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CYP2J2 and EETs Protect against Oxidative Stress and Apoptosis in Vivo and in Vitro Following Lung Ischemia/Reperfusion

Background: Cytochrome P450 epoxygenase 2J2 (CYP2J2) metabolizes arachidonic acids to epoxyeicosatrienoic acids (EETs). EETs exert various biological effects, including anti-inflammatory, anti-apoptotic, pro-proliferation, pro-angiogenesis, anti-oxidation, and anti-fibrosis effects. However, little is known about the role of CYP2J2 and EETs in lung ischemia/reperfusion injury. In this study, we examined the effects of exogenous EETs or CYP2J2 overexpression on lung ischemia/reperfusion injury in vivo and in vitro. Methods and Results: CYP2J2 gene was stably transfected into rat lungs via pcDNA3.1-CYP2J2 plasmid delivery, resulting in increased EETs levels in the serum and lung. A rat model of lung ischemia/reperfusion injury was developed by clamping the left lung hilum for 1 hour, followed by reperfusion for 2 hours. We found that CYP2J2 overexpression markedly decreased the levels of oxidative stress and cell apoptosis in lung tissues induced by ischemia/reperfusion. Moreover, we observed that exogenous EETs, or CYP2J2 overexpression, enhanced cell viability, decreased intracellular reactive oxygen species (ROS) generation, inhibited mitochondrial dysfunction, and attenuated several apoptotic signaling events in a human pulmonary artery endothelial cells (HPAECs)-based anoxia/reoxygenation model. These apoptotic events included activation of NADPH oxidase, collapse of mitochondrial transmembrane potential, and activation of pro-apoptotic proteins and caspase-3. These effects were mediated, at least partially, by the PI3K/Akt signaling pathway. Conclusion: These results reveal that CYP2J2 overexpression and exogenous EETs can protect against oxidative stress and apoptosis following lung ischemia/reperfusion in vivo and in vitro, suggesting that increasing the level of EETs may be a novel promising strategy to prevent and treat lung ischemia/reperfusion injury.


Introduction
Lung ischemia/reperfusion injury (LIRI) occurs in various conditions such as lung transplantation, cardiopulmonary bypass, cardiac bypass surgery, sleeve lobectomy, pulmonary embolism, resuscitation from circulatory arrest, and trauma [1][2][3]. Clinical studies have indicated that an increase of LIRI-associated mortality and morbidity results in prolonged ventilation, postoperative pulmonary hypertension, longer stay in the intensive care unit, and increased hospital costs [4]. LIRI is a complex pathophysiological process, accompanied by oxidative stress, intracellular calcium overload, release of proin lammatory mediators, leu ocyte activation, and upregulation of molecules on cell surface membrane [5,6]. Due to the important role in the pulmonary air-blood barrier, pulmonary artery endothelial cells are recognized as important mediators of LIRI [7]. However, there is no effective method currently available to prevent LIRI, and the precise mechanism of LIRI remains elusive.
Arachidonic acid (AA), the most widely distributed polyunsaturated fatty acid in humans, is located in cell membranes in an inactive state. It can be released by phospholipases A2 in response to various stimuli, such as ischemia [8]. Free AA can then be activated by three enzymatic pathways, including the cyclooxygenase, the lipoxygenase, and the cytochrome P450 (CYP) monooxygenase pathways, to generate numerous active metabolites [9]. CYP epoxygenases, a superfamily of enzymes including the CYP2C and CYP2J subfamilies, metabolize AA to four regioisomeric epoxyeicosatrienoic acids (5,8,11,and 14, [10,11]. Among these, CYP2J2 is expressed in humans, mainly in the cardiovascular system [12]. In previous studies, EETs have been reported to exert diverse biological effects in the cardiovascular and renal systems, such as anti-in lammatory, anti-apoptotic, proproliferation, pro-angiogenesis, anti-oxidation, anti-ibrosis, and anti-hypertension effects [13][14][15][16][17][18].
Either exogenous EETs or CYP2J2 overexpression has been reported to attenuate ischemia/reperfusion injury or hypoxia-reoxygenation injury in myocardium and cerebrum by decreasing infarct size and apoptosis [15,19], suggesting that EETs and/or CYP2J2 overexpression might also exert a protective role in LIRI. We therefore hypothesized that an increase in EETs may protect against lung ischemia/reperfusion injury in vivo and in vitro.
In this study, we examined the bene icial effects of CYP2J2 overexpression and exogenous EETs on ischemia/reperfusion or anoxia-reoxygenation induced oxidative stress and apoptosis in vivo and in vitro. Our data demonstrate that EETs alleviate the levels of oxidative stress and apoptosis induced by LIRI and these effects are mediated, at least in part, by activation of the PI3K/A t signaling pathway.

Construction of the gene delivery vector
The coding DNA fragment of CYP2J2 or green luorescent protein ( FP) was subcloned into the pcDNA3.1 plasmid as previously described [13,20]. The pcDNA3.1-CYP2J2 and pcDNA3.1-FP plasmids were puri ied using the IA EN Plasmid axi Kit ( IA EN, Inc., Chatsworth, CA) according to the manufacturer's instructions. Animal experimental protocols were approved by the Institutional Animal Research Committee of  Tongji edical College and complied with the National Institutes of Health uidelines for the Care and se  of Laboratory Animals. Fifty healthy male Wistar rats (280-350 g) were obtained from the Experimental Animal Center of Hubei Province. After 1-wee adaptation, rats were randomly divided into ive groups (ten in each group) blan control group ( lan ), sham group ( ham), lung ischemia/reperfusion group (IR), pcDNA3.1-FP gene transfection and lung ischemia/reperfusion group (IR+ FP), and pcDNA3.1-CYP2J2 gene transfection and lung ischemia/reperfusion group (IR+2J2). Animals received a tail vein injection of 0.9% NaCl ( lan , ham, IR group) or an e ual volume of plasmid (IR+ FP, IR+2J2 group) at a dose of 3mg/ g body weight, once a wee for two consecutive wee s.

Gene delivery and animal model of lung ischemia/reperfusion injury
One wee after the second vein injection, rats in the IR groups were subjected to left thoracotomy via the ifth intercostal space. The left pulmonary hilar, including the left main bronchus, pulmonary artery, and pulmonary vein, were dissected and then occluded for 1 hour with a non-invasive arterial clamp, resulting in complete left lung ischemia and anoxia. At the end of the ischemic time, the clamp was removed. The left lung was then ventilated and reperfused for an additional 2 hours [21,22]. Time-matched sham rats underwent the identical thoracotomy and hilar dissection, but without hilar occlusion. All operations were carried out under intraperitoneal anesthesia and mechanical ventilation. Animals in the blan group did not receive any other treatment. Animals were sacri iced at the end of experiment. lood samples were ac uired from the left atria prior to sacri ice and the left lungs were then removed for further analysis.
Immunohistochemistry Immunohistochemical (IHC) staining was performed to detect the expression of CYP2J2 in lung tissues using a treptavidin/Peroxidase immunohistochemical it (Fuzhou aixin iotechnology Co. Ltd, Fujian, China) according to the manufacturer ' s instruction [23]. The results were counted by two investigators in a double-blind fashion. At least ive randomly-selected ields ( 400) were examined.

Detection of 14,15-DHET
EETs are unstable, and can be enzymatically hydrated to the corresponding stable metabolite dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases (sEH) [10]. To evaluate the in vivo EETs level, an ELI A it (Detroit R D, Detroit, I) was used to determine the concentrations of 14,15-DHET in the rat serum and lung homogenates according to the manufacturer's instructions, as previously described [14,19,24].
TUNEL staining for assessment of apoptosis Apoptotic cell death in the lung was detected in situ by terminal deoxynucleotidyl transferase (TdT)-mediated d TP-biotin nic end-labeling (T NEL) staining of fragmented DNA using an In itu Cell Death Detection Kit, POD (Roche, annheim, ermany). The procedure was performed according to the manufacturer's instructions. Cells were identi ied as T NEL positive if they showed dar brown staining under a light microscope. ore than ive randomly-selected high-power ields ( 400) were examined for T NEL-positive cells. The number of T NEL positive cells was scored and the percentage of apoptotic cells was calculated from the total number of cells.

Cell culture and gene transfection
Human pulmonary artery endothelial cells (HPAECs) ( ciencell Research Laboratories, Carlsbad, CA, A) were cultured in endothelial cell medium supplemented with 5% fetal bovine serum and penicillin/ streptomycin ( ciencell Research Laboratories). Cells were incubated at 37 C in a humidi ied atmosphere with 5% CO 2 /95% air. Cells were grown to 60% con luency and then transfected with pcDNA3.1/ FP or pcDNA3.1/CYP2J2 plasmids (0.2 ug DNA/cm 2 ) with the Fugene HD Transfection Reagent (Roche olecular iochemicals, Indianapolis, IN). After48-72 hours of transfection, the transfection ef iciency was determined by examining pcDNA3.1/ FP-transfected cells for FP luorescence under a luorescence microscope (NIKON) and by low cytometric analysis. The expression of CYP2J2 was con irmed by Western blotting.

Model of anoxia-reoxygenation and treatment of cultured cells
HPAECs were grown to 80% con luence in 6-well plates under an atmosphere of 95% air and 5% CO 2 . For the experimental groups, the medium on the culture plates was replaced with fresh medium without serum, and then subjected to anoxia-reoxygenation (AR). Cells were maintained at 37 C under an atmosphere of 95% N 2 and 5% CO 2 (anoxia). After 8 h of anoxia, cells were given fresh medium with serum and maintained in normal conditions (reoxygenation) for the next 16 h. The oxygen content (FiO2) during anoxia was continuously monitored to ensure that it remained below 1% (Pro-Ox 110, iospherix Ltd, Red ield, NY). The cells were pretreated with two inhibitors, LY294002 (PI3K inhibitor, 20 u ) and 14,15-EEZE (EETs inhibitor, 10 u , Cayman Chemical Co.) for 60 min before addition of vehicle or EETs (1u ), which were applied 60 min prior to AR until the end of the experiment. Normal control cells were treated with the same procedure under normal culture conditions.

Cell proliferation assay
Cell proliferation was monitored using a Cell Counting it-8 (CCK-8, eyotime, Haimen, China) according to the manufacture ' s instruction. rie ly, cells were seeded in 96-well plates at a density of 2 10 3 per well in 100 ul of complete medium. After being treated with the corresponding drugs and anoxiareoxygenation as described above, the cells were further incubated with 10 l of the CCK-8 reagent for 0.5 to 4 hours. The absorbance of 450 nm was measured with a microplate reader ( io-Te EL 800, A).
Detection of reactive oxygen species 2,7-Dichlorodihydro luorescein diacetate (DCFH-DA) ( igma-Aldrich, t. Louis, O, A) was used to measure the uantity of reactive oxygen species (RO ) produced by HPAECs following anoxia-reoxygenation. Non-ionized DCFH-DA, which itself is non-luorescent, can diffuse freely through the cell membrane. Once it is intracellular, DCFH-DA is deacetylated by nonspeci ic esterases to form DCFH, and then further oxidized by RO to yield the luorescent compound DCF. Cells were incubated with 10 DCFH-DA at 37 C for 20 min. After staining, cells were harvested immediately to detect DCF luorescence by low cytometry.
Measurement of mitochondrial transmembrane potential 5, 5 , 6, 6 -tetrachloro-1, 1 , 3, 3 -tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a mitochondriaspeci ic membrane potential-sensitive luorescent probe, was used to determine mitochondrial membrane potential. JC-1 accumulates in the mitochondrial matrix when mitochondrial membrane potential is high, forming J-aggregates which emit a bright yellow-red luorescence. In contrast, JC-1 presents itself as a green luorescent monomer with low levels of aggregation when mitochondrial membrane potential is low. During this assay, cells were seeded on 6-well plates. After corresponding treatment in each group, the cells were loaded with 10 JC-1 for 20 min at 37 C, and then washed and viewed under a luorescence microscope with a green excitation ilter at 543 nm and a blue excitation ilter at 488 nm to visualize yellow-red and green luorescence, respectively. ubse uently, the cells were harvested to detect luorescence by low cytometry.
Cells in 6-well plates were harvested with trypsin/EDTA, resuspended in the binding buffer, and stained with Annexin V-FITC and propidium iodide (PI) using an apoptosis detection it (Key en, Nanjing, China) according to the manufacturer's protocol. Within 1 h, the cells were subjected to low cytometric analysis with a CycleTE T PL ( ecton Dic inson, an Jose, CA). The annexin V positive (FL1-H) cells were counted for early stages of apoptosis (the lower right uadrant) and late apoptotic or necrotic cells (the upper right uadrant). The PI positive cells were counted for necrotic(the upper left uadrant) and late apoptotic or necrotic cells. The results were then uantitated using Cell uest and odFit analysis software.

Cellular Physiology and Biochemistry
Cellular Physiology and Biochemistry everly, A, A) and CYP2J2 (Abcam Inc., Cambridge, A, A). The horseradish peroxidase-conjugated secondary antibodies (goat anti-rabbit and goat anti-mice) were purchased from Jac son ImmunoResearch Laboratories (West rove, PA, A). lotting bands were uanti ied by densitometry using uantity One software ( io-Rad, Hercules, CA).

Statistical analysis
All data were expressed as mean standard deviation ( D). Comparisons between groups were performed by a one-way analysis of variance (ANOVA). tatistical signi icance was de ined as p< 0.05.

Delivery of pcDNA3.1-CYP2J2 induces CYP2J2 overexpression in the lung and increases 14,15-DHET levels in the serum and lung
Rats were injected with either pcDNA3.1-FP or pcDNA3.1-CYP2J2 once a wee for two consecutive wee s. One wee after the second gene delivery, CYP2J2 protein levels were increased in the lung of pcDNA3.1-CYP2J2transgenic rats, as evaluated by Western blot (Fig. 1A). oreover, immunohistochemical staining showed that the expression of CYP2J2 protein was signi icantly increased in lung tissues of transgenic rats, especially in the cytoplasm of vascular endothelial cells and epithelial cells. CYP2J2+ cells were stained brown and con ined to the cytoplasm in transgenic rats (Fig. 1 ), as described previously [26]. CYP2J2 metabolizes arachidonic acids to form EETs, which can be uic ly hydrolyzed to their corresponding DHETs that exhibit much more stable biological activity. Therefore, we measured the levels of 14,15-DHET in the serum and lung. Interestingly, pcDNA3.1-CYP2J2 injection remar ably increased 14,15-DHET levels in both the serum and lung (Fig. 1C).

Cellular Physiology and Biochemistry
Cellular Physiology and Biochemistry

Effects of CYP2J2 overexpression on oxidative stress and apoptosis in vivo
To evaluate the effects of the ischemia/reperfusion model on oxidative stress in this study, we measured the expression of pro-oxidative and anti-oxidative enzymes in lung tissues. As depicted in Fig. 2A, the expression of the pro-oxidative enzymes gp91, p47, and p67 was signi icantly increased in the lung tissues following ischemia/reperfusion, a phenomenon which was reversed by CYP2J2 overexpression. In contrast, CYP2J2 delivery remar ably inhibited the ischemia/reperfusion-induced reduction in OD1, OD2 and catalase protein levels (Fig. 2 ).
Lung tissue apoptosis or necrosis is an important event following ischemia/reperfusion injury, which is associated with the development of post-operative lung dysfunction [6]. In  ( ) raph re-T NEL-positive cells as compared with the blan and sham groups, an effect which was reversed by pcDNA3.1-CYP2J2 transfection. oreover, the expression of cleaved caspase-3 was remar ably increased in lung tissues after ischemia/reperfusion, an effect that was bloc ed by CYP2J2 delivery (Fig. 2D).

Effects of exogenous EETs on viability of HPAECs
To assess the impact of anoxia/reoxygenation on cell viability, we developed three different anoxia/reoxygenation models. We found that treatment of HPAECs with anoxia conditions for 8 h, followed by reoxygenation for the next 16 h, led to a signi icant decrease in cell viability (Fig. 9A). To test effects of exogenous EETs on the viability of HPAECs, we examined proliferation of HPAECs under normoxia and anoxia/reoxygenation conditions, respectively. We found that EETs had no signi icant effects on cell viability under normal conditions (Fig. 9 ). However, the cell viability remar ably decreased following anoxia/ reoxygenation, which was reversed by 11,12-EET and 14,15-EET (Fig. 9C).

EETs and CYP2J2 overexpression suppress ROS production induced by anoxia/ reoxygenationin HPAECs
RO play an important role in lung ischemia-reperfusion injury [2, 6]. Therefore, we examined RO production by low cytometry in anoxia/reoxygenation-treated HPAECs. The mean luorescence intensity of DCFH-DA, as detected by low cytometry, represented the RO level. The results indicated that pre-incubation with EETs signi icantly attenuated the anoxia/reoxygenation-induced increase in the RO level, while EETs alone did not show this effect (Fig. 3A and ). However, the effects of EETs in the anoxia/reoxygenation condition were largely inhibited when cells were pretreated with 14,15-EEZE. Transfection with pcDNA3.1-CYP2J2 displayed similar protective effects on RO production in anoxia/ reoxygenation treated cells (Fig. 3C and D).

Effects of EETs and CYP2J2 overexpression on apoptosis and mitochondrial transmembrane
The effects of exogenous EETs and CYP2J2 overexpression on the apoptosis levels of HPAECs were assessed by low cytometry. Our results revealed that pre-incubation with presents the mean percentage of Annexin V-positive cells in each group. (n=3 for each experiment *p < 0.05 vs. control p < 0.05 vs. AR or AR+ D O $ p<0.05 vs. groups of AR+EETs) (C and D) Apoptosis of HPAECs induced by anoxia/reoxygenation is decreased by CYP2J2 overexpression and increased by LY294002 (PI3K inhibitor) and 14,15-EEZE. These data are representative of 3 independent assays. (*p < 0.05 vs. control p < 0.05 vs. AR or AR+ FP $ p<0.05 vs. AR+CYP2J2). itochondrial transmembrane potential was determined by JC-1 and analyzed by low cytometry. The upper right uadrant represents cells with normal mitochondrial function, which emit yellow-red luorescence. The lower right uadrant represents cells with a collapsed mitochondrial transmembrane potential, which emit green luorescence.  EETs signi icantly decreased apoptosis induced by anoxia/reoxygenation. This protective effect was notably inhibited by LY294002 or 14,15-EEZE (Fig. 4A and ). However, EETs did not show this anti-apoptotic effect in cells under normal conditions. Li ewise, CYP2J2 overexpression demonstrated similar effects on apoptosis induced by anoxia/reoxygenation ( Fig. 4C and D).
Previous studies have shown that collapse of mitochondrial transmembrane potential is a sign of early programmed cell death [27,28]. Therefore, we determined mitochondrial transmembrane potential by JC-1 and analyzed it by low cytometry. We found that mitochondrial transmembrane potential remar ably decreased in the anoxia/reoxygenation treated group as compared with the groups incubated under normal conditions, an effect which was reversed by 14,15-EET ( Fig. 4E and F). The protective effect of 14,15-EET, however, was mar edly suppressed by LY294002 or 14,15-EEZE. In addition, similar effects were found in the cells transfected with pcDNA3.1/CYP2J2 (Fig. 4 and H).

Effects of 14,15-EET and CYP2J2 overexpression on anti-oxidative and pro-oxidative enzymes in HPAECs
Oxidative stress plays a central role throughout the process of lung ischemia/reperfusion injury. We investigated the regulation of anti-oxidative and pro-oxidative enzymes by 14,15-  EET and CYP2J2 overexpression in HPAECs. The results indicated that 14,15-EET and CYP2J2 overexpression signi icantly attenuated the anoxia/reoxygenation-induced increase in gp91 (a transmembrane protein), p47, p67 and NO 4 protein levels (Fig. 5A, , D and E). Those effects, however, were reversed by LY294002. In contrast, 14,15-EET and CYP2J2 overexpression remar ably increased the expression of the anti-oxidative proteins OD1, OD2, and catalase, which were obviously decreased in the anoxia/reoxygenation condition ( Fig. 5C and F). However, these protective effects were mar edly decreased by LY294002. Collectively, these results suggest that the anti-oxidative effect of 14,15-EET and CYP2J2 overexpression might be mediated by PI3K activation.

Effects of 14,15-EET and CYP2J2 overexpression on apoptosis-related protein expression
The expression of the anti-apoptotic proteins cl-2 and cl-xl was decreased after anoxia/reoxygenation treatment in HPAECs, an effect which was reversed by 14,15-EET and CYP2J2 overexpression ( Fig. 6A and C). In contrast, the expression of ax was increased after the anoxia/reoxygenation treatment, while this increase was attenuated by 14,15-EET and CYP2J2 overexpression ( Fig. 6A and C). oreover, the expression of cleaved caspase-3 was also increased after anoxia/reoxygenation treatment, while this increase was inhibited by 14,15-EET and CYP2J2 overexpression ( Fig. 6 and D). Remar ably, the effects of 14,15-EET and CYP2J2 overexpression were bloc ed by LY294002, supporting a role of the PI3K/A t signaling pathway in EET-mediated regulation of apoptosis.
-Anoxia/reoxygenation treatment signi icantly inhibited the expression of PI3K and the PI3K-dependant A t phosphorylation in HPAECs, which were reversed by 14,15-EET and CYP2J2 overexpression (Fig. 7A and ). Interestingly, these effects of 14,15-EET and CYP2J2 overexpression were notably bloc ed by LY294002.
Ta en together, these data indicate that exogenous 14,15-EET and CYP2J2 overexpression activate the PI3K/A t signaling pathway, which may contribute to the anti-oxidative and anti-apoptotic effects observed in vivo and in vitro following lung ischemia/reperfusion.

Discussion
The current study tested the effect of the administration of exogenous EETs or CYP2J2 transfection on lung ischemia/reperfusion-induced oxidative stress and apoptosis in vivo and in vitro. It has been documented that CYP2J2 is predominantly expressed in human cardiomyocytes and endothelial cells [13,29]. ased on previous studies [13,14], we chose the pcDNA3.1 plasmid as a vector to transfect CYP2J2. Immunohistochemical staining con irmed that CYP2J2 expression was remar ably elevated in lung tissues of transgenic rats, especially in vascular endothelial cells and epithelial cells. Elevated CYP2J2 effectively increased the concentration of circulating EETs. CYP2J2 overexpression in the lung signi icantly inhibited apoptosis and the levels of oxidative stress via regulating the expression of pro-oxidative (NADPH oxidase) and anti-oxidative enzymes ( OD1, OD2, catalase). Therefore, we further investigated the effects of lung ischemia/reperfusion and its possible mechanisms in vitro. We found that anoxia/reoxygenation treatment attenuated the viability of human pulmonary artery endothelial cells in a time-dependent manner, an effect that was reversed by pre-incubation with exogenous EETs. Exogenous EETs, particularly 14,15-EET, and CYP2J2 overexpression, suppressed RO production, inhibited apoptosis, These effects were bloc ed by 14,15-EEZE and LY294002. oreover, 14,15-EET and CYP2J2 overexpression signi icantly attenuated the anoxia/reoxygenation-induced increase in the expression of the pro-oxidative proteins gp91, p47, p67, and NO 4, the apoptosis-related protein ax, and the activity of caspase-3, and inhibited the anoxia/reoxygenation-induced decrease in the expression of the anti-oxidative proteins OD1, OD2, and catalase, and the pro-survival proteins cl-2 and cl-xl. Furthermore, we demonstrated that these protective effects were mediated, at least in part, via the PI3K/A t signaling pathway. nli e other organs, lungs ac uire oxygen from both blood via a dual circulatory system and alveolar gas exchange. Hence, various animal models have been used to explore the mechanism and possible treatment of LIRI [30]. In this study, we developed a stable and reproducible lung ischemia/reperfusion model in rats, by clamping the left pulmonary hilum to create complete ischemia and anoxia. Due to the critical role of endothelial cells in LIRI [2, 31], we further developed a HPAECs-based anoxia/reoxygenation model in vitro.
Lung ischemia/reperfusion injury is a complex pathophysiologic process, in which oxidative stress and apoptosis play critical roles. During ischemia, endothelial cells and macrophages generate RO , activate nicotinamide adenine dinucleotide phosphate (NADPH), nitric oxide synthases (NO ), nuclear factor-(NF-), and pro-in lammatory cyto ines. When reperfusion occurs, RO activates neutrophils. These reactions result in vascular damage, characterized by increased pulmonary vascular resistance and microvascular permeability [2], eventually leading to pulmonary edema and gas exchange dysfunction with severe ventilation-perfusion imbalance immediately following reperfusion [32]. It has also been reported that RO cause an in lammatory response and dysfunction in remote organs, which further exacerbate ischemia/reperfusion injury [33]. In addition, apoptosis, which is only found during reperfusion, also plays an important role in the development of lung dysfunction [6,34]. The intrinsic pathway of apoptosis, nown as the mitochondrial pathway, is activated by oxidative stress [6]. Previous studies have shown that EETs can protect heart, brain, and aortic endothelial cells against ischemia/reperfusion injury [19,20,35]. However, the effect and mechanism of EETs in lung ischemia/reperfusion injury are still elusive.
RO , such as superoxide anions, hydrogen peroxide, hydroxyl radicals, and singlet oxygen, are products of normal cellular metabolism [36]. It is well recognized that RO play a dual role as both bene icial and harmful effects. RO exert bene icial effects at low or moderate concentrations, and involve physiological roles, such as in defence against infectious agents, induction of a mitogenic response, and regulation of several cellular signaling pathways [36,37]. On the contrary, overproduction of RO leads to oxidative stress, a harmful process resulting in cellular structural damage through lipid peroxidation, inactivation of various proteins and DNA damage [2,37]. Therefore, oxidative stress plays an important role in various human diseases.
During LIRI, mitochondria and NADPH oxidase are the most important pathways through which RO are generated [2]. itochondria are not only a major source of RO generation, but also sensitive targets for oxygen radical [38]. During lung ischemia/ reperfusion, mitochondrial damage occurs mainly during ischemia and not reperfusion [39]. During ischemia, the lac of oxygen ends ATP synthesis. The rapid depletion of ATP causes a decrease in mitochondrial membrane potential, triggering apoptotic process and RO overproduction [2]. Excessive RO causes mitochondrial outer membrane permeabilization, leading to release of cytochrome c and overexpression of pro-apoptotic proteins, which can further trigger caspase activation and cell apoptotis [38].
NADPH oxidase is present in endothelial cells, macrophages and vascular smooth muscle cells, including gp91 phox , p47 phox , p67 phox , p40 phox , p22 phox , and Rac subunits [40]. p91 phox , located in the plasma membrane, is the main functional subunit. NADPH oxidase is also a major source of RO in endothelial cells during lung ischemia [41,42]. The rapid depletion of ATP causes inactivation of ATP-sensitive K + channels, resulting in endothelial cell membrane depolarization, followed by the activation of endothelial cell-localized NADPH oxidase [43], with subse uent increased production of RO [44]. It has been demonstrated that inactivation of NADPH oxidase can attenuate LIRI in rat and pig models of lung ischemia/ reperfusion [45,46].
It is well nown that the PI3K/A t signaling pathway plays an important role in promoting cell proliferation and anti-apoptosis via regulating the expression of cl-2 family proteins, which is located in mitochondrial outer membrane [47,48]. Although lac of detailed mechanisms, previous studies have con irmed that EETs activate the PI3K/A t signaling pathway and protect against apoptosis in pulmonary artery endothelial cells [49,50]. Activation of the PI3K/A t pathway has emerged as a critical event for survival and proliferation in various ischemia/reperfusion models [19,51]. PI3K has also been proved to regulate NADPH oxidase assembly and activation via its products PtdIns(3,4)P2 and PtdIns3P binding directly to oxidase subunits [40]. It has been reported that melatonin bloc s the translocation p47 phox and p67 phox to the membrane via a PI3K/A t-dependent signaling pathway, inhibits the binding of p47 phox to gp91 phox , and impairs NADPH oxidase assembly, eventually resulting in inhibition of RO production [52]. Therefore, PI3K/A t inhibits apoptosis at a pre-mitochondrial level via regulating the activity of NADPH oxidase.
In the current study, we found that lung ischemia/reperfusion led to mitochondrial dysfunction and activation of NADPH oxidase, resulting in RO overproduction. Excessive RO further damaged mitochondria, triggering apoptotic process via regulating the expression of cl-2 family proteins and activation of caspase-3. These deleterious effects induced by lung ischemia/reperfusion were attenuated by either CYP2J2 overexpression or exogenous EETs. oreover, the protective effects of CYP2J2 and EETs were bloc ed by LY294002. Ta en together, these results indicated that the protective effects of EETs might be mediated, at least partially, by the PI3K/A t pathway. EETs might inhibit the activity of NADPH oxidase and promote the expression of anti-oxidative proteins following lung ischemia/reperfusion via activating the PI3K/A t pathway, leading to inhibition of RO production which further alleviated mitochondria dysfunction. This would decrease RO generation by mitochondria which further bloc ed apoptotic process [53]. In addition, EETs might promote cell proliferation and inhibit cell apoptosis via PI3K/A t activation, as previously reported [18].
ince lung IR injury is a complicated process, further studies are needed to clarify whether EETs also affect other events including in lammation, intracellular calcium overload, leu ocyte activation, and the corresponding signaling pathways, similar to its involvement with PI3K/A t following ischemia/reperfusion.
In conclusion, our results demonstrate the protective effects of CYP2J2 expression and its metabolites, EETs, on oxidative stress and apoptosis after lung ischemia/reperfusion. The mechanisms involved may include reduction of the intracellular RO levels, mitigation of mitochondrial dysfunction, and suppression of cell apoptosis. CYP2J2 overexpression or administration of exogenous EETs could be a novel strategy for the prevention and treatment of lung ischemia/reperfusion injury.

Disclosure Statement
The authors have no con lict of interests to declare.