BACKGROUND: Epoxyeicosatrienoic acids (EETs), which are metabolites of arachidonic acid catalyzed by cytochrome P450 epoxygenase, are degraded into inactive dihydroxyeicosatrienoic acids by soluble epoxide hydrolase (sEH). Many studies have revealed that sEH gene deletion exerts protective effects against diabetes. Vascular calcification is a common complication of diabetes, but the potential effects of sEH on diabetic vascular calcification are still unknown. METHODS: The level of aortic calcification in wild-type and Ephx2-/- C57BL/6 diabetic mice induced with streptozotocin was evaluated by measuring the aortic calcium content through alizarin red staining, immunohistochemistry staining, and immunofluorescence staining. Mouse vascular smooth muscle cell lines (MOVAS cells) treated with β-glycerol phosphate (0.01 mol/L) plus advanced glycation end products (50 mg/L) were used to investigate the effects of sEH inhibitors or sEH knockdown and EETs on the calcification of vascular smooth muscle cells, which was detected by Western blotting, alizarin red staining, and Von Kossa staining. RESULTS: sEH gene deletion significantly inhibited diabetic vascular calcification by increasing levels of EETs in the aortas of mice. EETs (especially 11,12-EET and 14,15-EET) efficiently prevented the osteogenic transdifferentiation of MOVAS cells by decreasing nidogen-2 (NID2) expression. Interestingly, suppressing sEH activity by small interfering ribonucleic acid or specific inhibitors did not block osteogenic transdifferentiation of MOVAS cells induced by β-glycerol phosphate and advanced glycation end products. NID2 overexpression significantly abolished the inhibitory effect of sEH gene deletion on diabetic vascular calcification. Moreover, NID2 overexpression mediated by adeno-associated virus 9 vectors markedly increased insulin-like growth factor 2 (IGF2) and phospho-ERK1/2 expression in MOVAS cells. Overall, sEH gene knockout inhibited diabetic vascular calcification by decreasing aortic NID2 expression and, then, inactivating the downstream IGF2-ERK1/2 signaling pathway. CONCLUSIONS sEH gene deletion markedly inhibited diabetic vascular calcification through repressed osteogenic transdifferentiation of vascular smooth muscle cells mediated by increased aortic EET levels, which was associated with decreased NID2 expression and inactivation of the downstream IGF2-ERK1/2 signaling pathway.
Animals ; Mice ; Vascular Calcification/metabolism* ; Mice, Inbred C57BL ; Epoxide Hydrolases/metabolism* ; Diabetes Mellitus, Experimental/genetics* ; Male ; Gene Deletion ; MAP Kinase Signaling System/genetics* ; Cell Line ; Immunohistochemistry ; Muscle, Smooth, Vascular/metabolism* ; Signal Transduction/genetics* ; Mice, Knockout

Soluble epoxide hydrolases (sEH) are enzymes present in all living organisms, metabolize epoxy fatty acids to 1,2-diols. sEH in the metabolism of polyunsaturated fatty acids plays a key role in inflammation. In addition, the endogenous lipid mediators in cardiovascular disease are also broken down to diols by the action of sEH that enhanced cardiovascular protection. In this study, sEH inhibitory guided fractionation led to the isolation of five phenolic compounds trans-resveratrol (1), trans-piceatannol (2), sulfuretin (3), (+)-balanophonin (4), and cassigarol E (5) from the ethanol extract of the seeds of Passiflora edulis Sims cultivated in Vietnam. The chemical structures of isolated compounds were determined by the interpretation of NMR spectral data, mass spectra, and comparison with data from the literature. The soluble epoxide hydrolase (sEH) inhibitory activity of isolated compounds was evaluated. Among them, trans-piceatannol (2) showed the most potent inhibitory activity on sEH with an IC₅₀ value of 3.4 µM. This study marks the first time that sulfuretin (3) was isolated from Passiflora edulis as well as (+)-balanophonin (4), and cassigarol E (5) were isolated from Passiflora genus.
Cardiovascular Diseases ; Epoxide Hydrolases ; Ethanol ; Fatty Acids ; Fatty Acids, Unsaturated ; Inflammation ; Metabolism ; Passiflora ; Passifloraceae ; Phenol ; Vietnam

OBJECTIVE: This study aimed to clone and characterize the oxiranedicarboxylate hydrolase (ORCH) from Labrys sp. WH-1. METHODS: Purification by column chromatography, characterization of enzymatic properties, gene cloning by protein terminal sequencing and polymerase chain reaction (PCR), and sequence analysis by secondary structure prediction and multiple sequence alignment were performed. RESULTS: The ORCH from Labrys sp. WH-1 was purified 26-fold with a yield of 12.7%. It is a monomer with an isoelectric point (pI) of 8.57 and molecular mass of 30.2 kDa. It was stable up to 55 °C with temperature at which the activity of the enzyme decreased by 50% in 15 min (T₅₀¹⁵) of 61 °C and the half-life at 50 °C (t_{1/2, 50 °C}) of 51 min and was also stable from pH 4 to 10, with maximum activity at 55 °C and pH 8.5. It is a metal-independent enzyme and strongly inhibited by Cu²⁺, Ag⁺, and anionic surfactants. Its kinetic parameters (K_m, k_cat, and k_cat/K_m) were 18.7 mmol/L, 222.3 s^-1, and 11.9 mmol/(L·s), respectively. The ORCH gene, which contained an open reading frame (ORF) of 825 bp encoding 274 amino acid residues, was overexpressed in Escherichia coli and the enzyme activity was 33 times higher than that of the wild strain. CONCLUSIONS The catalytic efficiency and thermal stability of the ORCH from Labrys sp. WH-1 were the best among the reported ORCHs, and it provides an alternative catalyst for preparation of L(+)-2,3-dihydrobutanedioic acid.
Alphaproteobacteria/enzymology* ; Cloning, Molecular ; Dicarboxylic Acids/metabolism* ; Enzyme Stability ; Epoxide Hydrolases/metabolism*

AIM: To investigate the relationship between cerebroprotection of pinocembrin and epoxyeicosatrienoic acids (EETs) and their regulating enzyme soluble epoxide hydrolase (sEH). METHODS: Rats underwent middle cerebral artery occlusion (MCAO) to mimic permanent focal ischemia, and pinocembrin was administrated via tail vein injection at 10 min, 4 h, 8 h and 23 h after MCAO. After 24 MCAO, rats were re-anesthetized, and the blood and brain were harvested and analyzed. RESULTS: Pinocembrin displayed significant protective effects on MCAO rats indicated by reduced neurological deficits and infarct volume. Importantly, co-administration of 0.2 mg·kg(-1) 14, 15-EEZE, a putative selective EET antagonist, weakened the beneficial effects of pinocembrin. 14, 15-EET levels in the blood and brain of rats after 24 h MCAO were elevated in the presence of pinocembrin. In an assay for hydrolase activity, pinocembrin significantly lowered brain sEH activity of MCAO rats and inhibited recombinant human sEH activity in a concentration-dependent manner (IC50, 2.58 μmol·L(-1)). In addition, Western blot and immunohistochemistry analysis showed that pinocembrin at doses of 10 mg·kg(-1) and 30 mg·kg(-1) significantly down-regulated sEH protein in rat brain, especially the hippocampus CA1 region of MCAO rats. CONCLUSION Inhibiting sEH and then increasing the potency of EETs may be one of the mechanisms through which pinocembrin provides cerebral protection.
Animals ; Arachidonic Acids ; metabolism ; Brain ; drug effects ; enzymology ; metabolism ; Brain Ischemia ; drug therapy ; enzymology ; genetics ; metabolism ; prevention & control ; Disease Models, Animal ; Epoxide Hydrolases ; genetics ; metabolism ; Flavanones ; administration & dosage ; Humans ; Male ; Protective Agents ; administration & dosage ; Rats ; Rats, Sprague-Dawley

To screen potential human soluble epoxide hydrolase (hsEH) inhibitors, a high-throughput screening model in 384-well microplate with total volume of 50 microL was established. Recombinant hsEH was cloned and expressed in E. coli. and its specific substrate PHOME was synthesized. The HTS model was based on fluorescence analysis with enhanced sensitivity and specificity (Z' = 0.65). A total of 47 360 samples (including 25 040 compounds and 22 320 natural products) were screened, of which 950 samples with inhibition greater than 80% were selected for further rescreening. Finally, two compounds with high inhibitory activity were identified, whose IC50 value were 8.56 and 4.31 micromol x L(-1), separately. The results indicated that the method was stable, sensitive, reproducible and also suitable for high-throughput screening.
Drug Evaluation, Preclinical ; methods ; Enzyme Inhibitors ; analysis ; chemistry ; Epoxide Hydrolases ; antagonists & inhibitors ; chemistry ; metabolism ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; methods ; Inhibitory Concentration 50 ; Recombinant Proteins ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Spectrometry, Fluorescence ; methods ; Substrate Specificity

OBJECTIVE: To investigate the effect of soluble epoxide hydrolase inhibitor (sEHi) tAUCB on the function of endothelial progenitor cells (EPCs) and expression of vascular endothelial growth factor (VEGF) in EPCs in patients with coronary heart disease (CHD). METHODS: Mononuclear cells, from the peripheral blood of CHD patients, were isolated by ficoll density gradient centrifugation and cultured. After 7 days of culture in vitro, EPCs were identified by double staining and flow cytometry. EPCs were then stimulated by 0, 10(-6), 10(-5), and 10(-4) mol/L of tAUCB for 24 h. Migration assay was performed in transwell chamber and tube formation assay was performed by Matrigel-Matrix in vitro model. The expression of VEGF in EPCs was measured by Western blot. EPCs from age and gender matched healthy subjects were also cultured as controls. RESULTS: The migration and tube formation activities of EPCs from CHD patients were obviously damaged compared with those from healthy controls (P<0.05). The tAUCB could dose-dependently increase the migration and tube formation activities and increase the expression of VEGF in EPCs compared with those from CHD patients without treatment. The 10(-6) mol/L tAUCB increased those activities of EPCs and the expression of VEGF with statistical difference. CONCLUSION sEHi can positively modulate the function of EPCs from CHD patients, suggesting the potential predictive significance of sEHi in the therapy of CHD.
Aged ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Coronary Disease ; pathology ; Endothelial Cells ; metabolism ; pathology ; physiology ; Enzyme Inhibitors ; pharmacology ; Epoxide Hydrolases ; antagonists & inhibitors ; Female ; Humans ; Leukocytes, Mononuclear ; pathology ; Male ; Middle Aged ; Solubility ; Stem Cells ; metabolism ; pathology ; physiology ; Vascular Endothelial Growth Factor A ; genetics ; metabolism

Wild-type biocatalysts usually show high activity and selectivity towards their native substrates. Since non-native substrates are often used in synthetically useful biocatalytic transformations, it is necessary to engineer enzymes for improved activity, stability and selectivity (chemo-, regio- and stereoselectivity). Herein we give an overview of the recent advances in engineering the enantioselectivity of biocatalysts, with an aim to stimulate further development of this important field in China.
Animals ; Biocatalysis ; Epoxide Hydrolases ; genetics ; metabolism ; Esterases ; genetics ; metabolism ; Humans ; Lipase ; genetics ; metabolism ; Protein Engineering ; methods ; Stereoisomerism

Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Arachidonate 5-Lipoxygenase ; metabolism ; therapeutic use ; Arachidonic Acid ; metabolism ; Cyclooxygenase Inhibitors ; therapeutic use ; Drug Delivery Systems ; methods ; Epoxide Hydrolases ; antagonists & inhibitors ; metabolism ; therapeutic use ; Humans ; Inflammation ; drug therapy ; Lipoxygenase Inhibitors ; Metabolic Networks and Pathways ; drug effects ; Phospholipase A2 Inhibitors ; Phospholipases A2 ; metabolism ; therapeutic use ; Prostaglandin-Endoperoxide Synthases ; metabolism

OBJECTIVETo investigate the associations of polymorphisms of metabolic enzyme genes with urinary 1-hydroxypyrene levels in coke oven workers.

METHODSOne hundred and forty-eight workers from a coke oven plant and 69 controls without occupational PAHs exposure were selected in this study. Urinary 1-hydroxypyrene was detected by high performance liquid chromatography with florescence detector. The genotypes at I462V site in exon 7 of CYP1A1 gene, GSTM1, GSTT1, I105V site in GSTP1gene, Pst1 and Dra1 sites in CYP2E1 gene, P187S site in NQO1 gene, Kpn1, BamH1 and Taq1 sites in NAT2 gene, and H113Y, R139H sites in mEH gene were determined by PCR-based methods. Personal information including occupational exposure history, age, sex, smoking and drinking status was collected by the questionnaire.

RESULTSThe level of urinary 1-hydroxypyrene in coke oven workers [(5.61 +/- 1.04) mol/mol Cr] was higher than that in control [(0.74 +/- 0.32) micro mol/mol Cr]. After adjusting external occupational exposure category and smoking, coke oven workers with variant homozygotes at H113Y site of mEH gene had significantly higher urinary 1-hydroxypyrene concentrations than those with heterozygotes, and wild homozygotes (6.41 +/- 1.09 vs. 6.24 +/- 1.08, and 4.62 +/- 0.95 micro mol/mol Cr, P < 0.05), and gene-gene interaction was found between CYP1A1 and mEH.

CONCLUSIONGenetic polymorphism of mEH gene could be a susceptible biomarker in coke oven workers which was involved in the individual susceptibility on metabolism of PAHs.

Coke ; adverse effects ; Cytochrome P-450 CYP1A1 ; genetics ; DNA Damage ; genetics ; Epoxide Hydrolases ; genetics ; Genetic Predisposition to Disease ; genetics ; Glutathione Transferase ; genetics ; Humans ; Male ; Occupational Exposure ; Polycyclic Aromatic Hydrocarbons ; poisoning ; Polymorphism, Genetic ; Pyrenes ; analysis ; metabolism

OBJECTIVETo investigate the association between polymorphisms of metabolic enzyme genes and chromosomal damage risk in peripheral blood lymphocytes among coke oven workers.

METHODSOne hundred and fourty-nine coke oven workers and 24 referents without occupational polycyclic aromatic hydrocarbons (PAH) exposure were recruited in this study. Urinary 1-hydroxypyrene levels were measured as the internal dose of PAH exposure. The 6 per 1 000 of micronucleus value was used as the cut-off value to determine whether the individual's chromosomal damage was positive. The genotypes of CYP1A1, GSTM1, GSTT1, GSTP1, CYP2E1, NQO1, NAT2 and mEH genes were determined by PCR-based methods. Multiple logistic regression was used to calculate the adjusted ORs and the 95% CI for the risk of chromosomal damage and to analyze the gene-gene interaction.

RESULTSIn 173 subjects, after adjusting the occupational exposure, age, sex, smoking and drinking status, the subjects with GSTM1 null genotype have significantly higher risk for chromosomal damage than subjects with GSTM1 positive genotype (adjusted OR = 2.01, 95% CI = 1.03 -3.91). Compared with the wild homozygotes at P187S site of NQO1 gene, the variant homozygotes have significantly higher risk for chromosomal damage (adjusted OR = 3.18, 95% CI = 1.18 - 8.62). The subjects with variant allele at H113Y site of mEH gene have significantly lower risk for chromosomal damage (adjusted OR = 0.40, 95% CI = 0.19 - 0.88). No significant associations were found for other gene polymorphisms and chromosomal damage risk. In addition, the gene-gene interactions were also found among GSTM1, NQO1 gene P187S and mEH gene H113Y polymorphisms for the risk of chromosomal damage risk.

CONCLUSIONSignificant associations between genetic polymorphisms in GSTM1, NQO1 and mEH gene and risk for chromosomal damage were found among occupational PAH-exposed workers, which related to the mechanism of PAH carcinogenesis.

Adult ; DNA Damage ; genetics ; Epoxide Hydrolases ; genetics ; Female ; Genetic Predisposition to Disease ; genetics ; Glutathione Transferase ; genetics ; Humans ; Logistic Models ; Lymphocytes ; metabolism ; Male ; Middle Aged ; Multivariate Analysis ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; Occupational Exposure ; Polycyclic Aromatic Hydrocarbons ; poisoning ; Polymorphism, Genetic ; Pyrenes ; analysis ; Risk Factors