Antinociception role of 14,15-epoxyeicosatrienoic acid in a central post-stroke pain model in rats mediated by anti-inflammation and anti-apoptosis effect.

Central post stroke pain (CPSP) is an intractable neuropathic pain syndrome that occurs after the acute focal lesion of the central nervous system (CNS) due to a cerebrovascular cause. Epoxyeicosatrienoic acids (EETs) exert many pharmacological effects in vivo and in vitro, such as anti-apoptosis, anti-inflammatory, and anti-oxidative stress. Neuroinflammation and apoptosis are the potential pathophysiological mechanisms of neuropathic pain. This study aimed to investigate whether 14,15-EET has an antinociception effect on CPSP rats through its anti-inflammation and anti-apoptosis mechanisms. Rats were treated with type IV collagenase (CPSP group) or saline (Sham group) via injection with a Hamilton syringe into the ventral posterior lateral nucleus (VPL) according to the stereotaxic coordinates. We first tested the mechanical withdrawal threshold, as well as neuroinflammation- and apoptosis-related protein expressions in the per-lesion site of CPSP and Sham rats. Sprague-Dawley rats were randomly divided into five groups, as follows: vehicle; EET at 0.025, 0.05, and 0.1 µg; and EET (0.1 µg) + EEZE (3.25 ng). EET or and vehicle were administered into VPL nuclei three consecutive days after hemorrhagic stroke. Immunostaining, ELISA, and Western blot were performed to evaluate neuroinflammation and apoptosis. Hemorrhagic stroke induced mechanical allodynia, glial activation, neuroinflammation, and apoptosis-related protein upregulation. However, early treatment with 14,15-EET inhibited glial cell activation, decreased proinflammatory cytokines and apoptosis-related protein, and alleviated the pain behavior of CPSP rats. Our results provided strong evidence that antinociception produced by 14,15-EET is partly mediated by the inhibition of neuroinflammation and apoptosis.

11,12 Epoxyeicosatrienoic Acid Rescues Deteriorated Wound Healing in Diabetes.

Epoxyeicosatrienoic acids (EET) facilitate regeneration in different tissues, and their benefit in dermal wound healing has been proven under normal conditions. In this study, we investigated the effect of 11,12 EET on dermal wound healing in diabetes. We induced diabetes by i.p. injection of streptozotocin 2 weeks prior to wound creation on the dorsal side of the mouse ear. 11,12 EET was applied every second day on the wound, whereas the control groups received only solvent. Epithelialization was monitored every second day intravitally up to wound closure. Wounds were stained for VEGF, CD31, TGF-ß, TNF-α, SDF-1α, NF-κB, and Ki-67, and fibroblasts were counted after hematoxylin-eosin stain on days 3, 6, 9, and 16 after wounding. After induction of diabetes, wounds closed on day 13.00 ± 2.20 standard deviation (SD). Local 11,12 ETT application improved wound closure significantly to day 8.40 ± 1.39 SD. EET treatment enhanced VEGF and CD31 expression in wounds on day 3. It also seemed to raise TNF-α level on all days investigated as well as TGF-ß level on days 3 and 6. A decrease in NF-κB could be observed on days 9 and 16 after EET application. The latter findings were not significant. SDF-1α expression was not influenced by EET application, and Ki-67 was significantly less in the EET group on day 9 after EET application. The number of fibroblasts was significantly increased on day 9 after the 11,12 EET application. 11,12 EET improve deteriorated wound healing in diabetes by enhancing neoangiogenesis, especially in the early phase of wound healing. Furthermore, they contribute to the dissolution of the initial inflammatory reaction, allowing the crucial transition from the inflammatory to proliferative phase in wound healing.

14,15-Epoxyeicosatrienoic Acid Alleviates Pathology in a Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the leading cause of late-onset dementia, and there exists an unmet medical need for effective treatments for AD. The accumulation of neurotoxic amyloid-ß (Aß) plaques contributes to the pathophysiology of AD. EPHX2 encoding soluble epoxide hydrolase (sEH)-a key enzyme for epoxyeicosatrienoic acid (EET) signaling that is mainly expressed in lysosomes of astrocytes in the adult brain-is cosited at a locus associated with AD, but it is unclear whether and how it contributes to the pathophysiology of AD. In this report, we show that the pharmacologic inhibition of sEH with 1-trifluoromethoxyphenyl- 3-(1-propionylpiperidin-4-yl) urea (TPPU) or the genetic deletion of Ephx2 reduces Aß deposition in the brains of both male and female familial Alzheimer's disease (5×FAD) model mice. The inhibition of sEH with TPPU or the genetic deletion of Ephx2 alleviated cognitive deficits and prevented astrocyte reactivation in the brains of 6-month-old male 5×FAD mice. 14,15-EET levels in the brains of these mice were also increased by sEH inhibition. In cultured adult astrocytes treated with TPPU or 14,15-EET, astrocyte Aß clearance was increased through enhanced lysosomal biogenesis. Infusion of 14,15-EET into the hippocampus of 5×FAD mice prevented the aggregation of Aß. Notably, a higher concentration of 14,15-EET (200 ng/ml) infusion into the hippocampus reversed Aß deposition in the brains of 6-month-old male 5×FAD mice. These results indicate that EET signaling, especially 14,15-EET, plays a key role in the pathophysiology of AD, and that targeting this pathway is a potential therapeutic strategy for the treatment of AD.SIGNIFICANCE STATEMENT There are limited treatment options for Alzheimer's disease (AD). EPHX2 encoding soluble epoxide hydrolase (sEH) is located at a locus that is linked to late-onset AD, but its contribution to the pathophysiology of AD is unclear. Here, we demonstrate that sEH inhibition or Ephx2 deletion alleviates pathology in familial Alzheimer's disease (5×FAD) mice. Inhibiting sEH or increasing 14,15-epoxyeicosatrienoic acid (EET) enhanced lysosomal biogenesis and amyloid-ß (Aß) clearance in cultured adult astrocytes. Moreover, the infusion of 14,15-EET into the hippocampus of 5×FAD mice not only prevented the aggregation of Aß, but also reversed the deposition of Aß. Thus, 14,15-EET plays a key role in the pathophysiology of AD and therapeutic strategies that target this pathway may be an effective treatment.

Lipidomic profiling of virus infection identifies mediators that resolve herpes simplex virus-induced corneal inflammatory lesions.

Lipid mediators (LMs) play a pivotal role in the induction and resolution of inflammation. To identify and elucidate their involvement during virus infection, multiple reaction monitoring (MRM) based liquid chromatography-tandem mass spectrometry lipidomic profiling of 62 lipid species was performed in this study. Results show that RAW264.7 macrophages differentially produce specific LMs signals depending on difference in virus pathogenicity. Integration of large-scale lipidomics with targeted gene expression data revealed mediators, such as RVD3, 18-HEPE, 11(12)-EET etc. correlated with the pathogenic phase of the infection. The herpes simplex virus (HSV)-induced keratitis model demonstrates that 11(12)-EET treatment represents a novel alternative for treating viral infection.

Epoxyeicosatrienoic acid prevents maladaptive remodeling in pressure overload by targeting calcineurin/NFAT and Smad-7.

BACKGROUND: Emerging evidence demonstrates that epoxyeicosatrienoic acids (EETs) as important active eicosanoids that regulate cardiovascular homeostasis, but the mechanisms underlying its favorable anti-hypertrophic benefits in overpressure model remain obscure. METHODS AND RESULTS: Four weeks after transverse aortic constriction (TAC), TAC mice developed maladaptive cardiac hypertrophy and consequent cardiac failure. Conversely, a cardiotropic adeno-associated viral vector (AAV9) encoding CYP2J2 prevented transverse aortic constriction-induced cardiac hypertrophy with preserved ejection fraction. EET also conferred protection against phenylephrine-induced hypertrophy in H9c2 cardiomyoblasts. Further investigations indicate CYP2J2/EET exerts protection against cardiac hypertrophy through opposing the increase of intracellular Ca2+ level and Ca2+-mediated calcineurin/NFATc3 signaling. Meanwhile, extended myocardial fibrosis in TAC mice was also effectively abolished with the administration of AAV9-2J2. Intriguingly, TAC mice display activated TGF-ß/Samd-3 signaling with decreased Smad-7 expression, whereas AAV9-2J2 attenuated the phosphorylation of Smad-3 without altering TGF-ß expression, whilst preservation of Smad-7. Subsequently, the differentiation of cardiac fibroblasts into myofibroblasts in the presence of TGF-ß1 stimulation was significantly disrupted with EET treatment, accompanied by declined Smad-3 activation and collagen production, whereas inhibition of Smad-7 with SiRNA Smad-7 substantially abrogated these effects of EET on cardiac fibroblasts. CONCLUSIONS: EET has synergistic actions on cardiomyocytes and cardiac fibroblasts, preventing cardiac hypertrophy through inhibition of Ca2+-mediated calcineurin/NFATc3 signaling cascades, and ameliorating myocardial fibrosis dependent on Smad-7. This work further extends the potential mechanisms of EET, providing a novel therapeutic approach for the treatment of pathological remodeling and heart failure.

Infarct size-limiting effect of epoxyeicosatrienoic acid analog EET-B is mediated by hypoxia-inducible factor-1α via downregulation of prolyl hydroxylase 3.

Epoxyeicosatrienoic acids (EETs) decrease cardiac ischemia-reperfusion injury; however, the mechanism of their protective effect remains elusive. Here, we investigated the cardioprotective action of a novel EET analog, EET-B, in reperfusion and the role of hypoxia-inducible factor (HIF)-1α in such action of EET-B. Adult male rats were subjected to 30 min of left coronary artery occlusion followed by 2 h of reperfusion. Administration of 14,15-EET (2.5 mg/kg) or EET-B (2.5 mg/kg) 5 min before reperfusion reduced infarct size expressed as a percentage of the area at risk from 64.3 ± 1.3% in control to 42.6 ± 1.9% and 46.0 ± 1.6%, respectively, and their coadministration did not provide any stronger effect. The 14,15-EET antagonist 14,15-epoxyeicosa-5( Z)-enoic acid (2.5 mg/kg) inhibited the infarct size-limiting effect of EET-B (62.5 ± 1.1%). Similarly, the HIF-1α inhibitors 2-methoxyestradiol (2.5 mg/kg) and acriflavine (2 mg/kg) completely abolished the cardioprotective effect of EET-B. In a separate set of experiments, the immunoreactivity of HIF-1α and its degrading enzyme prolyl hydroxylase domain protein 3 (PHD3) were analyzed in the ischemic areas and nonischemic septa. At the end of ischemia, the HIF-1α immunogenic signal markedly increased in the ischemic area compared with the septum (10.31 ± 0.78% vs. 0.34 ± 0.08%). After 20 min and 2 h of reperfusion, HIF-1α immunoreactivity decreased to 2.40 ± 0.48% and 1.85 ± 0.43%, respectively, in the controls. EET-B blunted the decrease of HIF-1α immunoreactivity (7.80 ± 0.69% and 6.44 ± 1.37%, respectively) and significantly reduced PHD3 immunogenic signal in ischemic tissue after reperfusion. In conclusion, EET-B provides an infarct size-limiting effect at reperfusion that is mediated by HIF-1α and downregulation of its degrading enzyme PHD3. NEW & NOTEWORTHY The present study shows that EET-B is an effective agonistic 14,15-epoxyeicosatrienoic acid analog, and its administration before reperfusion markedly reduced myocardial infarction in rats. Most importantly, we demonstrate that increased hypoxia-inducible factor-1α levels play a role in cardioprotection mediated by EET-B in reperfusion likely by mechanisms including downregulation of the hypoxia-inducible factor -1α-degrading enzyme prolyl hydroxylase domain protein 3.

Epoxyeicosatrienoic acid ameliorates cerebral ischemia-reperfusion injury by inhibiting inflammatory factors and pannexin-1.

Epoxyeicosatrienoic acid (EET) has wide applications due to the unique biological effects of anti­hyperlipidemia, inhibition of platelet aggregation, anti­inflammation, anti­cancer, anti­lipid oxidation and the promotion of brain tissue development. The present study investigated whether EET ameliorates cerebral ischemia­reperfusion injury (CIRI) by inhibiting inflammatory factors and pannexin. Specific pathogen­free 7­week­old male Sprague­Dawley rats were randomly divided into three groups: Sham, CIRI and EET. Neurological deficit scores, cerebral infarct volume and cerebral edema were assessed in CIRI rats. Enzyme­linked immunosorbent assays were performed to detect tumor necrosis factor­α, interleukin­6, nuclear factor­κB and inducible nitric oxide synthase (iNOS) levels, and western blot analysis was performed also used to assess cleaved caspase­3, phospholipase A2 (PLA2), cyclooxygenase­2 and prostaglandin E2 (PGE2) protein expression levels. EET ameliorated cerebral injury and CIRI­induced cleaved caspase­3 protein expression levels in rats. EET additionally suppressed CIRI­induced inflammation reactions and iNOS protein expression in rats. Furthermore, the protein expression levels of PLA2, PGE2 and pannexin­1 in CIRI rats were inhibited by treatment with EET. These results indicated that EET reduces CIRI by inhibiting inflammation and levels of cleaved caspase­3, PLA2, PGE2 and pannexin-1.

Soluble Epoxide Hydrolase Inhibitor Attenuates Lipopolysaccharide-Induced Acute Lung Injury and Improves Survival in Mice.

Acute lung injury (ALI) is characterized by rapid alveolar injury, vascular leakage, lung inflammation, neutrophil accumulation, and induced cytokines production leading to lung edema. The mortality rate of patients suffering from ALI remains high. Epoxyeicosatrienoic acids (EETs) are cytochrome P450-dependent derivatives of polyunsaturated fatty acid with antihypertensive, profibrinolytic, and anti-inflammatory functions. EETs are rapidly hydrated by soluble epoxide hydrolase (sEH) to their less potent diols. The aim of this study was to investigate the role of sEH inhibitor trifluoromethoxyphenyl propionylpiperidin urea (TPPU) and EETs in lipopolysaccharide (LPS)-induced ALI of mice. Our studies revealed that inhibition of sEH with TPPU attenuated the morphological changes in mice, decreased the neutrophil infiltration to the lung, pro-inflammatory cytokine levels (IL-1ß and TNF-α) in serum and bronchoalveolar lavage fluid (BALF), and alveolar capillary leakage (lung wet/dry ratio and total protein concentration in BALF). TPPU improved the survival rate of LPS-induced ALI. In addition, in vitro experiments revealed that both TPPU and EETs (11,12-EET and 14,15-EET) suppressed the expression of IL-1ß and TNF-α, and LDH release in RAW264.7 cells. These results indicate that EETs play a role in dampening LPS-induced acute lung inflammation, and suggest that sEH could be a valuable candidate for the treatment of ALI.

Epoxyeicosatrienoic acid analog attenuates the development of malignant hypertension, but does not reverse it once established: a study in Cyp1a1-Ren-2 transgenic rats.

OBJECTIVE: We evaluated the therapeutic effectiveness of a new, orally active epoxyeicosatrienoic acid analog (EET-A) in rats with angiotensin II (ANG II)-dependent malignant hypertension. METHODS: Malignant hypertension was induced in Cyp1a1-Ren-2 transgenic rats by activation of the renin gene using indole-3-carbinol (I3C), a natural xenobiotic. EET-A treatment was started either simultaneously with I3C induction process (early treatment) or 10 days later during established hypertension (late treatment). Blood pressure (BP) (radiotelemetry), indices of renal and cardiac injury, and plasma and kidney levels of the components of the renin-angiotensin system (RAS) were determined. RESULTS: In I3C-induced hypertensive rats, early EET-A treatment attenuated BP increase (to 175 ±â€Š3 versus 193 ±â€Š4 mmHg, P < 0.05, on day 13), reduced albuminuria (15 ±â€Š1 versus 28 ±â€Š2 mg/24 h, P < 0.05), and cardiac hypertrophy as compared with untreated I3C-induced rats. This was associated with suppression of plasma and kidney ANG II levels (48 ±â€Š6 versus 106 ±â€Š9 and 122 ±â€Š19 versus 346 ±â€Š11 fmol ml or g, respectively, P < 0.05) and increases in plasma and kidney angiotensin (1-7) concentrations (84 ±â€Š9 versus 37 ±â€Š6 and 199 ±â€Š12 versus 68 ±â€Š9 fmol/ml or g, respectively, P < 0.05). Remarkably, late EET-A treatment did not lower BP or improve renal and cardiac injury; indices of RAS activity were not affected. CONCLUSION: The new, orally active EET-A attenuated the development of experimental ANG II-dependent malignant hypertension, likely via suppression of the hypertensiogenic axis and augmentation of the vasodilatory/natriuretic axis of RAS.

Epoxyeicosanoid Signaling Provides Multi-target Protective Effects on Neurovascular Unit in Rats After Focal Ischemia.

Multiple players are involved in the highly complex pathophysiologic responses after stroke. Therefore, therapeutic approaches that target multiple cellular elements of the neurovascular unit in the damage cascade hold considerable promise for the treatment of stroke. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active eicosanoids called epoxyeicosatrienoic acids (EETs), which are further converted by soluble epoxide hydrolase (sEH) to less bioactive diols. EETs have been shown to exert direct cytoprotective effects upon several individual components of the neurovascular unit under simulated ischemic conditions in vitro. However, the cellular mechanism underlying EET-mediated neuroprotective effects after ischemia remains to be clarified. In this study, we investigated the effects of 14,15-EET and 12-(3-adamantan-1-yl-ureido)dodecanoic acid (AUDA), a selective inhibitor of sEH, on multiple elements of neurovascular unit of the rat brain after middle cerebral artery occlusion-induced focal ischemia. The results showed that exogenous administration of 14,15-EET or AUDA could suppress astrogliosis and glial scar formation, inhibit microglia activation and inflammatory response, promote angiogenesis, attenuate neuronal apoptosis and infarct volume, and further promote the behavioral function recovery after focal ischemia. The results suggest that epoxyeicosanoid signaling is a promising multi-mechanism therapeutic target for the treatment of stroke.

Dihomo-γ-linolenic acid prevents the development of atopic dermatitis through prostaglandin D1 production in NC/Tnd mice.

BACKGROUND: Atopic dermatitis (AD) is a chronic and relapsing skin disorder with pruritic skin symptoms. We previously reported that dihomo-γ-linolenic acid (DGLA) prevented the development of AD in NC/Tnd mice, though the mechanism remained unclear. OBJECTIVE: We attempted to investigate the mechanism of preventive effect of DGLA on AD development in NC/Tnd mice. METHODS: The clinical outcomes of NC/Tnd mice that were given diets containing DGLA, arachidonic acid, or eicosapentaenoic acid were compared. Lipid mediator contents in the skin in each group were also quantified. In addition, release of lipid mediators from RBL-2H3 mast cells treated with either DGLA or prostaglandin D1 (PGD1) was measured. Furthermore, effect of PGD1 on gene expression of thymic stromal lymphopoietin (TSLP) in PAM212 keratinocyte cells was determined. RESULTS: Only DGLA containing diet suppressed the development of dermatitis in vivo. By quantifying the 20-carbon fatty acid-derived eicosanoids in the skin, the application of DGLA was found to upregulate PGD1, which correlated with a better outcome in NC/Tnd mice. Moreover, we confirmed that mast cells produced PGD1 after DGLA exposure, thereby exerting a suppressive effect on immunoglobulin E-mediated degranulation. PGD1 also suppressed gene expression of TSLP in keratinocytes. CONCLUSION: These results suggest that oral administration of DGLA causes preventive effects on AD development in NC/Tnd mice by regulating the PGD1 supply.

Soluble Epoxide Hydrolase Deficiency or Inhibition Attenuates MPTP-Induced Parkinsonism.

Soluble epoxide hydrolase (sEH) inhibition has been demonstrated to have beneficial effects on various diseases, such as hypertension, diabetes, and brain ischemia. However, whether sEH inhibition has therapeutic potential in Parkinson's disease is still unknown. In this paper, we found that sEH expression is increased in 1-methyl-4-phenyl-1,2,3,6-tetrahydro pyridine (MPTP)-treated mice, and sEH deficiency and inhibition significantly attenuated tyrosine hydroxylase (TH)-positive cell loss and improved rotarod performance. The substrate of sEH, 14,15-epoxyeicosatrienoic acid (14,15-EET), protected TH-positive cells and alleviated the rotarod performance deficits of wild-type mice but not sEH-knockout mice. Moreover, the 14,15-EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE) abolished the neuronal protective effects of sEH deficiency. In primary cultured cortical neurons, MPP(+) induced significant Akt inactivation in neurons from sEH wild-type mice, and this effect was not observed in neurons from knockout mice. Our data indicate that sEH deficiency and inhibition increased 14,15-EET in MPTP-treated mice, which activated the Akt-mediated protection of TH-positive neurons and behavioral functioning. We also found that sEH deficiency attenuated TH-positive cell loss in a paraquat-induced mouse model of Parkinson's. Our data suggest that sEH inhibition might be a powerful tool to protect dopaminergic neurons in Parkinson's disease.

The protective effect of epoxyeicosatrienoic acids on cerebral ischemia/reperfusion injury is associated with PI3K/Akt pathway and ATP-sensitive potassium channels.

Epoxyeicosatrienoic acids (EETs), the cytochrome P450 epoxygenase metabolite of arachidonic acid, have been demonstrated to have neuroprotective effect. Phosphatidylinositol 3-kinase (PI3K)/Akt and ATP-sensitive potassium (KATP) channels are thought to be important factors that mediate neuroprotection. However, little is known about the role of PI3K/Akt and KATP channels in brain after EETs administration. In vitro experiment, oxygen-glucose deprivation (OGD) was performed in cultured rat cerebral microvascular smooth muscle cells (SMCs) for 4 h. The effect of 14,15-EET on OGD induced cell apoptosis was examined after reoxygenation. Western blot and real-time PCR were used to analyze the expression of Kir6.1, SUR2B (two subunits of KATP channels) and p-Akt on cerebral microvascular SMCs. In vivo experiments, we use 12-(3-adamantan-1-yl-ureido)-dodecanoic acid [AUDA, a specific soluble epoxide hydrolase (sEH) inhibitor] to confirm the effect of EETs indirectly. Rats were injected intraperitoneally with AUDA before being subjected to middle cerebral artery occlusion (MCAO). We detected the apoptosis and the expression of p-Akt, Kir6.1 and SUR2B in ischemic penumbra. The results showed that EETs protect against cerebral ischemia/reperfusion (I/R) injury and upregulated the expression of p-Akt and Kir6.1 in both of ischemic penumbra and OGD induced cerebral microvascular SMCs. The protective effect was inhibited by Wortmannin (a specific PI3K inhibitor) and Glib (a specific KATP inhibitor) respectively in vitro experiment. In conclusion, these results suggested that the protective effect of EETs on cerebral I/R injury is associated with PI3K/Akt pathway and KATP channels. Furthermore, the PI3K pathway may contribute to mediating KATP channels on cerebral microvascular SMCs.

The antitumor agent PBT-1 directly targets HSP90 and hnRNP A2/B1 and inhibits lung adenocarcinoma growth and metastasis.

Natural products are the major sources of currently available anticancer drugs. We recently reported that phenanthrene-based tylophorine derivative-1 (PBT-1) may be a potential antitumor agent for lung adenocarcinoma. We therefore examined the direct targets of PBT-1 and their effects in inhibiting lung adenocarcinoma. We found that PBT-1 reduced the level of Slug and inhibits the migration, invasion, and filopodia formation of lung adenocarcinoma CL1-5 cells in vitro. In addition, PBT-1 displayed in vivo antitumor and antimetastasis activities against subcutaneous and orthotopic xenografts of CL1-5 cells in nude mice. Chemical proteomics showed that heat shock protein 90 (HSP90) and heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNP A2/B1) bound PBT-1 in CL1-5 cells. Inhibition of HSP90 and hnRNP A2/B1 reduced the activation of AKT and Slug expression. Taken together, these findings suggest that PBT-1 binds to HSP90 and/or hnRNP A2/B1 and initiates antitumor activities by affecting Slug- and AKT-mediated metastasis and tumorigenesis.

Roles of endothelial nitric oxide synthase (eNOS) and mitochondrial permeability transition pore (MPTP) in epoxyeicosatrienoic acid (EET)-induced cardioprotection against infarction in intact rat hearts.

We previously demonstrated that 11,12 and 14,15-epoxeicosatrienoic acids (EETs) produce cardioprotection against ischemia-reperfusion injury in dogs and rats. Several signaling mechanisms have been implicated in the cardioprotective actions of the EETs; however, their mechanisms remain largely elusive. Since nitric oxide (NO) plays a significant role in cardioprotection and EETs have been demonstrated to induce NO production in various tissues, we hypothesized that NO is involved in mediating the EET actions in cardioprotection. To test this hypothesis, we used an in vivo rat model of infarction in which intact rat hearts were subjected to 30-min occlusion of the left coronary artery and 2-hr reperfusion. 11,12-EET or 14,15-EET (2.5mg/kg) administered 10min prior to the occlusion reduced infarct size, expressed as a percentage of the AAR (IS/AAR), from 63.9±0.8% (control) to 45.3±1.2% and 45.5±1.7%, respectively. A nonselective nitric oxide synthase (NOS) inhibitor, L-NAME (1.0mg/kg) or a selective endothelial NOS inhibitor, L-NIO (0.30mg/kg) alone did not affect IS/AAR but they completely abolished the cardioprotective effects of the EETs. On the other hand, a selective neuronal NOS inhibitor, nNOS I (0.03mg/kg) and a selective inducible NOS inhibitor, 1400W (0.10mg/kg) did not affect IS/AAR or block the cardioprotective effects of the EETs. Administration of 11,12-EET (2.5mg/kg) to the rats also transiently increased the plasma NO concentration. 14,15-EET (10µM) induced the phosphorylation of eNOS (Ser(1177)) as well as a transient increase of NO production in rat cardiomyoblast cell line (H9c2 cells). When 11,12-EET or 14,15-EET was administered at 5min prior to reperfusion, infarct size was also reduced to 42.8±2.2% and 42.6±1.9%, respectively. Interestingly, L-NAME (1.0mg/kg) and a mitochondrial KATP channel blocker, 5-HD (10mg/kg) did not abolish while a sarcolemmal KATP channel blocker, HMR 1098 (6.0mg/kg) and a mitochondrial permeability transition pore (MPTP) opener, atractyloside (5.0mg/kg) completely abolished the cardioprotection produced by the EETs. 14,15-EET (1.5mg/kg) with an inhibitor of MPTP opening, cyclosporin A (CsA, 1.0mg/kg) produced a greater reduction of infarct size than their individual administration. Conversely, an EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, 2.5mg/kg) completely abolished the cardioprotective effects of CsA, suggesting a role of MPTP in mediating the EET actions. Taken together, these results suggest that the cardioprotective effects of the EETs in an acute ischemia-reperfusion model are mediated by distinct mediators depending on the time of EET administration. The cardioprotective effects of EETs administered prior to ischemia were regulated by the activation of eNOS and increased NO production, while sarcKATP channels and MPTP were involved in the beneficial effects of the EETs when administered just prior to reperfusion.

Mechanisms of epoxyeicosatrienoic acids to improve cardiac remodeling in chronic renal failure disease.

Both clinical and basic science studies have demonstrated that cardiac remodeling in patients with chronic renal failure (CRF) is very common. It is a key feature during the course of heart failure and an important risk factor for subsequent cardiac mortality. Traditional drugs or therapies rarely have effects on cardiac regression of CRF and cardiovascular events are still the first cause of death. Epoxyeicosatrienoic acids (EETs) are the products of arachidonic acids metabolized by cytochrome P450 epoxygenases. It has been found that EETs have important biological effects including anti-hypertension and anti-inflammation. Recent data suggest that EETs are involved in regulating cardiomyocyte injury, renal dysfunction, chronic kidney disease (CKD)-related risk factors and signaling pathways, all of which play key roles in cardiac remodeling induced by CRF. This review analyzes the literature to identify the possible mechanisms for EETs to improve cardiac remodeling induced by CRF and indicates the therapeutic potential of EETs in it.

Multiple roles of dihomo-γ-linolenic acid against proliferation diseases.

Considerable arguments remain regarding the diverse biological activities of polyunsaturated fatty acids (PUFA). One of the most interesting but controversial dietary approaches focused on the diverse function of dihomo-dietary γ-linolenic acid (DGLA) in anti-inflammation and anti-proliferation diseases, especially for cancers. This strategy is based on the ability of DGLA to interfere in cellular lipid metabolism and eicosanoid (cyclooxygenase and lipoxygenase) biosynthesis. Subsequently, DGLA can be further converted by inflammatory cells to 15-(S)-hydroxy-8,11,13-eicosatrienoic acid and prostaglandin E1 (PGE1). This is noteworthy because these compounds possess both anti-inflammatory and anti-proliferative properties. PGE1 could also induce growth inhibition and differentiation of cancer cells. Although the mechanism of DGLA has not yet been elucidated, it is significant to anticipate the antitumor potential benefits from DGLA.

Evidence for a role of opioids in epoxyeicosatrienoic acid-induced cardioprotection in rat hearts.

We previously demonstrated that several epoxyeicosatrienoic acids (EETs) produce reductions in myocardial infarct size in rats and dogs. Since a recent study demonstrated the release of opioids in mediating the antinociceptive effect of 14,15-EET, we hypothesized that endogenous opioids may also be involved in mediating the cardioprotective effect of the EETs. To test this hypothesis, we used an in vivo rat model of infarction and a rat Langendorff model. In the infarct model, hearts were subjected to 30 min occlusion of the left coronary artery and 2 h reperfusion. Animals were treated with 11,12-EET or 14,15-EET (2.5 mg/kg) alone 15 min before occlusion or with opioid antagonists [naloxone, naltrindole, nor-binaltorphimine (nor-BNI), and d-Phe-Cys-Tyr-d-Trp-Om-Thr-Pen-Thr-NH(2) (CTOP), a nonselective, a selective delta, a selective kappa, and a selective mu receptor antagonist, respectively] 10 min before EET administration. In four separate groups, antiserum to Met- and Leu-enkephalin and dynorphin-A-(1-17) was administered 50 min before the 11,12-EET administration. Infarct size expressed as a percent of the area at risk (IS/AAR) was 63.5 + or - 1.2, 45.3 + or - 1.0, and 40.9 + or - 1.2% for control, 11,12-EET, and 14,15-EET, respectively. The protective effects of 11,12-EET were abolished by pretreatment with either naloxone (60.5 + or - 1.8%), naltrindole (60.8 + or - 1.0%), nor-BNI (62.3 + or - 2.8%), or Met-enkephalin antiserum (63.2 + or - 1.7%) but not CTOP (42.0 + or - 3.0%). In isolated heart experiments, 11,12-EET was administered to the perfusate 15 min before 20 min global ischemia followed by 45 min reperfusion in control hearts or in those pretreated with pertussis toxin (48 h). 11,12-EET increased the recovery of left ventricular developed pressure from 33 + or - 1 to 45 + or - 6% (P < 0.05) and reduced IS/AAR from 37 + or - 4 to 20 + or - 3% (P < 0.05). Both pertussis toxin and naloxone abolished these beneficial effects of 11,12-EET. Taken together, these results suggest that the major cardioprotective effects of the EETs depend on activation of a G(i/o) protein-coupled delta- and/or kappa-opioid receptor.

Anti-atherosclerotic effects of dihomo-gamma-linolenic acid in ApoE-deficient mice.

AIM: Dihomo-gamma-linolenic acid (DGLA) is an n-6 polyunsaturated fatty acid that is mainly metabolized to an anti-inflammatory eicosanoid, prostaglandin (PG) E1, via the cyclooxygenase (COX) pathway. We evaluated the effect of DGLA on atherosclerosis in apoE-deficient mice and studied the mechanism of the anti-atherosclerotic effect. METHODS: ApoE-deficient mice were fed a normal diet supplemented with 0.5% DGLA or vehicle for 6 months. ApoE-deficient mice were also fed a high-cholesterol diet supplemented with 0.5% DGLA or vehicle for 1 month. To clarify the influence of a COX inhibitor, naproxen, on the anti-atherosclerotic effect of DGLA, age-matched apoE-deficient mice fed a high-cholesterol diet supplemented with 0.5% DGLA were given oral naproxen for 1 month. RESULTS: In normal diet-fed mice, acetylcholine-induced vascular relaxation was significantly greater in the DGLA group than in the vehicle group. NADPH oxidase subunits, p22phox and gp91phox, intercellular adhesion molecule-1, and vascular cellular adhesion molecule-1 were significantly lower in the DGLA group than in the vehicle group, and DGLA significantly prevented atherosclerosis. In high-cholesterol diet-fed mice, DGLA also significantly prevented atherosclerosis, but the anti-atherosclerotic effect was attenuated by naproxen. CONCLUSION: DGLA may have an anti-atherosclerotic effect in apoE-deficient mice via PGE1 formation.