Cytochrome P450 Epoxygenase Research Articles

Epoxyeicosatrienoic Acids and Fibrosis: Recent Insights for the Novel Therapeutic Strategies.

Organ fibrosis often ends in eventual organ failure and leads to high mortality. Although researchers have identified many effector cells and molecular pathways, there are few effective therapies for fibrosis to date and the underlying mechanism needs to be examined and defined further. Epoxyeicosatrienoic acids (EETs) are endogenous lipid metabolites of arachidonic acid (ARA) synthesized by cytochrome P450 (CYP) epoxygenases. EETs are rapidly metabolized primarily via the soluble epoxide hydrolase (sEH) pathway. The sEH pathway produces dihydroxyeicosatrienoic acids (DHETs), which have lower activity. Stabilized or increased EETs levels exert several protective effects, including pro-angiogenesis, anti-inflammation, anti-apoptosis, and anti-senescence. Currently, intensive investigations are being carried out on their anti-fibrotic effects in the kidney, heart, lung, and liver. The present review provides an update on how the stabilized or increased production of EETs is a reasonable theoretical basis for fibrosis treatment.

Hydrolysis of glycerophosphocholine epoxides by human group IIA, V, and X secretory phospholipases A2.

This study was prompted by recent reports that epoxyeicosatrienoic (EET) and epoxyeicosatetraenoic (EEQ) acids accelerate tumor growth and metastasis by stimulation of angiogenesis, while eicosapentaenoic (EPA) and epoxydocosapentaenoic (EDP) acids inhibit angiogenesis, tumor growth, and metastasis. Cytochrome P450 epoxygenases convert arachidonic to EET, eicosapentaenoic acid to EEQ, and docosahexaenoic acid to EDP, which are found both in free form and esterified to glycerophosphocholine (GPC). Both free and esterified epoxy (EP) acids are also formed during lipid autoxidation. For biological activity, the GPC-EP requires hydrolysis, which we presumed could occur by sPLA2 s located in proximity of lipoproteins carrying the lipid epoxides. The plasma lipoproteins were isolated by ultracentrifugation and analyzed by LC/ESI-MS. The GPC-EPs were identified by reference to standards and to retention times of phospholipid masses. The GPC-EP monoepoxides (corrected for isobaric ether overlaps) in stored human LDL, HDL, HDL3 , or APHDL ranged from 0 to 1 nmol/mg protein, but during 4-h incubation at 37°C increased to 1-5 nmol/mg protein. An incubation of autoxidized LDL, HDL, or HDL3 with 1 μg/ml of group V or X sPLA2 resulted in complete hydrolysis of diacyl GPC epoxide esters. Group IIA sPLA2 at 1 μg/ml failed to produce significant hydrolysis in 4 h, but at 2.5 μg/ml in 8 h yielded almost 80% hydrolysis, which represented complete diacyl GPC-EP hydrolysis. The present study shows that group IIA, V, and X sPLA2 s are capable of extensive hydrolysis of PtdCho epoxides of autoxidized plasma lipoproteins. Therefore, all three human sPLA2 s were potentially capable of inducing epoxide biological activity in vivo.

Cytochrome P450-epoxygenated fatty acids inhibit M\xfcller glial inflammation

Free fatty acid dysregulation in diabetics may elicit the release of inflammatory cytokines from Müller cells (MC), promoting the onset and progression of diabetic retinopathy (DR). Palmitic acid (PA) is elevated in the sera of diabetics and stimulates the production of the DR-relevant cytokines by MC, including IL-1β, which induces the production of itself and other inflammatory cytokines in the retina as well. In this study we propose that experimental elevation of cytochrome P450 epoxygenase (CYP)-derived epoxygenated fatty acids, epoxyeicosatrienoic acid (EET) and epoxydocosapentaenoic acid (EDP), will reduce PA- and IL-1β-induced MC inflammation. Broad-spectrum CYP inhibition by SKF-525a increased MC expression of inflammatory cytokines. Exogenous 11,12-EET and 19,20-EDP significantly decreased PA- and IL-1β-induced MC expression of IL-1β and IL-6. Both epoxygenated fatty acids significantly decreased IL-8 expression in IL-1β-induced MC and TNFα in PA-induced MC. Interestingly, 11,12-EET and 19,20-EDP significantly increased TNFα in IL-1β-treated MC. GSK2256294, a soluble epoxide hydrolase (sEH) inhibitor, significantly reduced PA- and IL-1β-stimulated MC cytokine expression. 11,12-EET and 19,20-EDP were also found to decrease PA- and IL-1β-induced NFκB-dependent transcriptional activity. These data suggest that experimental elevation of 11,12-EET and 19,20-EDP decreases MC inflammation in part by blocking NFκB-dependent transcription and may represent a viable therapeutic strategy for inhibition of early retinal inflammation in DR.

Differential Effects of Combined Soluble Epoxide Hydrolase inhibitor t ‐TUCB and n‐3 Epoxide in Diet‐Induced Obesity

Brown adipose tissue (BAT) is a promising target for obesity treatment and prevention by increasing energy expenditure through nonshivering thermogenesis. N-3 epoxides, such as epoxyeicosatetraenoic acids (EEQs) and epoxydocosapentaenoic acids (EDPs), are produced by cytochrome P-450 epoxygenases from n-3 polyunsaturated fatty acids, which have shown many beneficial effects in vascular dilation, inflammation, and cell growth/differentiation. However, the epoxides are unstable and quickly metabolized to less active diols by soluble epoxide hydrolase (sEH), a cytosolic enzyme encoded by the Ephx2 gene. We have previously demonstrated that t-TUCB, a potent sEH inhibitor, decreased serum triglycerides (TG) level by increasing protein expression of lipid metabolic genes in the BAT of diet-induced obese mice. This study aims to investigate the preventive effects of t-TUCB alone or combined with 19,20-EDP or 17,18-EEQ on BAT activation in the development of diet-induced obesity and associated metabolic disorders. 8-week old male C57BL6/J mice were fed a high-fat diet (45% kcal from fat) and received either of the following treatment: the vehicle control, t-TUCB alone (T) (3 mg/kg/day), or t-TUCB combined with 19,20-EDP (0.05 mg/kg/day) (T+EDP) or 17,18-EEQ (0.05 mg/kg/day) (T+EEQ) (n=5-6 per group) via osmotic minipump delivery at the subcutaneous area near the interscapular BAT for 6 weeks. Mice were examined for changes in bodyweight, food intake, glucose and insulin tolerance tests, cold tolerance tests, and indirect calorimetry before the mice were euthanized for further biochemical analysis. Although there were no differences in food intake among groups, there were small but significant increases in bodyweight in both T and T+EDP groups. While there were no differences in insulin tolerance tests among groups, the T+EEQ group showed a trend of improvement in glucose tolerance tests, and both T+EEQ and T+EDP groups showed significant improvement in fasting glucose compared to the controls (208.8 ± 36.43 mg/dl vs. 214 ± 10.81 mg/dL vs. 262.8 ± 8.9 mg/dL). The core body temperature was not changed by t-TUCB (37.34 ± 0.08 °C), but were significantly increased in the T+EDP (37.85±0.06 °C) and T+EEQ (38.1± 0.09 °C) groups compared to the controls (37.30±0.14°C). Consistently, both T+EDP and T+EEQ groups showed significantly better cold tolerance in the cold tolerance tests. Furthermore, heat production measured by indirect calorimetry was significantly increased in the T+EEQ group but not in the T and T+EDP groups compared to the controls. In addition, there were no differences in serum total cholesterol and non-esterified free fatty acids levels among all groups, but serum TG levels were significantly decreased in the T+EDP (45.75± 2.23 mg/dL) and T+EEQ (38.32± 6.67 mg/dL) groups compared to the controls (69.73± 7.2 mg/dL). Our results suggest that sEH pharmacological inhibition by t-TUCB combined with n-3 epoxide may prevent high-fat diet-induced glucose and lipid disorders, in part through increased thermogenesis.

Serum Soluble Epoxide Hydrolase Related Oxylipins and Depression in Patients With Type 2 Diabetes: An Exploratory Lipidomic Study

Type 2 diabetes mellitus (T2DM) carries an increased risk for depression. Perturbations in polyunsaturated fatty acid (PUFA) metabolism have been observed in depression and T2DM. PUFAs can be converted by cytochrome P450 epoxygenases (CYP450s) into epoxides, which resolve inflammation, and epoxides are metabolized by soluble epoxide hydrolase (sEH) into inactive or cytotoxic diols. Here, we survey these epoxide and diol “oxylipins” in T2DM patients with and without depression.

The Role of Epoxyeicosatrienoic Acids in Cardiac Remodeling.

Epoxyeicosatrienoic acids (EETs) are metabolites of arachidonic acid by cytochrome P450 (CYP) epoxygenases, which include four regioisomers: 5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET. Each of them possesses beneficial effects against inflammation, fibrosis, and apoptosis, which could combat cardiovascular diseases. Numerous studies have demonstrated that elevation of EETs by overexpression of CYP2J2, inhibition of sEH, or treatment with EET analogs showed protective effects in various cardiovascular diseases, including hypertension, myocardial infarction, and heart failure. As is known to all, cardiac remodeling is the major pathogenesis of cardiovascular diseases. This review will begin with the introduction of EETs and their protective effects in cardiovascular diseases. In the following, the roles of EETs in cardiac remodeling, with a particular emphasis on myocardial hypertrophy, apoptosis, fibrosis, inflammation, and angiogenesis, will be summarized. Finally, it is suggested that upregulation of EETs is a potential therapeutic strategy for cardiovascular diseases. The EET-related drug development against cardiac remodeling is also discussed, including the overexpression of CYP2J2, inhibition of sEH, and the analogs of EET.

Extrahepatic cytochrome P450 epoxygenases: pathophysiology and clinical significance in human gastrointestinal cancers.

Cytochrome P450 (CYP) epoxygenases, a multi-gene superfamily of heme-containing enzymes, are commonly known to metabolize endogenous arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs). The role of CYPs is mostly studied in liver drugs metabolism, cardiac pathophysiology, and hypertension fields. Particularly, the biological functions of these enzymes have increasingly attracted a growing interest in cancer biology. Most published studies on CYPs in cancer have been limited to their role as drug metabolizing systems. The activity of these enzymes may affect drug pharmacokinetics and bioavailability as well as exogenous compounds turnover. Some CYP isoforms are selectively highly expressed in tumors, suggesting a potential mechanistic role in promoting resistance to chemotherapy. Majority of drugs elicit their effects in extrahepatic tissues whereby their metabolism can significantly determine treatment outcome. Nonetheless, the role of extrahepatic CYPs is not fully understood and targeting these enzymes as effective anti-cancer therapies are yet to be developed. This review article summarizes an up-to-date body of information from published studies on CYP enzymes expression levels and pathophysiological functions in human normal and malignant gastrointestinal (GI) tract tissues. Specifically, we reviewed and discussed the current research initiatives by emphasizing on the clinical significance and the pathological implication of CYPs in GI malignancies of esophagus, stomach, and colon.

Regulation of the cytochrome P450 epoxyeicosanoid pathway is associated with distinct histologic features in pediatric non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a significant health burden in obese children for which there is currently no specific therapy. Preclinical studies indicate that epoxyeicosanoids, a class of bioactive lipid mediators that are generated by cytochrome P450 (CYP) epoxygenases and inactivated by the soluble epoxide hydrolase (sEH), play a protective role in NAFLD. We performed a comprehensive lipidomics analysis using liver tissue and blood samples of 40 children with NAFLD. Proteomics was performed to determine CYP epoxygenase and sEH expressions. Hepatic epoxyeicosanoids significantly increased with higher grades of steatosis, while their precursor PUFAs were unaltered. Concomitantly, total CYP epoxygenase activity increased while protein level and activity of sEH decreased. In contrast, hepatic epoxyeicosanoids showed a strong decreasing trend with higher stages of fibrosis, accompanied by a decrease of CYP epoxygenase activity and protein expression. These findings suggest that the CYP epoxygenase/sEH pathway represents a potential pharmacologic target for the treatment of NAFLD.

The profiling of lipid metabolites in the nasal lavage fluid of a murine model of allergic rhinitis

Allergic rhinitis (AR) is a complex nasal disease accompanied with mast cell and/or eosinophils-induced allergic symptoms. Although several types of lipid mediators regulate the development of AR, the entire profile of their production is unknown. The aim of study is to describe the lipid profiling in nasal lavage fluid (NALF) of ovalbumin (OVA)-induced AR and to compare the profiling with that of non-allergic LPS-induced rhinitis in mice. AR or non-AR model were generated by repeated administration of OVA or by single administration of LPS intranasally to female BALB/c mice. The lipid profiling of NALF was analyzed with liquid chromatography tandem mass spectrometry (LC-MS/MS). Based on the severity of allergic symptoms, the lipid profile in AR was examined by using the NALF with 5 or 10 episodes of OVA challenges. LC-MS based comprehensive analysis identified 15 types of lipid metabolites in AR. Almost of all metabolites showed higher volume in AR than that in non-AR. The metabolites catalyzed by 12/15-lipoxygenase (LO) rather than cyclooxygenase, cytochrome P450 epoxygenase and auto-oxidation were predominantly produced in NALF with AR regardless of disease stage. 12/15-LO metabolites mainly composes a unique lipid profile of NALF in AR. Our analysis reveals the unique profile of lipid production in the nasal cavity with AR model mice and may help for the better understanding of disease mechanism.

Neuroinflammation is able to downregulate cytochrome P450 epoxygenases 2J3 and 2C11 in the rat brain

Cytochrome P450 (CYP) epoxygenases have been considered the main producers of epoxyeicosatrienoic acids (EETs) through the oxidation of arachidonic acid (AA). EETs display various biological properties, notably their powerful anti-inflammatory activities. In the brain, EETs have proven to be neuroprotective and to improve neuroinflammation. However, it is known that inflammation could modify CYP expression. We have previously reported that an inflammatory process in astrocytes is able to down-regulate CYP2J3 and CYP2C11 mRNA, protein levels, and activity (Navarro-Mabarak et al., 2019). In this work, we evaluated the effect of neuroinflammation in protein expression of CYP epoxygenases in the brain. Neuroinflammation was induced by the intraperitoneal administration of LPS (1 mg/kg) to male Wistar rats and was corroborated by IL-6, GFAP, and Iba-1 protein levels in the cortex over time. CYP2J3 and CYP2C11 protein levels were also evaluated in the cortex after 6, 12, 24, 48, and 72 h of LPS treatment. Our results show for the first time that neuroinflammation is able to downregulate CYP2J3 and CYP2C11 protein expression in the brain cortex.

NLRX1 knockout aggravates lipopolysaccharide (LPS)-induced heart injury and attenuates the anti-LPS cardioprotective effect of CYP2J2/11,12-EET by enhancing activation of NF-κB and NLRP3 inflammasome

NLRX1 weakens lipopolysaccharide (LPS)-induced NF-κB activation on immune cells. Cytochrome P450 epoxygenase 2J2 (CYP2J2) attenuates LPS-induced cardiac injury by inhibiting NF-κB activation. However, it is still unclear whether NLRX1 could reduce LPS-induced heart damage and whether it is involved in the anti-LPS cardioprotective effect of CYP2J2. In this study, we found that NLRX1 knockout further exacerbated LPS-induced heart injury and up-regulated the proinflammatory cytokines in serum and heart tissue, and weakened the inhibitory effect of CYP2J2 on the harmful effects caused by LPS. We also found that LPS treatment induced ubiquitination of NLRX1 and promoted its binding to IKKα/β in myocardial tissue, which should theoretically inhibit NF-κB activation. However, LPS eventually leads to activation of NF-κB and NLRP3 inflammasome. Under the action of LPS, CYP2J2 further promoted the ubiquitination of NLRX1 and its binding to IKKα/β, impaired NF-κB activation and NLRP3 inflammasome activation. NLRX1 knockout notably aggravated LPS-induced NF-κB activation and NLRP3 inflammasome activation, and attenuated the inhibitory effects of CYP2J2 on NF-κB signal and NLRP3 inflammasome. More, CYP2J2 reduced LPS-induced reactive oxygen species (ROS) production and mitochondrial depolarization in heart cells, thereby inhibiting NLRP3 inflammasome activation. NLRX1 knockdown aggravated mitochondrial depolarization induced by LPS and weakened the protective effect of CYP2J2 on mitochondrial potential, although it had no significant effect on reactive oxygen species production. Together, these findings demonstrated that NLRX1 knockout aggravated LPS-induced heart injury and weakened the anti-LPS cardioprotective effect of CYP2J2 by enhancing activation of NF-κB and NLRP3 inflammasome.

Effects of hemodialysis on plasma oxylipins.

Chronic kidney disease (CKD) is an important risk factor for cardiovascular and all‐cause mortality. Survival rates among end‐stage renal disease (ESRD) hemodialysis patients are poor and most deaths are related to cardiovascular disease. Oxylipins constitute a family of oxygenated natural products, formed from fatty acid by pathways involving at least one step of dioxygen‐dependent oxidation. They are derived from polyunsaturated fatty acids (PUFAs) by cyclooxygenase (COX) enzymes, by lipoxygenases (LOX) enzymes, or by cytochrome P450 epoxygenase. Oxylipins have physiological significance and some could be of regulatory importance. The effects of decreased renal function and dialysis treatment on oxylipin metabolism are unknown. We studied 15 healthy persons and 15 CKD patients undergoing regular hemodialysis treatments and measured oxylipins (HPLC‐MS lipidomics) derived from cytochrome P450 (CYP) monooxygenase and lipoxygenase (LOX)/CYP ω/(ω‐1)‐hydroxylase pathways in circulating blood. We found that all four subclasses of CYP epoxy metabolites were increased after the dialysis treatment. Rather than resulting from altered soluble epoxide hydrolase (sEH) activity, the oxylipins were released and accumulated in the circulation. Furthermore, hemodialysis did not change the majority of LOX/CYP ω/(ω‐1)‐hydroxylase metabolites. Our data support the idea that oxylipin profiles discriminate ESRD patients from normal controls and are influenced by renal replacement therapies.

Cytochrome P450 Epoxygenase Cyp2j13 Regulates Murine Brown Adipogenesis

Abstract Objectives Brown adipocytes have emerged as novel targets for obesity treatment and prevention. Cytochrome P450 (CYP) epoxygenases, primarily CYP2J and CYP2C isoforms, produce epoxy fatty acids (EpFAs), which have been suggested to play important roles in the regulation of white adipogenesis and obesity. However, the roles of CYP epoxygenases in brown adipogenesis remain unexplored. In this study, we sought to characterize mRNA expression patterns of Cyp2j and 2c subfamily members during adipogenesis of human and murine brown adipocytes and in obese mice and investigate the impact of modulating the expression of Cyp2j13 on brown adipogenesis. Methods The mRNA expression of various Cyp2j and Cyp2c isoforms were examined throughout murine and human brown adipocyte differentiation and in the brown adipose tissue (BAT) of diet-induced obese and control mice. To induce epoxygenase overexpression, stable transfection of murine brown preadipocytes with either Cyp2j13 or a vector control was performed. Protein and mRNA expression of Cyp2j13 and brown marker genes were analyzed. Results Expression of murine Cyp2j isoforms Cyp2j6, Cyp2j8, Cyp2j9, and Cyp2j13, and the human isoform CYP2J2 consistently decreased throughout brown adipocyte differentiation, while expression of Cyp2c isoforms did not elicit consistent patterns. Moreover, Cyp2j expression in BAT was enhanced in diet-induced obese mice compared to the controls. Due to its high relative abundance and significance, Cyp2j13 was selected for further investigation. Overexpression of Cyp2j13 significantly suppressed murine brown adipocyte differentiation as evaluated by lipid accumulation and brown marker gene UCP1 expression. Conclusions Our results suggest that CYP epoxygenases may play important roles in brown adipogenesis. Cyp2j13, in particular, may be a novel target for brown adipogenesis, and consequently, for obesity treatment and prevention. Further studies using CYP2J inhibitors and Cyp2j13 knockdown are warranted. Funding Sources The work was supported by NIH 1R15DK114790–01A1 (to L.Z.), K99DK100736 and R00DK100736 (to A.B.).

Cytochrome P450 Epoxygenase-Dependent Activation of TRPV4 Channel Participates in Enhanced Serotonin-Induced Pulmonary Vasoconstriction in Chronic Hypoxic Pulmonary Hypertension.

Transient receptor potential vanilloid 4 (TRPV4) is a multi-functional non-selective channel expressed in pulmonary vasculatures. TRPV4 contributes to serotonin- (5-HT-) induced pulmonary vasoconstriction and is responsible in part for the enhanced 5-HT response in pulmonary arteries (PAs) of chronic hypoxia mice. Epoxyeicosatrienoic acid (EET) is an endogenous agonist of TRPV4 and is known to regulate vasoreactivity. The levels of EETs, the expression of cytochrome P450 (CYP) epoxygenase for EET production, and epoxide hydrolase for EET degradation are altered by chronic hypoxia. Here, we examined the role of EET-dependent TRPV4 activation in the 5-HT-mediated PA contraction. In PAs of normoxic mice, inhibition of TRPV4 with a specific inhibitor HC-067047 caused a decrease in the sensitivity of 5-HT-induced PA contraction without affecting the maximal contractile response. Application of the cytochrome P450 epoxygenase inhibitor MS-PPOH had no effect on the vasoreactivity to 5-HT. In contrast, inhibition of CYP epoxygenase or TRPV4 both attenuated the 5-HT-elicited maximal contraction to a comparable level in PAs of chronic hypoxic mice. Moreover, the inhibitory effect of MS-PPOH on the 5-HT-induced contraction was obliterated in PAs of chronic hypoxic trpv4−/− mice. These results suggest that TRPV4 contributes to the enhanced 5-HT-induced vasoconstriction in chronic hypoxic PAs, in part via the CYP-EET-TRPV4 pathway. Our results further support the notion that manipulation of TRPV4 function may offer a novel therapeutic strategy for the treatment of hypoxia-related pulmonary hypertension.

CYP2J2-produced epoxyeicosatrienoic acids attenuate ischemia/reperfusion-induced acute kidney injury by activating the SIRT1-FoxO3a pathway

Cytochrome P450 (CYP) epoxygenases can metabolize arachidonic acids to epoxyeicosatrienoic acids (EETs), which play a protective role in the renal system, but their involvement in ischemia/reperfusion (I/R)-induced acute kidney injury remains unknown. Here, using a rat model, we demonstrated that forced CYP2J2 expression attenuated I/R-induced renal dysfunction and protected histological integrity. We showed that CYP2J2 significantly decreased I/R-induced upregulation of blood urea nitrogen and serum creatinine and enhanced autophagy during I/R treatment. In addition, we determined the protective effect of CYP2J2 against I/R-caused apoptosis. We demonstrated that CYP2J2 overexpression attenuated the downregulation of SIRT1 and FoxO3a by I/R-induced injury. Moreover, exogenous 11,12-EET addition obviously promoted I/R-induced autophagic flux and suppressed I/R-induced apoptosis through SIRT1–FoxO3a signaling activation. Our data indicate that CYP2J2-produced EETs improve I/R-caused kidney injury by activating the SIRT1–FoxO3a signaling pathway, which protects from renal I/R injury.

Altered brain levels of arachidonic acid-derived inflammatory eicosanoids in a rodent model of anorexia nervosa

Increasing evidence underline the role of inflammation in the behavioral, emotional and cognitive dysregulations displayed in anorexia nervosa (AN). Among the inflammatory mediators acting at both peripheral and central levels, growing attention receives a class of lipids derived from arachidonic acid (AA), called eicosanoids (eiCs), which exert a complex, multifaceted role in a wide range of neuroinflammatory processes, peripheral inflammation, and generally in immune system function. To date, little is known about their possible involvement in the neurobiological underpinnings of AN. The present study evaluated whether the activity-based model of AN (ABA) may alter AA-metabolic pathways by changing the levels of AA-derived eiCs in specific brain areas implicated in the development of the typical anorexic-like phenotype, i.e. in prefrontal cortex, cerebral cortex, nucleus accumbens, caudate putamen, amygdala, hippocampus, hypothalamus and cerebellum. Our results point to brain region-specific alterations of the cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 epoxygenase (CYP) metabolic pathways rendering altered levels of AA-derived eiCs (i.e. prostaglandins, thromboxanes and hydroxyeicosatetraenoic acids) in response to induction of and recovery from the ABA condition. These changes, supported by altered messenger RNA (mRNA) levels of genes coding for enzymes involved in eiCs-related methabolic pathways (i.e., PLA2, COX-2, 5-LOX and 15-LOX), underlie a widespread brain dysregulation of pro- and anti-inflammatory eiC-mediated processes in the ABA model of AN. These data suggest the importance of eiCs signaling within corticolimbic areas in regulating key neurobehavioral functions and highlight eiCs as biomarker candidates for monitoring the onset and development of AN, and/or as possible targets for pharmacological management.

Elevated levels of arachidonic acid metabolites in follicular fluid of PCOS patients.

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder in reproductive-age women usually accompanied by lipid metabolic disorders. However, it remains unknown whether arachidonic acid (AA) and its metabolites in follicular fluid (FF) were altered in PCOS patients. This study was intended to measure the levels of AA and its metabolites in the FF of non-obese PCOS patients that underwent in vitro fertilization (IVF) and to explore the possible causes of the alterations. Thirty-nine non-obese women with PCOS and 30 non-obese women without PCOS were enrolled. AA and its metabolites were measured by liquid chromatography-mass spectrometry. The levels of AA metabolites generated via cyclooxygenase-2 (COX-2) pathway and cytochrome P450 epoxygenase pathway but not lipoxygenase (LOX) pathway were significantly higher in the FF of PCOS patients. The metabolites generated via COX-2 pathway were significantly correlated with levels of testosterone and fasting insulin in serum. The in vitro study further demonstrated that insulin but not testosterone could promote the IL-1β and hCG-induced COX-2 expression and prostaglandin E2 (PGE2) secretion in primary human granulosa cells. In conclusion, there was an elevation in AA metabolites in FF of PCOS patients. Insulin played a pivotal role in the increased AA metabolites generated via COX-2, which could be interpreted as another novel molecular pathophysiological mechanism of PCOS.

Protease-activated receptor 2 protects against myocardial ischemia-reperfusion injury through the lipoxygenase pathway and TRPV1 channels.

This study tests the hypothesis that the lipoxygenase (LOX) pathway mediates protease-activated receptor (PAR) 2-induced activation of the transient receptor potential vanilloid receptor 1 (TRPV1) to protect the heart from ischemia/reperfusion (I/R) injury. SLIGRL, a PAR2 activating peptide, was administered prior to reperfusion following left anterior descending coronary artery ligation in wild type (WT) and TRPV1 knockout (TRPV1-/-) mice. In a Langendorffly perfused heart I/R model, hemodynamic parameters, including left ventricular end-diastolic pressure, left ventricular developed pressure, coronary blood flow and left ventricular peak +dP/dt were evaluated after I/R. SLIGRL reduced the cardiac infarct size in WT and TRPV1-/- mice with a greater effect in the former strain (P<0.05). SLIGRL increased plasma levels of calcitonin gene-related peptide (CGRP) and substance P in WT (both P<0.05) but not in TRPV1-/- mice. Pretreatment with CGRP8-37 (a CGRP receptor antagonist) or RP67580 (a neurokinin-1 receptor antagonist) alone had no effect on SLIGRL-induced cardiac protection in either strain. However, combined administration of CGRP8-37 and RP67580 abolished SLIGRL-induced cardiac protection in WT but not in TRPV1-/- mice. Nordihydroguaiaretic acid (a general LOX inhibitor) and baicalein (a 12-LOX inhibitor), but not indomethacin (a cyclooxygenase inhibitor) and hexanamide (a selective cytochrome P450 epoxygenase inhibitor), abolished the protective effects of SLIGRL in WT (all P<0.05) but not in TRPV1-/- hearts. These data suggested that PAR2, possibly via 12-LOX, activates TRPV1 and leads to CGRP and substance P release to prevent I/R injury in the heart, indicating that the 12-LOX-TRPV1 pathway conveys cardiac protection to alleviate myocardial infarction.

The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.

Glaucoma is a leading cause of irreversible blindness worldwide. Vascular factors play a substantial role in the pathogenesis of glaucoma. Expressed in the vascular endothelium, cytochrome P450 (CYP) 2J2 is one of the CYP epoxygenases that metabolize arachidonic acid to produce epoxyeicosatrienoic acids and exert pleiotropic protective effects on the vasculature. In the present study, we investigated whether endothelium-specific overexpression of CYP2J2 (tie2-CYP2J2-Tr) protects against retinal ganglion cell (RGC) loss induced by glaucoma and in what way retinal vessels are involved in this process. We used a glaucoma model of retinal ischemia-reperfusion (I/R) injury in rats and found that endothelium-specific overexpression of CYP2J2 attenuated RGC loss induced by retinal I/R. Moreover, retinal I/R triggered retinal vascular senescence, indicated by up-regulated senescence-related proteins p53, p16, and β-galactosidase activity. The senescent endothelial cells resulted in pericyte loss and increased endothelial secretion of matrix metallopeptidase 9, which further contributed to RGC loss. CYP2J2 overexpression alleviated vascular senescence, pericyte loss, and matrix metallopeptidase 9 secretion. CYP2J2 suppressed endothelial senescence by down-regulating senescence-associated proteins p53 and p16. These 2 proteins were positively regulated by microRNA-128-3p, which was inhibited by CYP2J2. These results suggest that CYP2J2 protects against endothelial senescence and RGC loss in glaucoma, a discovery that may lead to the development of a potential treatment strategy for glaucoma.-Huang, J., Zhao, Q., Li, M., Duan, Q., Zhao, Y., Zhang, H. The effects of endothelium-specific CYP2J2 overexpression on the attenuation of retinal ganglion cell apoptosis in a glaucoma rat model.

Role of NF-κB in cytochrome P450 epoxygenases down-regulation during an inflammatory process in astrocytes

Cytochrome P450 (CYP) epoxygenases and their metabolic products, epoxyeicosatrienoic acids (EETs), have been proposed as important therapeutic targets in the brain. However, CYP expression can be modified by the presence of diverse pro-inflammatory cytokines and the subsequent activation of the NF-κB pathway. It has been indicated that CYP epoxygenases are down-regulated by inflammation in the heart, kidney and liver. However, up to this point, there has been no evidence regarding regulation of CYP epoxygenases during inflammation in the brain. Therefore, in order to explore the effects of inflammation and NF-κB activation in CYP2J3 and CYP2C11 regulation, rat primary astrocytes cultures were treated with LPS with and without IMD-0354 (selective NF-κB inhibitor). Cyp2j3 and Cyp2c11 mRNA expression was determined by qRT-PCR; protein expression was determined by immunofluorescence and by Western Blot and total epoxygenase activity was determined by the quantification of EETs by ELISA. NF-κB binding sites in Cyp2j3 and Cyp2c11 promoter regions were bioinformatically predicted and Electrophoretic Mobility Shift Assays (EMSA) were performed to determine if each hypothetic response element was able to bind NF-κB complexes. Results shown that LPS treatment is able to down-regulate astrocyte CYP2J3 and CYP2C11 mRNA, protein and activity. Additionally, we have identified NK-κB as the transcription factor involved in this regulation.