| Background and purposeArachidonic acid (AA) is the most widely distributed and the most abundant polyunsaturated fatty in human body, in vivo, AA metabolic network is an important part of the multiple metabolic pathways and of endogenous bioactive substances, including oxidative stress network and inflammation signaling pathway. AA mainly metabolized through three different enzymatic pathways, including the cyclooxygenase (COX) pathway, the lipoxygenase (LOX) pathway and the third important pathway of cytochrome P450(CYP) pathway which play an important roles in numerous physiological&pathophysiological processes.Cytochrome P450epoxygenases(CYP2J and CYP2C) metabolize arachidonic acid (AA) to four different epoxyeicosatrienoic acids (EETs), namely5,6-EET,8,9-EET,11,12-EET and14,15-EET, which are converted to dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolase(sEH).The cytochrome P450enzymes comprise a large superfamily of proteins. CYP widely distributed in the body, the CYP epoxygenase including two types2C and2J, CYP has been found for6clones type of oxidase2J, which the type CYP2J2is only found in the human. After the years of research on CYP-EET system, evidence shows that CYP-EET system plays an important role in the human body.(1)EETs are with a strong expansion effects of Cardiovascular function and regulate blood pressure;(2) EETs can significantly alleviate the apoptosis in endothelial cells and cardiomyocytes induced by tumor necrosis factor-α(TNF-α);(3) EETs can inhibit inflammation, inhibiting endothelial cell expression of inflammatory adhesion molecules and protect the biological function of endothelial cells;(4)EETs can promote endothelial cell proliferation, inhibits apoptosis of endothelial cells and promotes tissue neovascularization,(5) EETs can activate plasminogen activator(tPA), which may play an important role to maintain thrombosis/thrombolysis balancing in vivo;(6) EETs can inhibit aortic smooth muscle cell migration induced by the transforming growth factor-P(TGF-β);(7) hypoxia-reoxygenation process can significantly inhibit CYP2J2gene expression in endothelial cells, By providing the exogenous EETs or CYP2J2overexpression in vivo can improve hypoxia-reoxygenation injury.Lung ischemia-reperfusion injury (LIRI) is an important issues in the face of thoracic surgery, It occurs in a variety of contexts such as lung transplantation, heart and lung transplantation, lobe sleeve resection, pulmonary artery angioplasty, cardiopulmonary bypass, pulmonary embolism, shock and cardiopulmonary resuscitation, Lung ischemia-reperfusion injury can result in the infiltration of inflammatory cells to increase, and Inflammatory cells produce and release large amounts of oxygen free radicals and thus damaging cells, relieves the ischemia-reperfusion injury can protect lung function, reduce the post-operative mortality and prolong the survival time of the transplanted organs, can reduce the occurrence of systemic inflammatory response syndrome and multiple organ dysfunction syndrome, especially to reduce acute respiratory distress syndrome(ARDS) caused by acute lung injury(ALI).In recent years, the incidence and mortality rates of lung cancer are rising rapidly in China; the incidence of lung cancer among malignant tumors has been ranked in the first. Removal of lesion tissue by surgical procedures is still the most thorough way to treat lung cancer. How to lessen IRI is a constantly focal point in lung cancer surgery study.LIRI is a complex phenomenon involving not only intracellular injury processes, but also injurious inflammatory responses and biochemical changes, During ischemia-reperfusion, such as leukocyte activation, a variety of promoting the release of inflammatory reaction medium, the release of inflammation-related protease, the occurrence of oxidative stress, intracellular calcium overload, activation of multiple transcription factors, membrane molecular raised, the biofilm injury and protective substances such as NO and decrease in pulmonary surfactant and other relevant factors, endothelial cells, and other immune cells to generate ROS Calcium/calmodulin dependent nitric oxide synthases(NOS), nuclear factor-KB (NF-κB), nicotinamide adenine dinucleotide phosphate (NADPH), and proinflammatory cytokines are activated, causing an upregulation of cell surface adhesion molecules on the endothelial of the lung, along with lung ischemia-reperfusion injury in the nature of deepening understanding, inflammation and oxidative stress mechanisms involved in lung ischemia-reperfusion injury and endothelial dysfunction, many studies have shown that IRI and in essence, is an inflammatory response to ischemia-reperfusion injury, inflammatory chemokine expression resulting in lung tissue inflammatory cell infiltration, infiltration a large number of inflammatory cells and lead to the occurrence of oxidative stress, these are lung ischemia-reperfusion injury formed an important start contributing factor.So, can CYP2J2be used for the prevention of lung ischemia-reperfusion injury? We suppose that through over-expression the CYP2J2gene in animals model of lung ischemia-reperfusion injury in order to increases the level of endogenous EETs;it could activate endogenous protective mechanisms of the vascular wall through the inhibition of inflammation and oxidative stress to relieve lung ischemia-reperfusion injury. Thus, we conducted experiments to confirm the effects of CYP2J2overexpression on the animal’s model of lung ischemia-reperfusion injury in rats, to explore CYP2J2ability to inhibit or block the lung ischemia-reperfusion injury in the inflammatory response and oxidative stress and the possible mechanisms which exist in the protective effects of CYP2J2overexpression.Methods and results Methods1Plasmid pcDNA3.1(+)-2J2and pcDNA3.1(+)-GFP were extracted and purificated by using endotoxin-free plasmid mention kit.2.50male Wistar rats,8weeks old, weight300-350g, subjected to a one-week adaptation period, were randomly divided into five groups, and each group has10rats:Blank group (Blank), Sham group (Sham), IR group (IR), IR+GFP group (IR+GFP), IR+2J2group (IR+2J2). The target gene pcDNA3.1-2J2and pcDNA3.1(+)-GFP as well as the equal volume of saline were injected into the bodies of rats via tail veins, and the dose of plasmid injection was3mg/kg body weight, injected once a week, a total of two injections. One week after the gene delivery, modeling surgery was performed. After the surgery was completed, the rats were sacrificed, Blood samples, bronchoalveolar lavage fluid(BALF) lung tissue samples were collected and part of lung tissue were placed in liquid nitrogen immediately, all samples transferred into the-80℃refrigerator, some of the lung tissues were fixed with formaldehyde solution, embedded in paraffin after dehydration at room temperature.3. Blank group:do not perform any surgical procedure, the animals were sacrificed after anesthesia; Sham group:left thoracotomymodel, before animals were sacrificed, we observe the animals180min; IR group:rats lung IRI model, the model were established by obstructing the left hilum of lung, ischemia1h and2h of reperfusion, then animals were sacrificed; IR+2J2group:same perform just like the IR group; IR+GFP group:same perform just like the IR group. Before sacrificed, half of animals were invented Millar catheter into right ventricle, in order to record right ventricular hemodynamic parameters.4. Proteins from frozen lung tissue were extracted for western blot methods to detect protein expression levels of CYP2J2protein. And ELIS A method was used to detect content of the lung tissue14,15-DHET.5.The ratio of lung wet/dry (W/D) weight were measured to assess lung edema after IRI; Serum/BALF protein concentration ratio was detection; the paraffin sections of lung were prepared to carry out HE stain, Immunohistochemical TUNEL staining was used to detect apoptosis of alveolar epithelial cell.6. Detection of serum and lung tissue Inflammatory-related cytokines:ELISA was used to detect TNF-α, IL-1β, IL-8, IL-10, sP-selectin, sE-selectin levels in serum; western blot to detect protein expression levels of ICAM-1and VCAM-1inlung tissue.7. Detection of oxidative stress in rat blood and lungtissue:Real-time quantitative PCRwas used to detect mRNA expression of oxidative stress related cytokines eNOS, XOD, NADPH p22phox and NADPH P67phox; ELISA method to detect the serum levels of COX.8. Western blot to detect protein expression levels of PI3K, Akt, p-Akt and NF κB-p65in rat lung tissue.Results:1. Western blot and ELISA results showed that after2weeks/2times CYP2J2gene delivery through tail vein into rat,the CYP2J2overexpression in rat lung;14,15-EET content in lung of CYP2J2overexpression rats were significantly higher than the control group (P<0.05).2. The results of TUNEL staining to detect apoptosis showed that, compared with Blank group and Sham group, the apoptotic index(AI) was significantly higher in IR group, IR+GFP group and IR+2J2group (P<0.01); The AI of IR+2J2group was significantly less than the IR group and IR+GFP group (P<0.05).CYP2J2overexpression significantly inhibited ischemia-reperfusion injury induced apoptosis.3. After1h ischemia and2h reperfusion, the ratio of W/D in IR+2J2group is (5.215±0.860), significantly lower than that (8.061±1.189) of IR group and (8.048±0.489) of IR+GFP group (P<0.05); both of three group were significantly higher than that in (4.075±0.729) of Blank group and (4.700±0.645) of Sham group (P<0.01), there was no significant difference between Blank group and Sham group, but also between IR group and IR+GFP group.After1h ischemia and2h reperfusion, the ratio of Serum/BALF protein concentration was significantly higher in IR+2J2(30.306±2.4956) than that in IR(17.727±1.870) and IR+GFP(18.414±1.993)(P<0.05); both of three group were significantly lower than that in Blank(44.463±4.023) and Sham (43.005±1.703)(P<0.01), there was no significant difference between Blank group and Sham group, also between IR group and IR+GFP group.4. Results of Inflammatory-related cytokines test:ELISA:detection of inflammatory cytokines in serum shows that CYP2J2overexpression significantly inhibited the serum levels of pro-inflammatory cytokinesThe serum levels of TNF-a in IR+2J2group (124.182±12.734pg/ml) is significantly lower than that in IR group(191.554±19.603pg/ml) and IR+GFP group(183.246±14.443pg/ml), all these three group are significantly higher than that Blank group(41.224±12.443pg/ml) and Sham group (93.004±12.701pg/ml);The serum levels of IL-1β in IR+2J2group (74.393±9.029pg/ml) is significantly lower than that in IR group(110.516±24.519pg/ml) and IR+GFP group(103.704±15.872pg/ml), all these three group are significantly higher than that Blank group(50.039±12.318pg/ml) and Sham group (62.018±12.319pg/ml);The serum levels of IL-8in IR+2J2group(55.330±6.608pg/ml) is significantly lower than that in IR group(67.754±8.797pg/ml) and IR+GFP group(65.008±7.670pg/ml), but are higher than that in Blank group(36.757±6.051pg/ml).The serum levels of IL-10in IR+2J2group (191.473±4.639pg/ml) is significantly higher than that in IR group(125.792±6.256pg/ml) and IR+GFP group(123.279±7.453pg/ml), all these three group are significantly higher than that in Blank group (46.828±5.010pg/ml) and Sham group(53.987±4.925pg/ml).The serum levels of sP-Selectin in IR+2J2group (38.297±3.3527ng/ml) is significantly lower than that in IR group (56.732±5.985ng/ml) and IR+GFP group (55.616±5.455ng/ml), all these three group are significantly higher than that in Blank group (6.259±1.631ng/ml) and Sham group (19.951±4.166ng/ml).The serum levels of sE-Selectin in IR+2J2group (1312.109±211.164pg/ml) is significantly lower than that in IR group (1779.279±322.441pg/ml) and IR+GFP group (1775.263±261.194pg/ml), all these three group are significantly higher than that in Blank group (500.193±162.362pg/ml) and Sham group (930.488±192.240pg/ml).Western blot test results and Real-time quantitative PCR data of the mRNA expression level showed that ICAM-1and VACM-1expression level was significantly upregulated after lung ischemia-reperfusion; CYP2J2gene overexpression significantly inhibited the effects.5. Results of oxidative stress related cytokines testReal-time quantitative PCR result showed that XOD, eNOS, NADPH P22phox, NADPH P67phox mRNA expression level was significantly upregulated after lung ischemia-reperfusion, CYP2J2gene overexpression can inhibited the effects.6. Western blot test results showed that:Compared with Sham and Sham group, PI3K, p-Akt protein expression level was obviously decreased after LIR, and NFicB-p65protein expression level was obviously increased after LIR. CYP2J2gene overexpression significantly inhibited the changes of related protein expression above-mentioned.7. Hemodynamic records showed that LIRI induced significant decreased the right ventricle Systolic blood pressure (RVSP)and the maximum rate of right ventricular pressure (±dp/dt min); CYP2J2overexpression reversed this effects induced by LIRIsignificantly; compared with other groups, CYP2J2overexpression decrease right ventricular end-diastolic pressure (RVEDP); LIRIinduced significant decrease in heart rate (HR), the CYP2J2overexpression did not show significant changes.Statistical AnalysisAll data were analyzed by SPSS19.0statistical software(IBM). Enumeration data were expressed as frequency or composition ratio, and the Chi-square test were used to evaluate the differences between two groups. Values were present as mean±standard deviation (x±SD).Analysis of variance (ANOVA) was used to compare group differences. Student’s t test was used to assess whether differences in the values of two groups were statistically significant. The level for statistical differences was set at P<0.05.Conclusion:1. Successfully constructed an animal model in vivo of left lung ischemia-reperfusion injury in Wister rats2. CYP2J2gene delivery in rat by plasmid tail vein injection received a stable expression, and increased the content of EETs in lung tissuesignificantly.3. CYP2J2overexpression significantly reduces the left lung ischemia-reperfusion injury and apoptosis in rats.4. CYP2J2gene overexpression significantly improves right ventricular systolic and diastolic function damage caused by the left lung ischemia-reperfusion injury.5. CYP2J2overexpression significantly decreased the serum levels of proinflammation cytokines and increased the levels of anti-inflammatory cytokines in the LIRI rats.6. CYP2J2overexpression inhibited the oxidative stress associated indicators (XOD, NADPH subunits) in blood, lung tissue significantly, and significantly upregulated expression of eNOS,7. A possible mechanism that CYP2J2anti-inflammatory and inhibit oxidative stress by activating PI3K/AKT/NF-κB signaling pathway. |
PDF Full Text Request