ABSTRACT
Cyclooxygenase and lipoxygenase derived lipid metabolites of polyunsaturated fatty acids (PUFAs), as well as their role in the inflammation, have been studied quite thoroughly. However, cytochrome P450 derived lipid mediators, as well as their participation in the regulation of the inflammation, need deeper understanding. In recent years, it has become known that PUFAs are oxidized by cytochrome P450 epoxygenases to epoxy fatty acids, which act as the extremely powerful lipid mediators involved in resolving inflammation. Recent studies have shown that the anti-inflammatory mechanisms of ω-3 PUFAs are also mediated by their conversion to the endocannabinoid epoxides. Thus, it is clear that a number of therapeutically relevant functions of PUFAs are due to their conversion to PUFA epoxides. However, with the participation of cytochrome P450 epoxygenases, not only PUFA epoxides, but also other metabolites are formed. They are further are converted by epoxide hydrolases into pro-inflammatory dihydroxy fatty acids and anti-inflammatory dihydroxyeicosatrienoic acids. The study of the role of PUFA epoxides in the regulation of the inflammation and pharmacological modeling of the activity of epoxide hydrolases are the promising strategies for the treatment of the inflammatory diseases. This review systematizes the current literature data of the fatty acid epoxides, in particular, the endocannabinoid epoxides. Their role in the regulation of inflammation is discussed.
Subject(s)
Epoxy Compounds , Fatty Acids, Omega-3 , Anti-Inflammatory Agents , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Endocannabinoids/metabolism , Epoxide Hydrolases/metabolism , Epoxy Compounds/metabolism , Epoxy Compounds/pharmacology , Fatty Acids , Fatty Acids, Omega-3/metabolism , Fatty Acids, Omega-3/pharmacology , Humans , Inflammation/drug therapyABSTRACT
OBJECTIVES: Khat, a natural amphetamine-like psychostimulant plant, are widely consumed globally. Concurrent intake of khat and xenobiotics may lead to herb-drug interactions and adverse drug reactions (ADRs). This study is a continuation of our previous study, targeted to evaluate the in vitro inhibitory effects of khat ethanol extract (KEE) on human cytochrome (CYP) 1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5, major human drug metabolizing enzymes. METHODS: In vitro fluorescence enzyme assays were employed to assess CYPs inhibition with the presence and absence of various KEE concentrations. RESULTS: KEE reversibly inhibited CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 but not CYP1A2 with IC50 values of 25.5, 99, 4.5, 21, 27, 17, and 10 µg/mL respectively. No irreversible inhibition of KEE on all the eight CYPs were identified. The Ki values of CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2 and CYP3A5 were 20.9, 85, 4.8, 18.3, 59.3, 3, and 21.7 µg/mL, respectively. KEE inhibited CYP2B6 via competitive or mixed inhibition; CYP2E1 via un-competitive or mixed inhibition; while CYP2A6, CYP2C8, CYP2C19, CYP2J2 and CYP3A5 via non-competitive or mixed inhibition. CONCLUSIONS: Caution should be taken by khat users who are on medications metabolized by CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, and CYP3A5.
Subject(s)
Catha , Cytochrome P-450 CYP2E1 , Catha/metabolism , Cytochrome P-450 CYP2A6/metabolism , Cytochrome P-450 CYP2B6/metabolism , Cytochrome P-450 CYP2C19/genetics , Cytochrome P-450 CYP2C19/metabolism , Cytochrome P-450 CYP2C8/metabolism , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 CYP2E1/pharmacology , Cytochrome P-450 CYP2J2 , Cytochrome P-450 CYP3A/metabolism , Cytochrome P-450 Enzyme System/metabolism , Ethanol/metabolism , Ethanol/pharmacology , Humans , Microsomes, Liver , Plant Extracts/pharmacologyABSTRACT
Cytochrome P450 2J2 (CYP2J2) enzyme attracts more attention because it not only metabolizes clinical drugs but also mediates the biotransformation of important endogenous substances and the regulation of physiologic function. Although CYP2J2 is very important, few animal models are available to study its function in vivo In particular, a CYP2J gene knockout (KO) rat model for drug metabolism and pharmacokinetics is not available. In this report, the CRISPR/Cas9 technology was used to delete rat CYP2J3/10, the orthologous genes of CYP2J2 in humans. The CYP2J3/10 KO rats were viable and fertile and showed no off-target effect. Compared with wild-type (WT) rats, the mRNA and protein expression of CYP2J3/10 in liver, small intestine, and heart of KO rats were completely absent. At the same time, CYP2J4 mRNA expression and protein expression were significantly decreased in these tissues. Further in vitro and in vivo metabolic studies of astemizole, a typical substrate of CYP2J, indicated that CYP2J was functionally inactive in KO rats. The heart function indexes of WT and KO rats were also measured and compared. The myocardial enzymes, including creatine kinase-muscle brain type (CK-MB), creatine kinase (CK), and CK-MB/CK ratio, of KO rats increased by nearly 140%, 80%, and 60%, respectively. In conclusion, this study successfully developed a new CYP2J3/10 KO rat model, which is a useful tool to study the function of CYP2J in drug metabolism and cardiovascular disease. SIGNIFICANCE STATEMENT: Human CYP2J2 is involved not only in clinical drug metabolism but also in the biotransformation of important endogenous substances. Therefore, it is very important to construct new animal models to study its function in vivo. This study successfully developed a new CYP2J knockout rat model by using CRISPR/Cas9 technology. This rat model provides a useful tool to study the role of CYP2J in drug metabolism and diseases.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Animals , Astemizole/pharmacokinetics , Biotransformation , CRISPR-Cas Systems/genetics , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/genetics , Drug Evaluation, Preclinical/methods , Feasibility Studies , Female , Gene Knockdown Techniques , Male , Models, Animal , Rats , Rats, TransgenicABSTRACT
AIMS: Vitamin D and its receptor, vitamin D receptor (VDR), have renoprotection effect against diabetic nephropathy (DN). But the exact mechanism has not been fully elucidated. Epoxyeicosatrienoic acids (EETs) are cytochrome P450 (CYP) epoxygenase-derived metabolites of arachidonic acid, protecting against diabetes and DN. Herein, we hypothesized that activation of VDR attenuated high glucose-induced cellular injury in renal tubular epithelial cells partially through up-regulating CYP2J5 expression. MAIN METHODS: Streptozotocin (STZ) was injected to induce diabetic in wild type and Vdr-/- mice. The effects of VDR knockout and an activator of VDR, paricalcitol, on the renal injury were detected. In vitro, a murine kidney proximal tubule epithelial cell line BU.MPT induced by high glucose were treated with or without paricalcitol (30â¯mM) for 12â¯h or 24â¯h. KEY FINDINGS: The expression of CYP2J5 was significantly decreased both in wild type and Vdr-/- diabetic mice induced by STZ. The STZ-induced kidney architecture damage and apoptosis rate in Vdr-/- mice were more severe. In vitro, high glucose treatment strongly reduced the CYP2J5 expression and the synthesis of 14,15-EET in BU.MPT cells. Supplement of 14,15-EET significantly reduced the lactate dehydrogenase (LDH) release induced by high glucose in BU.MPT cells. Furthermore, treatment with paricalcitol attenuated cellular injury and restored the expression of CYP2J5 reduced by high glucose in BU.MPT cells. SIGNIFICANCE: We conclude that activation of VDR attenuates high glucose-induced cellular injury partially dependent on CYP2J5 in murine renal tubule epithelial cells and paricalcitol may represent a potential therapy for DN.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Diabetes Mellitus, Experimental/complications , Diabetic Nephropathies/drug therapy , Diabetic Nephropathies/etiology , Ergocalciferols/pharmacology , Kidney Tubules, Proximal/drug effects , Receptors, Calcitriol/agonists , Animals , Cell Line , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Diabetes Mellitus, Experimental/drug therapy , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/genetics , Diabetic Nephropathies/pathology , Ergocalciferols/therapeutic use , Gene Deletion , Gene Expression Regulation/drug effects , Glucose/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Male , Mice , Mice, Knockout , Receptors, Calcitriol/genetics , Receptors, Calcitriol/metabolismABSTRACT
Therapeutics for arachidonic acid pathways began with the development of non-steroidal anti-inflammatory drugs that inhibit cyclooxygenase (COX). The enzymatic pathways and arachidonic acid metabolites and respective receptors have been successfully targeted and therapeutics developed for pain, inflammation, pulmonary and cardiovascular diseases. These drugs target the COX and lipoxygenase pathways but not the third branch for arachidonic acid metabolism, the cytochrome P450 (CYP) pathway. Small molecule compounds targeting enzymes and CYP epoxy-fatty acid metabolites have evolved rapidly over the last two decades. These therapeutics have primarily focused on inhibiting soluble epoxide hydrolase (sEH) or agonist mimetics for epoxyeicosatrienoic acids (EET). Based on preclinical animal model studies and human studies, major therapeutic indications for these sEH inhibitors and EET mimics/analogs are renal and cardiovascular diseases. Novel small molecules that inhibit sEH have advanced to human clinical trials and demonstrate promise for cardiovascular diseases. Challenges remain for sEH inhibitor and EET analog drug development; however, there is a high likelihood that a drug that acts on this third branch of arachidonic acid metabolism will be utilized to treat a cardiovascular or kidney disease in the next decade.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Arachidonic Acids/metabolism , Cardiovascular Diseases/drug therapy , Cytochrome P-450 Enzyme Inhibitors/therapeutic use , Cytochrome P-450 Enzyme System/metabolism , Drug Discovery/trends , Kidney Diseases/drug therapy , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Cardiovascular Diseases/enzymology , Clinical Trials as Topic , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors/chemistry , Drug Evaluation, Preclinical , Humans , Kidney Diseases/enzymologyABSTRACT
The use of Chinese herbal medicines and natural products has become increasingly popular in both China and Western societies as an alternative medicine for the treatment of diseases or as a health supplement. Danshen, the dried root of Salvia miltiorrhiza (Fam.Labiatae), which is rich in phenolic acids and tanshinones, is a widely used herbal medicine for the treatment of cardio-cerebrovascular diseases. The goal of this study was to examine the inhibitory effects of fifteen components derived from Danshen on CYP2C8 and CYP2J2, which are expressed both in human liver and cardiovascular systems. Recombinant CYP2C8 and CYP2J2 were used, and the mechanism, kinetics, and type of inhibition were determined. Taxol 6-hydroxylation and astemizole O-desmethyastemizole were determined as probe activities for CYP2C8 and CYP2J2, respectively. Metabolites formations were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that salvianolic acid A was a competitive inhibitor of CYP2C8 (Kiâ¯=â¯2.5⯵M) and mixed-type inhibitor of CYP2J2 (Kiâ¯=â¯7.44⯵M). Salvianolic acid C had moderate noncompetitive and mixed-type inhibitions on CYP2C8 (Kiâ¯=â¯4.82⯵M) and CYP2J2 (Kiâ¯=â¯5.75⯵M), respectively. Tanshinone IIA was a moderate competitive inhibitor of CYP2C8 (Kiâ¯=â¯1.18⯵M). Dihydrotanshinone I had moderate noncompetitive inhibition on CYP2J2 (Kiâ¯=â¯6.59⯵M), but mechanism-based inhibition on CYP2C8 (KIâ¯=â¯0.43⯵M, kinactâ¯=â¯0.097 min-1). Tanshinone I was a moderate competitive inhibitor of CYP2C8 (Kiâ¯=â¯4.20⯵M). These findings suggested that Danshen preparations appear not likely to pose a significant risk of drug interactions mediated by CYP2C8 after oral administration; but their inhibitory effects on intestinal CYP2J2 mediated drug metabolism should not be neglected when they are given orally in combination with other drugs. Additionally, this study provided novel insights into the underling pharmacological mechanisms of Danshen components from the perspective of CYP2C8 and CYP2J2 inhibition.
Subject(s)
Cytochrome P-450 CYP2C8/metabolism , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Drugs, Chinese Herbal/pharmacology , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors/chemistry , Drugs, Chinese Herbal/chemistry , Humans , Inhibitory Concentration 50 , Kinetics , Recombinant Proteins/metabolism , Salvia miltiorrhiza , Taxoids/metabolism , Time FactorsABSTRACT
BACKGROUND: Broussonetia papyrifera (L.) Ventenat, a traditional medicinal herb, has been applied as a folk medicine to treat various diseases. Broussochalcone A (BCA), a chalcone compound isolated from the cortex of Broussonetia papyrifera (L.) Ventenat, exhibits several biological activities including potent anti-oxidant, antiplatelet, and cytotoxic effects. PURPOSE: The purpose of this study is to elucidate the inhibitory effect of BCA against CYP2J2 enzyme which is predominantly expressed in human tumor tissues and carcinoma cell lines. STUDY DESIGN: The inhibitory effect of BCA on the activities of CYP2J2-mediated metabolism were investigated using human liver microsomes (HLMs), and its anti-cancer effect against human hepatoma HepG2 cells was also evaluated. METHODS: Two representative CYP2J2-specific probe substrates, astemizole and ebastine, were incubated in HLMs with BCA. After incubation, the samples were analyzed using liquid chromatography-tandem mass spectrometry. To investigate the binding model between BCA and CYP2J2, we carried out structure-based docking simulations by using software and scripts written in-house. RESULTS: BCA inhibited CYP2J2-mediated astemizole O-demethylation and ebastine hydroxylase activities in a concentration dependent manner with Ki values of 2.3 and 3.7 µM, respectively. It also showed cytotoxic effects against human hepatoma HepG2 cells in a dose-dependent manner with activation of apoptosis related proteins. CONCLUSION: Overall, this was the first report of the inhibitory effects of BCA on CYP2J2 in HLMs. The present data suggest that BCA is a potential candidate for further evaluation for its CYP2J2 targeting anti-cancer activities.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Chalcones/pharmacology , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Forkhead Box Protein O3/metabolism , Resorcinols/pharmacology , Antineoplastic Agents, Phytogenic/administration & dosage , Astemizole/metabolism , Butyrophenones/metabolism , Cell Proliferation/drug effects , Chalcones/administration & dosage , Chalcones/chemistry , Chromatography, Liquid , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors/administration & dosage , Cytochrome P-450 Enzyme System/chemistry , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Hep G2 Cells , Humans , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Docking Simulation , Piperidines/metabolism , Resorcinols/administration & dosage , Resorcinols/chemistry , Tandem Mass SpectrometryABSTRACT
This study aimed to investigate the effect and mechanism of ophiopogonin D (OP-D) on Ang â ¡-induced HUVECs apoptosis, in order to provide a reliable basis for the safety and efficacy of traditional Chinese medicines. The effect of Ang â ¡ on survival and total proteins content of HUVECs were measured by MTT and Western blotting. The effect of OP-D on Ang â ¡-induced lactate dehydrogenase (LDH) release rate in HUVECs was measured by enzyme standard instrument. The effects of OP-D and 11,12-EET on phosphorylation of JNK/c-Jun induced by Ang â ¡ were measured by Western blot and RT-PCR with the help of JNK specific inhibitor SP600125 and CYP450 isozymes selective inhibitor 6-(2-propargyloxyphenyl) hexanoic acid (PPOH). The cell apoptosis was assayed by flow cytometry. According to the results, different doses of Ang â ¡ had no significant effect on cell survival; treatment with Ang â ¡ at 1×10â»6 mol·L⻹ could increase the release of LDH (P<0.001), improve the JNK and c-Jun phosphorylation levels(P<0.01, P<0.001), increase the expression of caspase-3(P<0.01), and promote the apoptosis of HUVECs(P<0.001). The phosphorylation of JNK and c-Jun could be inhibited by the pre-treatment with SP600125, 11,12-EET and OP-D. Pre-treatment with OP-D could significantly reduce the release of LDH induced by Ang â ¡ stimulation, decrease the expression of caspase-3, and diminish the apoptosis of cells. The protective effect of OP-D was suppressed, when being pretreated with PPOH. The experimental results showed that the apoptosis of HUVECs induced by Ang â ¡ may be associated with JNK/c-Jun signaling pathway. OP-D-mediated CYP2J2 expression increased 11,12-EET levels, and could remarkably resist Ang â ¡-induced injury and apoptosis of cells, which is associated with the maintenance of endothelium homeostasis.
Subject(s)
Apoptosis , Arachidonic Acids/metabolism , Cytochrome P-450 Enzyme System/metabolism , Human Umbilical Vein Endothelial Cells/drug effects , Saponins/pharmacology , Spirostans/pharmacology , Angiotensin II , Cells, Cultured , Cytochrome P-450 CYP2J2 , Human Umbilical Vein Endothelial Cells/cytology , Humans , Phosphorylation , Signal TransductionABSTRACT
BACKGROUND: Cytochrome P450 2J2 (CYP2J2) is not only highly expressed in many kinds of human tumors, but also promotes tumor cell growth via regulating the metabolism of arachidonic acids. CYP2J2 inhibitors can significantly reduce proliferation, migration and promote apoptosis of tumor cells by inhibiting epoxyeicosatrienoic acids (EETs) biosynthesis. Therefore screening CYP2J2 inhibitors is a significant way for the development of anti-cancer drug. PURPOSE: The aim of this study was to identify a new CYP2J2 inhibitor from fifty natural compounds obtained from plants. STUDY DESIGN: CYP2J2 inhibitor was screened from a natural compounds library and further the inhibitory manner and mechanism were evaluated. Its cytotoxicity against HepG2 and SMMC-7721 cell lines was also estimated. METHODS: The inhibitory effect was evaluated in rat liver microsomes (RLMs), human liver microsomes (HLMs) and recombinant CYP2J2 (rCYP2J2), using astemizole as a probe substrate and inhibitory mechanism was illustrated through molecular docking. The cytotoxicity was detected using SRB. RESULTS: In all candidates, plumbagin showed the strongest inhibitory effect on the CYP2J2-mediated astemizole O-demethylation activity. Further study revealed that plumbagin potently inhibited CYP2J2 activity with IC50 value at 3.82⯵M, 3.37⯵M and 1.17⯵M in RLMs, HLMs and rCYP2J2, respectively. Enzyme kinetic studies showed that plumbagin was a mixed-type inhibitor of CYP2J2 in HLMs and rCYP2J2 with Ki value of 1.88⯵M and 0.92⯵M, respectively. Docking data presented that plumbagin interacted with CYP2J2 mainly through GLU 222 and ALA 223. Moreover, plumbagin showed strongly cytotoxic effects on hepatoma cell lines, such as HepG2 and SMMC-7721, with lower toxicity on rat primary hepatocytes. Plumbagin had no effect on the protein expression of CYP2J2 in HepG2 and SMMC-7721, while down-regulated the mRNA level of anti-apoptosis protein Bcl-2. CONCLUSION: This study found out a new CYP2J2 inhibitor plumbagin from fifty natural compounds. Plumbagin presented a potential of anti-cancer pharmacological activity.
Subject(s)
Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/chemistry , Cytochrome P-450 Enzyme System/metabolism , Naphthoquinones/pharmacology , Animals , Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Carcinoma, Hepatocellular/drug therapy , Cell Proliferation/drug effects , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme Inhibitors/chemistry , Drug Evaluation, Preclinical/methods , Hepatocytes/drug effects , Humans , Kinetics , Liver Neoplasms/drug therapy , Male , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Molecular Docking Simulation , Naphthoquinones/chemistry , Rats, Sprague-DawleyABSTRACT
Numerous benefits have been attributed to dietary long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs), including protection against cardiac arrhythmia, triglyceride-lowering, amelioration of inflammatory, and neurodegenerative disorders. This review covers recent findings indicating that a variety of these beneficial effects are mediated by "omega-3 epoxyeicosanoids", a class of novel n-3 LC-PUFA-derived lipid mediators, which are generated via the cytochrome P450 (CYP) epoxygenase pathway. CYP enzymes, previously identified as arachidonic acid (20:4n-6; AA) epoxygenases, accept eicosapentaenoic acid (20:5n-3; EPA) and docosahexaenoic acid (22:6n-3; DHA), the major fish oil n-3 LC-PUFAs, as efficient alternative substrates. In humans and rodents, dietary EPA/DHA supplementation causes a profound shift of the endogenous CYP-eicosanoid profile from AA- to EPA- and DHA-derived metabolites, increasing, in particular, the plasma and tissue levels of 17,18-epoxyeicosatetraenoic acid (17,18-EEQ) and 19,20-epoxydocosapentaenoic acid (19,20-EDP). Based on preclinical studies, these omega-3 epoxyeicosanoids display cardioprotective, vasodilatory, anti-inflammatory, and anti-allergic properties that contribute to the beneficial effects of n-3 LC-PUFAs in diverse disease conditions ranging from cardiac disease, bronchial disorders, and intraocular neovascularization, to allergic intestinal inflammation and inflammatory pain. Increasing evidence also suggests that background nutrition as well as genetic and disease state-related factors could limit the response to EPA/DHA-supplementation by reducing the formation and/or enhancing the degradation of omega-3 epoxyeicosanoids. Recently, metabolically robust synthetic analogs mimicking the biological activities of 17,18-EEQ have been developed. These drug candidates may overcome limitations of dietary EPA/DHA supplementation and provide novel options for the treatment of cardiovascular and inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents/therapeutic use , Cardiovascular Diseases/drug therapy , Eicosanoids/therapeutic use , Fatty Acids, Omega-3/therapeutic use , Animals , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Humans , Inflammation/drug therapyABSTRACT
Clinical studies suggest that diets rich in ω-3 polyunsaturated fatty acids (PUFAs) provide beneficial anti-inflammatory effects, in part through their conversion to bioactive metabolites. Here we report on the endogenous production of a previously unknown class of ω-3 PUFA-derived lipid metabolites that originate from the crosstalk between endocannabinoid and cytochrome P450 (CYP) epoxygenase metabolic pathways. The ω-3 endocannabinoid epoxides are derived from docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) to form epoxyeicosatetraenoic acid-ethanolamide (EEQ-EA) and epoxydocosapentaenoic acid-ethanolamide (EDP-EA), respectively. Both EEQ-EAs and EDP-EAs are endogenously present in rat brain and peripheral organs as determined via targeted lipidomics methods. These metabolites were directly produced by direct epoxygenation of the ω-3 endocannabinoids, docosahexanoyl ethanolamide (DHEA) and eicosapentaenoyl ethanolamide (EPEA) by activated BV-2 microglial cells, and by human CYP2J2. Neuroinflammation studies revealed that the terminal epoxides 17,18-EEQ-EA and 19,20-EDP-EA dose-dependently abated proinflammatory IL-6 cytokines while increasing anti-inflammatory IL-10 cytokines, in part through cannabinoid receptor-2 activation. Furthermore the ω-3 endocannabinoid epoxides 17,18-EEQ-EA and 19,20-EDP-EA exerted antiangiogenic effects in human microvascular endothelial cells (HMVEC) and vasodilatory actions on bovine coronary arteries and reciprocally regulated platelet aggregation in washed human platelets. Taken together, the ω-3 endocannabinoid epoxides' physiological effects are mediated through both endocannabinoid and epoxyeicosanoid signaling pathways. In summary, the ω-3 endocannabinoid epoxides are found at concentrations comparable to those of other endocannabinoids and are expected to play critical roles during inflammation in vivo; thus their identification may aid in the development of therapeutics for neuroinflammatory and cerebrovascular diseases.
Subject(s)
Anti-Inflammatory Agents/blood , Endocannabinoids/metabolism , Epoxy Compounds/blood , Ethanolamines/blood , Fatty Acids, Omega-3/metabolism , Amidohydrolases/metabolism , Animals , Brain/metabolism , Cattle , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Drug Evaluation, Preclinical , Epoxide Hydrolases/metabolism , Epoxy Compounds/pharmacology , Epoxy Compounds/therapeutic use , Ethanolamines/pharmacology , Ethanolamines/therapeutic use , Humans , Lipid Metabolism , Mice , Microglia/metabolism , Neovascularization, Pathologic/prevention & control , Platelet Aggregation/drug effects , Rats , Vasodilation/drug effectsABSTRACT
Reactive oxygen species (ROS) like hydrogen peroxide (H2O2) are involved in the in endothelium-derived hyperpolarization (EDH)-type relaxant responses of coronary and mesenteric arterioles. The role of ROS in kidney vascular function has mainly been investigated in the context of harmful ROS generation associated to kidney disease. The present study was sought to investigate whether H2O2 is involved in the endothelium-dependent relaxations of intrarenal arteries as well the possible endothelial sources of ROS generation involved in these responses. Under conditions of cyclooxygenase (COX) and nitric oxide (NO) synthase inhibition, acetylcholine (ACh) induced relaxations and stimulated H2O2 release that were reduced by catalase and by the glutathione peroxidase (GPx) mimetic ebselen in rat renal interlobar arteries, suggesting the involvement of H2O2 in the endothelium-dependent responses. ACh relaxations were also blunted by the CYP2C inhibitor sulfaphenazole and by the NADPH oxidase inhibitor apocynin. Acetylcholine stimulated both superoxide (O2â¢-) and H2O2 production that were reduced by sulfaphenazole and apocynin. Expression of the antioxidant enzyme CuZnSOD and of the H2O2 reducing enzymes catalase and GPx-1 was found in both intrarenal arteries and renal cortex. On the other hand, exogenous H2O2 relaxed renal arteries by decreasing vascular smooth muscle (VSM) intracellular calcium concentration [Ca2+]i and markedly enhanced endothelial KCa currents in freshly isolated renal endothelial cells. CYP2C11 and CYP2C23 epoxygenases were highly expressed in interlobar renal arteries and renal cortex, respectively, and were co-localized with eNOS in renal endothelial cells. These results demonstrate that H2O2 is involved in the EDH-type relaxant responses of renal arteries and that CYP 2C epoxygenases are physiologically relevant endothelial sources of vasodilator H2O2 in the kidney.
Subject(s)
Aryl Hydrocarbon Hydroxylases/metabolism , Cytochrome P-450 Enzyme System/metabolism , Cytochrome P450 Family 2/metabolism , Hydrogen Peroxide/metabolism , Kidney/metabolism , Muscle, Smooth, Vascular/metabolism , Steroid 16-alpha-Hydroxylase/metabolism , Acetophenones/administration & dosage , Acetylcholine/metabolism , Animals , Arteries/drug effects , Arteries/metabolism , Biological Factors/metabolism , Calcium/metabolism , Cytochrome P-450 CYP2J2 , Endothelium/drug effects , Endothelium/metabolism , Humans , Kidney/pathology , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/pathology , NADPH Oxidases/metabolism , Nitric Oxide Synthase/genetics , Nitric Oxide Synthase Type III , Prostaglandin-Endoperoxide Synthases/genetics , Prostaglandin-Endoperoxide Synthases/metabolism , Rats , Reactive Oxygen Species/metabolism , Relaxation , Sulfaphenazole/administration & dosage , Superoxides/metabolismABSTRACT
Members of the cytochrome P450 CYP2J subfamily are expressed in multiple tissues in mice and humans. These enzymes are active in the metabolism of fatty acids to generate bioactive compounds. Herein we report new methods and results for quantitative polymerase chain reaction (qPCR) analysis for the seven genes (Cyp2j5, Cyp2j6, Cyp2j8, Cyp2j9, Cyp2j11, Cyp2j12, and Cyp2j13) of the mouse Cyp2j subfamily. SYBR Green primer sets were developed and compared with commercially available TaqMan primer/probe assays for specificity toward mouse Cyp2j cDNA, and analysis of tissue distribution and regulation of Cyp2j genes. Each TaqMan primer/probe set and SYBR Green primer set were shown to be specific for their intended mouse Cyp2j cDNA. Tissue distribution of the mouse Cyp2j isoforms confirmed similar patterns of expression between the two qPCR methods. Cyp2j5 and Cyp2j13 were highly expressed in male kidneys, and Cyp2j11 was highly expressed in both male and female kidneys. Cyp2j6 was expressed in multiple tissues, with the highest expression in the small intestine and duodenum. Cyp2j8 was detected in various tissues, with highest expression found in the skin. Cyp2j9 was highly expressed in the brain, liver, and lung. Cyp2j12 was predominately expressed in the brain. We also determined the Cyp2j isoform expression in Cyp2j5 knockout mice to determine whether there was compensatory regulation of other Cyp2j isoforms, and we assessed Cyp2j isoform regulation during various inflammatory models, including influenza A, bacterial lipopolysaccharide, house dust mite allergen, and corn pollen. Both qPCR methods detected similar suppression of Cyp2j6 and Cyp2j9 during inflammation in the lung.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Animals , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/biosynthesis , DNA Primers , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Female , Gene Expression Regulation, Enzymologic/genetics , Hypersensitivity/enzymology , Hypersensitivity/genetics , Kidney/enzymology , Lung/enzymology , Male , Mice , Mice, Inbred C57BL , Orthomyxoviridae Infections/enzymology , Pollen/immunology , Polymerase Chain Reaction , Tissue Distribution , Zea mays/immunologyABSTRACT
We have previously demonstrated that a stable synthetic analog of 20-hydroxyeicosatetraenoic acid (20-HETE), N-[20-hydroxyeicosa-5(Z),14(Z)-dienoyl]glycine (5,14-HEDGE), prevents vascular hyporeactivity, hypotension, tachycardia, and inflammation in rats treated with lipopolysaccharide (LPS) and mortality in endotoxemic mice. These changes were attributed to decreased production of inducible nitric oxide (NO) synthase (iNOS)-derived NO, cyclooxygenase (COX)-2-derived vasodilator prostanoids, and proinflammatory mediators associated with increased cyctochrome P450 (CYP) 4A1-derived 20-HETE and CYP2C23-dependent antiinflammatory mediator formation. The aim of this study was to determine whether decreased expression and activity of iNOS, soluble guanylyl cyclase (sGC), protein kinase G (PKG), COX-2, gp91(phox) (NOX2; a superoxide generating NOX enzyme), and peroxynitrite production associated with increased expression of COX-1 and CYP4A1 and 20-HETE formation in renal and cardiovascular tissues of rats contributes to the effect of 5,14-HEDGE to prevent vasodilation, hypotension, tachycardia, and inflammation in response to systemic administration of LPS. Mean arterial pressure fell by 28mmHg and heart rate rose by 47beats/min in LPS (10mg/kg, i.p.)-treated rats. Administration of LPS also increased mRNA and protein expression of iNOS and COX-2 associated with a decrease in COX-1 and CYP4A1 mRNA and protein expression. Increased NOS activity, iNOS-heat shock protein 90 complex formation (an index for iNOS activity), protein expression of phosphorylated vasodilator stimulated phosphoprotein (an index for PKG activity), gp91(phox), p47(phox) (NOXO2; organizer subunit of gp91(phox)), and nitrotyrosine (an index for peroxynitrite production) as well as cGMP (an index for sGC activity), 6-keto-PGF1α (a stable metabolite PGI2) and PGE2 levels (indexes for COX activity), and nitrotyrosine levels by LPS were also associated with decreased CYP hydroxylase activity as measured by 20-HETE formation from arachidonic acid in renal microsomes of LPS-treated rats. These effects of LPS, except iNOS mRNA and COX-1 protein expression, were prevented by 5,14-HEDGE (30mg/kg, s.c.; 1h after LPS). A competitive antagonist of vasoconstrictor effects of 20-HETE, 20-hydroxyeicosa-6(Z),15(Z)-dienoic acid (30mg/kg, s.c.; 1h after LPS) reversed the effects of 5,14-HEDGE, except iNOS and COX-1 mRNA and protein expression as well as expression of CYP4A1 mRNA. These results suggest that increased CYP4A1 expression and 20-HETE formation associated with suppression of iNOS/sGC/PKG pathway, COX-2, and gp91(phox) participate in the protective effect of 5,14-HEDGE against vasodilation, hypotension, tachycardia, and inflammation in the rat model of septic shock.
Subject(s)
Lipopeptides/pharmacology , Nitric Oxide Synthase Type II/metabolism , Protective Agents/pharmacology , Shock, Septic/drug therapy , Shock, Septic/metabolism , Signal Transduction/drug effects , Animals , Cell Adhesion Molecules/metabolism , Cyclic GMP-Dependent Protein Kinases/metabolism , Cyclooxygenase 2/genetics , Cyclooxygenase 2/metabolism , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Disease Models, Animal , Guanylate Cyclase/metabolism , HSP90 Heat-Shock Proteins/metabolism , Hydroxyeicosatetraenoic Acids/pharmacology , Lipopolysaccharides/pharmacology , Male , Membrane Glycoproteins/metabolism , Microfilament Proteins/metabolism , NADPH Oxidase 2 , NADPH Oxidases/metabolism , Nitric Oxide Synthase Type II/genetics , Organ Specificity , Peroxynitrous Acid/metabolism , Phosphoproteins/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Random Allocation , Rats , Rats, Wistar , Receptors, Cytoplasmic and Nuclear/metabolism , Shock, Septic/enzymology , Shock, Septic/genetics , Soluble Guanylyl CyclaseABSTRACT
Fufang Xueshuantong Capsule (FXST) can reduce urinary albumin and whole blood viscosity in early diabetic nephropathy (DN) patients. This research aimed to investigate the effect of FXST on kidney function in DN rats and to identify the underlying molecular mechanisms. We performed Illumina RatRef-12 Expression BeadChip gene array analysis, and found that 3-month treatment with FXST significantly decreased 24-h urinary albumin, serum creatinine and blood urea nitrogen, and increased urinary creatinine in DN model rats. Kidney hypertrophy and glomerular mesangial matrix expansion were also ameliorated. Kidneys from the high-dose FXST group had 67 genes with significantly changed expression (34 increased, 33 decreased). DAVID analysis showed that the fold enrichment score of "collagen type 1" was the highest in all GO functional categories. DAVID function annotation cluster analysis indicated that the top annotation cluster included three GO function categories: "response to nutrient", "response to nutrient levels" and "response to extracellular stimulus". Based on KEGG pathway analysis, we found that the most two significant pathways were "metabolism of xenobiotics by cytochrome P450" and "drug metabolism". Real-time PCR showed that relative levels of Col1a1 (collagen type 1 alpha 1), Ctgf (connective tissue growth factor) and Tgfb1 (transforming growth factor beta 1) were significantly decreased in the FXST group, while Cyp2c23 (cytochrome P-450 family 2 subfamily C polypeptide 23) and Nphs1 (nephrin) were increased. The increased expressions of TGFß and collagen (type 1, α2) in the kidneys of DN rats were attenuated by FXST. Our data suggest that FXST can moderate kidney function in DN rats. The mechanism may involve the BMP2-TGFß-CTGF pathway, CYP2C23 and podocyte proteins.
Subject(s)
Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Drugs, Chinese Herbal/therapeutic use , Animals , Blood Glucose/drug effects , Body Weight/drug effects , Collagen Type I, alpha 1 Chain , Cytochrome P-450 CYP2J2 , Diabetes Mellitus, Experimental/blood , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/urine , Diabetic Nephropathies/blood , Diabetic Nephropathies/genetics , Diabetic Nephropathies/urine , Male , Rats , Rats, WistarABSTRACT
Fish oil omega-3 fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against arrhythmia and sudden cardiac death by largely unknown mechanisms. Recent in vitro and in vivo studies demonstrate that arachidonic acid (AA) metabolizing cytochrome P450-(CYP) enzymes accept EPA and DHA as efficient alternative substrates. Dietary EPA/DHA supplementation causes a profound shift of the cardiac CYP-eicosanoid profile from AA- to EPA- and DHA-derived epoxy- and hydroxy-metabolites. CYP2J2 and other CYP epoxygenases preferentially epoxidize the ω-3 double bond of EPA and DHA. The corresponding metabolites, 17,18-epoxy-EPA and 19,20-epoxy-DHA, dominate the CYP-eicosanoid profile of the rat heart after EPA/DHA supplementation. The (ω-3)-epoxyeicosanoids show highly potent antiarrhythmic properties in neonatal cardiomyocytes, suggesting that these metabolites may specifically contribute to the cardioprotective effects of omega-3 fatty acids. This hypothesis is discussed in the context of recent findings that revealed CYP-eicosanoid mediated mechanisms in cardiac ischemia-reperfusion injury and maladaptive cardiac hypertrophy.
Subject(s)
Arrhythmias, Cardiac/metabolism , Cardiomegaly/metabolism , Death, Sudden, Cardiac/prevention & control , Docosahexaenoic Acids/metabolism , Eicosapentaenoic Acid/metabolism , Fish Oils/metabolism , Myocardium/metabolism , Myocytes, Cardiac/enzymology , Animals , Animals, Newborn , Anti-Arrhythmia Agents/metabolism , Anti-Arrhythmia Agents/pharmacology , Arachidonic Acid/metabolism , Arrhythmias, Cardiac/physiopathology , Arrhythmias, Cardiac/prevention & control , Cardiomegaly/physiopathology , Cardiomegaly/prevention & control , Cardiotonic Agents/metabolism , Cardiotonic Agents/pharmacology , Cytochrome P-450 CYP2J2 , Cytochrome P-450 Enzyme System/metabolism , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/pharmacology , Fish Oils/pharmacology , Humans , Mice , Myocardium/pathology , Myocytes, Cardiac/cytology , Myocytes, Cardiac/drug effects , Rats , Reperfusion InjuryABSTRACT
Dietary fish oil omega-3 fatty acids (n-3 PUFAs), such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), protect against arrhythmia and sudden cardiac death using largely unknown mechanisms. EPA and DHA may serve as efficient alternative substrates of arachidonic acid (AA) metabolizing cytochrome P450 (CYP) enzymes. For many of the CYP isoforms, the n-3 PUFAs are the preferred substrates. Moreover, the CYP enzymes oxygenate EPA and DHA with largely different regioselectivities compared to AA. In particular, the omega-3 double bond that distinguishes EPA and DHA from AA is a preferred site of CYP-catalyzed epoxidation reactions. Given the pivotal role of CYP-dependent AA metabolites in the regulation of vascular, renal and cardiac functions, their replacement by unique sets of epoxy- and hydroxy-metabolites derived from EPA and DHA may have far-reaching physiological implications. The currently available data suggest that some of the vasculo- and cardioprotective effects attributed to dietary n-3 PUFAs may be mediated by CYP-dependent metabolites of EPA and DHA.
Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Fatty Acids, Omega-3/metabolism , Fatty Acids, Omega-6/metabolism , Animals , Arachidonic Acid/metabolism , Cardiotonic Agents/metabolism , Cardiovascular Diseases/prevention & control , Cytochrome P-450 CYP1A1/metabolism , Cytochrome P-450 CYP2E1/metabolism , Cytochrome P-450 CYP2J2 , Death, Sudden, Cardiac/prevention & control , Docosahexaenoic Acids/metabolism , Docosahexaenoic Acids/pharmacology , Eicosapentaenoic Acid/metabolism , Eicosapentaenoic Acid/pharmacology , Epoxide Hydrolases/metabolism , Fatty Acids, Omega-3/administration & dosage , Fatty Acids, Omega-6/administration & dosage , Humans , Hydroxyeicosatetraenoic Acids/metabolism , Hydroxyeicosatetraenoic Acids/pharmacologyABSTRACT
Cytochrome P450 (CYP) generated cardioprotective metabolites, epoxyeicosatrienoic acids (EETs), and cardiotoxic metabolites, hydroxyeicosatetraenoic acids (HETEs) levels are determined by many factors, including the induction or repression of the CYP enzymes, responsible for their formation. Therefore, we examined the effect of acute inflammation on the expression of CYP epoxygenases and CYP omega-hydroxylases in the heart, kidney, and liver and the cardiac CYP-mediated arachidonic acid metabolism. For this purpose, male Sprague-Dawley rats were injected intraperitoneally with LPS (1mg/kg). After 6, 12, or 24h, the tissues were harvested and the expression of CYP genes and protein levels were determined using real time-PCR, and Western blot analyses, respectively. Arachidonic acid metabolites formations were determined by liquid chromatography-electron spray ionization-mass spectrometry LC-ESI-MS. Our results showed that inflammation significantly decreased the CYP epoxygenases expression in the heart, kidney and liver with a concomitant decrease in the EETs produced by these enzymes. In contrast to CYP expoxygenses, inflammation differentially altered CYP omega-hydroxylases expression with a significant increase in 20-HETE formation. The present study demonstrates for the first time that acute inflammation decreases CYP epoxygenases and their associated cardioprotective metabolites, EETs while on the other hand increases CYP omega-hydroxylases and their associated cardiotoxic metabolites, 20-HETE. These changes may be involved in the development and/or progression of cardiovascular diseases by inflammation.
Subject(s)
Arachidonic Acid/metabolism , Cytochrome P-450 Enzyme System/metabolism , Inflammation/metabolism , Lipopolysaccharides/toxicity , Myocardium/enzymology , Animals , Blotting, Western , Cytochrome P-450 CYP2J2 , Cytochrome P-450 CYP4A/metabolism , DNA, Complementary/biosynthesis , DNA, Complementary/isolation & purification , Heart/drug effects , Hydroxyeicosatetraenoic Acids/metabolism , Inflammation/chemically induced , Inflammation/enzymology , Kidney/drug effects , Kidney/enzymology , Liver/drug effects , Liver/enzymology , Male , Microsomes/drug effects , Microsomes/enzymology , Myocardium/metabolism , RNA/biosynthesis , RNA/isolation & purification , Rats , Rats, Sprague-Dawley , Reverse Transcriptase Polymerase Chain Reaction , Spectrometry, Mass, Electrospray IonizationABSTRACT
Hyperhomocysteinemia (HHcy) is associated with atherosclerotic events involving the modulation of arachidonic acid (AA) metabolism and the activation of matrix metalloproteinase-9 (MMP-9). Cytochrome P450 (CYP) epoxygenase-2J2 (CYP2J2) is abundant in the heart endothelium, and its AA metabolites epoxyeicosatrienoic acids (EETs) mitigates inflammation through NF-kappabeta. However, the underlying molecular mechanisms for MMP-9 regulation by CYP2J2 in HHcy remain obscure. We sought to determine the molecular mechanisms by which P450 epoxygenase gene transfection or EETs supplementation attenuate homocysteine (Hcy)-induced MMP-9 activation. CYP2J2 was over-expressed in mouse aortic endothelial cells (MAECs) by transfection with the pcDNA3.1/CYP2J2 vector. The effects of P450 epoxygenase transfection or exogenous supplementation of EETs on NF-kappabeta-mediated MMP-9 regulation were evaluated using Western blot, in-gel gelatin zymography, electromobility shift assay, immunocytochemistry. The result suggested that Hcy downregulated CYP2J2 protein expression and dephosphorylated PI3K-dependent AKT signal. Hcy induced the nuclear translocation of NF-kappabeta via downregulation of IKbetaalpha (endogenous cytoplasmic inhibitor of NF-kappabeta). Hcy induced MMP-9 activation by increasing NF-kappabeta-DNA binding. Moreover, P450 epoxygenase transfection or exogenous addition of 8,9-EET phosphorylated the AKT and attenuated Hcy-induced MMP-9 activation. This occurred, in part, by the inhibition of NF-kappabeta nuclear translocation, NF-kappabeta-DNA binding and activation of IKbetaalpha. The study unequivocally suggested the pivotal role of EETs in the modulation of Hcy/MMP-9 signal.