cPLA2 Levels Research Articles

Investigation on the antipyretic mechanism of Chaiqin Qingning capsule for the treatment of fever based on network pharmacology, molecular docking, and in vitro experimental validation

AbstractChaiqin Qingning Capsule (CQQNC), a traditional Chinese patent medicine, can effectively shorten the duration of fever and significantly improve fever symptoms. However, the mechanism of its antipyretic effect needs to be further elucidated. Therefore, we aimed to investigate the molecular mechanism of CQQNC in the treatment of fever. We used the network pharmacology method to analyze the mechanism of action of CQQNC in the treatment of fever and validated our study primarily by molecular docking. Finally, the predictive results were verified by IL‐1β‐induced bEnd.3 cells. The results showed that quercetin, kaempferol, cubebin, chenodeoxycholic acid, isorhamnetin, bilirubin, cholic acid, and baicalin were the major components of CQQNC against fever. A total of 381 common targets have been crossed by CQQNC for the treatment of fever. Furthermore, we found that CQQNC targets several deregulated genes in fever such as AKT1, COX2, AVP, cAMP, IL6, IL1B, TNF, mPGES1, and PI3K, biological functions such as endopeptidase, cytokine receptor binding, and phosphatase activity, and signaling pathways such as the PI3K‐Akt pathway and the AGE‐RAGE signaling pathway in diabetic complications. The docking study revealed that the core components of CQQNC both had high affinity for hub targets, especially the targets of COX‐2, cAMP, mPGES1, and PI3K proteins. To further investigate the mechanism of CQQNC, an Elisa assay and Western blot detection were performed as part of an in vitro study. Elisa's result showed that CQQNC can significantly decrease the expression levels of cPLA2, sPLA2, PGE2, cAMP, and 15‐PGDH after stimulating IL‐1β to bEnd.3 cells in a dose‐dependent manner (p < .01, p < .001, p < .0001). In addition, detection of the PGE2/ COX /cMAP pathway via immunoblotting showed that CQQNC can significantly downregulate the protein expression of COX‐1, COX‐2, EP3, cAMP, and mPGES1 in bEnd.3 (p < .0001, p < .001, p < .01). In conclusion, our study confirmed that the antipyretic mechanism of CQQNC affects the synthesis and secretion processes of PGE2 via the PGE2/ COX/cAMP pathway, providing insight into the antipyretic mechanism of CQQNC in clinical application.

Co-exposure to Fe, Zn, and Cu induced neuronal ferroptosis with associated lipid metabolism disorder via the ERK/cPLA2/AA pathway

Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans. However, the precise mechanisms underlying the neurotoxicity of exposure to mixtures of Fe, Zn, and Cu are still mostly unclear. The research aimed to investigate the influence of co-exposure to iron, zinc and copper and the related mechanisms in HT22 murine hippocampal neuronal cells. Intracellular metal content, markers of oxidative damage, and biomarkers of ferroptosis were respectively detected. Afterward, metabolomic analyses were performed to obtain a comprehensive understanding of the metal mixtures on metabolism, and the functions of key enzymes on metabolic pathways were validated. The results showed that metal co-exposure resulted in cellular iron overload and increased lipid peroxidation, accompanied by significant pathological damage and mitochondrial abnormalities in HT22 cells. Meanwhile, it was found that GSH depletion, decreased GPX4, and increased expression of the lipid metabolism gene ACSL4 play important roles in ferroptosis induced by metal mixture. Further, metabolomic analysis revealed metal co-exposure induced significant alterations in metabolite levels, especially in the glycerophospholipid metabolism pathway and the arachidonic acid metabolism pathway. The levels of cPLA2 and its metabolite, arachidonic acid, were significantly increased after metal co-exposure. Then, inhibition of cPLA2 decreased the level of arachidonic acid and attenuated ferroptosis in neuronal cells. Collectively, our findings unveiled ferroptosis induced by metal co-exposure associated with crucial molecular changes in neuronal cells, providing a novel perspective on the comprehensive toxicity risk assessment of metal mixtures.

Phospholipase A2 enzymes differently impact PUFA release and oxylipin formation ex vivo in rat hearts.

Phospholipase A2 (PLA2) enzymes cleave cell membrane phospholipids and release polyunsaturated fatty acids (PUFA), which can be converted into oxylipins. However, little is known about PLA2 preference for PUFA, and even less is known about how this further impacts oxylipin formation. Therefore, we investigated the role of different PLA2 groups in PUFA release and oxylipin formation in rat hearts. Sprague-Dawley rat heart homogenates were incubated without or with varespladib (VAR), methyl arachidonyl fluorophosphonate (MAFP) or EDTA. Free PUFA and oxylipins were determined by HPLC-MS/MS, and isoform expressions by RT-qPCR. Inhibition of sPLA2 IIA and/or V by VAR reduced the release of ARA and DHA, but only DHA oxylipins were inhibited. MAFP reduced the release of ARA, DHA, ALA, and EPA, and the formation of ARA, LA, DGLA, DHA, ALA, and EPA oxylipins. Interestingly, cyclooxygenase and 12-lipoxygenase oxylipins were not inhibited. mRNA expression levels of sPLA2 and iPLA2 isoforms were highest whereas levels of cPLA2 were low, consistent with activity. In conclusion, sPLA2 enzymes lead to the formation of DHA oxylipins, while iPLA2 is likely responsible for the formation of most other oxylipins in healthy rat hearts. Oxylipin formation cannot be implied from PUFA release, thus, both should be evaluated in PLA2 activity studies.

Gypenoside L and Gypenoside LI Inhibit Proliferation in Renal Cell Carcinoma via Regulation of the MAPK and Arachidonic Acid Metabolism Pathways.

Renal cell carcinoma (RCC) has the highest mortality rate of all urological malignancies. Clear cell renal cell carcinoma (ccRCC) accounts for approximately 80% of all RCC cases and is often accompanied by the accumulation of lipid droplets. Growing evidence indicates that ccRCC is a metabolism-related disease. Gypenosides are commonly used for the clinical treatment of hyperlipidemia, and their antitumor activity has also been recognized. However, the potential inhibitory effects and mechanisms of action of gypenoside L (Gyp L) and gypenoside LI (Gyp LI) in ccRCC remain unclear. In this study, we confirmed that Gyp L and Gyp LI significantly inhibited proliferation and induced apoptosis in ccRCC cells in vitro. We performed network pharmacology and RNA-seq, and verified the results by Western blotting, RT-qPCR, and immunofluorescence experiments. Our results demonstrated that Gyp L and Gyp LI upregulate the expression of COX2 and downregulate the expression levels of cPLA2 and CYP1A1, resulting in reduced arachidonic acid and apoptosis. Gyp L and Gyp LI upregulated the protein levels of DUSP1, p-JUN, and p-JNK, and downregulated p-MEK1/2, p-ERK, and p-P38 levels. Moreover, gypenosides significantly inhibited tumor growth in vivo, and gypenosides significantly reduced cPLA2 and CYP1A1 expression. Furthermore, we performed absolute quantification of arachidonic acid (AA) content in ccRCC cells and tumor tissues by HPLC-MS, and found that the arachidonic acid content was significantly reduced after Gyp L, Gyp LI, and gypenoside intervention. In conclusion, our data suggest that Gyp L, Gyp LI, and gypenosides decrease the content of arachidonic acid in ccRCC cells and tumor tissues, but do not have cytotoxic effects on nude mice. Thus, Gyp L, Gyp LI, and total gypenosides extracted from Gynostemma pentaphyllum exhibited antitumor activities against ccRCC.

Altered hippocampal expression and function of cytosolic phospholipase A2 (cPLA2) in temporal lobe epilepsy (TLE)

ABSTRACT Objectives Temporal lobe epilepsy (TLE) is the most common form of drug-resistant epilepsy. Blood–brain barrier (BBB) leakage occurs during epileptogenesis and several pieces of evidence suggest that this might contribute to the progression of epilepsy. Seizures trigger a pathway involving glutamate signalling through cytosolic phospholipase A2 (cPLA2). This pathway leads to BBB leakage and induces the expression of drug efflux transporters, leading to drug resistance. Therefore, this study aims to determine the mRNA and protein levels of cPLA2, along with its functional activity, in the hippocampus of pilocarpine model of TLE as well as in the surgically resected hippocampal samples of patients with TLE. Methods mRNA levels and protein levels of cPLA2 were evaluated by real-time PCR and western blot analysis respectively in animal model of TLE as well as surgically resected hippocampal tissue specimens of TLE. cPLA2 functional activity was measured spectrophotometrically. Results Significant up-regulation of cPLA2 mRNA was observed in the hippocampal samples obtained from TLE rats (p < 0.05) and-TLE patients (p < 0.01). Increased protein expression of cPLA2 was also demonstrated in the hippocampal samples of TLE rats (p < 0.01) as well as TLE patients (p < 0.01). Similarly, functional activity of cPLA2 was found to be up-regulated in the hippocampus of pilocarpine model of TLE rats (p < 0.01) as well as in the TLE patients (p < 0.01). Discussion These findings suggest that alterations in cPLA2 expression and activity level in the hippocampus could potentially be a part of dynamic changes associated with TLE.

Epigenetic Regulation of the COX-2 Pathway Modulates Macrophage Inflammation in Diabetic Wound Repair

Abstract Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to proliferative phase of healing. In pathologic conditions, such as type 2 diabetes (T2D), wounds fail to heal due to impaired resolution of inflammation. There is increasing evidence that epigenetic-based mechanisms control macrophage function. The cyclooxygenase (COX)-2/Prostaglandin E2 (PGE2) axis, as well as upstream pathways including cytosolic phospholipase A2 (c(PL)A2), have been associated with chronic inflammation, however the underlying mechanisms of COX-2/PGE2 regulation in wound repair have not been examined. Here, we find in human and murine diabetic wounds, that PGE2 production is elevated in wound macrophages and regulates inflammation. Further, epigenetic modification of the COX-2/PGE2 pathway was regulated by MLL1, a methyltransferase, that increased H3K4 trimethylation on the cPLA2 promoter in diabetic wound macrophages and human T2D monocytes resulting in increased cPLA2 expression. Myeloid-specific deletion of MLL1 restored basal cPLA2 levels. Separately, increased TGFβ1 in diabetic wounds augmented miR-29b and DNMT 3a/3b in wound macrophages, resulting in hypomethylation of the Cox-2 gene promoter and overexpression of COX-2/PGE2. Inhibition of the PGE2 pathway, with a macrophage-specific nanocarrier containing a COX-2 inhibitor improved diabetic healing. Taken together, our results indicate the COX-2/PGE2 pathway is a critical regulator of macrophage-mediated inflammation in diabetic wounds and therapeutic manipulation of this pathway improves pathologic wound repair.

Cytosolic phospholipase A2 and F2 isoprostanes are involved in semen quality and human infertility-A study on leucocytospermia, varicocele and idiopathic infertility.

Phospholipase A2 (PLA2 ) is involved in eicosanoid release, and F2 -isoprostanes (F2 -IsoPs), as free radical-generated eicosanoids released by PLA2 , are indicators of oxidative stress in different human conditions. This study investigated the interplay between cytosolic PLA2 (cPLA2 ), F2 -IsoPs and sperm features in male infertility, when the involvement of oxidative stress has been reported. Semen evaluation was performed following WHO guidelines, sperm ultrastructure was detected by transmission electron microscopy indicating a fertility index, and the percentages of sperm immaturity, apoptosis and necrosis. In sperm cells and seminal plasma, cPLA2 levels were determined by immunological method, whereas F2 -IsoPs by mass spectrometry. Sperm concentration, morphology, vitality and fertility index values were significantly lower in infertile groups compared with fertile men. An increase in sperm apoptosis and necrosis (p<.01), apoptosis (p<.01) and immaturity (p<.001) was detected in leucocytospermia, idiopathic infertility and varicocele, respectively. Seminal cPLA2 showed the highest value in varicocele group (p<.05), whereas seminal F2 -IsoPs increased in varicocele (p<.001) and leucocytospermia (p<.05) groups. In the whole population, F2 -IsoP and cPLA2 levels were positively correlated (p<.05). On the contrary, F2 -IsoPs and cPLA2 were not significantly different when investigated in sperm cells. Our data indicate that fatty acid oxidation/metabolism plays a role in different male reproductive pathological conditions.

PARP inhibition in vivo blocks alcohol-induced brain neurodegeneration and neuroinflammatory cytosolic phospholipase A2 elevations

Chronic alcoholism promotes brain damage that impairs memory and cognition. High binge alcohol levels in adult rats also cause substantial neurodamage to memory-linked regions, notably, the hippocampus (HC) and entorhinal cortex (ECX). Concurrent with neurodegeneration, alcohol elevates poly (ADP-ribose) polymerase-1 (PARP-1) and cytosolic phospholipase A2 (cPLA2) levels. PARP-1 triggers necrosis when excessively activated, while cPLA2 liberates neuroinflammatory ω-6 arachidonic acid. Inhibitors of PARP exert in vitro neuroprotection while suppressing cPLA2 elevations in alcohol-treated HC-ECX slice cultures. Here, we examined in vivo neuroprotection and cPLA2 suppression by the PARP inhibitor, veliparib, in a recognized adult rat model of alcohol-binging. Adult male rats received Vanilla Ensure containing alcohol (ethanol, 7.1 ± 0.3 g/kg/day), or control (dextrose) ± veliparib (25 mg/kg/day), by gavage 3x daily for 4 days. Rats were sacrificed on the morning after the final binge. HC and ECX neurodegeneration was assessed in fixed sections by Fluoro-Jade B (FJB) staining. Dorsal HC, ventral HC, and ECX cPLA2 levels were quantified by immunoblotting. Like other studies using this model, alcohol binges elevated FJB staining in the HC (dentate gyrus) and ECX, indicating neurodegeneration. Veliparib co-treatment significantly reduced dentate gyrus and ECX neurodegeneration by 79% and 66%, respectively. Alcohol binges increased cPLA2 in the ventral HC by 34% and ECX by 72%, which veliparib co-treatment largely prevented. Dorsal HC cPLA2 levels remained unaffected by alcohol binges, consistent with negligible FJB staining in this brain region. These in vivo results support an emerging key role for PARP in binge alcohol-induced neurodegeneration and cPLA2-related neuroinflammation.

Neuro-Behavioral Status and the Hippocampal Expression of Metabolic Associated Genes in Wild-Type Rat Following a Ketogenic Diet.

While a ketogenic diet (KD) is a well-established therapy for medically intractable epilepsy, clinical evidence of relevant adverse events of a KD has also been reported. We asked whether this kind of diet would have deleterious effects on wild-type brain function by evaluating KD-induced biochemical changes in the hippocampus as well as neurobehavioral changes occurring in wild-type rats. Fifty-four Sprague-Dawley rats were randomly assigned to three groups on postnatal day 28 (P28): wild-type rats fed with a KD qd (daily for 4 weeks, KD) or qod (every other day for 4 weeks, KOD), and wild-type rats fed with standard normal laboratory diet (ND). Neurobehavioral changes were observed on P35, P42, and P49. The hippocampal mossy fiber sprouting, the expression levels of zinc transporters (ZnTs) and lipid metabolism related genes were detected by Timm staining, RT-qPCR and western blot analysis, respectively, on P58. The KD-treated KOD and KD groups showed a significant delay of negative geotaxis reflex on P35, but not on P42 or P49. In the open field test, daily KD treatment only led to a reduction in exploratory activity and increased grooming times but induced no significant changes in the scores of vertical activity or delay time. KD qod treated rats (KOD) displayed a slight delay in the place navigation test on P35 compared with the KD group. There were no significant differences in Timm staining among the three groups. In parallel with these changes, KD treatment (both KD and KOD) induced significantly downregulated mRNA levels of Apoa1, Pdk4, and upregulated expression of ApoE, ANXN7, and cPLA2 in the hippocampus when compared with the ND group (except in the case of ApoE in the KOD group). Notably, both the mRNA and protein levels of cPLA2 in the KOD rats were significantly downregulated compared with the KD group but still markedly higher than in the ND group. No significant difference was found in ZnTs among the three groups. Our data suggest that early-life KD can provoke minor neurobehavioral effects in particular a delay in negative geotaxis reflex and an increase in grooming activity. The hippocampal lipid metabolism signaling pathway, especially cPLA2, may be the target of the protective effect of KD on long-term brain injury after developmental seizures.

Changes in the levels of 12/15-lipoxygenase, apoptosis-related proteins and inflammatory factors in the cortex of diabetic rats and the neuroprotection of baicalein

This study was designed to investigate the neuroprotective effects of baicalein and the effect of the cortical 12/15-lipoxygenase (12/15-LOX) pathway on diabetic cognitive dysfunction. Our results showed that spatial learning and memory ability, as well as cortex neurons, were significantly impaired after the onset of diabetes. The fasting blood glucose and random blood glucose levels in the model group were significantly higher than those in the normal group. The levels of TG and TC in the plasma of the model group were significantly increased, but there was no significant difference in the LDL level. The insulin content in the plasma of diabetic rats was significantly lower than that of the normal group. The levels of inflammatory factors and 12(S)-HETE were significantly increased in diabetic rats, as were the protein expression levels of cPLA2, 12/15-LOX, p38MAPK, phospho-p38MAPK, caspase-3, caspase-9 and Aβ1–42; by contrast, protein expression of Bcl-2 was significantly decreased. Administration of baicalein was shown to improve the spatial learning and memory ability and significantly decrease the levels of inflammatory cytokines. However, baicalein did not significantly influence the levels of blood glucose, lipids or insulin in rats. Baicalein treatment significantly protected diabetes rats from neuron death; significantly attenuated the overexpression of cPLA2, 12/15-LOX, p38MAPK, phospho-p38MAPK, caspase-3, caspase-9 and Aβ1–42; and upregulated the expression of Bcl-2. These findings suggest that baicalein improves the cognitive function of diabetic rats by directly acting in the brain rather than by regulating the levels of blood glucose, lipids or insulin. In addition, baicalein can protect rat cortical neurons from damage caused by diabetes via inhibiting the 12/15-LOX pathway and relieving inflammation and apoptosis of the central nervous system.

Abstract 1123: Sphingolipids transfer proteins (GLTP and CPTP) regulate the neoplastic progression of colon and breast cancer cells

Abstract Sphingolipids have previously been shown to affect cancer progression by regulating cell death (apoptosis) and survival (autophagy). While apoptosis is promoted by elevations in sphingosine and ceramide, increases in sphingosine-1-phosphate and ceramide-1-phosphate can tip the balance toward survival. Changes in the expression of sphingolipid transfer proteins such as GLTP (glycolipid transfer protein) and CPTP (ceramide-1-phosphate transfer protein) have been shown to modulate cell shape change and regulate pro-inflammatory cytokine release, respectively, in human cancer epithelial cell lines. To gain a better understanding of the situation, we investigated further. We discovered that GLTP overexpression inhibits the growth of human colon carcinoma cell lines (HT-29; HCT-116), but spares normal colonic epithelial cells (HCEC) largely due to growth arrest at the G0/G1 cell cycle checkpoint. Mechanistically, we found that GLTP overexpression modulated cell cycle progression by upregulating Kip1/p27 and Cip1/p21 protein and mRNA levels, while decreasing CDK2, CDK4, cyclin E and cyclin D1 protein levels. The cell cycle arrest and growth inhibition induced by GLTP overexpression led to apoptotic cell death of HT-29 cells. Neither GLTP nor CPTP overexpression induced apoptosis in breast cancer cell lines (T47D and HTB-126). However, depletion of CPTP, but not GLTP, induced autophagy as indicated by increased LC3 transcript and autophagosome levels (GFP-LC3-II puncta). In the breast cancer cell lines, increased transcript levels of cPLA2 and CERK were detected consistent with the earlier pro-inflammatory effect reported for CPTP depletion in A549 lung epithelial carcinoma cells. Our findings shed light on the mechanistic ways by which GLTP and CPTP affect apoptosis and autophagy to impact cancer progression. Future in vivo studies are planned to ascertain the role(s) of these new players in cancer development and therapy. [Support: NIH GM45928, HL-125353, Paint-the-Town-Pink (PTTP) Cancer Funds, Hormel Fdn] Citation Format: Shrawan K. Mishra, Rhoderick E. Brown. Sphingolipids transfer proteins (GLTP and CPTP) regulate the neoplastic progression of colon and breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1123. doi:10.1158/1538-7445.AM2017-1123

Dietary eicosapentaenoic acid normalizes hippocampal omega-3 and 6 polyunsaturated fatty acid profile, attenuates glial activation and regulates BDNF function in a rodent model of neuroinflammation induced by central interleukin-1β administration

Interleukin (IL)-1β can activate glial cells to trigger neuroinflammation and neurodegeneration. Lower omega (n)-3 polyunsaturated fatty acids (PUFAs) and lower n-3/n-6 PUFA ratios occur in the brain of patients with Alzheimer's disease (AD). We have previously reported that an n-3 PUFA, eicosapentaenoic acid (EPA), can improve memory and attenuate neurodegeneration-like changes in animal models of AD. However, whether and how EPA modulates glial cell activity and functions remains unclear. The aim of this study was to test the hypothesis that EPA may attenuate neuroinflammation by inhibiting microglial activation and microglia-produced proinflammatory cytokines, and by enhancing the expression of astrocytes-produced neurotrophins and their receptors. Male Long-Evans rats were fed either palm oil supplemented diet or EPA supplemented diet for 42days. On day 36 of diet feeding, rats received an intracerebroventricular injection of IL-1β or saline for 7days. The glial activation, the expression of amyloid precursor protein (APP), calcium-dependent phospholipase (cPL) A2, brain-derived neurotrophic factor (BDNF) and its receptor, and PUFA profile in the hippocampus were analyzed. IL-1β elevated biomarkers of microglial CD11b and astrocyte GFAP expression, increased the expression of APP, tumor-necrosis factor (TNF)-α, but reduced BDNF and its receptor (TrKB). IL-1β also lowered n-3 EPA and docosapentaenoic acid concentrations but increased n-6 PUFAs and cPLA2 activity in the hippocampus. EPA supplement normalized the n-3 and n-6 PUFA profiles and cPLA2 levels, inhibited glial activation, reduced APP and TNF-α expression, as well as up-regulated BDNF and TrKB. Supplementation with EPA appear to have potential effects on improving glial over-activation, n3/n6 imbalance and BDNF down-regulation, which contribute to anti-inflammatory and may provide beneficial effects on inflammation-associated disease such as AD.

Thyroxine therapy ameliorates serum levels of eicosanoids in Chinese subclinical hypothyroidism patients.

The eicosanoids derived from phospholipids play key roles in inflammation. However, the profiles of serum eicosanoids in subclinical hypothyroidism (SH) patients and the effects of thyroxine replacement therapy (TRT) on these eicosanoids remain unclear. Many studies show that TSH regulates lipid metabolism. As eicosanoids derived from phospholipids play key roles in oxidative stress and immune function and inflammatory process, it was necessary to explore the profiles of serum eicosanoids in SH patients and the effects of thyroxine replacement therapy (TRT) on the eicosanoids. A total of 50 Chinese SH patients and 22 healthy volunteers were recruited. SH patients received TRT (L-T4, 25 and 50 mcg/d for patients with TSH≤10.0 mIU/L and TSH>10.0 mIU/L, respectively) for 3 months. Serum levels of major eicosanoids and cPLA2 were analyzed using LC-MS and clinical biochemical assays. The serum levels of cPLA2, eicosanoids (8-isoPGF2a, 11-dehydroTXB2 and 12-HETE) and 11-dehydroTXB2/6-Keto-PGF1a were significantly elevated in SH patients. The serum TSH levels were significantly correlated with the levels of cPLA2 (r=+0.65), 11-dehydroTXB2 (r=+0.32) and 11-dehydroTXB2/6-Keto-PGF1a (r=+0.37). After 3-month TRT, the serum levels of TSH, cPLA2 and the above-mentioned eicosanoids in SH patients were significantly decreased. The metabolism of eicosanoids is significantly altered in Chinese SH patients, and TRT can ameliorate the abnormalities of serum eicosanoid levels.

Gastroprotective Effects of PMK-S005 against Ethanol-Induced Acute Gastric Damage in Rats.

Background/AimsThis study aimed to examine the gastroprotective effects of PMK-S005, which is a synthetic S-allyl-l-cysteine (SAC; a sulfur-containing amino acid), against acute ethanol-induced gastric damage in rats.MethodsSprague-Dawley rats were divided into six groups, including a nonethanol group, groups treated with absolute ethanol 1 hour after pretreatment with various doses of PMK-S005 (1, 5, and 10 mg/kg) or rebamipide (50 mg/kg), and an absolute ethanol-only group. Ethanol-induced gross ulcer and mucus levels were measured. Myeloperoxidase, tumor necrosis factor α, interleukin 1β, PGE2, LTB4, cPLA2, COX-1, and COX-2 levels were estimated by enzyme-linked immunosorbent assay or Western blot analysis. Furthermore, the protein expression levels of antioxidant enzymes, including heme oxygenase-1 (HO-1), NAD(P)H:quinine oxidoreductase 1 (NQO-1), GCLC, and GCLM, were assessed.ResultsPMK-S005 significantly attenuated the ethanol-induced gastric damage; it reduced mucosal inflammatory cytokine production and increased mucus levels. The expression levels of cPLA2, COX-1, and COX-2 were decreased by PMK-S005. PMK-S005 did not affect PGE2 synthesis, but LTB4 production was significantly suppressed. In addition, long-term administration of PMK-S005 significantly increased the expression of HO-1, NQO-1, GCLC, and GCLM.ConclusionsThese results strongly suggest that PMK-S005 prevents gastric mucosal damage and that these gastroprotective activities are due to anti-inflammatory effects and enhancement of the gastric defense system, including antioxidant enzymes.

An in vitro investigation of the actions of reproductive hormones on the cervix of the ewe in the follicular stage: The effects of 17β-estradiol, oxytocin, FSH, and arachidonic acid on the cervical pathway for the synthesis of prostaglandin E2

During the periovulatory period, the cervix of the ewe relaxes and this mechanism is thought to be mediated by oxytocin and prostaglandin E2 (PGE2) in response to increased concentrations of 17β-estradiol and perhaps FSH. The aim of the study was to determine the in vitro effects of 17β-estradiol, FSH, oxytocin, and arachidonic acid (AA) on the synthesis of PGE2 and on the expression of oxytocin receptor (OTR), cytoplasmic phospholipase A2 (cPLA2), and cyclooxygenase 2 (COX-2) in explants of cervical tissue collected from ewes in the periovulatory phase of the estrous cycle. Cervical minces from ewes in the follicular phase of the estrous cycle were cultured in supplemented Eagle's Minimum Essential Medium for 48 hours with 17β-estradiol, FSH, oxytocin, or AA. After incubation, the tissue was stored at −80 °C and the media at −20 °C. Western immunoblotting was used to determine relative levels of OTR, cPLA2, and COX-2 in cervical tissue, and the media was analyzed by RIA, to determine the concentration of PGE2. The addition of 17β-estradiol increased the concentration of PGE2 in the media (P = 0.001), the levels of COX-2 (P = 0.02) and OTR (P = 0.006) but not those of cPLA2 (P = 0.15). The addition of FSH increased the levels of COX-2 (P = 0.01) but, it had no effect on the concentration of PGE2 (P = 0.08) or on the levels of OTR (P = 0.07) and cPLA2 (P = 0.15). Oxytocin did not increase the levels of COX-2 (P = 0.38) but increased those of OTR (P = 0.001) and cPLA2 (P = 0.01) but not on the concentration of PGE2 in the media. Arachidonic acid increased the levels of cPLA2 (P = 0.01) and those of COX-2 (P = 0.02) but not the concentration of PGE2 in the media. Our findings suggest that the PGE2-mediated mechanisms of cervical relaxation in the ewe during the follicular phase are stimulated by FSH, 17β-estradiol, oxytocin, and AA. They all appear to act by inducing receptors and enzymes along the synthetic pathway for PGE2.

111 EXPRESSION OF PHOSPHOLIPASE A2 ISOFORMS IN EQUINE ENDOMETRIUM DURING THE ESTROUS CYCLE AND EARLY PREGNANCY

Phospholipase A2 (PLA2) is involved in the synthesis of prostaglandins (PG) as it releases arachidonic acid from the membrane phospholipids to be a precursor for cyclooxygenase enzymes. Therefore, it is critically important during luteolysis and at the time of maternal recognition of pregnancy. The embryo must attenuate endometrial PGF2α production, whereas PGE2 is considered to be luteoprotective. Furthermore, implantation also requires the action of PGs. A balance should be maintained in PG synthesis in the endometrium. The objective of this study was to evaluate the expression of isoforms of PLA2 (cytosolic [cPLA2], secretory [PLA2], and calcium independent [iPLA2]) in equine endometrium during the oestrous cycle and early pregnancy. Biopsies were obtained from mares on the day of ovulation (d0, n = 4), late diestrus (LD, n = 4, high progesterone [P4]), and after luteolysis in the beginning of oestrus phase (AL, n = 4, &lt;1 ng mL–1 P4) of the cycle. Biopsies were also taken on days 14 (P14, n = 4), 18 (P18, n = 4), and 22 (P22; n = 4) of pregnancy. Relative mRNA expression levels of genes were quantified using real-time quantitative RT-qPCR. Data were analysed using one-way ANOVA and l.s.d. test. Compared with the day of oestrus (d0), steady-state levels of cPLA2 and sPLA2 were down-regulated at LD, where P4 was high, and expression of both were up-regulated again at AL. In contrast, iPLA2 expression was higher at LD and then decreased again at AL. Pregnancy decreased expression of cPLA2 and sPLA2 mRNA at P14 and P18 compared with their respective cycle days. Late diestrus elevation in the expression of iPLA2 was suppressed by pregnancy at P14; however, it was up-regulated later in pregnancy at P22. The results suggest that expression of cPLA2 and sPLA2 is negatively correlated with circulating progesterone concentrations. Pregnancy further inhibits their expression in the equine endometrium. However, iPLA2 expression seems to be positively correlated by progesterone presence and its expression increased as equine pregnancy advanced. This project was partially funded by TUBITAK 107O035 and SUBAP to AG.

The expression of ERα, OTR, cPLA2, COX-2, and PPARγ in the cervix of the ewe during the estrous cycle

The ovine cervix relaxes at estrus allowing easier entry of spermatozoa into the uterus. The mechanism responsible for this relaxation is not fully elucidated and we hypothesized that cervical relaxation at estrus is induced by ovarian and pituitary hormones stimulating the local production of prostaglandin E2 via a biosynthetic pathway involving a number of mediators including oxytocin, phospholipase A2 (cPLA2), cyclooxygenase-2 (COX-2), and peroxisome proliferator-activated receptor gamma (PPARγ). The aim of this study was to investigate the cervical expression of estradiol receptor alpha (ERα), oxytocin receptor (OTR), cPLA2, COX-2, and PPARγ at three stages of the estrous cycle (the luteal phase and two times during the follicular phase, just before and just after the LH surge). An experiment was conducted during the breeding season, in 25 ewes to test this hypothesis. Samples of cervical tissue were collected from groups of ewes at three stages of the estrous cycle: the luteal (N = 8), “pre-LH surge” (N = 8), and “post-LH surge” (N = 9) stages. Cervical tissue from uterine, mid, and vaginal regions of the cervix were analyzed by Western immunoblot analysis for ERα, OTR, cPLA2, COX-2, and PPARγ. The results showed that the levels of all five proteins were lowest during the luteal phase of the estrous cycle in all regions of the cervix. The levels of all except cPLA2, increased significantly during the “pre-LH surge” stage. The levels of cPLA2 and ERα increased in the “post-LH surge” stage and those for OTR and PPARγ were unchanged and those for COX-2 were lower. These data show that the cervical levels of all five of the intermediates in the synthesis of prostaglandin E2 that were examined in this study were higher in the “pre-” and “post-LH surge” stages compared with the luteal phase of the estrous cycle and these findings are consistent with our hypothesis.

4-hydroxy-2-nonenal upregulates and phosphorylates cytosolic phospholipase A2 in cultured Ra2 microglial cells via MAPK pathways

Several studies have suggested the involvement of neuroinflammation in the pathomechanism of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS). We recently demonstrated increased levels of protein-bound 4-hydroxy-2-nonenal (HNE) as a highly reactive lipid peroxidation product and cytosolic phospholipase A(2) (cPLA(2)) as a proinflammatory enzyme in glial cells as well as motor neurons in the spinal cord of sporadic ALS patients. However, a link between HNE and cPLA(2) in ALS remains to be determined. To address this issue, we investigated effects of HNE stimulation on the state of cPLA(2) expression in cultured microglial cell line (Ra2). Exposure of Ra2 cells to HNE significantly increased expression levels of cPLA(2) and its activated form phosphorylated at amino acid residue S(505) (p-cPLA(2)) on immunoblots. Pretreatment of Ra2 cells with the antioxidant N-acetylcysteine, the extracellular signal-regulated kinase (ERK) inhibitor PD98059 or the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 prevented the HNE-induced increased expression of cPLA(2) and p-cPLA(2). Immunocytochemical analysis revealed that staining for p-cPLA(2) in Ra2 cells was localized in the cytoplasm and more intense in the HNE-stimulated group than in the vehicle group. The present results provide in vitro evidence that HNE upregulates and phosphorylates cPLA(2) in microglia via the ERK and p38 MAPK pathways.

Study on the relationship of cPLA2, CK-MB, and membrane phospholipid content in acute myocardial infarction

Increased plasma creatine kinase-myoglobin (CK-MB) occurs in patients with acute myocardial infarction (AMI), but its underlying relationship with cytosolic phospholipase A2 (cPLA2) is poorly understood. In the present study we sought to determine cPLA2 activation and its relationship with plasma and myocardial CK-MB level and membrane phospholipids (PLs) in an animal model of AMI. AMI was induced in 60 male Sprague-Dawley rats by ligating the left anterior descending of coronary artery. Rats were randomized into six groups at 0 (sham group), 1, 2, 4, 6, and 12 h after AMI onset (ten rats in each group). cPLA2 was detected by reverse transcription polymerase chain reaction and immunohistochemical staining for mRNA and protein expression, respectively. Plasma and myocardial CK-MB activity were measured by inhibition kinetics, and membrane PLs were determined by phosphorus quantitative method. Myocardial cPLA2 expression increased from 1 h and reached a peak at 2 h after AMI onset (p < 0.01) followed by a decrease but still remained high, whereas plasma CK-MB significantly increased in rats from 4 h after the onset of AMI (p < 0.05). During the first 6 h of AMI, a negative correlation existed between myocardial cPLA2 and membrane PLs (r = -0.504, p < 0.01), whereas myocardial cPLA2 levels were positively associated with plasma CK-MB (r = 0.741, p < 0.01) but negatively correlated with myocardial CK-MB in AMI groups (r = -0.785, p < 0.01). Myocardial cPLA2 level increased and is positively correlated with plasma CK-MB activity and negatively correlated with membrane phospholipid content in AMI rats at early stage.

Dietary Soy Protein Selectively Reduces Renal Prostanoids and Cyclooxygenases in Polycystic Kidney Disease

Increasing evidence in human chronic kidney disease and in animal models indicates the potential utility of dietary soy protein in the treatment of this disorder. A model in which a beneficial soy protein effect has been consistently demonstrated is the Han:SPRD-cy rat model of polycystic kidney disease. Therefore, since dietary soy protein alters renal hemodynamics and prostanoid production, the effects of dietary soy protein on renal prostanoids and related rate-limiting enzymes were examined. Normal and diseased weanling rats were given diets containing casein or soy protein for 7 wk. At 10 wk of age, renal levels of thromboxane B(2) (TXB(2), stable metabolite of TXA(2)), prostaglandin E(2) (PGE(2)) and 6-keto PGF(1alpha) (stable metabolite of PGI(2)) and activities of cyclooxygenase 1 (COX1) and COX2 were elevated in diseased compared to normal kidneys. Soy protein feeding resulted in 49% lower in vitro steady-state levels of TXB(2), and 76% less 6-keto PGF(1alpha) produced by COX1 activity in diseased kidneys, while not altering these parameters in normal kidneys. It also resulted in 47% less TXB(2) and 36% lower 6-keto PGF(1alpha) produced by COX2 activity in diseased kidneys. The relative effect of soy protein feeding on COX2 activity was in the order of TXB(2) > 6-keto PGF(1alpha) > PGE(2). Diseased kidneys had elevated protein and mRNA levels of cytosolic phospholipase A(2) (cPLA(2)) and COX1 and lower levels of COX2. Dietary soy protein attenuated the protein levels of cPLA(2) in diseased kidneys, and reduced COX2 mRNA expression in both normal and diseased kidneys. Dietary soy protein therefore reduced the levels of specific renal prostanoids, cPLA(2) and COX enzymes in this model of polycystic kidney disease, a model in which soy protein has been demonstrated to reduce disease progression.