6-keto PGF1α Research Articles

Generation of monoclonal antibody against 6-Keto PGF1α and development of ELISA for its quantification in culture medium

Prostacyclin or prostaglandin I2 (PGI2), a metabolite of arachidonic cyclooxygenase pathway, has been demonstrated as an effector of adipocyte differentiation. However, due to its instability in biological fluid, it is difficult to evaluate the role of PGI2 in regulating adipocyte differentiation in different stages in culture. Therefore, this study aimed to establish a simple and rapid method for the production of monoclonal antibody against 6-Keto PGF1α, a stable PGI2 metabolite, and its quantification to determine the role of PGI2 in culture medium. Eight-week-old female BALB/c mice were immunized with the hapten of 6-Keto PGF1α and BSA for several weeks until a higher antibody titer (absorbance value > 0.9 at 1000-times dilution) against 6-Keto PGF1α was found. Then, fusion of antibody-producing spleen lymphocytes with SP-2 myeloma cells and thymocytes was performed and cultured in HAT-medium supplemented with hypoxanthine, aminopterin, and thymine. Specific antibody-producing cells (M2-A4-B8-D10) against 6-Keto PGF1α were identified and separated. A standard ELISA calibration curve was developed with 100% reactivity for 6-Keto-PGF 1 α ranging from 0.26 pg to 6.44 ng corresponding to 90% and 10% of the maximum binding capacity for the immobilized antigen respectively. This method can easily be applied to monitor PGI2 regulation in different stages of cultured adipocytes to reveal the regulatory roles of PGI2 in maintaining homeostasis and adipocyte differentiation.

Nicotinic acetylcholine receptor-mediated effects of varenicline on LPS-elevated prostaglandin and cyclooxygenase levels in RAW 264.7 macrophages.

Introduction: The purpose of this study is to delineate anti-inflammatory and antioxidant potential of varenicline, a cigarette smoking cessation aid, on decreasing lipopolysaccharide (LPS)-elevated proinflammatory cytokines in RAW 264.7 murine macrophage cultures which we showed earlier to occur via cholinergic anti-inflammatory pathway (CAP) activation. To this end, we investigated the possible suppressive capacity of varenicline on LPS-regulated cyclooxygenase (COX-1 and COX-2) via α7 nicotinic acetylcholine receptor (α7nAChR) activation using the same in vitro model. Materials and Methods: In order to test anti-inflammatory effectiveness of varenicline, the levels of COX isoforms and products (PGE2, 6-keto PGF1α, a stable analog of PGI2, and TXA2) altered after LPS administration were determined by Enzyme Linked Immunosorbent Assay (ELISA). The antioxidant effects of varenicline were assessed by measuring reductions in reactive oxygen species (ROS) using a fluorometric intracellular ROS assay kit. We further investigated the contribution of nAChR subtypes by using non-selective and/or selective α7nAChR antagonists. The results were compared with that of conventional anti-inflammatory medications, such as ibuprofen, celecoxib and dexamethasone. Results: Varenicline significantly reduced LPS-induced COX-1, COX-2 and prostaglandin levels and ROS to an extent similar to that observed with anti-inflammatory agents used. Discussion: Significant downregulation in LPS-induced COX isoforms and associated decreases in PGE2, 6-keto PGF1α, and TXA2 levels along with reduction in ROS may be partly mediated via varenicline-activated α7nAChRs.

Effect of omega-3 polyunsaturated fatty acid supplementation on oxidative stress parameters and sex hormone levels of modern genotype sows.

Sows are exposed to severe stress and hormonal challenges during their whole productive life. As polyunsaturated fatty acids play an important role in immune and reproductive functions, with a better understanding of their role in breeding sows' nutrition, improved performance and more sustainable pig production can be achieved. In this study, we investigated the effects of omega-6 and omega-3 fatty acid supplementation on the antioxidant status and hormone levels of sows. A total of 48 Danish Large white × Danish Landrace sows were supplemented either with sunflower oil (SO) as a control group or with fish oil (FO) as experimental group at the same dose of 10g/kg feed. Blood samples were collected on day 14 of lactation, 5 days after weaning (insemination), and 30 days after insemination. To estimate antioxidant and reproductive effects, the amounts of reduced glutathione (GSH), thiobarbituric acid reactive substance, the activity of glutathione peroxidase (GPx), serum 17β oestradiol (E2), progesterone (P4), and 6-keto prostaglandin F1α (6-keto PGF1α) levels were investigated. FO-based supplementation increased GPx activity on day 14 of lactation. Five days after weaning, the concentration of GSH in FO-fed sows was significantly higher than that in SO-fed sows. The E2 content of blood was significantly lower in the experimental group than in the control group for two of the three examined periods (day 14 of lactation and 30 days after insemination), whereas P4 levels were significantly higher in the experimental group 5 days after weaning. We found that 6-keto PGF1α levels were systematically lower in the experimental group throughout the trial. This study provides evidence of the major impact of omega-6 and -3 fatty acids on the tested hormone levels, which serve as precursors for the production of E2 and P4 but have an opposite effect on PGF2α production.

Prostacyclin Synthesis and Prostacyclin Receptor Expression in the Porcine Myometrium: Prostacyclin Potential to Regulate Fatty Acid Transporters, Cytokines and Contractility-Related Factors.

Simple SummaryProstacyclin (prostaglandin I2; PGI2) is an important modulator of vascular functions and is involved in various reproductive processes. PGI2 was also described as a modulator of uterine contractility in several species, including the pig. However, its synthesis and role in the myometrium of the porcine uterus are still not fully described. The objective of this study was to evaluate profiles of PGI2 synthesis and PGI2 receptor expression in the myometrium of gilts throughout the estrous cycle and during early pregnancy and to investigate the in vitro effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. The obtained results showed that the synthesis of PGI2 changes in the myometrium of pigs during both the estrous cycle and early pregnancy, resulting in much greater concentrations of PGI2 in cyclic than in pregnant gilts. Moreover, PGI2 stimulated the expression of fatty acid transporters and contractility-related calponin 1 and caldesmon 1, whereas it decreased cytokine expression. This study indicates that PGI2 may participate in the regulation of myometrial functions modulating the availability of factors involved in smooth muscle activity and inflammatory reaction in the uterus of pigs.Although prostacyclin (PGI2) has been well described as a regulator of smooth muscle activity, limited data are available concerning its role in the myometrium of pigs. The present research aimed to examine profiles of PGI2 synthase (PTGIS) and PGI2 receptor (PTGIR) expression and 6-keto PGF1α (a PGI2 metabolite) concentrations in the myometrium of gilts throughout the estrous cycle and during early pregnancy using qPCR, Western blot, and/or ELISA methods. Furthermore, myometrial explants were exposed to iloprost (a stable PGI2 analog) to investigate the effect of PGI2 on the mRNA expression of factors engaged in smooth muscle contraction, nutrient transport, prostaglandin synthesis and action, and inflammatory response. PTGIS mRNA expression was greater in cyclic than in pregnant gilts on days 11–12 after estrus and was accompanied by greater concentrations of 6-keto PGF1α detected in cyclic than in pregnant animals on days 11–20. Iloprost stimulated fatty acid transporters and contractility-related calponin 1 and caldesmon 1 mRNA expression and decreased interleukin 1β and tumor necrosis factor transcript abundance. The obtained results indicate a physiologically relevant role of PGI2 during the estrous cycle in the porcine myometrium with its importance for regulating the expression of contractility-, nutrient transport- and inflammatory response-related factors.

Innate Immune System Stimulation During Pregnancy Upregulates Thromboxane Synthesis in Rat Maternal Heart

BackgroundInfections during pregnancy are associated with adverse maternal and fetal outcomes. We previously showed that exposure to immunostimulatory CpG oligonucleotides (ODN2395, synthetic Toll‐like receptor 9 agonist) during pregnancy induces maternal vascular inflammation and enhances vascular tone in pregnant rats. These outcomes were mediated in part by activation of the cyclooxygenase/thromboxane A2 (COX/TxA2) pathway. The objective of this study was to investigate the impact of ODN2395‐induced immune system stimulation on maternal hearts during pregnancy.HypothesesExposure to TLR9‐mediated immune system activation during pregnancy upregulates the COX/TxA2signaling pathway in maternal cardiac tissues.MethodsRats were treated with a synthetic CpG DNA (ODN2395, 1 mg/kg, intraperitoneal injection) or vehicle (saline) in late pregnancy (gestational day (GD) 14, 16, and 18) and euthanized on GD 20 (term 22‐23). Fetoplacental biometrics were recorded after euthanasia and maternal hearts were collected to assess COX‐1 and COX‐2 expression via western blotting and 6‐keto PGF1α (PGI2 metabolic byproduct) and TxB2 (TxA2 metabolic byproduct) production use ELISA.ResultsMaternal heart weights did not differ between groups (ODN2395: 0.26 g/100 g BW vs. Vehicle: 0.23 g/100 g BW, n≥7, p=0.11). Left ventricular tissues from dams treated with ODN2395 released higher concentrations of TxB2compared to cardiac tissues from vehicle‐treated dams (ODN2395: 0.56 ± 0.06 ng/mg total protein vs. Vehicle: 0.31 ± 0.04 ng/mg total protein, n≥5, p=0.0041) but there were no differences in cardiac 6‐keto PGF1α release between groups (p=0.16). COX‐2 expression was lower in left ventricles from ODN2395‐treated rats compared to vehicle‐treated rats (p=0.009). There were no differences in cardiac COX‐1 expression between groups (p=0.27). Exposure to ODN2395 during pregnancy did not affect fetal weights (ODN2395: 2.28 ± 0.03 g vs. Vehicle: 2.25 ± 0.06 g, n≥7, p=0.9) or placenta weights (ODN2395: 0.44 ± 0.02 g vs. Vehicle 0.49 ± 0.01 g, n≥7, p=0.06), but increased fetal‐placental weight ratio (ODN2395: 5.3 ± 0.22 vs. Vehicle: 4.7 ± 0.15, p = 0.04). COX‐2 expression was greater in placental tissues from ODN2395‐treated rats (p=0.004) but there were no differences in placental 6‐keto PGF1α(p=0.51) and TxB2 production (p=0.32).ConclusionExposure to immunostimulatory CpG ODN during pregnancy induces upregulation of TxB2 synthesis in maternal cardiac tissues coupled with a reduction in COX‐2 expression. We suggest that cardiac inflammation during pregnancy may increase maternal risk for future cardiovascular events.

Coordinated action of microsomal prostaglandin E synthase-1 and prostacyclin synthase on contact hypersensitivity

Microsomal prostaglandin (PG) E synthase-1 (mPGES-1) and prostacyclin (PGI2) synthase (PGIS) are PG terminal synthases that work downstream of cyclooxygenase and synthesize PGE2 and PGI2, respectively. Although the involvement of PG receptors in acquired cutaneous immune responses was recently shown, the roles of these PG terminal synthases remain unclear. To identify the pathophysiological roles of mPGES-1 and PGIS in cutaneous immune systems, we applied contact hypersensitivity (CHS) to mPGES-1 and PGIS knockout (KO) mice as a model of acquired immune responses. Mice were treated with 1-fluoro-2,4-dinitrobenzene (DNFB) and evaluated for ear thickness and histopathological features. The results showed that the severity of ear swelling in both gene-deficient mice was much lower than that in wild-type (WT) mice. Histological examination of DNFB-treated ears showed that inflammatory cell infiltration and edema in the dermis were also less apparent in both genotypic mice. LC-MS analysis further showed that the increment in PGE2 levels in DNFB-treated ear tissue was reduced in mPGES-1 KO mice, and that 6-keto PGF1α (a stable metabolite of PGI2) was not detected in PGIS KO mice. Furthermore, we made bone marrow (BM) chimera and found that transplantation of WT mouse-derived BM cells restored the impaired CHS response in mPGES-1 KO mice but did not restore the response in PGIS KO mice. These results indicated that mPGES-1 in BM-derived cells and PGIS in non-BM-derived cells might play critical roles in DNFB-induced CHS. mPGES-1-derived PGE2 and PGIS-derived PGI2 might coordinately promote acquired cutaneous immune responses.

1667-P: Diabetes Incidence following Adoptive Transfer of Splenocytes Correlates with Increased Generation of Proinflammatory Lipids

Type 1 diabetes (T1D) is a T-cell mediated autoimmune disease characterized by the destruction of insulin-producing pancreatic islet β-cells. An understudied area is the role of lipid signaling. We find that the Ca2+-independent phospholipase A2β (iPLA2β) is induced under a diabetic milieu and that mitigation of iPLA2β activity attenuates β-cell death. iPLA2β hydrolyzes the sn-2 substituent from glycerophospholipid substrates to yield a free fatty acid. When the fatty acid is arachidonic acid, it can be metabolized to bioactive eicosanoids, many of which are pro-inflammatory. To address our overarching hypothesis that iPLA2β-derived lipids (iDLs) contribute to T1D development, we investigated the role of immune cell-iDLs in this process by utilizing a splenocyte adoptive transfer protocol. Splenocytes were prepared from 12-week-old female spontaneous diabetes prone non-obese diabetic (NOD, WT) and NOD.iPLA2β+/- (HET) donor mice and transferred into 5-week-old female immunodeficient (NOD.scid) recipient mice. Our overall findings revealed that: (1) splenocyte-iPLA2β mRNA in HET donor mice was ca. 40% of that in WT donor mice, (2) temporal IPGTTs revealed that the recipient mice of the HET genotype were more glucose tolerant, compared to recipients of the WT splenocytes; (3) weekly blood glucose monitoring revealed that all WT recipient mice developed diabetes between 11-16 weeks of age. In contrast, onset of diabetes in the HET recipient mice was delayed 3 weeks and by 16 weeks of age, 50% of these mice remained diabetes-free, (4) HET donor T-cell differentiation to Th1 phenotype is mitigated, (5) HET donor T-cell production of select pro-inflammatory lipids (i.e., 6-keto PGF1α, PGE2, and DHETs) is decreased, relative to WT donors, and (5) similar lipid signatures are evident in the terminal plasma of recipient mice. These findings suggest that immune T-cell-iPLA2β is a critical contributor to T1D development and could be targeted for therapeutics. Disclosure T. White: None. C. Chalfant: None. S. Ramanadham: None. Funding National Institute of Diabetes and Digestive and Kidney Diseases (R01DK110292); National Institute of Allergy and Infectious Diseases (R21AI146743)

A Walnut-Enriched Diet Modifies the Oxylipin Profile in Liver and Brain of Aged Mice — Impact on Inflammation Markers

Abstract Objectives Neuroinflammation contributes to brain-aging which may be mitigated by anti-inflammatory oxylipins. Based on our previous findings that a 6% walnut-enriched diet alone, and additional physical activity (PA), enhanced cognition in 18 months old NMRI, we now investigated the effects of this diet on oxylipin- and inflammatory marker levels in liver and brain. Methods 18 months and 3 months old female NMRI mice were fed with a 6% walnut-enriched diet. Oxylipins were determined in brain and liver sections using LC-MS. Expression of IL1β gene was determined by qRT-PCR. Results The walnut diet compensates for the age related increase in IL1β gene expression in the liver of mice, whereas expression in the brain was not affected. Basal levels of oxylipins in brain and liver samples isolated from young mice were generally lower compared to aged mice. The walnut diet further increased oxylipin levels of walnut specific fatty acids in liver and brain of aged mice. Enrichment of linoleic acid (LA) and α-linolenic acid (ALA) derived oxylipin levels were quantitatively higher in the liver compared to the brain (P < 0.0001). Hydroxy-oxylipins (HO) based on fatty acid LA were significantly increased in brain (P < 0.001) and liver (P < 0.0001) compared to control mice, while ALA based HO were only detected in the brains of walnut fed mice. The walnut diet in combination with physical activity (PA) reduced ARA based oxylipin levels (P < 0.05). Across all groups, concentrations of prostanoids were higher in the brain as compared to liver (P < 0.001). In the liver, walnuts tended to decrease PGD2 and TxB2 levels while increasing 6-keto PGF1α. The latter, as well as TxB2 tended to be decreased in the brain. Other ARA based prostanoids were unaffected. Effects of PA were contrary to each other, tending to increase ARA based prostanoids in the liver while decreasing them in the brain. PA further enhanced this effect in the brain, but tended to increase the inflammatory response in the liver. Conclusions A walnut diet differentially affects the oxylipin profile of liver and brain in aged mice. Production of oxylipins based on walnut fatty acids is generally increased. Attenuation of age-related, chronic inflammation in might be one of walnut's benefits and may contribute to a healthier aging of the brain. Funding Sources Research was supported by grants from California Walnut Commission.

Macrophage polarization is linked to Ca2+-independent phospholipase A2β-derived lipids and cross-cell signaling in mice

Phospholipases A2 (PLA2s) catalyze hydrolysis of the sn-2 substituent from glycerophospholipids to yield a free fatty acid (i.e., arachidonic acid), which can be metabolized to pro- or anti-inflammatory eicosanoids. Macrophages modulate inflammatory responses and are affected by Ca2+-independent phospholipase A2 (PLA2)β (iPLA2β). Here, we assessed the link between iPLA2β-derived lipids (iDLs) and macrophage polarization. Macrophages from WT and KO (iPLA2β-/-) mice were classically M1 pro-inflammatory phenotype activated or alternatively M2 anti-inflammatory phenotype activated, and eicosanoid production was determined by ultra-performance LC ESI-MS/MS. As a genotypic control, we performed similar analyses on macrophages from RIP.iPLA2β.Tg mice with selective iPLA2β overexpression in β-cells. Compared with WT, generation of select pro-inflammatory prostaglandins (PGs) was lower in iPLA2β-/- , and that of a specialized pro-resolving lipid mediator (SPM), resolvin D2, was higher; both changes are consistent with the M2 phenotype. Conversely, macrophages from RIP.iPLA2β.Tg mice exhibited an opposite landscape, one associated with the M1 phenotype: namely, increased production of pro-inflammatory eicosanoids (6-keto PGF1α, PGE2, leukotriene B4) and decreased ability to generate resolvin D2. These changes were not linked with secretory PLA2 or cytosolic PLA2α or with leakage of the transgene. Thus, we report previously unidentified links between select iPLA2β-derived eicosanoids, an SPM, and macrophage polarization. Importantly, our findings reveal for the first time that β-cell iPLA2β-derived signaling can predispose macrophage responses. These findings suggest that iDLs play critical roles in macrophage polarization, and we posit that they could be targeted therapeutically to counter inflammation-based disorders.

Acute changes in colonic PGE2 levels as a biomarker of efficacy after treatment of the Pirc (F344/NTac-Apcam1137) rat with celecoxib.

This study sought to evaluate short-term treatment with COX-2 inhibitors and acute changes in colonic PGE2 levels as predictors of long-term efficacy in a genetic model of colorectal cancer. Celecoxib oral suspension (40 mg/kg BID) was dosed to Apc-mutant Pirc (F344/NTac-Apcam1137) rats for 4 days (short-term group), or the equivalent dose of 1500 ppm celecoxib was administered in the diet for 4 months (long-term group). Percent inhibition of colonic PGE2 was calculated, and the reduction in colonic PGE2 was assessed in relation to suppression of adenomatous colon polyps. Colonic mucosa PGE2 was fourfold higher in Pirc than in F344 wild-type rats (21 vs. 5.6 pg/mg epithelial tissue), due at least in part to higher COX-2 expression, and this was confirmed by elevated PGE2-d11 levels in Pirc colonic S9 incubations. In the 4-day study, dose-dependent reductions in PGE2 were observed in colonic epithelium (-33% (P>0.05) and -57% (P=0.0012)), after low- and high-dose celecoxib treatments of 4 mg/kg and 40 mg/kg (bid), respectively. In the 4-month study, 1500 ppm celecoxib suppressed colonic epithelium PGE2 by 43.5%, and tumor multiplicity by 80% (P<0.0015). Suppression of plasma 6-keto PGF1α also was corroborated following long-term treatment with 1500 ppm celecoxib (P<0.05). Acute changes in colonic mucosa PGE2 provided a rapid means of predicting long-term chemopreventive effects from celecoxib, and might be useful for screening of new COX-2 inhibitor compounds.

Oxaliplatin induces prostaglandin E2 release in vascular endothelial cells.

Oxaliplatin (L-OHP) is known to induce adverse reactions at the injection site, including vascular pain, but the underlying mechanisms have not been clarified. Vascular pain during intravenous L-OHP administration can be inhibited by taking non-steroidal anti-inflammatory drugs (NSAIDs). In this study, we investigated the involvement of the arachidonic acid cascade and prostaglandin (PG) E2 and 15d-PGJ2 in vascular pain sensation during intravenous delivery of L-OHP. Cultured normal human umbilical cord vein endothelial cells (HUVECs) were treated with L-OHP or L-OHP + NSAID flurbiprofen for 2h and analyzed for the release of PGE2 and 15d-PGJ2 into culture supernatant by ELISA. The results showed that L-OHP significantly and dose-dependently increased PGE2 secretion by HUVECs; however, flurbiprofen effectively prevented PGE2 increase. On the other hand, cisplatin, another platinum anticancer drug, did not stimulate PGE2 production. Other PGs, including 15d-PGJ2, 6-keto PGF1α, PGF2α, and PGD2 were not increased by L-OHP or cisplatin. Protein expression analysis revealed that cyclooxygenase 1 and cytoplasmic PGE synthase involved in constitutive PG metabolism were expressed in HUVECs but not affected by L-OHP exposure. This study indicates that L-OHP treatment specifically upregulated PGE2 secretion by vascular endothelial cells, which may contribute to vascular pain, and that NSAIDs can be used to inhibit PGE2 release and attenuate L-OHP-induced hyperalgesia.

Mannitol triggers mast cell–dependent contractions of human small bronchi and prostacyclin bronchoprotection

Clinical research supports that exercise-induced bronchoconstriction (EIB) is caused by hyperosmolar triggering of mast cells. The reaction can be mimicked by inhalation of mannitol, but it has paradoxically previously not been possible to replicate this mode of action of mannitol in isolated airways. We sought to establish an exvivo model of EIB in human small bronchi. Small bronchi (inner diameter, 0.5-2mm) from macroscopically healthy human lung tissue were obtained from 48 patients and mounted in organ baths. Contractions and mediator release were analyzed after challenge with hyperosmolar mannitol (850 mOsm). Ten minutes of exposure to mannitol caused a small initial contraction (12%±1% of maximum) that was followed by a second and much larger contraction (maximum effect [Emax], 47%±5%) when mannitol was washed out. The mast cell stabilizer cromolyn reduced the second contraction (Emax,27%±3%). Furthermore, this main contraction was abolished by the combination of antagonists of histamine and cysteinyl leukotrienes in the presence of indomethacin. Mannitol increased the release of the mast cell mediators histamine (9.0-fold), cysteinyl leukotrienes (4.5-fold), and prostaglandin (PG) D2 (5.4-fold), as well as PGE2 (6.3-fold) and the prostacyclin metabolite 6-keto PGF1α (5.7-fold). In contrast, indomethacin alone enhanced the bronchoconstriction (Emax,68%±6%). Likewise, receptor antagonists for PGE2 (EP2 and EP4) and prostacyclin (IP)also enhanced the mannitol-induced bronchoconstriction (Emax, 67%±5%, 66%±4%, and 68%±3%, respectively). In bronchi precontracted by carbachol, the IP receptor agonist cicaprost induced profound relaxation. This new protocol established an invitro model for studies of EIB in isolated human bronchi. The IP receptor might be a new target for asthma treatment.

Long-Term Aspirin Administration Has No Effect on Erectile Function: Evidence from Adult Rats and Ageing Rat Model

As the broad spectrum pharmacological action, aspirin has been one of the most widely used medicines since its initial synthesis; however, the association between aspirin and erectile function is still controversial. We aim to explore whether long-term aspirin administration deteriorates or preserves erectile function from adult rats and ageing rat model. Twenty adult rats (10 weeks of age) and twenty ageing rats (80 weeks of age) were randomly divided into four groups as follows: Adult-Control (normal saline [NS]), Adult-Aspirin (aspirin, 10 mg/kg/d), Ageing-Control (NS), and Ageing-Aspirin (aspirin, 10 mg/kg/d) groups (n = 10 per group). For all rats, erectile function was assessed by maximum intracavernous pressure (ICP), total area under ICP curve (AUC), ICP/mean arterial pressure (MAP) ratio, and MAP. The total treatment duration was one month. Protein expression levels of cyclooxygenase-1 (COX-1), COX-2, endothelial nitric oxide synthase (eNOS), and nNOS of the corpus cavernosum were detected by Western blot. ELISA kits were used to determine 6-keto PGF1a, PGE2, TXB2, cyclic adenosine monophosphate (cAMP), and cyclic guanosine monophosphate (cGMP) levels. Total nitric oxide (NO) concentration was measured using a fluorometric assay kit. As a result, Ageing-Control rats revealed significantly decreased ICP, AUC, and ICP/MAP ratios compared to Adult-Control rats, and these effects were accompanied by reduced eNOS protein expression and lower total NO and cGMP levels; however, no difference was found in nNOS protein expression. For adult rat groups, aspirin significantly inhibited the production of 6-keto PGF1a, PGE2, and TXB2; however, it neither changed the ICP, AUC, or ICP/ MAP ratios nor altered the protein expression of eNOS, nNOS, COX-1, and COX-2. Meanwhile, aspirin did not influence the concentrations of total NO, cAMP, or cGMP. The same tendency was also found in the ageing rat model, which confirmed that aspirin did not alter erectile function. Our data suggested that long-term aspirin administration did not strengthen or weaken erectile function in adult rats or ageing rat model. Thus, it had no impact on erectile function.

Divergent roles of prostacyclin and PGE2 in human tendinopathy

BackgroundTendon disease is a significant global healthcare burden whereby patients experience pain and disability; however, the mechanisms that underlie inflammation and pain are poorly understood. Herein, we investigated the role of prostaglandins as important mediators of inflammation and pain in tissues and cells derived from patients with tendinopathy.MethodsWe studied supraspinatus and Achilles tendon biopsies from symptomatic patients with tendinopathy or rupture. Tendon-derived stromal cells (CD45negCD34neg) isolated from tendons were cultured and treated with interleukin-1β (IL-1β) to investigate prostaglandin production.ResultsDiseased tendon tissues showed increased expression of prostacyclin receptor (IP) and enzymes catalyzing the biosynthesis of prostaglandins, including cyclooxygenase-1 (COX-1), COX-2, prostacyclin synthase (PGIS), and microsomal prostaglandin E synthase-1 (mPGES-1). PGIS co-localized with cells expressing Podoplanin, a marker of stromal fibroblast activation, and the nociceptive neuromodulator NMDAR-1. Treatment with IL-1β induced release of the prostacyclin metabolite 6-keto PGF1α in tendon cells isolated from diseased supraspinatus and Achilles tendons but not in cells from healthy comparator tendons. The same treatment induced profound prostaglandin E2 (PGE2) release in tendon cells derived from patients with supraspinatus tendon tears. Incubation of IL-1β treated diseased tendon cells with selective mPGES-1 inhibitor Compound III, reduced PGE2, and simultaneously increased 6-keto PGF1α production. Conversely, COX blockade with naproxen or NS-398 inhibited both PGE2 and 6-keto PGF1α production. Tendon biopsies from patients in whom symptoms had resolved showed increased PTGIS compared to biopsies from patients with persistent tendinopathy.ConclusionsOur results suggest that PGE2 sustains inflammation and pain while prostacyclin may have a protective role in human tendon disease.

The Effects of Exercise, Aspirin, and Celecoxib in an Atherogenic Environment.

Optimal vascular function is a hallmark of cardiovascular health. Specifically, the balance of vasoconstricting and vasodilating substances is recognized as a marker of vascular health. One of the greatest challenges to vascular health and vasodilatory balance is tumor necrosis factor alpha (TNFα)-mediated inflammation. Uncovering effective strategies that maintain a vascular environment that is more vasodilatory and antithrombotic in the face of an inflammatory challenge is favorable. To test the ability of various antithrombotic and provasodilatory treatments, as well as combinations thereof, to prevent unfavorable changes in markers of endothelial dysfunction in human umbilical vein endothelial cells when presented with an inflammatory challenge. Human umbilical vein endothelial cells were pretreated with exercise-like levels of laminar shear stress (LSS), aspirin, celecoxib, and their combination before a TNFα challenge. Western blot analysis as well as colorimetric assays were used to determine levels of endothelial nitric oxide synthase (eNOS) and prostacyclin (6-keto PGF1α)/thromboxane (TXB2) metabolite ratio, respectively. Neither aspirin nor celecoxib were effective in preventing TNFα-induced reduction in eNOS. Further, aspirin was unable to maintain baseline levels of prostacyclin/thromboxane ratio in the face of the inflammatory challenge. Laminar shear stress, aspirin/LSS combination, and celecoxib/LSS combination were all able to prevent TNFα-induced alterations in eNOS levels and prostacyclin/thromboxane ratio. Effective strategies to maintain a healthy endothelium, and therefore resistance vessel health, need to include exercise-levels of shear stress to be effective.

Investigation of the therapy targets of Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu recipe on type 2 diabetes by serum proteome labeled with iTRAQ

Ethnopharmacology relevanceBased on basic theories of Chinese medicine, Yi-Qi-Yang-Yin-Hua-Tan-Qu-Yu (YQYYHTQY) recipe was constituted by eleven kinds of Chinese herbs and effective in treatment of type 2 diabetes (T2DM). But the therapy target was unclear. ObjectiveIn this study, we used the serum proteome labeled by iTRAQ to find therapy target of YQYYHTQY recipe on T2DM. Materials and methodsThe rat model was induced by high-fat diet (HFD) and streptozotocin (STZ, 30 mg/kg). Drugs were administered to rats once daily for 14 days. Related laboratory parameters were observed. Serum proteome were compared between T2DM and YQYYHTQY group using the iTRAQ labeling quantitative proteomics technique. Functional differential proteins were analysis by STRING software. Target proteins were confirmed by ELISA kits. ResultsHyperglycemia, hyperinsulinemia, insulin resistance, decrease of glucose transporter, depilation, less activity, flock together, depression, ecchymosis of tongue and tail appearance, the typical diabetic patients “a little more than three” symptoms, as well as the decrease of grip strength, serum cyclic adenosine monophosphate (cAMP)/ cyclic guanosine monophosphate (cGMP) ratio, serum high density lipoprotein-cholesterol (HDL-C) and the increase of serum triglyceride (TG), total cholesterol (TC), low density lipoprotein-cholesterol (LDL-C), thromboxane B2 (TXB2)/ 6-keto prostaglandin F1α (6-keto PGF1α) ratio, endothelin-1 (ET-1) levels were found in T2DM group. After drugs treatment, all the above indexes almost were improved in different degrees and effect of YQYYHTQY recipe was superior to pioglitazone hydrochloride. In addition, there were 23 differential proteins, 5 up-regulated and 18 down-regulated proteins. Of them, there were 4 proteins related with diabetes, blood and behavior. Cell division control protein 42 homolog (CDC42) and Ras homolog gene family member A (RhoA) were the therapy targets of YQYYHTQY recipe on T2DM. ConclusionsYQYYHTQY recipe showed therapy effect on T2DM. CDC42 and RhoA proteins were the therapy targets of YQYYHTQY recipe.

Influence of the selective COX-2 inhibitor celecoxib on sex differences in blood pressure and albuminuria in spontaneously hypertensive rats

We previously reported that female spontaneously hypertensive rats (SHR) have greater cyclooxygenase-2 (COX-2) expression in the renal medulla and enhanced urinary excretion of prostaglandin (PG) E2 (PGE2) metabolites compared to male SHR. Based on the role of COX-2-derived prostanoids in the regulation of cardiovascular health, the aim of the current study was to test the hypothesis that blood pressure (BP) in female SHR is more sensitive to COX-2 inhibition than in males. Seven week old male and female SHR were implanted with telemetry transmitters for continuous BP recording. After one week of baseline BP recording, male and female SHR were randomized to receive the selective COX-2 inhibitor celecoxib (10 mg/kg/day) or vehicle for six weeks (from 9 to 14 weeks of age). Female SHR had lower BP and albuminuria compared to male SHR as well as enhanced urinary excretion of PGE metabolite (PGEM), 6-keto PGF1α and thromboxane B2, indicators of PGE2, PGI2 and TXA2, respectively. Treatment with celecoxib did not significantly alter BP or albuminuria in either female or male SHR. Celecoxib did not change PGs metabolites excretion in male SHR; however, excretion levels of PGEM and 6-keto PGF1α were reduced in female SHR. COX-2 derived PG can also induce oxidative stress. Markers of oxidative stress (thiobarbituric acid reactive substances (TBARs) and H2O2 excretion) were lesser in female SHR versus male SHR. Celecoxib treatment did not significantly change markers of oxidative stress in female SHR, however, urinary TBARs excretion was significantly reduced in male SHR after 6 weeks of treatment with celecoxib. Therefore, although celecoxib treatment appears to have distinct effects on prostanoids levels in female SHR vs. males, it is unlikely that COX-2 contributes to established sex differences in BP in SHR.

Leptin upregulates COX-2 and its downstream products in aortic endothelial cells.

The adipocyte-derived hormone leptin is associated with hypertension. The involvement of cyclooxygenase-2 (COX-2) and its downstream vasomotor products prostaglandin (PG) and thromboxane (TX)A2 in the mechanisms of action of leptin have remained elusive. The aim of the present study was to investigate the effects of leptin on the expression of COX-2 by rat aortic endothelial cells (RAECs) and the concentration of its products, represented by 6-keto PGF1α and TXB2, in the culture media. RAECs were isolated, cultured and identified by immunofluorescence staining. The RAECs were incubated with different concentrations of leptin (10-10, 10-9 and 10-8 M) for various durations (36 or 48 h). COX-2 mRNA and protein expression in the cells was detected by reverse-transcription quantitative PCR and western blot analysis, respectively. The vasodilator 6-keto PGF1α and the vasoconstrictor TXB2 were detected in the supernatant by ELISA. The cultured cells displayed specific factor VIII expression in the cytoplasm. Compared with the PBS-treated control group, leptin significantly increased the expression of COX-2 mRNA and protein in a time- and dose-dependent manner (P<0.01). Furthermore, the vasodilator 6-keto PGF1α was increased and the TXB2/6-keto PGF1α ratio decreased only with relatively high concentrations of leptin (10-9 or 10-8 M; P<0.01), but TXB2 levels were not affected (P>0.05). In conclusion, leptin significantly increased the expression of inflammatory marker COX-2 and its downstream product 6-keto PGF1α, while also decreasing the TXB2/6-keto PGF1α ratio in vitro. These observations suggested that COX-2 may have an important role in the effects of leptin on inflammation, such as the low-inflammatory disease hypertension. However, selective COX-2 inhibitors may increase the risk of hypertension due to inhibiting 6-keto PGF1α, the vasodilator product of COX-2.

Reduced platelet aggregation in women after intercourse: a possible role for the cyclooxygenase pathway.

We hypothesise that molecules in the cyclooxygenase pathway affect platelet activity when seminal fluid (SF) is present. We considered the influence of SF on platelet aggregation in women, and believe that the prostanoids in SF signalling are significant. Thirty-one female subjects were studied, 20 of whom were sexually active. Male partners were given either aspirin or indomethacin to inhibit cyclooxygenase. The 6-keto prostaglandin F1α (6-keto PGF1α) and prostaglandin E metabolite (PGE-M) in SF were measured by competitive assay. Platelets and prostanoids were evaluated in women, periodically, before and after intercourse. The platelets were tested with adenosine diphosphate (ADP) and arachidonic acid (AA). To block the interaction between the uterus and SF, some couples used condoms. We found that the 6-keto prostaglandin F1α in urine at 2hours post-intercourse (1418.75pg/mL, Std 688.39) was greater than pre-intercourse (772.68pg/mL, Std 116.54). Post-intercourse, a transient decrease in platelet aggregation was observed in women whose partners did not use condoms. Averages for platelet aggregation were 20.16% with ADP, and more significantly, 37.79% with AA after 2hours. In contrast, couples using condoms showed no changes, averaging 64.02% with ADP and 72.06% with AA. Women whose partners were taking aspirin or indomethacin also showed no changes. SF from men taking aspirin or indomethacin led to no reduction in platelet aggregometry in their partners. These results indicate that in cases of exposure to SF, the transient change in women's platelet activity could be related to the cyclooxygenase pathway.

Reduced expression of adipose triglyceride lipase decreases arachidonic acid release and prostacyclin secretion in human aortic endothelial cells

Background: Vascular endothelial cells represent an important source of arachidonic acid (AA)-derived mediators involved in the generation of anti- or proatherogenic environments. Evidence emerged (in mast cells), that in addition to phospholipases, neutral lipid hydrolases as adipose triglyceride lipase (ATGL) also participate in this process.Objective: To examine the impact of ATGL on AA-release from cellular phospholipids (PL) and on prostacyclin secretion in human aortic endothelial cells (HAEC).Methods and results: siRNA-mediated silencing of ATGL promoted lipid droplet formation and TG accumulation in HAEC (nile red stain). ATGL knockdown decreased the basal and A23187 (calcium ionophore)-induced release of 14C-AA from (14C-AA-labeled) HAEC. In A23187-stimulated ATGL silenced cells, this was accompanied by a decreased content of 14C-AA in cellular PL and a decreased secretion of prostacyclin (determined by 6-keto PGF1α EIA).Conclusions: In vascular endothelial cells, the efficiency of stimulus-induced AA release and prostacyclin secretion is dependent on ATGL.