CYP2J2-produced epoxyeicosatrienoic acids contribute to the ferroptosis resistance of pancreatic ductal adenocarcinoma in a PPARγ-dependent manner / 中南大学学报(医学版)

Objective: Pancreatic ductal adenocarcinoma (PDAC) is one of the most malignant digestive tract tumors with a poor prognosis and high recurrence rate. Recently, ferroptosis resistance has been found in PDAC. However, the underlying mechanism of ferroptosis resistance has not been fully elucidated. Cytochrome P4502J2 (CYP2J2) is the main enzyme which mediates arachidonic acid to produce epoxyeicosatrienoic acids (EETs) in human tissues. It has been reported that EETs involve in the development of cancer, while the roles of EETs in PDAC and ferroptosis remain unclear. This study aims to explore the effect of CYP2J2/EETs on ferroptosis of human pancreatic ductal adenocarcinoma cells PANC-1 cells and the underlying mechanisms.Methods: The tumor tissues and para-carcinoma tissues of 9 patients with PDAC were collected and the expression of CYP2J2 was detected with real-time PCR and Western blotting. Enzyme-linked immunosorbent assay (ELISA) was used to detect the level of 8,9-dihydroxyeicosatrienoic acid (8,9-DHET), and the degradation product of 8,9-epoxyeicosa-trienoic acid (8, 9-EET). PANC-1 cells were used in this study. The ferroptosis inducer erastin was used to induce ferroptosis. The intracellular long-chain acyl-CoA synthetase 4 (ACSL4) protein level, lactate dehydrogenase (LDH) activity, malondialdehyde (MDA) content, Fe2+concentration, and cell survival were detected. The 8, 9-EET was pretreated to observe its effect on erastin-induced ferroptosis in PANC-1 cells. Lentivirus was used to construct a CYP2J2 knockdown cell line to observe its effect on the ferroptosis of PANC-1 cells induced by erastin. A peroxisome proliferation-activated receptor γ (PPARγ) blocker was used to observe the effect of 8, 9-EET on erastin-induced glutathione peroxidase 4 (GPX4) and MDA content in PANC-1 cells.Results: High expression of CYP2J2 was found in PDAC, accompanied by an increased level of 8, 9-DHET. The 8, 9-EET pretreatment significantly attenuated the PANC-1 cell death induced by erastin. The 8, 9-EET reduced the Fe2+ concentration, LDH activity and MDA content, and ACSL4 protein expression in erastin-treated PANC-1 cells. The 8,9-EET also restored the ferroportin (FPN) and ferroptosis suppressor protein 1 (FSP1) mRNA expressions in erastin-treated PANC-1 cells. But CYP2J2 knockdown exacerbated the erastin-induced ferroptosis in PANC-1 cells. Besides, CYP2J2 knockdown furtherly down-regulated the gene expression of FPN and FSP1. The 8, 9-EET increased the expression of GPX4 in the erastin-treated PANC-1 cells, which was eliminated by a PPARγ blocker GW9662. And GW9662 abolished the anti-ferroptosis effects of 8,9-EET. Conclusion: CYP2J2/EETs are highly expressed in PDAC tissues. EETs inhibit the ferroptosis via up-regulation of GPX4 in a PPARγ-dependent manner, which contributes to the ferroptosis resistance of PDAC.

Effect of 14,15-EET on Release of BDNF from Astrocytes and Neuronal Apoptosis in a Co-culture System after Oxygen-glucose Deprivation and Reperfusion / 华中科技大学学报(医学版)

Objective@#To investigate whether post-treatment of cultured astrocytes with 14,15-epoxyeicosatrienoic acids（14,15-EET）would increase the brain derived neurotrophic factor（BDNF）secretion after oxygen-glucose deprivation and reperfusion（OGD/R）, and if this effect would subsequently protect neurons during reperfusion after OGD in the co-cultured system.@*Methods@#Astrocytes and neurons were subjected to OGD/R. Exogenous 14,15-EET were applied to astrocytes in the reperfusion period and ELISA was then performed to measure BDNF secretion from astrocytes at different time points following OGD/R. After that，the OGD neurons were co-cultured with the astrocytes that were previously incubated with DMSO or 14,15-EET.Tunnel staining was used to detect neuronal apoptosis.@*Results@#BDNF secretion was significantly promoted by application of 14,15-EET on astrocytes in the reperfusion stage after OGD. Exposure of OGD neurons to astrocyte media previously conditioned with 14,15-EET reduced the neuronal apoptosis，but the pro-survival effect could be partly reversed by TrkB inhibitor k252a.@*Conclusion@#Exogenous administration of 14,15-EET augments BDNF secretion from astrocytes，which increases TrkB receptor occupancy on neurons and promotes neuronal survival after OGD/R.

Epoxyeicosatrienoic acids and metabolic syndrome :a literature review / 中华老年医学杂志

Metabolic syndrome including four components of central obesity ,hypertension , dyslipidemia ,and hyperglycemia ,is a complex syndrome. Along with the improvement of life conditions and population aging ,elderly people become a high risk population for metabolic syndrome , in whom the risks for cardio-and cerebro-vascular events are obviously increased. For the past few years ,epoxyeicosatrienoic acids (EETs)are found to improve patient's condition of hypertension , myocardial ischemia ,and atherosclerosis.Moreover ,EETs can up-regulate HO-1 expression.EETs and HO-1 interaction attenuates inflammatory response and oxidative stress ,regulates vascular endothelial function ,enhances insulin sensitivity ,and improves insulin resistance. This review takes aim at providing a new approach to diagnosis of ,and a new target for therapeutic intervention of metabolic syndrome.

Effect of 14,15-EET on Inflammatory Responses of BV2 Cells After Oxygen and Glucose Depriviation/Reoxygenation / 医药导报

Objective To explore the effect of 14,15-epoxyeicosatrienoic acids (14,15-EET) on the inflammatory response of BV2 cells under oxygen and glucose depriviation/reoxygenation (OGD/R) conditions.Methods BV2 cells were randomly divided into three groups,blank control group,vehicle control group,and 14,15-EET group.Under treatment of 14,15-EET,the concentration of inflammatory factor in BV2 cell culture media was detected by ELISA at different time points (reoxygenation for 0,3,6,12,24 h) after OGD1h.The viability of BV2 cells was detected by MTT assay at different time points.At the same conditions,using Transwell migration experiment,migration ability of BV2 cells was observed.Results The 14,15-EET group had the lower levels of inflammatory factor secretion,lower viability and weaker ability of migration than the vehicle control group.The above results were most statistically significant at OGD1h/R12h.Conclusion 14,15-EET can inhibit the inflammation of BV2 cells induced by the injury of OGD reperfusion.

CYP2C8-derived epoxyeicosatrienoic acids decrease oxidative stress-induced endothelial apoptosis in development of atherosclerosis: Role of Nrf2 activation / 华中科技大学学报（医学）（英德文版）

The aim of the present study is to investigate how cytochrome P450 enzymes (CYP) 2C8-derived epoxyeicosatrienoic acids (EETs) regulate the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway and protect against oxidative stress-induced endothelial injuries in the development and progression of atherosclerosis. In this study, cultured human umbilical vein endothelial cells (HUVECs) were transfected with CYP2C8 or pretreated with exogenous EETs (1 μmol/L) before TNF-α (20 ng/mL) stimulation. Apoptosis and intracellular ROS production were determined by flow cytometry. The expression levels of ROS-associated NAD(P)H subunits gp91 and p47, the anti-oxidative enzyme catalase (CAT), Nrf2, heme oxygenase-1 (HO-1) and endothelial nitric oxide synthase (eNOS) were detected by Western blotting. The results showed that CYP2C8-derived EETs decreased apoptosis of HUVECs treated with TNF-α. Pretreatment with 11, 12-EET also significantly blocked TNF-α-induced ROS production. In addition, 11, 12-EET decreased oxidative stress-induced apoptosis. Furthermore, the ability of 11, 12-EET to protect cells against TNF-α-induced apoptosis via oxidative stress was abrogated by transient transfection with Nrf2-specific small interfering RNA (siRNA). In conclusion, CYP2C8-derived EETs prevented TNF-α-induced HUVECs apoptosis via inhibition of oxidative stress associated with the Nrf2 signaling.

P-450-dependent Epoxygenase Pathway of Arachidonic Acid Is Involved in Myeloma-induced Angiogenesis of Endothelial Cells / 华中科技大学学报（医学）（英德文版）

P-450-dependent epoxygenase pathway of arachidonic acid and the products of epoxyeicosatrienoic acids (EETs) have been demonstrated to be involved in angiogenesis and tumor progression.This study examined the expression of EETs and the role of the pathway in the angiogenesis of multiple myeloma (MM).MM cell lines of U266 and RPMI8226 were cultured,and the EETs levels (11,12-EET and 14,15-EET) in the supematant were determined by ELISA.Human umbilical vein endothelial cells (HUVECs) were cultured and used for analysis of the angiogenesis activity of the two MM cell lines,which was examined both in vitro and in vivo by employing MTT,chemotaxis,tube formation and matrigel plug assays.11,12-EET and 14,15-EET were found in the supematant of the cultured MM cells.The levels of the two EETs in the supernatant of U266 cells were significantly higher than those in the RPMI8226 cell supematant (P＜0.05),and the levels paralleled the respective angiogenesis activity of the two different MM cell lines.17-octadecynoic acid (17-ODYA),as a specific inhibitor of P450 enzyme,suppressed HUVECs proliferation and tube formation induced by MM cells.Furthermore,17-ODYA decreased the EET levels in the supernatant of MM cells.These results suggest that EETs may play an important role in the angiogenesis of MM,and the inhibitor 17-ODYA suppresses this effect.

Arachidonic acid cytochrome P450 epoxygenases protect vessel endothelial cells against apoptosis induced by tumor necrosis factor-? / 中国病理生理杂志

AIM: To investigate whether endogenous endothelium-derived hyperpolarizing factors(EHDFs) produced by CYP epoxygenases BM3?F87V,2C11OR or CYP2J2 transfection was able to protect endothelial cells against apoptosis induced by tumor necrosis factor alpha.METHODS: Three or four passages of cultured bovine aortic endothelial cells(BAECs) were transfected with epoxygenases or the empty vector(pCB6).Cell viability was detected by MTT assay.Apoptosis of transfected endothelial cells was evaluated by DNA ladder assay,flow cytometry and morphological observations under fluorescence microscopy.RESULTS: Overexpression of CYP epoxygenases BM3?F87V,2C11OR,CYP2J2 increased cell viability respectively observed by MTT assay.The percentage of cells undergoing apoptosis was decreased in 2C11OR-,BM3F87V-or 2J2-transfected cells compared to the vector as evaluated by DNA fragment assay,flow cytometry analysis and morphological observations under fluorescence microscopy.CONCLUSION: Overexpression of CYP epoxygenases BM3?F87V,2C11OR or 2J2 increases cell viability and protects endothelial cells against TNF-?-induced apoptosis.These findings suggest new targets to investigate the endothelium-associated disorders and provide novel therapeutic strategies to treat them by modulating cytochrome P450 epoxygenases.

Relationship between delayed cardioprotection effect of 11,12-EET and phosphorylated ERK during ischemia and reperfusion in the rat myocardium / 中国病理生理杂志

AIM: In order to study the relationship of the activation of ERK and delayed cardioprotection of 11,12-EET.METHODS: A rat ischemia/reperfusion(I/R) model was replicated by ligating left anterior descending coronary artery 30 min followed by 60 min.The expression of ERK was detected with Western blotting,and the change of heart function during reperfusion was observed.RESULTS: The difference of myocardial function was prominent at (24 h) in I/R group compared with sham group,EET+I/R and EET+PD098059+I/R group.The activity of ERK at(24 h) in EET+I/R group was higher than sham group, and the activity of ERK in EET+PD098059+I/R group was lower than that in EET+(I/R) group;the expression of phosphorylated ERK1/ERK2 at(24 h) in EET+I/R group was more than that in I/R group,and the expression of phosphorylated ERK1/ERK2 in EET+PD098059+I/R group was less than EET+I/R group.CONCLUSION: 11,12-EET has a delayed cardioprotection effect,and this protection effect is involved in the activity of ERK and expression of phosphorylated ERK1/ERK2.