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Cytochrome P450 (CYP) 2J2 is responsible for the epoxidation of arachidonic acid, producing epoxyeicosatrienoic acids (EETs) that are known to enhance tumorigenesis. CYP2J2 is prominently expressed in the heart and also found in the lungs. Furthermore, the expression level of CYP2J2 in tumour tissues is higher than that in adjacent normal tissues. Non-small cell lung carcinoma is a common cancer, and tyrosine kinase inhibitors (TKIs) are powerful tools for its treatment. This study aimed to elucidate the inhibitory effects of 17 TKIs on CYP2J2 activity using LC-MS/MS. Seventeen TKIs exhibited different inhibitory effects on CYP2J2-catalysed astemizole O-demethylation in recombinant CYP2J2. Pralsetinib and selpercatinib showed strong competitive inhibition, with inhibition constant values of 0.48 and 1.1 µM, respectively. They also inhibited other CYP2J2 activities, including arachidonic acid epoxidation, hydroxyebastine carboxylation, and rivaroxaban hydroxylation. In conclusion, we showed that pralsetinib and selpercatinib strongly inhibit CYP2J2 activity. Inhibition of 14,15-EET production by these TKIs may be a novel mechanism for suppressing tumour growth and proliferation. Additionally, when these TKIs are co-administered with a CYP2J2 substrate, we may consider the possibility of drug–drug interactions via CYP2J2 inhibition. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) is widely distributed in many organs and can be metabolized in several ways into small lipid molecules: eicosanoides that perform a physiological and/or pathophysiological role. Among them, epoxyeicosatrienoic acids (EET) and hydroxyeicosatetraenoic acids (HETE), which are produced by cytochrome P450 (CYP) enzymes, have attracted much attention. AA metabolites play an important role in such cellular processes as proliferation, differentiation, and apoptosis and are also involved in the development of organs and tissues as well as in the pathogenetic processes underlying the development of various diseases; in particular, AA metabolites formed with the participation of CYP are involved in normal and pathological processes associated with the functioning of the cardiovascular system. This review focuses on the formation and cardiovascular effects of CYP-generated AA metabolites and summarizes current literature data on the physiological and pathophysiological significance of the CYP-dependent AA metabolic pathway and its relevance to the cardiovascular system in both health and disease. [ABSTRACT FROM AUTHOR]

Objective: Gastrointestinal dysfunction are often associated with type 2 diabetes mellitus (T2DM), a complicated metabolic illness. Contributing factors have been proposed, including genetic predisposition, gene environmental, and lifestyle interactions, but the pathophysiology remains unknown. Methods: We aim to explore the possible causes behind gastrointestinal dysfunction caused by type 2 diabetes in this study. A comprehensive analysis of the gastric sinus metabolome, transcriptome, and proteome in db/db mice with gastrointestinal dysfunction was conducted. Results: The model group of mice had considerably lower small intestine propulsion and gastric emptying rates, higher blood glucose levels, and were significantly obese compared to the control group. We identified 297 genes, 350 proteins, and 1,001 metabolites exhibiting significant differences between db/db and control mice (p < 0.05). Moreover, multi-omics analysis revealed that the genes, proteins, and metabolites in the T2DM-induced gastroenteropathy mice group were involved in arachidonic acid metabolism, glycerophospholipid metabolism and vitamin digestion and absorption. Specifically, Cbr3, Etnppl, and Apob were the major mRNAs associated with T2DM-induced gastrointestinal dysfunction, while Cyp2b10, Cyp2b19, Pgs1, Gpat3, Apoa4, and Tcn2 were the major proteins associated with T2DM-induced gastrointestinal injury, and 16(R)-HET, 5-HETE, LysoPC (22:0), and Pantothenic acid were the major metabolites associated with T2DM-induced gastrointestinal disorders. Conclusion: The mechanism of action of diabetic gastroenteropathy may be related to vitamin digestion and absorption, glycerophospholipid metabolism, and arachidonic acid metabolism. [ABSTRACT FROM AUTHOR]

Background: Doxorubicin (DOX) is a potent chemotherapy widely used in treating various neoplastic diseases. However, the clinical use of DOX is limited due to its potential toxic effect on the cardiovascular system. Thus, identifying the pathway involved in this toxicity may help minimize chemotherapy risk and improve cancer patients' quality of life. Recent studies suggest that Endothelial-to-Mesenchymal transition (EndMT) and endothelial toxicity contribute to the pathogenesis of DOX-induced cardiovascular toxicity. However, the molecular mechanism is yet unknown. Given that arachidonic acid and associated cytochrome P450 (CYP) epoxygenase have been involved in endothelial and cardiovascular function, we aimed to examine the effect of suppressing CYP epoxygenases on DOX-induced EndMT and cardiovascular toxicity in vitro and in vivo. Methods and Results: To test this, human endothelial cells were treated with DOX, with or without CYP epoxygenase inhibitor, MSPPOH. We also investigated the effect of MSPPOH on the cardiovascular system in our zebrafish model of DOX-induced cardiotoxicity. Our results showed that MSPPOH exacerbated DOX-induced EndMT, inflammation, oxidative stress, and apoptosis in our endothelial cells. Furthermore, we also show that MSPPOH increased cardiac edema, lowered vascular blood flow velocity, and worsened the expression of EndMT and cardiac injury markers in our zebrafish model of DOX-induced cardiotoxicity. Conclusion: Our data indicate that a selective CYP epoxygenase inhibitor, MSPPOH, induces EndMT and endothelial toxicity to contribute to DOX-induced cardiovascular toxicity. [ABSTRACT FROM AUTHOR]

Nonylphenol (NP), an endocrine-disrupting chemical, is an environmental contaminant, and many notorious effects on male fertility have been reported in animal models and wild-type species. Here, we evaluated the effects of NP in follicle-stimulating hormone (FSH) signal transduction pathways and lipid metabolism using an in vitro model of rat Sertoli cell (SC) primary culture. Results show that an acute (1 h) SC exposure to NP (10 µM) increased the intraand extra-cellular concentrations of free fatty acids (FFAs), mainly arachidonic acid (20:4n-6). Phosphatidylinositol seemed to be the major phospholipid source of this 20:4n-6 release by activation of the protein kinase A (PKA)/cytoplasmic phospholipase A2 (cPLA2) pathway. NP also increased diacylglycerols (DAG) levels and the expression (mRNA) of cyclooxygenase 2 (Cox2) and prostaglandin E2 (PGE2) levels. It is noteworthy that accumulation of lipid droplets took place after 24 h NP exposition, which was prevented by both a PKA inhibitor and a PLA2 inhibitor. Like FSH, NP triggers the release of 20:4n-6, which is a substrate for PGE2 synthesis via PKA/PLA2 activation. In addition, NP induces the formation of DAG, which could be required as a cofactor of the PKC-mediated activation of the COX2 inflammatory pathway. Our findings suggest that NP alters lipid homeostasis in SCs by inducing the activation of pro-inflammatory pathways that may trigger adverse effects in testis physiology over time. Concomitantly, the SC enhances the acylation of surplus FFAs (including 20:4n-6) in neutral lipids as a protective mechanism to shield itself from lipotoxicity and pro-inflammatory signals. [ABSTRACT FROM AUTHOR]

Inhibitors of soluble epoxide hydrolase (sEH), which catalyzes the hydrolysis of various natural epoxides to their corresponding diols, present an opportunity for developing oral drugs for a range of human cardiovascular and inflammatory diseases, including, among others, diabetes and neuropathic pain. However, some evidence suggests that their administration may precipitate the development of pulmonary hypertension (PH). We thus evaluated the impact of chronic oral administration of the sEH inhibitor TPPU (N-[1-(1-Oxopropyl)-4-piperidinyl]-N'-[4-(trifluoromethoxy)phenyl]-urea) on hemodynamics, pulmonary vascular reactivity, and remodeling, as well as on right ventricular (RV) dimension and function at baseline and in the Sugen (SU5416) + hypoxia (SuHx) rat model of severe PH. Treatment with TPPU started 5 weeks after SU5416 injection for 3 weeks. No differences regarding the increase in pulmonary vascular resistance, remodeling, and inflammation, nor the abolishment of phenylephrine-induced pulmonary artery constriction, were noted in SuHx rats. In addition, TPPU did not modify the development of RV dysfunction, hypertrophy, and fibrosis in SuHx rats. Similarly, none of these parameters were affected by TPPU in normoxic rats. Complementary in vitro data demonstrated that TPPU reduced the proliferation of cultured human pulmonary artery-smooth muscle cells (PA-SMCs). This study demonstrates that inhibition of sEH does not induce nor aggravate the development of PH and RV dysfunction in SuHx rats. In contrast, a potential beneficial effect against pulmonary artery remodeling in humans is suggested.

Inflammation plays a crucial role in progression of fibrosis. Epoxyeicosatrienoic acids (EET) have multiple protective effects in different diseases, but their ability to inhibit the development of LPS-induced fibrosis remains unknown. The potential therapeutic effects of 11,12-EET were studied in in vitro model of LPS-induced fibrosis. Mouse embryonic fibroblast cells NIH/3T3 were pre-incubated with 1 μM 11,12-EET and/or a structural analogue and selective EET antagonist 14,15-epoxyeicosa-5(Z)-enoic acid before exposing to LPS. The effect of EET was evaluated by the protein and mRNA expression of NF-κB, collagens I and III, and α-smooth muscle actin by Western blotting and quantitative reverse transcription PCR, respectively. LPS provoked inflammation and fibrosis-like changes accompanied by elevated expression of NF-κB and collagens in NIH/3T3 cells. We also studied the effects of 11,12-EET on the A2AR and PI3K/Akt signaling pathways in intact and LPS-treated NIH/3T3 cells. 11,12-EET prevented inflammation and fibrosis-like changes through up-regulation of A2AR and PI3K/Akt signaling pathways. Our findings demonstrate the potential antifibrotic effects of 11,12-EET, which can be natural antagonists of tissue fibrosis. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) has three main metabolic pathways: the cycloxygenases (COXs) pathway, the lipoxygenases (LOXs) pathway, and the cytochrome P450s (CYPs) pathway. AA produces epoxyeicosatrienoic acids (EETs) through the CYPs pathway. EETs are very unstable in vivo and can be degraded in seconds to minutes. EETs have multiple degradation pathways, but are mainly degraded in the presence of soluble epoxide hydrolase (sEH). sEH is an enzyme of bifunctional nature, and current research focuses on the activity of its C-terminal epoxide hydrolase (sEH-H), which hydrolyzes the EETs to the corresponding inactive or low activity diol. Previous studies have reported that EETs have cardiovascular protective effects, and the activity of sEH-H plays a role by degrading EETs and inhibiting their protective effects. The activity of sEH-H plays a different role in different cells, such as inhibiting endothelial cell proliferation and migration, but promoting vascular smooth muscle cell proliferation and migration. Therefore, it is of interest whether the activity of sEH-H is involved in the initiation and progression of cardiovascular diseases by affecting the function of different cells through EETs. [ABSTRACT FROM AUTHOR]

Objective(s): Utilization of doxorubicin (DOX) as a chemotherapy medication is limited due to its cardiotoxic effects. Carnosic acid exerts antioxidant, anti-inflammatory, besides cytoprotective effects. The objective of this study was to investigate the ability of carnosic acid to protect rat hearts and the MCF7 cell line against cardiotoxicity induced by DOX. Materials and Methods: The study involved the classification of male Wistar rats into seven groups: 1) Control 2) DOX (2 mg/kg, every 48h, IP, 12d), 3-5) Carnosic acid (10, 20, 40 mg/kg/day, IP, 16d)+ DOX, 6) Vitamin E (200 mg/kg, every 48h, IP, 16d)+ DOX 7) Carnosic acid (40 mg/kg/day, IP, 16d). Finally, cardiac histopathological alterations, ECG factors, carotid blood pressure, left ventricular function, heart-to-body weight ratio, oxidative (MDA, GSH), inflammatory (IL-1β, TNF-α), plus apoptosis (caspase 3, 8, 9, Bcl-2, Bax) markers were evaluated. DOX toxicity and carnosic acid ameliorative effect were evaluated on MCF7 cells using the MTT assay. Results: DOX augmented the QRS duration, QA, RRI, STI, and heart-to-body weight ratio, and reduced HR, LVDP, Min dP/dt, Max dP/dt, blood pressure, boosted MDA, TNF-a, IL1-ß, caspase 3,8,9, Bax/Bcl-2 ratio, decreased GSH content, caused fibrosis, necrosis, and cytoplasmic vacuolization in cardiac tissue but carnosic acid administration reduced the toxic effects of DOX. The cytotoxic effects of DOX were not affected by carnosic acid at concentrations of 5 and 10 μM. Conclusion: Carnosic acid as an anti-inflammatory and antioxidant substance is effective in reducing DOX-induced damage by enhancing antioxidant defense and modifying inflammatory signal pathway activity and can be used as an adjunct in treating DOX cardiotoxicity. [ABSTRACT FROM AUTHOR]

Arachidonic acid (AA) is a main component of cell membrane lipids. AA is mainly metabolized by three enzymes: cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP450). Esterified AA is hydrolysed by phospholipase A2 into a free form that is further metabolized by COX, LOX and CYP450 to a wide range of bioactive mediators, including prostaglandins, lipoxins, thromboxanes, leukotrienes, hydroxyeicosatetraenoic acids and epoxyeicosatrienoic acids. Increased mitochondrial oxidative stress is considered to be a central mechanism in the pathophysiology of the kidney. Along with increased oxidative stress, apoptosis, inflammation and tissue fibrosis drive the progressive loss of kidney function, affecting the glomerular filtration barrier and the tubulointerstitium. Recent studies have shown that AA and its active derivative eicosanoids play important roles in the regulation of physiological kidney function and the pathogenesis of kidney disease. These factors are potentially novel biomarkers, especially in the context of their involvement in inflammatory processes and oxidative stress. In this review, we introduce the three main metabolic pathways of AA and discuss the molecular mechanisms by which these pathways affect the progression of acute kidney injury (AKI), diabetic nephropathy (DN) and renal cell carcinoma (RCC). This review may provide new therapeutic targets for the identification of AKI to CKD continuum. [ABSTRACT FROM AUTHOR]

In recent years, numerous experimental studies have underscored the pivotal role of soluble epoxide hydrolase (sEH) in renal diseases, demonstrating the reno-protective effects of sEH inhibitors. The nexus between sEH and renalassociated diseases has garnered escalating attention. This review endeavors to elucidate the potential molecular mechanisms of sEH in renal diseases and emphasize the critical role of sEH inhibitors as a prospective treatment modality. Initially, we expound upon the correlation between sEH and Epoxyeicosatrienoic acids (EETs) and also addressing the impact of sEH on other epoxy fatty acids, delineate prevalent EPHX2 single nucleotide polymorphisms (SNPs) associated with renal diseases, and delve into sEHmediated potential mechanisms, encompassing oxidative stress, inflammation, ER stress, and autophagy. Subsequently, we delineate clinical research pertaining to sEH inhibition or co-inhibition of sEH with other inhibitors for the regulation of renal-associated diseases, covering conditions such as acute kidney injury, chronic kidney diseases, diabetic nephropathy, and hypertension-induced renal injury. Our objective is to validate the potential role of sEH inhibitors in the treatment of renal injuries. We contend that a comprehensive comprehension of the salient attributes of sEH, coupled with insights from clinical experiments, provides invaluable guidance for clinicians and presents promising therapeutic avenues for patients suffering from renal diseases. [ABSTRACT FROM AUTHOR]

Drug withdrawal post-marketing due to cardiotoxicity is a major concern for drug developers, regulatory agencies, and patients. One common mechanism of cardiotoxicity is through inhibition of cardiac ion channels, leading to prolongation of the QT interval and sometimes fatal arrythmias. Recently, oxylipin signaling compounds have been shown to bind to and alter ion channel function, and disruption in their cardiac levels may contribute to QT prolongation. Cytochrome P450 2J2 (CYP2J2) is the predominant CYP isoform expressed in cardiomyocytes, where it oxidizes arachidonic acid to cardioprotective epoxyeicosatrienoic acids (EETs). In addition to roles in vasodilation and angiogenesis, EETs bind to and activate various ion channels. CYP2J2 inhibition can lower EET levels and decrease their ability to preserve cardiac rhythm. In this review, we investigated the ability of known CYP inhibitors to cause QT prolongation using Certara's Drug Interaction Database. We discovered that among the multiple CYP isozymes, CYP2J2 inhibitors were more likely to also be QT-prolonging drugs (by approximately 2-fold). We explored potential binding interactions between these inhibitors and CYP2J2 using molecular docking and identified four amino acid residues (Phe61, Ala223, Asn231, and Leu402) predicted to interact with QT-prolonging drugs. The four residues are located near the opening of egress channel 2, highlighting the potential importance of this channel in CYP2J2 binding and inhibition. These findings suggest that if a drug inhibits CYP2J2 and interacts with one of these four residues, then it may have a higher risk of QT prolongation and more preclinical studies are warranted to assess cardiovascular safety. [ABSTRACT FROM AUTHOR]

Aim: To determine whether the crustacean Rh1 protein functions as a dual CO2/ammonia transporter and investigate its role in branchial ammonia excretion and acid–base regulation. Methods: Sequence analysis of decapod Rh1 proteins was used to determine the conservation of amino acid residues putatively involved in ammonia transport and CO2 binding in human and bacterial Rh proteins. Using the Carcinus maenas Rh1 protein (CmRh1) as a representative of decapod Rh1 proteins, we test the ammonia and CO2 transport capabilities of CmRh1 through heterologous expression in yeast and Xenopus oocytes coupled with site‐directed mutagenesis. Quantitative PCR was used to assess the distribution of CmRh1 mRNA in various tissues. Western blotting was used to assess CmRh1 protein expression changes in response to high environmental ammonia and CO2. Further, immunohistochemistry was used to assess sub‐cellular localization of CmRh1 and a membrane‐bound carbonic anhydrase (CmCAg). Results: Sequence analysis of decapod Rh proteins revealed high conservation of several amino acid residues putatively involved in conducting ammonia transport and CO2 binding. Expression of CmRh1 in Xenopus oocytes enhanced both ammonia and CO2 transport which was nullified in CmRh1 D180N mutant oocytes. Transport of the ammonia analog methylamine by CmRh1 is dependent on both ionized and un‐ionized ammonia/methylamine species. CmRh1 was co‐localized with CmCAg to the apical membrane of the crustacean gill and only experienced decreased protein expression in the anterior gills when exposed to high environmental ammonia. Conclusion: CmRh1 is the first identified apical transporter‐mediated route for ammonia and CO2 excretion in the crustacean gill. Our findings shed further light on the potential universality of dual ammonia and CO2 transport capacity of Rhesus glycoproteins in both vertebrates and invertebrates. [ABSTRACT FROM AUTHOR]

Soluble epoxide hydrolase (sEH) is an enzyme involved in the metabolism of bioactive lipid signaling molecules. sEH converts epoxyeicosatrienoic acids (EET) to virtually inactive dihydroxyeicosatrienoic acids (DHET). The first acids are "medicinal" molecules, the second increase the inflammatory infiltration of cells. Mitogen-activated protein kinases (p38 MAPKs) are key protein kinases involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α) and cyclooxygenase-2 (COX-2). p38 MAPK signaling plays an important role in the regulation of cellular processes, especially inflammation. The proto-oncogenic serine/threonine protein kinase Raf (c-Raf) is a major component of the mitogen-activated protein kinase (MAPK) pathway: ERK1/2 signaling. Normal cellular Raf genes can also mutate and become oncogenes, overloading the activity of MEK1/2 and ERK1/2. The development of multitarget inhibitors is a promising strategy for the treatment of socially dangerous diseases. We synthesized 1,3-disubstituted ureas and diureas containing a dichloroadamantyl moiety. The results of computational methods show that soluble epoxide hydrolase inhibitors can act on two more targets in different signaling pathways of mitogen-activated protein kinases p38 MAPK and c-Raf. The two chlorine atoms in the adamantyl moiety may provide additional Cl-π interactions in the active site of human sEH. Molecular dynamics studies have shown that the stability of ligand–protein complexes largely depends on the "spacer effect." The compound containing a bridge between the chloroadamantyl fragment and the ureide group forms more stable ligand–protein complexes with sEH and p38 MAPK, which indicates a better conformational ability of the molecule in the active sites of these targets. In turn, a compound containing two chlorine atoms forms a more stable complex with c-Raf, probably due to the presence of additional halogen bonds of chlorine atoms with amino acid residues. [ABSTRACT FROM AUTHOR]

The soluble epoxide hydrolase (sEH) is a potential target to treat cardiovascular, renal and neuronal diseases. A series of sEH inhibitors containing naturally occurring lipophilic groups (originating from camphor and fenchone) were developed. Inhibitory potency ranging from 0.7 nM to 6.47 μM was obtained. It was discovered that ureas derived from L-camphor were more active against sEH (2.3-fold average) than the corresponding analogues derived from D-camphor indicating enantiomeric preference of sEH. Ureas derived from fenchone possess lower activity against sEH (ca. 80-fold on average) than their camphor-derived analogs due to the specific structure of the lipophilic fragment and show less enantiomeric preference (1.75-fold on average). Moreover, fenchone-derived ureas show no consistency in enantiomeric preference. Endo/exo-form of compound L-3a derived from L-camphor is 4-fold more potent than the corresponding analogue prepared from D-camphor (IC50 = 0.7 nM vs. 2.8 nM) making it the most promising sEH inhibitor among the tested series.

Alzheimer's disease (AD) is the most common neurodegenerative disorder characterized by dementia. Inhibition of soluble epoxide hydrolase (sEH) regulates inflammation involving in central nervous system (CNS) diseases. However, the exactly mechanism of sEH in AD is still unclear. In this study, we evaluated the vital role of sEH in amyloid beta (Aβ)-induced AD mice, and revealed a possible molecular mechanism for inhibition of sEH in the treatment of AD. The results showed that the sEH expression and activity were remarkably increased in the hippocampus of Aβ-induced AD mice. Chemical inhibition of sEH by TPPU, a selective sEH inhibitor, alleviated spatial learning and memory deficits, and elevated levels of neurotransmitters in Aβ-induced AD mice. Furthermore, inhibition of sEH could ameliorate neuroinflammation, neuronal death, and oxidative stress via stabilizing the in vivo level of epoxyeicosatrienoic acids (EETs), especially 8,9-EET and 14,15-EET, further resulting in the anti-AD effect through the regulation of GSK3β-mediated NF-κB, p53, and Nrf2 signaling pathways. These findings revealed the underlying mechanism of sEH as a potential therapeutic target in treatment of AD.

Abstract Background Recent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron’s anti-breast carcinoma (BC) function. Methods Bioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo. Results Quercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice. Conclusion Quercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development.

Epoxyeicosatrienoic acids (EETs), which exert multiple endogenous protective effects, are hydrolyzed into less active dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase (sEH). However, commercial drugs related to EETs or sEH are not yet in clinical use. Firstly, the plasma concentration of EETs and DHETs of 316 patients with heart failure (HF) were detected and quantitated by liquid chromatography-tandem mass spectrometry. Then, transverse aortic constriction (TAC)-induced HF was introduced in cardiomyocyte-specific Ephx2−/− mice. Moreover, Western blot, real-time PCR, luciferase reporter, ChIP assays were employed to explore the underlying mechanism. Finally, multiple sEH inhibitors were designed, synthesized, and validated in vitro and in vivo. The ratios of DHETs/EETs were increased in the plasma from patients with HF. Meanwhile, the expression of sEH was upregulated in the heart of patients and mice with HF, especially in cardiomyocytes. Cardiomyocyte-specific Ephx2−/− mice ameliorated cardiac dysfunction induced by TAC. Consistently, Ephx2 knockdown protected Angiotensin II (AngII)-treated cardiomyocytes via increasing EETs in vitro. Mechanistically, AngII could enhance the expression of transcript factor Krüppel-like factor 15 (KLF15), which in turn upregulated sEH. Importantly, glimepiride was identified as a novel sEH inhibitor, which benefited from the elevated EETs during HF. Glimepiride attenuates HF in mice in part by increasing EETs. Clinical trial identifier: NCT03461107 (https://clinicaltrials.gov). [Display omitted] • The plasma concentration of EETs is decreased in patients with heart failure as quantified by LC-MS/MS. • Enhanced activity and expression of sEH in cardiomyocyte are observed in both human and murine hearts with heart failure. • Specific deletion of the Ephx2 gene in cardiomyocytes ameliorates cardiac dysfunction induced by TAC. • AngII stimulation results in elevated expression of the transcript factor KLF15, subsequently leading to sEH upregulation. • Glimepiride, a novel sEH inhibitor, shows potential clinical application in HF treatment by increasing EET levels. [ABSTRACT FROM AUTHOR]

Background: Inflammatory response of human vascular smooth muscle cells (hVSMCs) is a driving factor in hypertension progression. It has been reported that miR-3646 was significantly up-regulated in serum samples from patients with coronary artery disease and acute myocardial infarction mice. However, its role and underlying molecular mechanism related to inflammatory response of angiotensin II (Ang II)-induced hVSMCs remain unclear. Objective: We aimed to explore the potential molecular mechanisms related to inflammatory response of angiotensin II (Ang II)-induced hVSMCs. Methods: Ang II-induced hypertension model was established after hVSMCs treated with 1 µM Ang II at 24 h. The interaction between microRNA 3646 (miR-3646) and cytochrome P450 2J2 (CYP2J2) was assessed by dual-luciferase reporter gene assay. MTS assay, Lipid Peroxidation MDA Assay Kit, ELISA, Western blot, and qRT-PCR were performed to examine viability, malondialdehyde (MDA) level, inflammatory cytokine levels, and the level of genes and proteins. Results: Our findings illustrated that miR-3646 was up-regulated but CYP2J2 was down-regulated in Ang II-induced hVSMCs. Mechanically, miR-3646 negatively targeted to CYP2J2 in Ang II-induced hVSMCs. These findings indicated that miR-3646 regulated inflammatory response of Ang II-induced hVSMCs via targeting CYP2J2. Moreover, functional researches showed that CYP2J2 overexpression alleviated inflammatory response of Ang II-induced hVSMCs via epoxyeicosatrienoic acids/peroxisome proliferatoractivated receptor-γ (EETs/PPARγ) axis, and miR-3646 aggravated inflammatory response of Ang II-induced hVSMCs via mediating CYP2J2/EETs axis. Conclusion: MiR-3646 accelerated inflammatory response of Ang II-induced hVSMCs via CYP2J2/EETs axis. Our findings illustrated the specific molecular mechanism of miR-3646 regulating hypertension. [ABSTRACT FROM AUTHOR]

Fatty acid binding proteins (FABPs) govern intracellular lipid transport to cytosolic organelles and nuclear receptors. More recently, FABP5 has emerged as a key regulator of synaptic endocannabinoid signaling, suggesting that FABPs may broadly regulate the signaling of neuroactive lipids in the brain. Herein, we demonstrate that brain-expressed FABPs (FABP3, FABP5, and FABP7) interact with epoxyeicosatrienoic acids (EETs) and the peroxisome proliferator-activated receptor gamma agonist 15-deoxy-Δ12,14-Prostaglandin J2 (15d-PGJ2). Among these lipids, EETs displayed highest affinities for FABP3 and FABP5, and 11,12-EET was identified as the preferred FABP ligand. Similarly, 15d-PGJ2 interacted with FABP3 and FABP5 while binding to FABP7 was markedly lower. Molecular modeling revealed unique binding interactions of the ligands within the FABP binding pockets and highlighted major contributions of van der Waals clashes and acyl chain solvent exposure in dictating FABP affinity and specificity. Functional studies demonstrated that endogenous EETs gate the strength of CA1 hippocampal glutamate synapses and that this function was impaired following FABP inhibition. As such, the present study reveals that FABPs control EET-mediated synaptic gating, thereby expanding the functional roles of this protein family in regulating neuronal lipid signaling. [ABSTRACT FROM AUTHOR]

Background: The association between the oxidative stress gene polymorphism and chronic obstructive pulmonary disease (COPD) risk has been extensively studied but the results have been controversial. This study aimed to investigate the overall association between the oxidative stress gene including glutathione S-transferase (GST), epoxide hydrolase exon (EPHX), superoxide dismutase (SOD), catalase (CAT), cytochrome P450 system (CYP) and heme oxygenase (HO-1) polymorphism and the risk of COPD. Methods: We searched the PubMed and EMBASE database to identify studies that investigated the association between the oxidative stress gene polymorphism and risk of COPD. The relevant data were extracted and statistical analyses were performed using the Revman 5.4 and STATA 12 software. Dominant genetic model, recessive model, co-dominant model, heterozygote model, and allele model were analyzed. Venice criteria and publication bias were conducted to access the credibility and reliability. Results: In total, 63 publications including 14,733 patients and 50,570 controls were included in the meta-analysis.15 genetic variants of 6 genes were analyzed, and 7 SNPs in GSTP1, CAT, CYP, SOD were first analyses until now. In our study, EPHX T113C C allele, GSTM1 null, GSTT1 null, GSTP1 A313G G and C341T T allele, CYP1A1 MspI C allele, SOD3 A213G G allele and L type in Ho-1 showed increased COPD risk, especially in Asians. T allele in CAT C262T and C allele in SOD2 Val 9 Ala were associated with decreased COPD risk. To avoid high heterogeneity and publications bias, subgroups analysis was performed in accord with HWE and ethnicity. Publication bias was assessed by Begg's funnel plots and Egger's test, and no publication bias were found for recessive models. 4 variants were identified with strong levels of epidemiological evidence of associations with the COPD risk. Conclusions: Our results confirm that oxidative stress gene polymorphism was associated with COPD risk. These finding can improve human understanding of this disease gene molecular level and enable early intervention and prevention of COPD. Well-designed studies with large sample sizes are essential to clarify the association of these significant variants with the susceptibility to COPD. [ABSTRACT FROM AUTHOR]

Neuropathic pain (NP) is a typical symptom of peripheral nerve disorders, including painful neuropathy. The biological mechanisms that control ion channels are important for many cell activities and are also therapeutic targets. Disruption of the cellular mechanisms that govern ion channel activity can contribute to pain pathophysiology. The voltage-gated sodium channel (VGSC) is the most researched ion channel in terms of NP; however, VGSC impairment is detected in only <20% of painful neuropathy patients. Here, we discuss the potential role of the other peripheral ion channels involved in sensory signaling (transient receptor potential cation channels), neuronal excitation regulation (potassium channels), involuntary action potential generation (hyperpolarization-activated cyclic nucleotide-gated channels), thermal pain (anoctamins), pH modulation (acid sensing ion channels), and neurotransmitter release (calcium channels) related to pain and their prospective role as therapeutic targets for painful neuropathy. [ABSTRACT FROM AUTHOR]

Pancreatic β-cells are crucial regulators of systemic glucose homeostasis, and their dysfunction and loss are central features in type 2 diabetes. Interventions that rectify β-cell dysfunction and loss are essential to combat this deadly malady. In the current study, we sought to delineate the role of soluble epoxide hydrolase (sEH) in β-cells under diet-induced metabolic stress. The expression of sEH was upregulated in murine and macaque diabetes models and islets of diabetic human patients. We postulated that hyperglycemia-induced elevation in sEH leads to a reduction in its substrates, epoxyeicosatrienoic acids (EETs), and attenuates the function of β-cells. Genetic deficiency of sEH potentiated glucose-stimulated insulin secretion in mice, likely in a cell-autonomous manner, contributing to better systemic glucose control. Consistent with this observation, genetic and pharmacological inactivation of sEH and the treatment with EETs exhibited insulinotropic effects in isolated murine islets ex vivo. Additionally, sEH deficiency enhanced glucose sensing and metabolism with elevated ATP and cAMP concentrations. This phenotype was associated with attenuated oxidative stress and diminished β-cell death in sEH deficient islets. Moreover, pharmacological inhibition of sEH in vivo mitigated, albeit partly, high fat diet-induced β-cell loss and dedifferentiation. The current observations provide new insights into the role of sEH in β-cells and information that may be leveraged for the development of a mechanism-based intervention to rectify β-cell dysfunction and loss.

Background: Recent years have witnessed impressive growth in applying natural medicine in tumor treatment. Saffron is reported to elicit an inhibitory property against BC. Herein, we sought to explore the specific components and mechanistic basis of saffron's anti-breast carcinoma (BC) function. Methods: Bioinformatics analysis was employed to analyze saffron components' anti-BC activity and screen the corresponding target genes involved in BC. Then, the roles of the main saffron ingredient quercetin in the activity of BC cells were examined using CCK-8, MTS, flow cytometry, colony formation, Transwell, and Gelatin zymogram assays. Additionally, the interactions among Quercetin, EET, and Stat3 were assessed by immunofluorescence and Western blot, and LC–MS/MS determined the levels of AA, EETs, and CYP3A. Finally, BC xenograft mouse models were established to verify the anti-BC function of Quercetin in vivo. Results: Quercetin, the main active component of saffron, inhibited BC progression. Quercetin suppressed BC cell growth, migration, and invasion and inhibited CYP3A4 expression and activity in BC. Mechanistically, Quercetin down-regulated CYP3A4 to block the nuclear translocation of Stat3 by decreasing the metabolization of AA to EETs, thereby alleviating BC. Moreover, exogenously added EETs counteracted the anti-tumor effect of Quercetin on BC. Quercetin also inhibited the tumor growth of tumor-bearing nude mice. Conclusion: Quercetin could inhibit the activity of CYP3A to down-regulate AA metabolites EETs, consequently hampering p-Stat3 and nuclear translocation, thus impeding BC development. [ABSTRACT FROM AUTHOR]

Human soluble epoxide hydrolase (sEH), a dual-functioning homodimeric enzyme with hydrolase and phosphatase activities, is known for its pivotal role in the hydrolysis of epoxyeicosatrienoic acids. Inhibitors targeting sEH have shown promising potential in the treatment of various life-threatening diseases. In this study, we employed a range of in silico modeling approaches to investigate a diverse dataset of structurally distinct sEH inhibitors. Our primary aim was to develop predictive and validated models while gaining insights into the structural requirements necessary for achieving higher inhibitory potential. To accomplish this, we initially calculated molecular descriptors using nine different descriptor-calculating tools, coupled with stochastic and non-stochastic feature selection strategies, to identify the most statistically significant linear 2D-QSAR model. The resulting model highlighted the critical roles played by topological characteristics, 2D pharmacophore features, and specific physicochemical properties in enhancing inhibitory potential. In addition to conventional 2D-QSAR modeling, we implemented the Transformer-CNN methodology to develop QSAR models, enabling us to obtain structural interpretations based on the Layer-wise Relevance Propagation (LRP) algorithm. Moreover, a comprehensive 3D-QSAR analysis provided additional insights into the structural requirements of these compounds as potent sEH inhibitors. To validate the findings from the QSAR modeling studies, we performed molecular dynamics (MD) simulations using selected compounds from the dataset. The simulation results offered crucial insights into receptor–ligand interactions, supporting the predictions obtained from the QSAR models. Collectively, our work serves as an essential guideline for the rational design of novel sEH inhibitors with enhanced therapeutic potential. Importantly, all the in silico studies were performed using open-access tools to ensure reproducibility and accessibility. [ABSTRACT FROM AUTHOR]

Glutathione (GSH) and cysteine are important antioxidants in maintaining the oxidation-reduction balance. 14,15-epoxyeicosatrienoic acid (14,15-EET) and 15-hydroxyeicosatetraenoic acid (15-HETE) are biological active compounds metabolized from arachidonic acid. To dynamically monitor GSH, cysteine, 14,15-EET, and 15-HETE, an ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS) method was developed. The derivative GSH, cysteine, and underivatized 14,15-EET, 15-HETE were determined by electrospray ionization source with multiple reaction monitoring. The developed method was well validated and applied to monitor the extracellular concentration of GSH, cysteine, 14,15-EET, and 15-HETE in paraquat (PQ) poisoning LO2 cells. It was shown that the derivation of GSH and cysteine is complete and specific. There was a good linear relationship among GSH, cysteine, 14,15-EET, and 15-HETE. The relative standard deviations of precision and accuracy were all within 15%. After PQ poisoning, the extracellular concentrations of GSH and cysteine were increased at 72 h. 14,15-EET and 15-HETE started to increase at 24 h, earlier than GSH and cysteine. Irreversible pathological alterations appeared at 48 h. In conclusion, a fast and specific UPLC-MS/MS method for the determination of GSH, cysteine, 14,15-EET, and 15-HETE has been developed and validated. PQ induced the increase of 14,15-EET and 15-HETE earlier than that of GSH and cysteine in LO2 cells culture medium. [ABSTRACT FROM AUTHOR]

Platelets play a modulatory role in inflammatory response by secreting a vast array of granules and disintegrating into membrane-bound microparticles upon activation. The interplay between eosinophils and platelets is postulated to be implicated in the pathology of allergic airway inflammation. In this study, we investigated whether activated platelets can induce eosinophil extracellular trap (EET) formation, a cellular process by which activated eosinophils release net-like DNA fibers. Platelets were stimulated with the calcium ionophore, A23187, and the platelet agonists, thrombin and adenosine diphosphate (ADP). Platelet cultures were fractionated into conditioned medium (CM) and pellet, which were then overlaid on eosinophils to examine EET formation. The CM and pellet from A23187-activated platelets stimulated eosinophils to generate EET, whereas those from thrombin- or ADP-activated platelets failed to induce such generation. The EET-inducing activity of the A23187-activated platelet culture was linearly proportional to the number of activated platelets. Interestingly, while EET formation induced by the direct stimulation of eosinophils with A23187 was NADPH oxidase (NOX)-dependent, EET formation induced by A23187-activated platelets was NOX-independent and significantly inhibited by necroptosis pathway inhibitors. Activated platelets and their products may induce EET formation, thereby potentiating their role in eosinophilic airway inflammation. [Display omitted] [ABSTRACT FROM AUTHOR]

A series of inhibitors of the soluble epoxide hydrolase (sEH) containing imidazolidine-2,4,5-trione or pirimidine-2,4,6-trione has been synthesized. Inhibition potency of the described compounds ranges from 8.4 μM to 0.4 nM. The tested compounds possess higher water solubility than their preceding ureas. Molecular docking indicates new bond between the triones and the active site of sEH that in part explain the observed potency of the new pharmacophores. While less potent than the corresponding ureas, the modifications of urea group reported herein yield compounds with higher water solubility, thus permitting easier formulation.

This study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes endothelium-derived epoxyeicosatrienoic acids, plays a role in vascular calcification. The sEH inhibitor trans-4-(4-(3-adamantan-1-yl-ureido)-cyclohexyloxy)-benzoic acid (t-AUCB) potentiated the increase in calcium deposition of rat aortic rings cultured in high-phosphate conditions. This was associated with increased tissue-nonspecific alkaline phosphatase activity and mRNA expression level of the osteochondrogenic marker Runx2. The procalcifying effect of t-AUCB was prevented by mechanical aortic deendothelialization or inhibition of the production and action of epoxyeicosatrienoic acids using the cytochrome P450 inhibitor fluconazole and the antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE), respectively. Similarly, exogenous epoxyeicosatrienoic acids potentiated the calcification of rat aortic rings through a protein kinase A (PKA)-dependent mechanism and of human aortic vascular smooth muscle cells when sEH was inhibited by t-AUCB. Finally, a global gene expression profiling analysis revealed that the mRNA expression level of sEH was decreased in human carotid calcified plaques compared to adjacent lesion-free sites and was inversely correlated with Runx2 expression. These results show that sEH hydrolase plays a protective role against vascular calcification by reducing the bioavailability of epoxyeicosatrienoic acids.

The veterinary pharmacopeia available to treat pain and inflammation is limited in number, target of action and efficacy. Inhibitors of soluble epoxide hydrolase (sEH) are a new class of anti-inflammatory, pro-resolving and analgesic drugs being tested in humans that have demonstrated efficacy in laboratory animals. They block the hydrolysis, and thus, increase endogenous concentrations of analgesic and anti-inflammatory signaling molecules called epoxy-fatty acids. Here, we screened a library of 2,300 inhibitors of the sEH human against partially purified feline, canine and equine hepatic sEH to identify inhibitors that are broadly potent among species. Six very potent sEH inhibitors (IC50 < 1 nM for each enzyme tested) were identified. Their microsomal stability was then measured in hepatic extracts from cat, dog and horse, as well as their solubility in solvents suitable for the formulation of drugs. The trans-4-{4-[3-(4-trifluoromethoxy-phenyl)-ureido]-cyclohexyloxy}-benzoic acid (t-TUCB, 1,728) appears to be the best compromise between stability and potency across species. Thus, it was selected for further testing in veterinary clinical trials of pain and inflammation in animals.

Cytochrome P4502J2 (CYP2J2) metabolizes arachidonic acid (AA) to cardioprotective epoxyeicosatrienoic acids (EETs). Dronedarone, an antiarrhythmic drug prescribed for treatment of atrial fibrillation (AF) induces cardiac adverse effects (AEs) with poorly understood mechanisms. We previously demonstrated that dronedarone inactivates CYP2J2 potently and irreversibly, disrupts AA-EET pathway leading to cardiac mitochondrial toxicity rescuable via EET enrichment. In this study, we investigated if mitigation of CYP2J2 inhibition prevents dronedarone-induced cardiac AEs. We first synthesized a deuterated analogue of dronedarone (termed poyendarone) and demonstrated that it neither inactivates CYP2J2, disrupts AA-EETs metabolism nor causes cardiac mitochondrial toxicity in vitro. Our patch-clamp experiments demonstrated that pharmacoelectrophysiology of dronedarone is unaffected by deuteration. Next, we show that dronedarone treatment or CYP2J2 knockdown in spontaneously beating cardiomyocytes indicative of depleted CYP2J2 activity exacerbates beat-to-beat (BTB) variability reflective of proarrhythmic phenotype. In contrast, poyendarone treatment yields significantly lower BTB variability compared to dronedarone in cardiomyocytes indicative of preserved CYP2J2 activity. Importantly, poyendarone and dronedarone display similar antiarrhythmic properties in the canine model of persistent AF, while poyendarone substantially reduces beat-to-beat variability of repolarization duration suggestive of diminished proarrhythmic risk. Our findings prove that deuteration of dronedarone prevents CYP2J2 inactivation and mitigates dronedarone-induced cardiac AEs.

Aim: To explore the genetic effects of CYP2C8, CYP2C9, CYP2J2, and EPHX2, the key genes involved in epoxyeicosatrienoic acid processing and degradation pathways in gestational diabetes mellitus (GDM) and metabolic traits in Chinese pregnant women. Methods: A total of 2548 unrelated pregnant women were included, of which 938 had GDM and 1610 were considered as controls. Common variants were genotyped using the Infinium Asian Screening Array. Association studies of single nucleotide polymorphisms (SNPs) with GDM and related traits were performed using logistic regression and multivariable linear regression analyses. A genetic risk score (GRS) model based on 12 independent target SNPs associated with GDM was constructed. Logistic regression was used to estimate odds ratios and 95% confidence intervals, adjusting for potential confounders including age, pre-pregnancy body mass index, history of polycystic ovarian syndrome, history of GDM, and family history of diabetes, with GRS entered both as a continuous variable and categorized groups. The relationship between GRS and quantitative traits was also evaluated. Results: The 12 SNPs in CYP2C8, CYP2C9, CYP2J2, and EPHX2 were significantly associated with GDM after adjusting for covariates (all P < 0.05). The GRS generated from these SNPs significantly correlated with GDM. Furthermore, a significant interaction between CYP2J2 and CYP2C8 in GDM (PInteraction = 0.014, ORInteraction= 0.61, 95%CI 0.41–0.90) was observed. Conclusion: We found significant associations between GDM susceptibility and 12 SNPs of the four genes involved in epoxyeicosatrienoic acid processing and degradation pathways in a Chinese population. Subjects with a higher GRS showed higher GDM susceptibility with higher fasting plasma glucose and area under the curve of glucose and poorer β-cell function. [ABSTRACT FROM AUTHOR]

Background and Purpose: Epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFA) are lipid mediators that are rapidly inactivated by soluble epoxide hydrolase (sEH). Uncontrolled and chronic inflammatory disorders fail to sufficiently activate endogenous regulatory pathways, including the production of specialized pro‐resolving mediators (SPMs). Here, we addressed the relationship between SPMs and the EET/sEH axis and explored the effects of sEH inhibition on resolving macrophage phenotype. Experimental Approach: Mice were treated with a sEH inhibitor, EETs, or sEH inhibitor + EETs (combination) before ligature placement to induce experimental periodontitis. Using RT‐qPCR, gingival samples were used to examine SPM receptors and osteolytic and inflammatory biomarkers. Maxillary alveolar bone loss was quantified by micro‐CT and methylene blue staining. SPM levels were analysed by salivary metabolo‐lipidomics. Gingival macrophage phenotype plasticity was determined by RT‐qPCR and flow cytometry. Effects of sEH inhibition on macrophage polarization and SPM production were assessed with bone marrow‐derived macrophages (BMDMs). Key Results: Pharmacological inhibition of sEH suppressed bone resorption and the inflammatory cytokine storm in experimental periodontitis. Lipidomic analysis revealed that sEH inhibition augmented levels of LXA4, RvE1, RvE2, and 4‐HDoHE, concomitant with up‐regulation of LTB4R1, CMKLR1/ChemR23, and ALX/FPR2 SPM receptors. Notably, there is an impact on gingival macrophage plasticity was affected suggesting an inflammation resolving phenotype with sEH inhibition. In BMDMs, sEH inhibition reduced inflammatory macrophage activation, and resolving macrophages were triggered to produce SPMs. Conclusion and Implications: Pharmacological sEH inhibition increased SPM synthesis associated with resolving macrophages, suggesting a potential target to control osteolytic inflammatory disorders. [ABSTRACT FROM AUTHOR]

Introduction: In clinical practice, warfarin is often combined with Compound Danshen dripping pill (CDDP) for the treatment of cardiovascular diseases. However, warfarin has a narrow therapeutic index, wide interindividual variability (genetic and non-genetic factors), and is susceptible to drug-drug interactions. Our previous study indicated that CDDP might interact with warfarin in individuals with the epoxide hydrolase gene (EPHX1; singlenucleotide polymorphism: rs2292566) A/A subtype. We sought to clarify the interaction between CDDP and warfarin associated with EPHX1 in a comprehensive and accurate manner. Methods: Here, EPHX1 A and EPHX1 G cell lines were established. Expression of microsomal epoxide hydrolase (mEH), vitamin K epoxide reductase (VKOR), and vitamin K-dependent clotting factors (FII, FVII, FIX, FX) was measured by western blotting upon incubation with CDDP and warfarin. mEH activity was evaluated by measuring the transformation of epoxyeicosatrienoic acids into dihydroxyeicosatrienoic acids. Then, healthy volunteers (HVs) with the EPHX1 A/A genotype were recruited and administered warfarin and CDDP to investigate the pharmacokinetics and pharmacodynamics of warfarin. Results: CDDP combined with warfarin could decrease expression of mEH and VKOR, and increase protein expression of FII, FVII, FIX, and FX, in EPHX1 A cells. CDDP could slightly influence the pharmacokinetics/pharmacodynamics of warfarin in HVs with the EPHX1 A/A genotype. Discussion: Rational combination of CDDP and warfarin was safe with no risk of bleeding, but the therapeutic management is also needed. The clinical study is posted in the China Clinical Trial Registry (ChiCTR190002434). [ABSTRACT FROM AUTHOR]

Fibrates are widely used hypolipidaemic agents that act as ligands of the peroxisome proliferator-activated receptor α (PPARα). p38 is a protein kinase that is mainly activated by environmental and genotoxic stress. We investigated the effect of the PPARα activators fenofibrate and WY-14643 and the PPARα inhibitor GW6471 on the levels of activated p38 (p-p38) in the colorectal cancer cell lines HT-29 and Caco2 in relation to their differentiation status. Fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased. HT-29 cells showed p-p38 predominantly in the cytoplasm, whereas Caco2 cells showed higher nuclear positivity. The effect of fibrates may depend on the differentiation status of the cell, as differentiated HT-29 and undifferentiated Caco2 cells share similar characteristics in terms of villin, CYP2J2, and soluble epoxide hydrolase (sEH) expression. In human colorectal carcinoma, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium, p-p38 showed nuclear positivity. The decrease in p-p38 positivity was associated with a decrease in sEH, consistent with in vitro results. In conclusion, fibrates affect the level of p-p38, but its exact role in the process of carcinogenesis remains unclear and further research is needed in this area. [ABSTRACT FROM AUTHOR]

Chronic non healing wounds in diabetic patients still impose a major problem in modern medicine. Especially additional peripheral vascular disease complicates treatment success in these patients. Thus, we analyzed the effects of 11,12 epoxyeicosatrienoic acid (EET) in a combined model of hyperglycemia and ischemia in mice. Hyperglycemia was induced by Streptozotozin 2 weeks prior to wounding. 3 days before wound creation 2 of the 3 suppling vessels of the moue ear were cautherized for ischemia. Either 11,12 EET or solvent for control was applied. Wound closure as well as TNF-α, TGF-β, SDF-1α, VEGF, CD31, and Ki67 were measured. The wounds closed on day 14.4 ± 0.4 standard deviation (SD). 11,12 EET treatment enhanced healing to 9.8 ± 0.6 SD. TNF-α level was augmented on day 9 compared to control and receded on day 18. TGF-β seemed to be elevated all days observed after 11,12 EET treatment. SDF-1α was enhanced on day 6 and 9 by 11,12 EET, and VEGF on day 6 and 18 as well as CD13 on day 3, 6, and 18. 11,12 EET did not alter Ki67. 11,12 EET are able to rescue deteriorated wound healing in a combined model of hyperglycamia and ischemia by resolution of inflammation, augmentation of neovascularization and increasing expression of TGF-β as well as SDF-1α. [ABSTRACT FROM AUTHOR]

Cytochromes P450 metabolize arachidonic acid to epoxyeicosatrienoic acids (EETs) which have numerous effects. After cardiac ischemia, EET-induced coronary vasodilation increases delivery of oxygen/nutrients to the myocardium, and EETinduced signaling protects cardiomyocytes against postischemic mitochondrial damage. Soluble epoxide hydrolase 2 (EPHX2) diminishes the benefits of EETs through hydrolysis to less active dihydroxyeicosatrienoic acids. EPHX2 inhibition or genetic disruption improves recovery of cardiac function after ischemia. Immunohistochemical staining revealed EPHX2 expression in cardiomyocytes and some endothelial cells but little expression in cardiac smooth muscle cells or fibroblasts. To determine specific roles of EPHX2 in cardiac cell types, we generated mice with cell-specific disruption of Ephx2 in endothelial cells (Ephx2fx/fx/Tek-cre) or cardiomyocytes (Ephx2fx/fx/Myh6-cre) to compare to global Ephx2-deficient mice (global Ephx2-/-) and WT (Ephx2fx/fx) mice in expression, EET hydrolase activity, and heart function studies. Most cardiac EPHX2 expression and activity is in cardiomyocytes with substantially less activity in endothelial cells. Ephx2fx/fx/Tek-cre hearts have similar EPHX2 expression, hydrolase activity, and postischemic cardiac function as control Ephx2fx/fx hearts. However, Ephx2fx/fx/Myh6-cre hearts were similar to global Ephx2-/- hearts with significantly diminished EPHX2 expression, decreased hydrolase activity, and enhanced postischemic cardiac function compared to Ephx2fx/fx hearts. During reperfusion, Ephx2fx/fx/Myh6-cre hearts displayed increased ERK activation compared to Ephx2fx/fx hearts, which could be reversed by EEZE treatment. EPHX2 did not regulate coronary vasodilation in this model. We conclude that EPHX2 is primarily expressed in cardiomyocytes where it regulates EET hydrolysis and postischemic cardiac function, whereas endothelial EPHX2 does not play a significant role in these processes. [ABSTRACT FROM AUTHOR]

Patients with chronic kidney disease (CKD) stage 3–5 are polypharmacy patients. Many of these drugs are metabolized by cytochrome P450 (CYP450) and CYP450. Genetic polymorphism is well known to result in altered drug metabolism capacity. This study determined the added value of pharmacogenetic testing to the routine medication evaluation in polypharmacy patients with CKD. In adult outpatient polypharmacy patients with CKD3-5 disease, a pharmacogenetic profile was determined. Then, automated medication surveillance for gene–drug interactions was performed based on the pharmacogenetic profile and the patients' current prescriptions. Of all identified gene–drug interactions, the hospital pharmacist and the treating nephrologist together assessed clinical relevance and necessity of a pharmacotherapeutic intervention. The primary endpoint of the study was the total number of applied pharmacotherapeutic interventions based on a relevant gene–drug interaction. A total of 61 patients were enrolled in the study. Medication surveillance resulted in a total of 66 gene–drug interactions, of which 26 (39%) were considered clinically relevant. This resulted in 26 applied pharmacotherapeutic interventions in 20 patients. Systematic pharmacogenetic testing enables pharmacotherapeutic interventions based on relevant gene–drug interactions. This study showed that pharmacogenetic testing adds to routine medication evaluation and could lead to optimized pharmacotherapy in CKD patients. [ABSTRACT FROM AUTHOR]

Alveolar epithelial cell (AEC) senescence is a key driver of a variety of chronic lung diseases. It remains a challenge how to alleviate AEC senescence and mitigate disease progression. Our study identified a critical role of epoxyeicosatrienoic acids (EETs), downstream metabolites of arachidonic acid (ARA) by cytochrome p450 (CYP), in alleviating AEC senescence. In vitro, we found that 14,15-EET content was significantly decreased in senescent AECs. Exogenous EETs supplementation, overexpression of CYP2J2, or inhibition of EETs degrading enzyme soluble epoxide hydrolase (sEH) to increase EETs alleviated AECs' senescence. Mechanistically, 14,15-EET promoted the expression of Trim25 to ubiquitinate and degrade Keap1 and promoted Nrf2 to enter the nucleus to exert an anti-oxidant effect, thereby inhibiting endoplasmic reticulum stress (ERS) and alleviating AEC senescence. Furthermore, in D-galactose (D-gal)-induced premature aging mouse model, inhibiting the degradation of EETs by Trifluoromethoxyphenyl propionylpiperidin urea (TPPU, an inhibitor of sEH) significantly inhibited the protein expression of p16, p21, and γH2AX. Meanwhile, TPPU reduced the degree of age-related pulmonary fibrosis in mice. Our study has confirmed that EETs are novel anti-senescence substances for AECs, providing new targets for the treatment of chronic lung diseases.

BackgroundThis pathophysiological study addressed the hypothesis that soluble epoxide hydrolase (sEH), which metabolizes the vasodilator and anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs), contributes to conduit artery endothelial dysfunction in type 2 diabetes.Methods and resultsRadial artery endothelium-dependent flow-mediated dilatation in response to hand skin heating was reduced in essential hypertensive patients (n = 9) and type 2 diabetic subjects with (n = 19) or without hypertension (n = 10) compared to healthy subjects (n = 36), taking into consideration cardiovascular risk factors, flow stimulus and endothelium-independent dilatation to glyceryl trinitrate. Diabetic patients but not non-diabetic hypertensive subjects displayed elevated whole blood reactive oxygen species levels and loss of NO release during heating, assessed by measuring local plasma nitrite variation. Moreover, plasma levels of EET regioisomers increased during heating in healthy subjects, did not change in hypertensive patients and decreased in diabetic patients. Correlation analysis showed in the overall population that the less NO and EETs bioavailability increases during heating, the more flow-mediated dilatation is reduced. The expression and activity of sEH, measured in isolated peripheral blood mononuclear cells, was elevated in diabetic but not hypertensive patients, leading to increased EETs conversion to DHETs. Finally, hyperglycemic and hyperinsulinemic euglycemic clamps induced a decrease in flow-mediated dilatation in healthy subjects and this was associated with an altered EETs release during heating.ConclusionsThese results demonstrate that an increased EETs degradation by sEH and altered NO bioavailability are associated with conduit artery endothelial dysfunction in type 2 diabetic patients independently from their hypertensive status. The hyperinsulinemic and hyperglycemic state in these patients may contribute to these alterations. Trial registration NCT02311075. Registered December 8, 2014.

Neuroinflammation is a physiologic response aimed at protecting the central nervous system during injury. However, unresolved and chronic neuroinflammation can lead to long term damage and eventually neurologic disease including Parkinson's disease, Alzheimer's disease and dementia. Recently, enhancing the concentration of epoxyeicosatrienoic acids (EETs) through blocking their hydrolytic degradation by soluble epoxide hydrolase (sEH) has been applied towards reducing the long-term damage associated with central neurologic insults. Evidence suggests this protective effect is mediated, at least in part, through polarization of microglia to an anti-inflammatory phenotype that blocks the inflammatory actions of prostaglandins and promotes wound repair. This mini-review overviews the epidemiologic basis for using sEH inhibition towards neuroinflammatory disease and pharmacologic studies testing sEH inhibition in several neurologic diseases. Additionally, the combination of sEH inhibition with other eicosanoid signaling pathways is considered as an enhanced approach for developing potent neuroprotectants.

Epoxyeicosatrienoic acids (EETs), metabolites of arachidonic acid, play a crucial role in cytoprotection by attenuating oxidative stress, inflammation and apoptosis. EETs are rapidly metabolised in vivo by the soluble epoxide hydrolase (sEH). Increasing the half life of EETs by inhibiting the sEH enzyme is a novel strategy for neuroprotection. In the present study, sEH inhibitors APAU was screened in silico and further evaluated for their antiparkinson activity against rotenone (ROT) induced neurodegeneration in N27 dopaminergic cell line and Drosophila melanogaster model of Parkinson disease (PD). In the in vitro study cell viability (MTT and LDH release assay), oxidative stress parameters (total intracellular ROS, hydroperoxides, protein oxidation, lipid peroxidation, superoxide dismutase, catalase, glutathione peroxidise, glutathione reductase, glutathione, total antioxidant status, mitochondrial complex-1activity and mitochondrial membrane potential), inflammatory markers (IL-6, COX-1 and COX-2), and apoptotic markers (JNK, phospho-JNK, c-jun, phospho-c-jun, pro and active caspase-3) were assessed to study the neuroprotective effects. In vivo activity of APAU was assessed in Drosophila melanogaster by measuring survival rate, negative geotaxis, oxidative stress parameters (total intracellular ROS, hydroperoxides, glutathione levels) were measured. Dopamine and its metabolites were estimated by LC-MS/MS analysis. In the in silico study the molecule, APAU showed good binding interaction at the active site of sEH (PDB: 1VJ5). In the in vitro study, APAU significantly attenuated ROT induced changes in oxidative, pro-inflammatory and apoptotic parameters. In the in vivo study, APAU significantly attenuates ROT induced changes in survival rate, negative geotaxis, oxidative stress, dopamine and its metabolites levels (p

The arachidonic acid pathway metabolite 20-hydroxyeicosatetraenoic acid (20- HETE) contributes to ischemia/reperfusion brain injury. Inhibition of 20-HETE formation can protect the developing brain from global ischemia. In previous studies, we have found that treatment with the 20-HETE synthesis inhibitor N-hydroxy-N-4-butyl-2-methylphenylformamidine (HET0016) can protect the immature brain from traumatic brain injury (TBI), but its hydrophobic nature limits its full potential. We designed a reactive oxygen species-responsive HET0016 prodrug, which consists of a thioketal link between HET0016 and stearyl alcohol (HET-TK-SA), and used the nanoprodrug strategy to successfully synthesize liposomes HET0016 prodrug liposomes (HPLs) to facilitate the application of HET0016 in protection from TBI. HPLs demonstrated spherical shape, size of about 127.8 nm, a zeta potential of -28.8 mv, a narrow particle size distribution and good stability. Male rats at postnatal day 16-17 underwent controlled cortical impact (CCI) followed by intravenous injection with vehicle or HET0016 (1 mg/kg, 2 h post-injury, once/day for 3 days). The results of the in vivo demonstrated that HPLs has good biosafety and can pass through the blood-brain barrier. Not only that compared with HET0016, HPLs better-inhibited inflammation and improved neuronal degeneration, which further led to lesion volume reduction, upgraded behavioral task performance, and ameliorated the degree of TBI impairment. Our results demonstrated HPLs could be a new strategy for juvenile TBI therapy. [ABSTRACT FROM AUTHOR]

We explored the shared pathophysiological mechanisms between COVID‐19 and non‐alcoholic fatty liver disease (NAFLD) by integrating multi‐omics data. We studied common genetic risk factors and underlying biological processes using functional enrichment analysis. To understand the sex‐specific pathways involved in the clinical course of SARS‐CoV‐2 infection, we processed sex‐stratified data from COVID‐19 genome‐wide association datasets. We further explored the transcriptional signature of the liver cells in healthy and COVID‐19 tissue specimens. We also integrated genetic and metabolomic information. We found that COVID‐19 and NAFLD share biological disease mechanisms, including pathways that regulate the inflammatory and lipopolysaccharide response. Single‐cell transcriptomics revealed enrichment of complement‐related pathways in Kupffer cells, syndecan‐mediated signalling in plasma cells, and epithelial‐to‐mesenchymal transition in hepatic stellate cells. The strategy of pathway‐level analysis of genomic and metabolomic data uncovered l‐lactic acid, Krebs cycle intermediate compounds, arachidonic acid and cortisol among the most prominent shared metabolites. [ABSTRACT FROM AUTHOR]

Introduction: The role of eicosanoids, metabolites of arachidonic acid with cardio-renal activity, remains unclear in human heart failure (HF). Methods: We enrolled 50 patients with HF to measure plasma 14,15-EET and 14,15-DHET levels using commercial ELISA kits and compared them with 25 age- and sex-matched controls. Results: Both of the measured eicosanoids were significantly higher in the HF group: 14,15-EET (91.3 ±25.7 ng/ml vs. 64.95 ±35.4 ng/ml) and 14,15-DHET (10.58 ±2.06 ng/ml vs. 9.07 ±1.60 ng/ml), p for both < 0.001. Conclusions: We found that peripheral plasma eicosanoid (14,15-EET, 14,15-DHET) levels are raised in patients with HF compared to age- and sex-matched controls. [ABSTRACT FROM AUTHOR]

The incidence of heart failure (HF) is generally preceded by cardiac hypertrophy (CH), which is the enlargement of cardiac myocytes in response to stress. During CH, the metabolism of arachidonic acid (AA), which is present in the cell membrane phospholipids, is modulated. Metabolism of AA gives rise to hydroxyeicosatetraenoic acids (HETEs) and epoxyeicosatrienoic acids (EETs) via cytochrome P450 (CYP) ω-hydroxylases and CYP epoxygenases, respectively. A plethora of studies demonstrated the involvement of CYP-mediated AA metabolites in the pathogenesis of CH. Also, inflammation is known to be a characteristic hallmark of CH. In this review, our aim is to highlight the impact of inflammation on CYP-derived AA metabolites and CH. Inflammation is shown to modulate the expression of various CYP ω-hydroxylases and CYP epoxygenases and their respective metabolites in the heart. In general, HETEs such as 20-HETE and mid-chain HETEs are pro-inflammatory, while EETs are characterized by their anti-inflammatory and cardioprotective properties. Several mechanisms are implicated in inflammation-induced CH, including the modulation of NF-κB and MAPK. This review demonstrated the inflammatory modulation of cardiac CYPs and their metabolites in the context of CH and the anti-inflammatory strategies that can be employed in the treatment of CH and HF. [ABSTRACT FROM AUTHOR]

Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites with biological effects, including antiapoptotic, anti-inflammatory, and antifibrotic functions. Soluble epoxide hydrolase (sEH)-mediated hydrolysis of EETs to dihydroxyeicosatrienoic acids (DHETs) attenuates these effects. Recent studies have demonstrated that inhibition of sEH prevents renal tubulointerstitial fibrosis and inflammation in the chronic kidney disease model. Given the pathophysiological role of the EET pathway in chronic kidney disease, we investigated if administration of EET regioisomers and/or sEH inhibition will promote antifibrotic and renoprotective effects in renal fi- brosis following unilateral ureteral obstruction (UUO). EETs administration abolished tubulointerstitial fibrogenesis, as demonstrated by reduced fibroblast activation and collagen deposition after UUO. The inflammatory response was prevented as demonstrated by decreased neutrophil and macrophage infiltration and expression of cytokines in EET-administered UUO kidneys. EET administration and/or sEH inhibition significantly reduced M1 macrophage markers, whereas M2 macrophage markers were highly upregulated. Furthermore, UUO-induced oxidative stress, tubular injury, and apoptosis were all downregulated following EET administration. Combined EET administration and sEH inhibition, however, had no additive effect in attenuating inflammation and renal interstitial fibrogenesis after UUO. Taken together, our findings provide a mechanistic understanding of how EETs prevent kidney fibrogenesis during obstructive nephropathy and suggest EET treatment as a potential therapeutic strategy to treat fibrotic diseases. [ABSTRACT FROM AUTHOR]

Introduction: The disruption of podocyte structure and function are the main pathological mechanism of membranous nephropathy (MN). Phospholipases A2, Group XII B (PLA2G12B) was reported involved in the regulation of MN by interfering with arachidonic acid (AA) metabolism, but there is a lack of sufficient evidence. In this study, we investigated the role and molecular mechanism of PLA2G12B in MN. Methods: C57BL/6 mice were used to establish MN model to extract primary podocytes, then divided into control, model, si-phospholipases A2 receptor (PLA2R), PLA2G12B, PLA2G12B + si-PLA2R, PLA2G12B + nuclear factor kappa-B (NF-κB) inhibitor, PLA2G12B + NF-κB inhibitor + si-PLA2R groups. Hematoxylin-eosin staining and immunofluorescence were used to detect kidney histological arrangement, serum levels of cholesterol-related indices, and AA. Genes and proteins associated with metabolism and inflammatory factors were detected by quantitative real-time PCR and Western blot. Results: The results revealed that AA metabolites were activated in the MN model mice, and the expression of PLA2G12B and NF-κB pathway levels were elevated. Besides, cellular experiments demonstrated that prostaglandin I2 (PGI2), thromboxane A2 (TXA2), leukotriene B4 (LTB4), and NF-κB pathway were significantly increased in the PLA2G12B group. Also, PLA2G12B promotes apoptosis and suppresses cell activity in podocytes, and these effects could be antagonized by NF-κB inhibitors. Furthermore, with the inference of si-PLA2R, the NF-κB inhibitors' effects were reversed. Conclusion: Promotional effects of PLA2G12B in primary MN are associated with the regulation of AA metabolism and NF-κB pathway. [ABSTRACT FROM AUTHOR]

The renal response to acid-base disturbances involves phenotypic and remodeling changes in the collecting duct. This study examines whether the proximal tubule controls these responses. We examined mice with genetic deletion of proteins present only in the proximal tubule, either the A variant or both A and B variants of isoform 1 of the electrogenic Naþ-bicarbonate cotransporter (NBCe1). Both knockout (KO) mice have spontaneous metabolic acidosis. We then determined the collecting duct phenotypic responses to this acidosis and the remodeling responses to exogenous acid loading. Despite the spontaneous acidosis in NBCe1-A KO mice, type A intercalated cells in the inner stripe of the outer medullary collecting duct (OMCDis) exhibited decreased height and reduced expression of Hþ-ATPase, anion exchanger 1, Rhesus B glycoprotein, and Rhesus C glycoprotein. Combined kidney-specific NBCe1-A/B deletion induced similar changes. Ultrastructural imaging showed decreased apical plasma membrane and increased vesicular Hþ-ATPase in OMCDis type A intercalated cell in NBCe1-A KO mice. Next, we examined the collecting duct remodeling response to acidosis. In wild-type mice, acid loading increased the proportion of type A intercalated cells in the connecting tubule (CNT) and OMCDis, and it decreased the proportion of non-A, non-B intercalated cells in the connecting tubule, and type B intercalated cells in the cortical collecting duct (CCD). These changes were absent in NBCe1-A KO mice. We conclude that the collecting duct phenotypic and remodeling responses depend on proximal tubule-dependent signaling mechanisms blocked by constitutive deletion of proximal tubule NBCe1 proteins. [ABSTRACT FROM AUTHOR]