| Background and ObjectiveLung ischemia/reperfusion injury (LIRI) remains a life-threatening but inevitable complication in various clinical conditions, such as lung transplantation, cardiopulmonary bypass, sleeve lobectomy, bronchovascular sleeve resection and reconstruction, resuscitation for circulatory arrest, and pulmonary embolism. It is one of the most important factors that may affect the recent recovery and long-term prognosis in these conditions. Re-establishment of perfusion in ischemic lung is a double-edged sword, which is not only essential to restore tissue function, but also triggers a complex cascade of events leading to lung injury and damage, characterized by increased pulmonary vascular permeability, pulmonary edema, microvascular damage, release of inflammatory mediators, production of reactive oxygen species. LIRI is a complex pathophysiological process, accompanied by oxidative stress, intracellular calcium overload, release of pro-inflammatory mediators, leukocyte activation, and upregulation of molecules on cell surface membrane. Unfortunately, the consequences of lung ischemia/reperfusion injury are not limited to lungs. In many cases, this local inflammation leads to systemic inflammatory response typically resulting in multiple organ dysfunction syndrome, which frequently leads to death. However, there is no effective method currently available to prevent LIRI. Further studies are needed to clear the precise mechanism and explore novel treatments.Endothelial cells are recognized as the most important mediators of lung ischemia/reperfusion injury. During ischemia, nuclear factor-KB (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH), calmodulin-dependent nitric oxide synthases (NOS), and proinflammatory cytokines are activated, and ROS are generated by endothelial cells, leading to an increased expression of cell-surface adhesion molecules on the endothelial side of the lung. All of these reactions may cause directly or indirectly several physiological changes in the microcirculation. These changes translate markedly into increased pulmonary vascular resistance (PVR), pulmonary microvascular permeability, and lung edema which leads to gas exchange abnormality owing to severe ventilation-perfusion mismatch following lung reperfusion. During the early stage of LIRI, the morphological response of the endothelium is initially apoptotic, rather than necrotic.Cytochrome P450epoxygenase (CYP) is a large oxidase superfamily, including CYP2C and CYP2J. Among these, only CYP2J2is expressed in humans and mainly expressed in the cardiovascular system. It is well documented that CYP2J2metabolizes arachidonic acid (AA), the most widely distributed polyunsaturated fatty acid in humans, to four regioisomeric epoxyeicosatrienoic acids (5,6-EET,8,9-EET,11,12-EET, and14,15-EET). In previous studies, EETs have been reported to exert various biological effects in normal and pathophysiological conditions, such as anti-inflammation, anti-apoptosis, pro-proliferation, anti-oxidation, anti-fibrosis, pro-angiogenesis, and anti-hypertension. Increasing evidences demonstrate that EETs protect organs against ischemia/reperfusion injury. For example, CYP2J2overpression and exogenous EETs protect against global cerebral ischemia in transgene mice via increase of regional cerebral blood flow and reduction of apoptosis, and protect the myocardium by enhancing cardiac functional recovery and decreasing the infarct size following ischemia/reperfusion. Preconditioning of mouse pulmonary arteries in vitro with11,12-EET has also been shown to reduce caspase-3activity. Although without clear mechanism, EETs have been reported to remarkably reduce endothelial permeability in isolated rat lungs following ischemia/reperfusion.Based on previous studies, we hypothesized that increased circulating levels of EETs by CYP2J2overexpression in vivo and in vitro may protect against lung IR injury via anti-inflammation, anti-oxidation, and anti-apoptosis. In this study, we overexpressed CYP2J2in rats, and successfully constructed a rat model of lung ischemia/reperfusion injury to investigate the effect and possible mechanism of CYP2J2overexpression during lung ischemia/reperfusion injury. We further explored the effect and possible signal pathway of CYP2J2overexpression and exogenous EETs precondition during hypoxia/reoxygenation in human pulmanory artery endothelial cells (HPAECs), to clarify the mechanisms of lung ischemia/reperfusion injury at the level of animal and cell, and the effect and corresponding signal pathway of CYP2J2-EETs during LIRI, providing new theoretical and experimental basis for prevention and treatment of LIRI.Part I. CYP2J2and EETs protect against lung ischemia/reperfusion injury via anti-inflammation in vivo and in vitroMethods:In vivo study1. Extraction and purification of pcDNA3.1-CYP2J2and pcDNA3.1-GFP.2. Animal grouping and gene delivery Male Wistar rats weighing280-350g were obtained from the Experimental Animal Center of Hubei Province, transported in sterile conditions and housed at25℃with12h light/dark cycles. After a week adaptation, animals were randomly assigned into five groups (n=10in each group):Blank (blank control group), Sham (sham group), IR (lung ischemia/reperfusion group), IR+GFP (lung ischemia/reperfusion and pcDNA3.1-GFP gene transfection group), and IR+CYP2J2(lung ischemia/reperfusion and pcDNA3.1-CYP2J2gene transfection group). Animals from IR+GFP and IR+CYP2J2groups received a volume of plasmid at a dose of3mg/kg body weight via tail vein, once a week for two consecutive weeks. Animals from the other three groups received an equal volume of saline at the same time.3. Animal model of lung ischemia/reperfusion injuryOne week after the second vein injection, rats in the IR groups underwent left thoracotomy via the fifth intercostals. The left pulmonary hilar, including the left main bronchus, pulmonary artery, and pulmonary vein, were dissected and then occluded for1hour 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 additional2hours. 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 blank group did not receive any other treatment and animals were sacrificed at the end of experiment. Blood samples were acquired from the left atria prior to sacrifice and the left lungs were then removed for further analysis.During the whole procedure, rats were placed on a warm-bed and under a heating light to keep warm. The wounds were kept covered with warm, saline-soaked gauze to prevent excessive fluid loss. Animals were sacrificed at the end of the experiment. Blood samples were obtained from the inferior vena cava just prior to sacrifice and the left lung was then removed for further analysis as below.4. Detection of CYP2J2in lungs, and14,15-DHET in serum and lung tissues.5. Measurement of lung weight/body weight (LW/BW) and lung wet weight/dry weight (W/D) ratio.6. Detection of protein concentration in bronchoalveolar lavage fluid.7. Lung histopathologic examination.8. Determination of inflammatory mediators in rat serum by ELISA.9. Determination of IκBα, NF-κB p65and PPARy in lung tissues by western blot. In vitro study 10. Cell culture and gene transfectionHuman pulmonary artery endothelial cells (HPAECs) were purchased from ScienCell Research Laboratories and cultured according to the manufacturer’s protocol. Cells were plated in6-well plates, and transfected respectively with pcDNA3.1-GFP or pcDNA3.1-CYP2J2after～60%confluence with Fugene HD Transfection Reagent. The transfection efficiency was detected by examining pcDNA3.1-GFP-transfected cells for GFP fluorescence under a fluorescence microscope and flow cytometric analysis. The expression of CYP2J2was confirmed by immunoblotting.11. Induction of hypoxia-reoxygenation in vitro and cell treatmentHypoxia-reoxygenation (HR) model is established and used based on previous study. Briefly, cells in6-well plates were grown to～80%in a normoxic incubator (5%CO2,95%air,37℃,95%humidity). Cells were pretreated with GW9662(PPARy inhibitor, lumol/L) for30min before addition of EET (lumol/L) or vehicle, which were added1hour before HR to the end of experiments. For HR groups, culture plates were replaced with fresh endothelial cell medium (ScienCell Research Laboratories) without serum, and subjected to hypoxia (5%CO2,95%N2,37℃) for8hours, followed by reoxygenation (5%CO2,95%air) for an additional16hours. Before reoxygenation, culture plates were replaced with complete endothelial cell medium. Normal control cells received the identical processes for24hours except for hypoxia/reoxygenation.12. Determination of inflammatory mediators in cell cultured media by ELISA.13. Detection of IκBα, NF-κB p65and PPARy in HPAECs by western blot.Results:1. Delivery of pcDNA3.1-CYP2J2induces CYP2J2overexpression in vivo and in vitro, and increases circulating EETs level.2. Effects of CYP2J2overexpression on lung W/D and LW/BW ratios.The lung W/D and LW/BW ratios notably increased following lung ischemia/reperfusion (IR) compared with control groups without IR, while the effect was reversed by CYP2J2 overexpression. These results show that lung IR leads to pulmonary inflammation and edema, which can be partially inhibited by CYP2J2overexpression.3. Effect of CYP2J2overexpression on protein concentration in bronchoalveolar lavage fluid.The total protein concentration in bronchoalveolar lavage was markedly higher in IR groups than those in control groups without IR, while CYP2J2overexpression inhibited the increase. The result indicates that lung IR induces increased pulmonary vascular permeability, which is extenuated by CYP2J2overexpression.4. Effects of CYP2J2overexpression on lung histopathology.Lung tissues in the blank and sham groups under light microscopy showed normal architecture and no inflammatory cell infiltration. In IR groups, there was significant interstitial and perivascular edema, inflammatory cell infiltration in interstitium and alveolar spaces, intra-alveolar fibrin deposition and hemorrhage. Alveolar septum notablely widened by edema, erythrocyte extravasation and fibrin deposition. However, CYP2J2overexpression effectively extenuated pulmonary edema and inflammatory cell infiltration. The lung injury scores further confirmed the protective role of CYP2J2overexpression against acute IR injury.5. Effects of CYP2J2overexpression on inflammatory mediators in serum and inflammation-related protein expression in vivo.In this study, we found that IR notably increased serum levels of proinflammatory mediators, including IL-1β, IL-8, TNF-α, sP-and sE-selectin, but significantly decreased the serum level of IL-10, an anti-inflammatory cytokine. Interestingly, the changes of inflammatory cytokines after lung IR were remarkably inhibited by CYP2J2overexpression. Moreover, the levels of PPARy and IκBα in the cytoplasm were markedly decreased after IR, while CYP2J2overexpression attenuated the effect. Furthermore, expression of NF-κB p65in the nucleus after IR significantly increased compared to control groups without IR, an effect that was reversed by CYP2J2overexpression. These results suggest that CYP2J2overexpression is associated with reduced lung inflammation, which might be possibly mediated by PPARy activation.6. Effects of CYP2J2overexpression and exogenous11,12-EET on inflammatory mediators and inflammation-related protein expression in HPAECs.The concentrations of proinflammatory cytokines in cell cultured media, including IL-1β and IL-6, were significantly increased after hypoxia/reoxygenation compared with these in the control and vehicle groups. Interestingly, CYP2J2overexpression remarkably extenuated these increases. However, these effects were notably reversed by GW9662, an selective inhibitor of PPARy. On the contrary, the level of IL-10was reduced following hypoxia/reoxygenation. CYP2J2overexpression significantly attenuated the decrease, an effect that was inhibited by GW9662. In addition, no significant difference was observed among HR groups on the concentrations of VCAM-1and ICAM-1.Moreover, consistent with observation in the in vivo lung IR model, hypoxia/reoxygenation treatment markedly reduced PPARy expression in HPAECs, and the effect was notably reversed by CYP2J2overexpression and11,12-EET pretreatment. Furthermore, hypoxia/reoxygenation treatment decreased the expression of IκBα in the cytoplasm and led to a remarkable elevation in the nuclear translocation of NF-κB p65. As expected, pretreatment with CYP2J2overexpression or11,12-EET significantly suppressed hypoxia/reoxygenation-induced NF-κB p65activation, an effect that was reversed by GW9662.Part II. CYP2J2and EETs protect against lung ischemia/reperfusion injury via anti-oxidation and anti-apoptosis in vivo and in vitroMethods:In vivo study1. Animal grouping and gene delivery:the same as part I.2. Animal model of lung ischemia/reperfusion injury:the same as part I. 3. TUNEL staining for assessment of apoptosisCell death by apoptosis in the lung was detected in situ by TUNEL staining of fragmented DNA. Cells were identified as TUNEL positive if they showed dark brown staining under a light microscope. More than five randomly-selected high-power fields (×400) were examined for TUNEL-positive cells. The number of TUNEL positive cells was scored and the percentage of apoptotic cells was calculated from the total number of cells.4. Expression of oxidative stress-related proteins, including SOD1, SOD2, catalase, gp91, p47and p67were detected by western blot.In vitro study5. Cell culture and gene transfection:the same as part I.6. Model of hypoxia-reoxygenation and cell treatment:the same as part I.7. Effect of exogenous EETs on cell proliferation was determined by CCK8. Cell proliferation was detected using a Cell Counting kit-8(CCK-8) according to the manufacture’s instruction. Briefly, cells were seeded in96-well plates at a density of2x103per well in100ul complete medium. After being treated with the corresponding drugs and hypoxia-reoxygenation as described above,10μl of the CCK-8reagent was added to the cells in each well, and the cells were further incubated at37℃for0.5to4hours. The absorbance in each well at a wavelength of450nm was measured with a microplate reader.8. Effect of CYP2J2overexpression and exogenous EETs on ROS was monitored by flow cytometry and a fluorescence microscope.9. Effect of CYP2J2overexpression and exogenous EETs on mitochondrial transmembrane potential was measured by flow cytometry.5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), a mitochondria-specific membrane potential-sensitive fluorescent probe, was used to determine the mitochondrial membrane potential of cells. JC-1accumulates in the mitochondrial matrix when the mitochondrial membrane potential is high, forming J-aggregates which emit a bright yellow-red fluorescence; in contrast, JC-1presents itself as a green fluorescent monomer with low levels of aggregation when mitochondrial membrane potential is low. During the assay, cells were seeded on6-well plates. After corresponding treatment in each group, the cells were loaded with10μmol/L JC-1for20min at37℃, then washed and harvested to detect fluorescence by flow cytometry.10. Effect of CYP2J2overexpression and exogenous EETs on cell apoptosis was detected by flow cytometry.13. Expression of PI3-Kinase (PI3K), phosphor-Thr308-Akt (p-Akt), Akt, Bcl-2, Bcl-xl, Bax, SOD1, SOD2, catalase, p67-phox, gp91-phox, p47-phox, and caspase-3were analyzed by western blot.Results1. Effect of CYP2J2overexpression on oxidative stress in vivo.In this sudy, the expression of the pro-oxidative enzymes gp91, p47, and p67significantly increased in the lung tissues following ischemia/reperfusion, an effect that was reversed by CYP2J2overexpression. In contrast, CYP2J2delivery remarkably inhibited the ischemia/reperfusion-induced reduction in SOD1, SOD2and catalase protein levels.2. Effect of CYP2J2overexpression on apoptosis in vivo.In the current study, treatment with ischemia/reperfusion remarkably increased the number of TUNEL-positive cells compared with the blank and sham groups, an effect that was reversed by pcDNA3.1-CYP2J2transfection.3. Effects of exogenous EETs on cell proliferation of HPAECs.We found that treatment of HPAECs with hypoxia conditions for8hours, followed by reoxygenation for the next16hours, led to a significant decrease in cell viability. To test effects of exogenous EETs on the viability of HPAECs, we examined proliferation of HPAECs under normoxia and hypoxia/reoxygenation conditions, respectively. We found that EETs had no significant effects on cell viability under normal conditions. However, the cell viability significantly decreased following hypoxia/reoxygenation, an effect that was reversed by11,12-EET and14,15-EET. 4. Exogenous EETs and CYP2J2overexpression suppressed ROS production induced by hypoxia/reoxygenation in HPAECs.In this sudy, we found that pre-incubation with EETs significantly attenuated the hypoxia/reoxygenation-induced increase in the ROS level, while EETs alone did not show these effect. However, the effects of EETs in the hypoxia/reoxygenation condition were significantly inhibited when cells were pretreated with14,15-EEZE (a selective EET antagonist). Transfection with pcDNA3.1-CYP2J2displayed similar protective effects on ROS production in hypoxia/reoxygenation treated cells. Furthermore, similar results were obtained by observation of DCFH-DA under a fluorescence microscope.5. Effects of EETs and CYP2J2overexpression on apoptosis in HPAECs following hypoxia/reoxygenation.Our results revealed that pre-incubation with EETs significantly decreased apoptosis induced by hypoxia/reoxygenation. This protective effect was notably inhibited by LY294002or14,15-EEZE. However, EETs did not show this anti-apoptotic effect in cells under normal conditions. Likewise, CYP2J2overexpression displayed similar effects on apoptosis induced by hypoxia/reoxygenation.6. Effects of EETs and CYP2J2overexpression on mitochondrial transmembrane potential in HPAECs following hypoxia/reoxygenation.We found that the mitochondrial transmembrane potential remarkably decreased in the hypoxia/reoxygenation treated group compared with groups incubated under normal conditions, an effect which was reversed by14,15-EET. The protective effect of14,15-EET, however, was markedly suppressed by LY294002or14,15-EEZE. In addition, similar effects were found in cells transfected with pcDNA3.1/CYP2J2.7. Effects of14,15-EET and CYP2J2overexpression on oxidative-related enzymes in HPAECs.The results indicated that14,15-EET and CYP2J2overexpression significantly attenuated the hypoxia/reoxygenation-induced increase in gp91(a transmembrane protein), p47, p67 and NOX4protein levels. Those effects, however, were reversed by LY294002. In contrast,14,15-EET and CYP2J2overexpression remarkably increased the expression of the anti-oxidative proteins SOD1, SOD2, and catalase, which were obviously decreased in the hypoxia/reoxygenation condition. However, these protective effects were markedly decreased by14,15-EEZE treatment. These results suggest that the anti-oxidative effect of14,15-EET and CYP2J2overexpression might be mediated by PI3K activation.8. Effects of14,15-EET and CYP2J2overexpression on apoptosis-related protein in HPAECs following hypoxia/reoxygenation.The expression of the anti-apoptotic proteins Bcl-2and Bcl-xl decreased after hypoxia/reoxygenation treatment in HPAECs, an effect that was reversed by14,15-EET and CYP2J2overexpression. In contrast, Bax increased after the hypoxia/reoxygenation treatment, while this increase was attenuated by14,15-EET and CYP2J2overexpression. Moreover, the expression of cleaved caspase-3also increased after hypoxia/reoxygenation treatment, while this increase was inhibited by14,15-EET and CYP2J2overexpression. Remarkably, the effects of14,15-EET and CYP2J2overexpression were blocked by LY294002, supporting a role of the PI3K/Akt signaling pathway in EET-mediated regulation of apoptosis.9. Effects of14,15-EET and CYP2J2overexpression on the PI3K/Akt pathway in HPAECs following hypoxia/reoxygenation.Hypoxia/reoxygenation treatment significantly inhibited the expression of PI3K and the PI3K-dependant Akt phosphorylation in HPAECs, effects which were reversed by14,15-EET and CYP2J2overexpression. Interestingly, these effects of14,15-EET and CYP2J2overexpression were notably blocked by LY294002.Statistical analysisAll data were analyzed by SPSS15.0statistical software. Values were presented as mean±standard deviation (SD). Comparisons between groups were performed by a one-way analysis of variance (ANOVA). Statistical significance was defined as p＜0.05. Conclusion1. CYP2J2gene can be stably transfected in lung tissues of Wistar rats and in HPAECs, leading to overexpression of CYP2J2protein and increasing level of circulating EETs.2. CYP2J2overexpression significantly attenuates pulmonary edema and inflammatory cell infiltration induced by ischemia/reperfusion.3. CYP2J2overexpression markedly reduces inflammation in rats after lung ischemia/reperfusion, including inhibiting the release of pro-inflammatory mediators and nuclear translocation of NF-κB p65.4. CYP2J2overexpression remarkably suppresses oxidative stress induced by lung ischemia/reperfusion.5. CYP2J2overexpression notably extenuates lung ischemia/reperfusion-induced apoptosis in rats.6. CYP2J2overexpression and exogenous EETs significantly attenuate hypoxia/reoxygenation-induced inflammation in HPAECs, including inhibiting the release of pro-inflammatory mediators and nuclear translocation of NF-κB p65, an effect that may be mediated by PPARy activation.7. CYP2J2overexpression and exogenous EETs remarkably alleviate hypoxia/reoxygenation-induced oxidative stress in HPAECs, including suppressing ROS production, inhibiting the expression of pro-oxidative proteins, and promoting the expression of anti-oxidative proteins.8. CYP2J2overexpression and exogenous EETs markedly reduce apoptotic level induced by hypoxia/reoxygenation in HPAECs, including inhibiting collapse of mitochondrial transmembrane potential, reducing apoptosis, and regulating the expression of apoptosis-related proteins, an effect that may be mediated by activation of PI3K/Akt pathway.Collectively, CYP2J2overexpression and exogenous EETs protect against lung ischemia/reperfusion injury via anti-inflammation, anti-oxidation, and anti-apoptosis in vivo and in vitro, which may be mediated by activation of PPARy and PI3K/Akt pathway. |
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