Upregulated Nuclear Expression of Soluble Epoxide Hydrolase Predicts Poor Outcome in Breast Cancer Patients: Importance of the Digital Pathology Approach.

Breast cancer (BC) is the most common cancer in women, with incidence rates increasing globally in recent years. Therefore, it is important to find new molecules with prognostic and therapeutic value to improve therapeutic response and quality of life. The polyunsaturated fatty acids (PUFAs) metabolic pathway participates in various physiological processes, as well as in the development of malignancies. Although aberrancies in the PUFAs metabolic pathway have been implicated in carcinogenesis, the functional and clinical relevance of this pathway has not been well explored in BC. To evaluate the clinical significance of soluble epoxide hydrolase (EPHX2) expression in Mexican patients with BC using tissue microarrays (TMAs) and digital pathology (DP). Immunohistochemical analyses were performed on 11 TMAs with 267 BC samples to quantify this enzyme. Using DP, EPHX2 protein expression was evaluated solely in tumor areas. The association of EPHX2 with overall survival (OS) was detected through bioinformatic analysis in public databases and confirmed in our cohort via Cox regression analysis. Clear nuclear expression of EPHX2 was identified. Receiver operating characteristics (ROC) curves revealed the optimal cutoff point at 2.847062 × 10-3 pixels, with sensitivity of 69.2% and specificity of 67%. Stratification based on this cutoff value showed elevated EPHX2 expression in multiple clinicopathological features, including older age and nuclear grade, human epidermal growth factor receptor 2 (HER2) and triple negative breast cancer (TNBC) subtypes, and recurrence. Kaplan-Meier curves demonstrated how higher nuclear expression of EPHX2 predicts shorter OS. Consistently, multivariate analysis confirmed EPHX2 as an independent predictor of OS, with a hazard ratio (HR) of 3.483 and a 95% confidence interval of 1.804-6.724 (p < 0.001). Our study demonstrates for the first time that nuclear overexpression of EPHX2 is a predictor of poor prognosis in BC patients. The DP approach was instrumental in identifying this significant association. Our study provides valuable insights into the potential clinical utility of EPHX2 as a prognostic biomarker and therapeutic target in BC.

WTAP promotes the progression of ulcerative colitis by silencing the expression of CES2 through m6A modification.

OBJECTIVE: This study will explore the function of WTAP, the critical segment of m6A methyltransferase complex, in UC and its regulation on immune response. METHODS: The expression levels of key proteins were detected in colon tissues which were derived from UC patients and mice. Macrophage polarization and CD4+ T cell infiltration were detected by flow cytometry and IF staining. ELISA assay was utilized to analyze the level of the inflammatory cytokines. m6A-RIP-PCR, actinomycin D test, and RIP assays were utilized to detect the m6A level, stability, and bound proteins of CES2 mRNA. A dual luciferase reporter assay was conducted to confirm the transcriptional interactions between genes. A co-culture system of intestinal epithelium-like organs was constructed to detect the primary mouse intestinal epithelial cells (PMIEC) differentiation. The interaction between proteins was detected via Co-IP assay. RESULTS: The expression of WTAP and CES2 in UC tissues was increased and decreased, respectively. Knockdown of WTAP inhibited the progression of UC in mice by inhibiting M1 macrophage polarization and CD4+ T cell infiltration. WTAP combined YTHDF2 to promote the m6A modification of CES2 mRNA and inhibited its expression. CES2 co-expressed with EPHX2 and overexpression of CES2 promoted the differentiation of PMIEC. The inhibitory effect of WTAP knockdown on the progress of UC was partially abrogated by CES2 knockdown. CONCLUSION: WTAP/YTHDF2 silences CES2 by promoting its m6A modification and then promotes the progression of UC. WTAP could be a promoting therapy target of UC.

Inhibition of the STAT3-EPHX2 axis promotes regression of ulcerative colitis by treatment with novel porphyrin derivative.

LD4, a novel porphyrin derivative, has attracted much attention for its excellent anti-inflammatory properties. It can promote the healing of colonic mucosa, reduce inflammatory response, regulate oxidative stress, and thus improve ulcerative colitis (UC) symptoms. However, the specific signaling pathways of LD4-PDT involved in UC have not been explored. The present study aimed to elucidate the effects of LD4 on UC and to investigate the underlying mechanisms both in vivo and in vitro. We classified and screened the LD4-PDT proteomic data to obtain key targets. Proteomic data revealed that EPHX2 and STAT3 are key targets of LD4-PDT for UC. Moreover, transcription factor STAT3 positively regulates the expression of EPHX2. Inhibiting EPHX2 can prevent the activation of NF-κB signaling pathway. Next, through pharmacological inhibition experiments, we confirmed that LD4-PDT can reduce intestinal inflammation by inhibiting STAT3-EPHX2 axis. However, by treating normal intestinal epithelial cells and colon cancer cells with TPPU and Stattic, our data confirmed that the STAT3-EPHX2 axis does not exist in colon cancer. In this study, we demonstrated that the transcription factor STAT3 can positively regulate the expression of EPHX2 in normal colon. LD4 can alleviate UC by inhibiting the STAT3-EPHX2 axis, but this axis does not exist in colon cancer. LD4-PDT may become a new and effective method for treating UC.

Overexpression of soluble epoxide hydrolase reduces post-ischemic recovery of cardiac contractile function.

Cytochromes P450 can metabolize endogenous fatty acids, such as arachidonic acid, to bioactive lipids such as epoxyeicosatrienoic acids (EETs) that have beneficial effects. EETs protect hearts against ischemic damage, heart failure or fibrosis; however, their effects are limited by hydrolysis to less active dihydroxy oxylipins by soluble epoxide hydrolase (sEH), encoded by the epoxide hydrolase 2 gene (EPHX2, EC 3.3.2.10). Pharmacological inhibition or genetic disruption of sEH/EPHX2 have been widely studied for their impact on cardiovascular diseases. Less well studied is the role of increased EPHX2 expression, which occurs in a substantial human population that carries the EPHX2 K55R polymorphism or after induction by inflammatory stimuli. Herein, we developed a mouse model with cardiomyocyte-selective expression of human EPHX2 (Myh6-EPHX2) that has significantly increased total EPHX2 expression and activity. Myh6-EPHX2 hearts exhibit strong, cardiomyocyte-selective expression of EPHX2. EPHX2 mRNA, protein, and epoxide hydrolysis measurements suggest that Myh6-EPHX2 hearts have 12-fold increase in epoxide hydrolase activity relative to wild type (WT) hearts. This increased activity significantly decreased epoxide:diol ratios in vivo. Isolated, perfused Myh6-EPHX2 hearts were not significantly different from WT hearts in basal parameters of cardiac function; however, compared to WT hearts, Myh6-EPHX2 hearts demonstrated reduced recovery of heart contractile function after ischemia and reperfusion (I/R). This impaired recovery after I/R correlated with reduced activation of PI3K/AKT and GSK3ß signaling pathways in Myh6-EPHX2 hearts compared to WT hearts. In summary, the Myh6-EPHX2 mouse line represents a novel model of cardiomyocyte-selective overexpression of EPHX2 that has detrimental effects on cardiac function.

Association between CYP4A11 and EPHX2 genetic polymorphisms and chronic kidney disease progression in hypertensive patients.

BACKGROUND: There are evidence indicating that some metabolites of arachidonic acid produced by cytochromes P450 (CYP) and epoxide hydroxylase (EPHX2), such as hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatrienoic acids (EETs) or dihydroxyeicosatrienoic acids (DHETEs), play an important role in blood pressure regulation and they could contribute to the development of hypertension (HT) and kidney damage. Therefore, the main aim of the study was to evaluate whether the genetic polymorphisms of CYP2C8, CYP2C9, CYP2J2, CYP4F2, CYP4F11 and EPHX2, responsible for the formation of HETEs, EETs and DHETEs, are related to the progression of impaired renal function in a group of patients with hypertension. METHODS: 151HT patients from a hospital nephrology service were included in the study. Additionally, a group of 87 normotensive subjects were involved in the study as control group. For HT patients, a general biochemistry analysis, estimated glomerular filtration rate and genotyping for different CYPs and EPHX2 variant alleles was performed. RESULTS: CYP4A11 rs3890011, rs9332982 and EPHX2 rs41507953 polymorphisms, according to the dominant model, presented a high risk of impaired kidney function, with odds ratios (OR) of 2.07 (1.00-4.32; P=0.049) 3.02 (1.11-8.23; P=0.030) and 3.59 (1.37-9.41; P=0.009), respectively, and the EPHX2 rs1042032 polymorphism a greater risk according to the recessive model (OR=6.23; 95% CI=1.50-25.95; P=0.007). However, no significant differences in allele frequencies between HT patients and in normotensive subjects for any of the SNP analysed. In addition, the patients with diagnosis of dyslipidemia (n=90) presented higher frequencies of EPHX2 K55R (rs41507953) and *35A>G (rs1042032) variants than patients without dyslipidemia, 4% vs. 14% (P=0.005) and 16 vs. 27% (P=0.02), respectively. CONCLUSIONS: In this study has been found higher odds of impaired renal function progression associated with rs3890011 and rs9332982 (CYP4A11) and rs41507953 and rs1042032 (EPHX2) polymorphisms, which may serve as biomarkers for improve clinical interventions aimed at avoiding or delaying, in chronic kidney disease patients, progress to end-stage kidney disease needing dialysis or kidney transplant.

Antioxidant Genes Variants and Their Association with Sperm DNA Fragmentation.

Semen possesses a variety of antioxidant defense mechanisms which protect sperm DNA from the damaging effects of oxidative stress. Correlation between antioxidant genes variants and sperm DNA fragmentation (SDF) level is not sufficiently studied. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs): CYP1A1 (rs1048943A > G), CYP4F2 (rs2108622G > A), NRF2 (rs6721961C > A), PON1 (rs662A > G), NOS3 (rs1799983G > T), GSTM1 (null), CAT (rs1001179C > T), SOD2 (rs4880A > G), GSTP1 (rs1695A > G), PON2 (rs7493G > C), EPHX2 (rs1042064T > C), and AHR (rs2066853G > A) and elevated SDF. The study employed a case-control design where, the allele and genotype frequencies of the selected SNPs were compared between 75 semen samples with abnormal SDF (the cases) and 75 samples with normal SDF (the controls). DNA was extracted from the semen samples and allele-specific PCR (AS-PCR) was used for genotyping the SNPs. Relevant data were collected from the patients' records et al.-Basma Fertility Center. Suitable statistical tests and multifactorial dimensionality reduction (MDR) test were used to anticipate SNP-SNP interactions. Comparison of semen parameters revealed significant differences between cases and controls in terms of liquefaction time, sperm total motility, and normal form. Genotype frequencies of NOS3 G > T (GT), SOD2 A > G (AA and AG), EPHX2 T > C (CC and CT), and AHR G > A (GA and GG) were significantly different between cases and controls. Allele frequencies of SOD2 (G-allele), and EPHX2 (T-allele) also significantly varied between cases and controls. MDR analysis revealed that the NOS3, SOD2, and EPHX2 SNPs combination has the highest impact on SDF. The study findings suggest that genetic variations in genes involved antioxidant defenses contribute to abnormal SDF.

Proteomic analysis discovers potential biomarkers of early traumatic axonal injury in the brainstem.

OBJECTIVE: Application of Tandem Mass Tags (TMT)-based LC-MS/MS analysis to screen for differentially expressed proteins (DEPs) in traumatic axonal injury (TAI) of the brainstem and to predict potential biomarkers and key molecular mechanisms of brainstem TAI. METHODS: A modified impact acceleration injury model was used to establish a brainstem TAI model in Sprague-Dawley rats, and the model was evaluated in terms of both functional changes (vital sign measurements) andstructural changes (HE staining, silver-plating staining and ß-APP immunohistochemical staining). TMT combined with LC-MS/MS was used to analyse the DEPs in brainstem tissues from TAI and Sham groups. The biological functions of DEPs and potential molecular mechanisms in the hyperacute phase of TAI were analysed by bioinformatics techniques, and candidate biomarkers were validated using western blotting and immunohistochemistry on brainstem tissues from animal models and humans. RESULTS: Based on the successful establishment of the brainstem TAI model in rats, TMT-based proteomics identified 65 DEPs, and bioinformatics analysis indicated that the hyperacute phase of TAI involves multiple stages of biological processes including inflammation, oxidative stress, energy metabolism, neuronal excitotoxicity and apoptosis. Three DEPs, CBR1, EPHX2 and CYP2U1, were selected as candidate biomarkers and all three proteins were found to be significantly expressed in brainstem tissue 30 min-7 days after TAI in both animal models and humans. CONCLUSION: Using TMT combined with LC-MS/MS analysis for proteomic study of early TAI in rat brainstem, we report for the first time that CBR1, EPHX2 and CYP2U1 can be used as biomarkers of early TAI in brainstem by means of western blotting and immunohistochemical staining, compensating for the limitations of silver-plating staining and ß-APP immunohistochemical staining, especially in the case of very short survival time after TAI (shorter than 30 min). A number of other proteins that also have a potential marker role are also presented, providing new insights into the molecular mechanisms, therapeutic targets and forensic identification of early TAI in brainstem.

CYP metabolic pathway related gene polymorphism increases the risk of embolic and atherothrombotic stroke and vulnerable carotid plaque in southeast China.

OBJECTIVE: To investigate the association of CYP metabolic pathway-related genetic polymorphisms with the susceptibility to ischemic stroke and stability of carotid plaque in southeast China. METHODS: We consecutively enrolled 294 acute ischemic stroke patients with carotid plaque and 282 controls from Wenling First People's Hospital. The patients were divided into the carotid vulnerable plaque group and stable plaque group according to the results of carotid B-mode ultrasonography. Polymorphisms of CYP3A5 (G6986A, rs776746), CYP2C9*2 (C430T, rs1799853), CYP2C9*3 (A1075C, rs1057910), and EPHX2 (G860A, rs751141) were determined using polymerase chain reaction and mass spectrometry analysis. RESULTS: EPHX2 GG may reduce the susceptibility to ischemic stroke (OR = 0.520, 95% CI: 0.288 ∼ 0.940, P = 0.030) and AA+AG may increase the risk for ischemic stroke (OR = 1.748, 95% CI: 1.001 ∼ 3.052, P = 0.050). The distribution of CYP3A5 genotypes showed significant differences between the vulnerable plaque and stable plaque groups (P = 0.026). Multivariate logistic regression analysis found that CYP3A5 GG could reduce the risk of vulnerable plaques (OR = 0.405, 95% CI: 0.178 ∼ 0.920, P = 0.031). CONCLUSION: EPHX2 G860A polymorphism may reduce the stroke susceptibility, while other SNPs of CYP genes are not associated with ischemic stroke in southeast China. Furthermore CYP3A5 polymorphism was related with carotid plaque instability.

Soluble epoxide hydrolase inhibitor promotes the healing of oral ulcers.

OBJECTIVE: Oral ulcers are a lesion in the oral mucosa that impacts chewing or drinking. Epoxyeicosatrienoic Acids (EETs) have enhanced angiogenic, regenerative, anti-inflammatory, and analgesic effects. The present study aims to evaluate the effects of 1-Trifluoromethoxyphenyl-3-(1-Propionylpiperidin-4-yl) Urea (TPPU), a soluble epoxide hydrolase inhibitor for increasing EETs level, on the healing of oral ulcers. METHODS: The chemically-induced oral ulcers were established in Sprague Dawley rats. The ulcer area was treated with TPPU to evaluate the healing time and pain threshold of ulcers. The expression of angiogenesis and cell proliferation-related protein in the ulcer area was detected using immunohistochemical staining. The effects of TPPU on migration and angiogenesis capability were measured with scratch assay and tube formation. RESULTS: Compared with the control group, TPPU promoted wound healing of oral ulcers with a shorter healing time, and raised pain thresholds. Immunohistochemical staining showed that TPPU increased the expression of angiogenesis and cell proliferation-related protein with reduced inflammatory cell infiltration in the ulcer area. TPPU enhanced cell migration and tube-forming potential in vitro. CONCLUSIONS: The present results support the potential of TPPU with multiple biological effects for the treatment of oral ulcers by targeting soluble epoxide hydrolase.

Disruption of Ephx2 in cardiomyocytes but not endothelial cells improves functional recovery after ischemia-reperfusion in isolated mouse hearts.

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 EET-induced 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.

Wide-spread enhancer effect of SNP rs2279590 on regulating epoxide hydrolase-2 and protein tyrosine kinase 2-beta gene expression.

Polymorphisms in the PTK2B-CLU locus have been associated with various neurodegenerative disorders including pseudoexfoliation glaucoma, Alzheimer's and Parkinson's. Many of these genomic variants are within enhancer elements and modulate genes associated with the disease pathogenesis. However, mechanisms by which they control the gene expression is unknown. Previously, we have shown that clusterin enhancer element surrounding rs2279590 intronic variant, a risk factor in the pathogenesis of pseudoexfoliation glaucoma modulates gene expression of clusterin (CLU), protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase 2 (EPHX2). Here, we explored the mechanism by which rs2279590 enhancer regulates their gene expression through chromosome conformation capture assays. 3C assays revealed a strong enhancer-promoter chromatin interaction between rs2279590 enhancer and promoters of genes CLU, PTK2B and EPHX2 in the HEK293 wild type cells. Moreover, genomic knockout of rs2279590 element significantly decreases the chromatin-chromatin cross-linking frequency suggesting gene regulation at transcriptional level through formation of chromatin loop. In addition, molecular assays showed a significantly decreased expression of EPHX2 but not PTK2B at both mRNA and protein level in the lens capsule of pseudoexfoliation affected patients in comparison to control subjects implying a role of EPHX2 in the pathogenesis of pseudoexfoliation.

Soluble epoxide hydrolase inhibitor promotes the healing of oral ulcers

Abstract Objective Oral ulcers are a lesion in the oral mucosa that impacts chewing or drinking. Epoxyeicosatrienoic Acids (EETs) have enhanced angiogenic, regenerative, anti-inflammatory, and analgesic effects. The present study aims to evaluate the effects of 1-Trifluoromethoxyphenyl-3-(1-Propionylpiperidin-4-yl) Urea (TPPU), a soluble epoxide hydrolase inhibitor for increasing EETs level, on the healing of oral ulcers. Methods The chemically-induced oral ulcers were established in Sprague Dawley rats. The ulcer area was treated with TPPU to evaluate the healing time and pain threshold of ulcers. The expression of angiogenesis and cell proliferation-related protein in the ulcer area was detected using immunohistochemical staining. The effects of TPPU on migration and angiogenesis capability were measured with scratch assay and tube formation. Results Compared with the control group, TPPU promoted wound healing of oral ulcers with a shorter healing time, and raised pain thresholds. Immunohistochemical staining showed that TPPU increased the expression of angiogenesis and cell proliferation-related protein with reduced inflammatory cell infiltration in the ulcer area. TPPU enhanced cell migration and tube-forming potential in vitro. Conclusions The present results support the potential of TPPU with multiple biological effects for the treatment of oral ulcers by targeting soluble epoxide hydrolase.

Cardiac Disease Alters Myocardial Tissue Levels of Epoxyeicosatrienoic Acids and Key Proteins Involved in Their Biosynthesis and Degradation.

CYP2J2 is the main epoxygenase in the heart that is responsible for oxidizing arachidonic acid to cis-epoxyeicosatrienoic acids (EETs). Once formed, EETs can then be hydrolyzed by soluble epoxide hydrolase (sEH, encoded by EPHX2) or re-esterified back to the membrane. EETs have several cardioprotective properties and higher levels are usually associated with better cardiac outcomes/prognosis. This study investigates how cardiovascular disease (CVD) can influence total EET levels by altering protein expression and activity of enzymes involved in their biosynthesis and degradation. Diseased ventricular cardiac tissues were collected from patients receiving Left Ventricular Assist Device (LVAD) or heart transplants and compared to ventricular tissue from controls free of CVD. EETs, and enzymes involved in EETs biosynthesis and degradation, were measured using mass spectrometric assays. Terfenadine hydroxylation was used to probe CYP2J2 activity. Significantly higher cis- and trans-EET levels were observed in control cardiac tissue (n = 17) relative to diseased tissue (n = 24). Control cardiac tissue had higher CYP2J2 protein levels, which resulted in higher rate of terfenadine hydroxylation, compared to diseased cardiac tissues. In addition, levels of both NADPH-Cytochrome P450 oxidoreductase (POR) and sEH proteins were significantly higher in control versus diseased cardiac tissue. Overall, alterations in protein and activity of enzymes involved in the biosynthesis and degradation of EETs provide a mechanistic understanding for decreased EET levels in diseased tissues.

EPHX2 Inhibits Colon Cancer Progression by Promoting Fatty Acid Degradation.

Tumor cells use metabolic reprogramming to keep up with the need for bioenergy, biosynthesis, and oxidation balance needed for rapid tumor division. This phenomenon is considered a marker of tumors, including colon cancer (CRC). As an important pathway of cellular energy metabolism, fatty acid metabolism plays an important role in cellular energy supply and oxidation balance, but presently, our understanding of the exact role of fatty acid metabolism in CRC is limited. Currently, no lipid metabolism therapy is available for the treatment of CRC. The establishment of a lipidmetabolism model regulated by oncogenes/tumor suppressor genes and associated with the clinical characteristics of CRC is necessary to further understand the mechanism of fatty acid metabolism in CRC. In this study, through multi-data combined with bioinformatic analysis and basic experiments, we introduced a tumor suppressor gene, EPHX2, which is rarely reported in CRC, and confirmed that its inhibitory effect on CRC is related to fatty acid degradation.

A hypothesis-driven study to comprehensively investigate the association between genetic polymorphisms in EPHX2 gene and cardiovascular diseases: Findings from the UK Biobank.

BACKGROUND: Epoxyeicosatrienoic acids (EETs) are protective factors against cardiovascular diseases (CVDs) because of their vasodilatory, cholesterol-lowering, and anti-inflammatory effects. Soluble epoxide hydrolase (sEH), encoded by the EPHX2 gene, degrades EETs into less biologically active metabolites. EPHX2 is highly polymorphic, and genetic polymorphisms in EPHX2 have been linked to various types of CVDs, such as coronary heart disease, essential hypertension, and atrial fibrillation recurrence. METHODS: Based on a priori hypothesis that EPHX2 genetic polymorphisms play an important role in the pathogenesis of CVDs, we comprehensively investigated the associations between 210 genetic polymorphisms in the EPHX2 gene and an array of 118 diseases in the circulatory system using a large sample from the UK Biobank (N = 307,516). The diseases in electronic health records were mapped to the phecode system, which was more representative of independent phenotypes. Survival analyses were employed to examine the effects of EPHX2 variants on CVD incidence, and a phenome-wide association study was conducted to study the impact of EPHX2 polymorphisms on 62 traits, including blood pressure, blood lipid levels, and inflammatory indicators. RESULTS: A novel association between the intronic variant rs116932590 and the phenotype "aneurysm and dissection of heart" was identified. In addition, the rs149467044 and rs200286838 variants showed nominal evidence of association with arterial aneurysm and cerebrovascular disease, respectively. Furthermore, the variant rs751141, which was linked with a lower hydrolase activity of sEH, was significantly associated with metabolic traits, including blood levels of triglycerides, creatinine, and urate. CONCLUSIONS: Multiple novel associations observed in the present study highlight the important role of EPHX2 genetic variation in the pathogenesis of CVDs.

The Expression and Functional Roles of Epoxide Hydrolase 2 in Hepatocellular Carcinoma / 中国生物化学与分子生物学报

The expression, function and prognostic significance of epoxide hydrolase 2 (EPHX2) in hepatocellular carcinoma (HCC) were comprehensively analyzed through collecting HCC tissues and public database. The GEO and MitoCarta databases were used to identify the mitochondria-related differentially expressed genes (DEGs) in HCC. The Cancer Genome Atlas (TCGA) database was applied to analyze the expression levels of DEGs in HCC, including EPHX2 and its co-expressed genes. The R package was applied to draw the Kaplan-Meier survival curve and gene function enrichment analysis. The STRING database and GSEA software were used to analyze the protein-protein interaction (PPI) network and gene set enrichment analysis. qPCR and GEO database were applied to verify the expression level of EPHX2 in HCC. In the present study, a total of 15 mitochondria-related DEGs were identified in HCC. The expression of EPHX2 in HCC was significantly decreased compared to the normal liver tissues (P < 0. 01). The expression of EPHX2 was related to gender, tumor stage and grade in HCC, but not associated with age, T stage, et al in HCC. Moreover, compared with the patients with lower expression of EPHX2, patients with higher expression of EPHX2 had a better prognosis. EPHX2 was associated with fatty acid degradation. In addition, PPI results indicated that HAO1, AGXT, ACOX1, GSTκ1, SCP-2, CAT, CYP2C8, CYP2C9, CYP2B6, and CYP2J2 were co-expressed with EPXH2 in HCC. Furthermore, GSEA results showed that the group with lower expression of EPHX2 was positively correlated with the gene set of liver cancer cell proliferation and liver cancer recurrence. qPCR and GEO database results verified that the expression of EPHX2 was significantly decreased in HCC. The expression of EPHX2 was decreased in HCC, strongly suggesting that EPHX2 might function as a tumor suppressor gene in HCC. However, the potential mechanism of EPHX2 in HCC needs to be further verified.

Soluble epoxide hydrolase (Ephx2) silencing attenuates the hydrogen peroxide-induced oxidative damage in IEC-6 cells.

INTRODUCTION: Oxidative stress can cause intestinal disease. Soluble epoxide hydrolase (sEH, Ephx2) is related to cell apoptosis. The effect of Ephx2 on the H2O2-induced oxidative damage remains unclear. Thus, we aimed to explore the effect of Ephx2 on oxidative damage and the underlying potential mechanism. MATERIAL AND METHODS: The cell viability was determined using cell counting kit-8 (CCK-8) assay. The reactive oxygen species (ROS), apoptosis, and mitochondrial membrane potential (MMP) were examined using flow cytometry analysis. Commercial kits were applied to respectively determine the lactate dehydrogenase (LDH) leakage, malondialdehyde (MDA) content, and superoxide dismutase (SOD) activity. The expressions of target factors were measured by conducting quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blot. RESULTS: We found that knockdown of Ephx2 enhanced the viability of H2O2-treated IEC-6 cells, and that si-Ephx2 reduced the ROS level, MMP loss, and apoptosis in comparison to the H2O2 model group. Knockdown of Ephx2 was found to decrease LDH activity and MDA content, and to improve the SOD activity in comparison to those in the H2O2 model group. Knockdown of Ephx2 reduced the expressions of Fas, Fasl, Bax, and cleavedcaspase-3 and elevated the expression of Bcl-2 in H2O2-treated IEC-6 cells. Furthermore, we observed that knockdown of Ephx2 enhanced the phosphorylation of PI3K, Akt, and GSK3ß, which were reduced by the treatment of H2O2. In addition, the anti-apoptotic effect of si-Ephx2 was enhanced in the presence of AUDA-pharmacological Ephx2 inhibitor. CONCLUSIONS: Ephx2 silencing inhibited H2O2-induced oxidative damage. The PI3K/Akt/GSK3ß pathway was related to the effect of si-Ephx2. Our study provided a potential target for the prevention of intestinal injury.

A Soluble Epoxide Hydrolase Inhibitor, 1-TrifluoromethoxyPhenyl-3-(1-Propionylpiperidin-4-yl) Urea, Ameliorates Experimental Autoimmune Encephalomyelitis.

Polyunsaturated fatty acids (PUFAs) are essential FAs for human health. Cytochrome P450 oxygenates PUFAs to produce anti-inflammatory and pain-resolving epoxy fatty acids (EpFAs) and other oxylipins whose epoxide ring is opened by the soluble epoxide hydrolase (sEH/Ephx2), resulting in the formation of toxic and pro-inflammatory vicinal diols (dihydroxy-FAs). Pharmacological inhibition of sEH is a promising strategy for the treatment of pain, inflammation, cardiovascular diseases, and other conditions. We tested the efficacy of a potent, selective sEH inhibitor, 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl) urea (TPPU), in an animal model of multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE). Prophylactic TPPU treatment significantly ameliorated EAE without affecting circulating white blood cell counts. TPPU accumulated in the spinal cords (SCs), which was correlated with plasma TPPU concentration. Targeted lipidomics in EAE SCs and plasma identified that TPPU blocked production of dihydroxy-FAs efficiently and increased some EpFA species including 12(13)-epoxy-octadecenoic acid (12(13)-EpOME) and 17(18)-epoxy-eicosatrienoic acid (17(18)-EpETE). TPPU did not alter levels of cyclooxygenase (COX-1/2) metabolites, while it increased 12-hydroxyeicosatetraenoic acid (12-HETE) and other 12/15-lipoxygenase metabolites. These analytical results are consistent with sEH inhibitors that reduce neuroinflammation and accelerate anti-inflammatory responses, providing the possibility that sEH inhibitors could be used as a disease modifying therapy, as well as for MS-associated pain relief.

Epoxide hydrolase 3 (Ephx3) gene disruption reduces ceramide linoleate epoxide hydrolysis and impairs skin barrier function.

The mammalian epoxide hydrolase (EPHX)3 is known from in vitro experiments to efficiently hydrolyze the linoleate epoxides 9,10-epoxyoctadecamonoenoic acid (EpOME) and epoxyalcohol 9R,10R-trans-epoxy-11E-13R-hydroxy-octadecenoate to corresponding diols and triols, respectively. Herein we examined the physiological relevance of EPHX3 to hydrolysis of both substrates in vivo. Ephx3-/- mice show no deficiency in EpOME-derived plasma diols, discounting a role for EPHX3 in their formation, whereas epoxyalcohol-derived triols esterified in acylceramides of the epidermal 12R-lipoxygenase pathway are reduced. Although the Ephx3-/- pups appear normal, measurements of transepidermal water loss detected a modest and statistically significant increase compared with the wild-type or heterozygote mice, reflecting a skin barrier impairment that was not evident in the knockouts of mouse microsomal (EPHX1/microsomal epoxide hydrolase) or soluble (EPHX2/sEH). This barrier phenotype in the Ephx3-/- pups was associated with a significant decrease in the covalently bound ceramides in the epidermis (40% reduction, p < 0.05), indicating a corresponding structural impairment in the integrity of the water barrier. Quantitative LC-MS analysis of the esterified linoleate-derived triols in the murine epidermis revealed a marked and isomer-specific reduction (∼85%) in the Ephx3-/- epidermis of the major trihydroxy isomer 9R,10S,13R-trihydroxy-11E-octadecenoate. We conclude that EPHX3 (and not EPHX1 or EPHX2) catalyzes hydrolysis of the 12R-LOX/eLOX3-derived epoxyalcohol esterified in acylceramide and may function to control flux through the alternative and crucial route of metabolism via the dehydrogenation pathway of SDR9C7. Importantly, our findings also identify a functional role for EPHX3 in transformation of a naturally esterified epoxide substrate, pointing to its potential contribution in other tissues.

The Human Genetic Variants CYP2J2 rs2280275 and EPHX2 rs751141 and Risk of Diabetic Nephropathy in Egyptian Type 2 Diabetic Patients.

BACKGROUND: Diabetic nephropathy (DN), the primary driver of end-stage kidney disease, is a problem with serious consequences for society's health. Single nucleotide polymorphisms (SNPs) can define differences in susceptibility to DN and aid in development of personalized treatment. Giving the importance of epoxyeicosatrienoic acids (EETs) in kidney health, we aimed to study the association between two SNPs in the genes controlling synthesis and degradation of EETs (CYP2J2 rs2280275 and EPHX2 rs751141 respectively) and susceptibility of type 2 diabetes mellitus (T2DM) patients to develop DN. PATIENTS AND METHODS: Two hundred subjects were enrolled and categorized into three groups: group I (80 T2DM patients with DN), group II (60 T2DM patients without DN) and group III (60 healthy controls). Urea, creatinine, albumin/creatinine ratio (ACR), and eGFR were measured for all participants. Genotyping of CYP2J2 rs2280275 and EPHX2 rs751141 was done by real time PCR. RESULTS: There was no significant difference between the studied groups regarding CYP2J2 rs2280275. In contrast, EPHX2 rs751141 was associated with increased risk of DN under a dominant model (GG vs GA+AA: OR=0.375; 95% CI (0.19-0.75), P=0.006) in unadjusted model and after adjustment for age and sex (OR=0.440; 95% CI (0.21-0.92), P=0.029), recessive model (AA vs GG+GA: OR=0.195; 95% CI (0.05-0.74), P=0.017) and additive model (GA vs GG+AA): OR=0.195; 95% CI (0.05-0.74), P=0.017). CONCLUSION: CYP2J2 rs2280275 was not associated with DN predisposition. However, EPHX2 rs751141 could be a genetic marker for development and progression of DN among Egyptian T2DM patients.