropriate amount of time to different pharmacological treatments. The tissues were then collected for cAMP assay and PGF2α content determination by radioimmunoassay. MAIN RESULTS AND THE ROLE OF CHANCE: In vitro treatment of uteri explants from 7-day pregnant BALB/c or CD1 (WT or CB1-KO) mice with LPS induced an increased production of PGF2α (P < 0.05) and a reduction of the tissue content of cAMP (P < 0.05). These effects were mediated by CB2 receptors since exposure to AM630 (a specific CB2 receptor antagonist) prevented these LPS-induced effects (P < 0.05). Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues. LIMITATIONS, REASONS FOR CAUTION: Since our experimental design involves in vitro experiments of uterine explants, the extrapolation of the results presented here to humans is limited. WIDER IMPLICATIONS OF THE FINDINGS: Our findings provide evidence for the role of CB2 receptors in reproductive events as well as their participation as a mediator of LPS deleterious effects on reproductive tissues. LARGE SCALE DATA: None. STUDY FUNDING AND COMPETING INTEREST(S): Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). Dr Carlos Davio was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2013/2050). The authors have no competing interests.

Prostaglandin synthesis by the porcine corpus luteum: effect of tumor necrosis factor-α.

The porcine corpus luteum (CL) displays delayed sensitivity to PGF-2α (luteolytic sensitivity, [LS]) until days 12 to 13 of cycle. The control of LS is unknown, but it is temporally associated with macrophage (which secrete tumor necrosis factor-α; TNF-α) infiltration into the CL. Other studies showed that TNF-α induces LS in vitro and that prostaglandins (PGs) may be involved in this mechanism. In experiment 1, PGF-2α and PGE secretion by luteal cells (LCs) was measured on days 4 to 14 of the estrous cycle, and the expression of PTGFS/AKR1B1 and PTGES/mPGES-1, determined by Western blot, before (day 7) vs after (day 13) the onset of LS. Results showed that the PGF-2α:PGE ratio increased significantly (P < 0.05) from day 4 to 13-14, and PTGFS/AKR1B1 and PTGES/mPGES-1 were significantly increased (P < 0.05) on day 13 (vs day 7). In experiment 2, LCs were collected from porcine CL at early (∼days 4-6) or mid (∼days 7-12) stages of the estrous cycle and cultured with 0, 0.1, 1, or 10 ng/mL TNF-α. Results showed that TNF-α significantly increased (P < 0.05) messenger RNA (mRNA) expression of cyclooxygenase (COX)-2 and mPGES-1 but not AKR1B1. TNF-α had no significant effects on AKR1B1 or mPGES protein abundance. TNF-α significantly increased (P < 0.05) PGE-2 but had no effect on PGF-2α secretion or on the PGF-2α:PGE2 ratio. In conclusion, although TNF-α increased COX2 and mPGES-1 mRNA, and PGE-2 secretion in vitro, it did not increase the PGF-2α:PGE2 ratio. Studies are currently directed toward exploring other pathways (eg, FP receptor signaling) by which TNF-α induces LS in the porcine CL.

Allopurinol attenuates rhabdomyolysis-associated acute kidney injury: Renal and muscular protection.

BACKGROUND: Acute kidney injury (AKI) is the most severe complication of rhabdomyolysis. Allopurinol (Allo), a xanthine oxidase inhibitor, has been in the spotlight in the last decade due to new therapeutic applications related to its potent antioxidant effect. The aim of this study was to evaluate the efficacy of Allo in the prevention and treatment of rhabdomyolysis-associated AKI. METHODS: Male Wistar rats were divided into five groups: saline control group; prophylactic Allo (300mg/L of drinking water, 7 days); glycerol (50%, 5ml/kg, IM); prophylactic Allo + glycerol; and therapeutic Allo (50mg/Kg, IV, 30min after glycerol injection) + glycerol. RESULTS: Glycerol-injected rats showed markedly reduced glomerular filtration rate associated with renal vasoconstriction, renal tubular damage, increased oxidative stress, apoptosis and inflammation. Allo ameliorated all these alterations. We found 8-isoprostane-PGF2a (F2-IsoP) as a main factor involved in the oxidative stress-mediated renal vasoconstriction following rhabdomyolysis. Allo reduced F2-IsoP renal expression and restored renal blood flow. Allo also reduced oxidative stress in the damaged muscle, attenuated muscle lesion/inflammation and accelerated muscular recovery. Moreover, we showed new insights into the pathogenesis of rhabdomyolysis-associated AKI, whereas Allo treatment reduced renal inflammation by decreasing renal tissue uric acid levels and consequently inhibiting the inflammasome cascade. CONCLUSIONS: Allo treatment attenuates renal dysfunction in a model of rhabdomyolysis-associated AKI by reducing oxidative stress (systemic, renal and muscular), apoptosis and inflammation. This may represent a new therapeutic approach for rhabdomyolysis-associated AKI - a new use for an old and widely available medication.

PPAR ligand association with prostaglandin F2α and E2 synthesis in the pig corpus luteum-An in vitro study.

The present study evaluated the involvement of PPARs in prostaglandin (PG) E2 and F2α production in the corpus luteum (CL) of pigs on days 10-12 and 14-16 of the estrous cycle or pregnancy. The tissue explants were incubated for 6h in the presence of PPARα, PPARß, PPARγ ligands. The concentration of PGs in the incubation media was determined by radioimmunoassay, while mRNA abundance of PG synthetases (PGES and PGFS) was analyzed by quantitative real-time PCR. It was found that L-165,045 and rosiglitazone stimulated PGES synthesis on days 10-12 of the estrous cycle, whereas all factors that were assessed did not affect PGE2 release. The PGFS mRNA abundance in the CL did not change in the presence of PPAR ligands during the assessment periods. However, PPARß agonist inhibited PGF2α secretion on days 10-12 of the estrous cycle and on days 14-16 of pregnancy. Interestingly, PPAR antagonists, MK 886, GW 9662 or T0070907 decreased PGF2α release by the slices on days 10-12 of the estrous cycle. It is concluded that the CL has a different susceptibility (greatest during mid-luteal phase of the estrous cycle) to the PPAR ligands, which is related to the physiological status of animal. The inhibition of PGF2α release and augmentation of PGES mRNA concentration during mid-luteal phase of the estrous cycle might suggest luteotropic properties of PPAR ligands.

Expression of nerve growth factor and its receptors in the uterus of rabbits: functional involvement in prostaglandin synthesis.

The aim of the present study was to evaluate: (1) the presence of nerve growth factor (NGF), neurotrophic tyrosine kinase receptor 1 (NTRK1), and nerve growth factor receptor (NGFR) in the rabbit uterus; and (2) the in vitro effects of NGF on PGF2α and PGE2 synthesis and on the PGE2-9-ketoreductase (PGE2-9-K) activity by the rabbit uterus. Nerve growth factor, NTRK1, and NGFR were immunolocalized in the luminal and glandular epithelium and stroma cells of the endometrium. reverse transcriptase polymerase chain reaction indicated the presence of messenger RNA for NGF, NTRK1, and NGFR in the uterus. Nerve growth factor increased (P < 0.01) in vitro secretions of PGF2α and PGE2 but coincubation with either NTRK1 or oxide nitric synthase (NOS) inhibitors reduced (P < 0.01) PGF2α production and blocked (P < 0.01) PGE2 secretion. Prostaglandins releases were lower (P < 0.01) than control when uterine samples were treated with NGF plus cyclooxygenase inhibitor. However, addition of NGFR inhibitor reduced (P < 0.01) PGF2α secretion less efficiently than NTRK1 or NOS inhibitors but had no effect on PGE2 yield. Nerve growth factor increased (P < 0.01) the activity of PGE2-9-K, whereas coincubation with NTRK1 or NOS inhibitors abolished (P < 0.01) this increase in PGE2-9-K activity. However, cotreatment with either cyclooxygenase or NGFR inhibitors had no effect on PGE2-9-K activity. This is the first study to document the distribution of NGF/NTRK1 and NGFR systems and their effects on prostaglandin synthesis in the rabbit uterus. NGF/NTRK1 increases PGF2α and PGE2 productions by upregulating NOS and PGE2-9-K activities, whereas NGF/NGFR augments only PGF2α secretion, through an intracellular mechanism that is still unknown.

Copper metabolism domain-containing 1 represses the mediators involved in the terminal effector pathways of human labour and delivery.

STUDY HYPOTHESIS: Does Copper Metabolism MURR1 Domain 1 (COMMD1) play a role in regulating the mediators involved in the terminal processes of human labour and delivery? STUDY FINDING: COMMD1 plays a critical role in the termination of nuclear factor-κB (NF-κB) activity and the control of pro-inflammatory and pro-labour mediators. WHAT IS KNOWN ALREADY: Inflammation and infection are the biggest aetiological factors associated with preterm birth. NF-κB drives the transcription of pro-inflammatory mediators involved in the terminal effector pathways of human labour and delivery. In non-gestational tissues, COMMD1 is a negative regulator of NF-κB-induced inflammation. STUDY DESIGN, SAMPLES/MATERIALS, METHODS: The mRNA and/or protein level of COMMD1 was assessed in myometrium (n = 8 per group) and fetal membranes (n = 8 per group) obtained from term non-labouring and labouring women at term, and fetal membranes (n = 8 per group) at preterm with and without histological chorioamnionitis. Primary human myometrial cells were used to determine the effect of pro-inflammatory mediators on COMMD1 level, and the effect of COMMD1 small interfering RNA (siRNA) on pro-labour mediators. Statistical significance was ascribed to a P < 0.05. MAIN RESULTS AND THE ROLE OF CHANCE: COMMD1 expression was significantly decreased with spontaneous term labour in myometrium; in fetal membranes with histologically confirmed chorioamnionitis and in myometrial cells treated with pro-inflammatory cytokines interleukin (IL)-1ß and tumour necrosis factor (TNF)-α, the bacterial product fibroblast-stimulating lipopeptide and the viral double stranded RNA analogue polyinosinic polycytidilic acid. Loss-of-function studies revealed an increase in inflammation- and infection-induced TNF-α, IL-1α, IL-1ß, IL-6, IL-8 and/or monocyte chemoattractant protein-1 mRNA abundance and/or release; and cyclo-oxygenase-2 mRNA level, release of prostaglandin (PG) F2α and mRNA level of the PGF2α receptor FP. In addition, siRNA knockdown of COMMD1 was associated with significantly increased NF-κB activation as evidenced by increased IL-1ß-induced IκB-α protein degradation and NF-κB DNA binding activity. LIMITATIONS, REASONS FOR CAUTION: The conclusions are based on in vitro experiments with cells isolated from myometrium. Animal models, however, will be required to establish whether COMMD1 activators can prevent spontaneous preterm birth in vivo. WIDER IMPLICATIONS OF THE FINDINGS: The control of COMMD1 activation may provide an alternative therapeutic strategy for reducing the release of pro-labour mediators in spontaneous preterm labour. LARGE SCALE DATA: Not applicable. STUDY FUNDING AND COMPETING INTERESTS: Associate Professor Martha Lappas is supported by a Career Development Fellowship from the National Health and Medical Research Council (NHMRC; grant no. 1047025). Additional funding was provided by the Medical Research Foundation for Women and Babies and the Mercy Research Foundation. The author has no conflict of interest.