Effects of leptin on hypoxia-induced apoptosis in cultured alveolar typeⅡ cells of fetal rat and its mechanism

AIM: To investigate the effects of leptin (LEP) on the alveolar type Ⅱ cells(AECⅡ) apoptosis induced by Na₂S₂O₄ and explore the molecular mechanisms. METHODS: Primary AECⅡ culture was prepared according to a specific immunosorption procedure with slight modification and the cells were identified by transmission electron microscope and immunocytochemistry. AECⅡ damage was induced by 5 mmol/L Na₂S₂O₄. LEP group cells were treated with LEP at concentrations from 100 μg/L to 1 600 μg/L. The cell survival rate was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Cell cycle and apoptosis were analyzed by flow cytometry and the level of caspase-3 was measured by Western blotting. RESULTS: Highly purified AECⅡ, obtained by the method of modified immunosorption, were identified with the positive expression of SP-A and intracellular lamellarbodies were found under electron micrography. The cells, exposed to 5 mmol/L Na₂S₂O₄, showed characteristic changes of apoptosis and activation of caspase 3. These damages were relieved by the treatment of LEP (100-1 600 μg/L), with survival increasing, apoptosis peak decreasing, cell morphology restoring and caspase 3 activation inhibiting.CONCLUSION: Leptin prevents AECⅡ from apoptosis induced by Na₂S₂O₄ or hypoxia. The potential mechanism of its action may be related to promoting cell cycle from G₁ phase to S phase and inhibiting the activating of caspase 3.     

Sevoflurane preconditioning inhibits brain injury in hypoxic mice

AIM: To observe the effects of sevoflurane preconditioning on brain injury in hypoxic mice and its possible mechanism. METHODS: Male C57BL/6J mice were randomly divided into control (C) group, hypoxia (H) group, 2% sevoflurane preconditioning for 30 min + hypoxia (S1+H) group, 2% sevoflurane preconditioning for 60 min+hypoxia (S2+H) group and 4% sevoflurane preconditioning for 30 min + hypoxia (S3+H) group. The hypoxia model was established by continuous inhalation of (6.5±0.1)% O₂ for 24 h. The sevoflurane preconditioning treatments, S1, S2 and S3, were conducted by inhalation of 2% sevoflurane for 30 min, 2% sevoflurane for 60 min and 4% sevoflurane for 30 min, respectively, with the carrier of (21.0±0.5)% O₂, followed by washout for 15 min and then hypoxia treatment. The histological changes of the hippocampal CA1 area were observed under light microscope and transmission electron microscope (TEM), and serum lactate dehydrogenase (LDH) activity was measured by colorimetric method. Furthermore, the protein levels of erythropoietin (EPO) and vascular endothelial growth factor (VEGF) in brain tissue homogenate were examined by ELISA, and the content of malondialdehyde (MDA) and the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPx) were measured by microplate reader. RESULTS: After hypoxia for 24 h, cell edema or pyknosis in the hippocampal CA1 area was observed in H group. Sevoflurane preconditioning reduced hypoxic injury, and the cell ultrastructure under TEM was significantly improved in S2+H group. Compared with C group, the serum LDH activity and the levels of EPO, VEGF and MDA in brain tissues were significantly increased in H group, while the activity of SOD and GPx decreased. After sevoflurane pretreatment, the serum LDH activity and the levels of EPO and VEGF in brain tissues were lower than those in H group, and the most significant difference was observed in S2+H group. Moreover, the MDA content and SOD activity decreased, and the GPx activity increased in the sevoflurane preconditioning groups. CONCLUSION: Sevoflurane preconditioning attenuates brain injury in hypoxic mice by regulating antihypoxic protein synthesis and reducing oxidative stress.     

Effect of ghrelin on iNOS expression in alveolar macrophages and lung tissues in rats with sepsis-associated lung injury

AIM: To investigate the effect of ghrelin on inducible nitric oxide synthase (iNOS) expression in alveolar macrophages and lung tissues in sepsis-induced acute lung injury (ALI) rats. METHODS: The septic rat model was established by cecal ligation and puncture (CLP). Male SD rats were divided into sham group, CLP group and CLP+ghrelin group. The rats in the former 2 groups were further divided into 3 subgroups, which were 6 h, 12 h and 20 h post-operation groups. Ghrelin was administered by intraperitoneal injection at 3 h and 15 h after operation in ghrelin group. The samples were harvested 20 h after operation. The mRNA expression of iNOS in alveolar macrophages collected from bronchoalveolar lavage was detected by RT-PCR. The protein levels of lung iNOS were measured by Western blotting. The lung pathological examination was performed 20 h after operation. RESULTS: In CLP group, the mRNA expression levels of iNOS in the alveolar macrophages were 1.33±0.05, 1.44±0.08, 1.57±0.11 at 6 h, 12 h and 20 h after CLP, respectively, which were higher than that in sham group, but did not show time correlation. However, it was lower in CLP group than that in CLP+ghrelin group at 20 h after CLP (2.27±0.37, P<0.05). At 20 h after CLP, the protein level of lung iNOS was decreased in CLP+ghrelin group (0.87± 0.03) as compared with CLP group (1.08±0.05). Compared with sham group, the histopathological score was increased in both CLP group and CLP+ghrelin group, but it was lower in CLP+ghrelin group (5.83±0.477) than that in CLP group (7.83±0.75). CONCLUSION: Ghrelin treatment improves the degree of ALI. During 6 h to 20 h after CLP, the mRNA expression of iNOS in alveolar macrophages was elevated, but the difference was not seen as the time went on. Ghrelin up-regulates the mRNA expression of iNOS in alveolar macrophages and inhibits iNOS expression in lungs of septic rats.     

Effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats

AIM:To study the effect of cilazapril on pulmonary vascular endothelial dysfunction in hypoxic rats. METHODS:The structure and function of endothelium in hypoxic rats were studied by biochemical analysis, radioimmunoassay, transmission electron microscope and correlated with hemodynamic. RESULTS:1) The change and damage of ultrastructure in endothelial cell (EC) were obsevered in hypoxic rats. 2) The contents of plasma nitric oxide (NO) and superoxide dismutase (SOD) activity in blood as well as endothelial nitric oxide synthase (eNOS) activity in the lung tissue were significantly lower in the hypoxic rat than those in contral animals. The concentrations of plasma endothelin-1(ET-1) and angiotensin converting enzyme(ACE) as well as malondialdehyde(MDA) were significantly higher in the hypoxic rat than these in contral animals. The relaxing and contracting factors had a significant positive/negative correlation with mean pulmonary artery pressure (mPAP). 3) Cilazapril significantly decreased the level of ET-1 and ACE and significantly increased the level of NO and activity of eNOS and SOD. At the same time, cilazapril extenuated hypoxia-induced injuries of EC. CONCLUSION:The results indicate that damaging structure and dysfunction of EC existes in hypoxic rats. The cilazapril effectively preventes and treates the chronic hypoxic PH by relieving the injury and improving secretion in EC.     

Progress on hypoxic regulatory factor miR-210

The expression of miR-210, a much concerned hypoxia-induced molecule, is significantly changed under the condition of hypoxic microenvironment. The up-regulation of miR-210 depends on the expression of hypoxia-inducible factor in different types of hypoxic tumor cells and normal cells. This review article summarizes the recent progress on the regulation of miR-210 expression and its biological function under hypoxic condition.     

Protective effect of hydrogen sulfide on hypoxia-induced injury of rat cortical neurons

AIM:To explore the role of hydrogen sulfide (H 2S) in cortial neuronal injury induced by hypoxia.METHODS:The SD rat cortical neurons were cultured in hypoxic conditions (2% O 2, 5% CO 2 and 93% N 2 at 37 °C) to establish the hypoxic model. Sodium hydrosulfide (NaHS) was used as the donor of H 2S and neuronal viability was detected by CCK-8 assay. Neuronal content of reactive oxygen species (ROS) was determined by DCFH-DA method, and mitochondrial membrane potential (MMP) was detected using Rh123 staining. Lactate dehydrogenase (LDH) release rate was measured by a commercial kit to reflect the degree of neuronal injury. RESULTS:Hypoxic treatment increased ROS content and the release rate of LDH in the neurons. However, NaHS pretreatment significantly inhibited the hypoxia-induced increases in ROS content and LDH release. Hypoxia decreased MMP and cell viability. Pretreatment with NaHS and N-acetyl-L-cysteine (NAC), a ROS scavenger, significantly inhibited the decreases in MMP and viability of the neurons. CONCLUSION:Hypoxia induces ROS generation in the neurons, thereby decreases MMP and neuronal viability. H 2S significantly attenuates hypoxia-induced neuronal injury by its antioxygenation.     

Effects of EGLN1 siRNA on growth of rat pulmonary artery smooth muscle cells under hypoxic condition

AIM: To observe whether EGLN1 gene is involved in the growth of pulmonary arterial smooth muscle cells (PASMCs) during hypoxia when EGLN1 gene expression was interference by siRNA. METHODS: The rat primary pulmonary arterial smooth muscle cells were cultured, and the specific lipidosome of EGLN1 siRNA was constructed and transfected into the PASMCs. The transfected PASMCs were cultured under hypoxia or normoxia conditions, respectively. The viability of the PASMCs was detected by CCK-8 assay. The protein expression of EGLN1 and vascular endothelial growth factor (VEGF) was determined by Western blot. RESULTS: The viability of the PASMCs was increased and the protein expression of VEGF was up-regulated in the PASMCs under hypoxic condition in a time-dependent manner. In hypoxia or normoxia condition, the viability and VEGF protein expression of the PASMCs were suppressed by EGLN1 siRNA. CONCLUSION: EGLN1 gene may involve in the growth of rat PASMCs by regulating VEGF protein level under hypoxic condition.     

Effect of Panax notoginoside on pulmonary arterial pressure and expression of p38 MAPK in lung tissues of hypoxic rats

AIM: To observe the effect of Panax notoginoside (PNS) on the pulmonary artery pressure and the p38 mitogen-activated protein kinase(p38 MAPK) in lung tissues of rats treated with hypoxia. METHODS: Thirty adult male SD rats were randomly divided into 3 groups. The rats in normal control group were exposed to normal conditions, the rats in hypoxia group were exposed to isobaric hypoxia, and the rats in hypoxia+PNS group were treated with PNS under the condition of hypoxia. After 4 weeks of treatment, the mean pulmonary arterial pressure (mPAP) and the mean carotid arterial pressure (mCAP) were measured by cardiac catheterization. The heart was isolated, and the right ventricle (RV), left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S).The quantity of phospho-p38 MAPK(p-p38 MAPK) in rat pulmonary arterioles was determined by the method of immunohistochemistry and the mRNA content of p38 MAPK was tested by RT-PCR. RESULTS: The mPAP and RV/(LV+S) in hypoxia group were higher than those in normal control group. The expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues were higher than those in normal control group (P<0.05). The mPAP, RV/(LV+S), the expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues in hypoxia+PNS group were significantly lower than those in hypoxia group (P<0.05).CONCLUSION: PNS possesses the preventive and therapeutic effect on hypoxic pulmonary hypertension by decreasing p-p38 MAPK and down-regulation of p38 MAPK mRNA in the lungs.     

Effect of hypercapnia on lysyl oxidase-dependent collagen cross-linking and hypoxia-induced pulmonary hypertension in rat

AIM: To investigate the effect of hypercapnia on hypoxia-induced pulmonary hypertension and the changes of lysyl oxidase (LOX) and extracellular matrix collagen cross-links in the rat. METHODS: Sprague-Dawley rats were randomly divided into 4 groups:normoxia group, hypoxia group, hypercapnia group and hypoxia+hypercapnia group. LOX activity was detected by fluorescence spectrophotometry. LOX protein expression was detected by immunohistochemistry and Western blot. The mRNA expression of LOX in the pulmonary artery was detected by real-time PCR. RESULTS: The levels of mean pulmonary artery pressure (mPAP), RV/(LV+S) and WA/TA in hypoxia group were significantly higher than those in normoxia group (P<0.01). Moreover, the levels of mPAP and RV/(LV+S) in hypoxia+hypercapnia group were significantly lower than those in hypoxia group (P<0.01). However, no significant difference of mPAP and RV/(LV+S) between hypercapnia group and normoxia group was observed. In hypoxia group, the collagen cross-links in the lung tissue was significantly higher than that in normoxia group and hypercapnia group (P<0.01). Importantly, collagen cross-links in the lung tissue of hypoxia+hypercapnia group was significantly lower than that in hypoxia group (P<0.01). There was no significant difference in collagen cross-links between hypercapnia group and normoxia group. The expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries of hypoxia group were significantly increased as compared with normoxia group (P<0.01). Furthermore, the expression of LOX at mRNA and protein levels and its activity in the pulmonary arteries in hypoxia+hypercapnia group were lower than those in hypoxia group (P<0.01). CONCLUSION: Hypoxia not only up-regulates LOX but also promotes collagen cross-linking in the rat lung, which contributes to the development of pulmonary hypertension. Hypercapnia inhibits hypoxia-induced LOX expression and collagen cross-linking, therefore impairing the progress in hypoxia-induced pulmonary hypertension.     

Effect of nitric oxide on apoptosis in lung injury following ischemia-reperfusion of hind limbs

AIM: To investigate the injury of lung and the role of cell apoptosis in the pathogenesis of acute lung injury following ischemia-reperfusion of hind limbs and the influence of nitric oxide (NO) to apoptosis.METHODS: Referring to our laboratory normal method,the model rats,which underwent 4 hours ischemia and 4 hours reperfusion of hind limbs were made.L-arginine (L-Arg) and N-nitro-L-arginine methyl ester (L-NAME) was administrated respectively to these rats before the experiment.Apoptosis was detected by TdT-mediated dUTP nick end labeling (TUNEL),respectively.The radioimmunoassay (RIA) was used to detect level in the expression of TNF-α.The immunohistochemistry (IHC) method was used to detect the level in the expression of Bcl-2,Bax,caspase-3 and TNF-α.The morphologic changes were observed under microscope,respectively.The results of the RIA and the IHC were analyzed quantitatively by relative computer analytical system.RESULTS: After rats’s hind limbs suffered from ischemia-reperfusion,the apoptosis in alveolar epithelial cells and pulmonary vascular endothelial cells was found.The expression of TNF-α,caspase-3 and Bax increased.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were not obvious in the L-Arg administrated group,but the expression of Bcl-2 was obvious in that group.Compared with IR rats,the expressions of TNF-α,caspase-3 and Bax were obvious in the L-NAME administrated group,but the expression of Bcl-2 was not obvious in that group even weaker than normal ones.CONCLUSION: Apoptosis participated in acute lung injury following ischemia-reperfusion of hind limbs.The excess expression of TNF-α related with apoptosis of alveolar epithelial cells and pulmonary vascular endothelial cells.NO may reduce the occurrence of apoptosis and other lung injury through down-regulating the level in the expression of TNF-α.     

Effect of microRNA-132 transfection on lipopolysaccharide-induced inflammation in rat alveolar macrophages

AIM: To investigate the effect of microRNA-132 (miR-132) transfection on the lipopolysaccharide (LPS)-induced inflammation in rat alveolar macrophages. METHODS: The rat alveolar macrophage NR8383 cultured without pyrogen in vitrowere divided into blank control group, negative control group and transfected group. The cells in the 3 groups were transfected with phosphate buffer solution (PBS), Lipofectamine 2000 and synthesized miR-132 mimic respectively. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Real-time PCR was used to detect the expression of miR-132 in the cells. After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group. The growth of NR8383 cells in transfected group was significantly inhibited compared with blank control group and negative control group (P<0.05). After the cells were stimulated with LPS, the productions of NF-κB, TNF-α and IL-6 in transfected NR8383 cells were decreased compared with blank control group and negative control group (P<0.05).CONCLUSION: Transfection of alveolar macrophages with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS.     

Effect of DADLE on lung injury in rats with acute global cerebral ischemia-reperfusion

AIM：To observe the effects of δ opioid receptor agonist DADLE on acute lung injury (ALI) induced by acute global cerebral ischemia-reperfusion in rats. METHODS：SD rats (n=30) were randomly divided into sham group, model (I/R) group and DADLE treatment group. Global cerebral ischemia-reperfusion model was established by a modified 2-vessel occlusion plus hypotension. DADLE (5 mg/kg) treatment was performed via the left jugular injection before reperfusion. After 120-min reperfusion, the pathological changes of the lung tissues were observed under light microscope and electronic microscope. The activity of superoxide dismutase (SOD) and malondialdehyde (MDA) level were detected. The partial pressure of arterial oxygen (PaO2) was also measured. RESULTS：In I/R group, widened alveolar septum, capillary dilatation and congestion, endovascular and perivascular cells in the lung with neutrophil infiltration, and significantly reduced type II epithelial cell surface microvilli, alveolar lumen cavity and trachea with serous exudate were observed. SOD activity decreased, but the MDA level increased. Compared with I/R group, the SOD activity increased and MDA level decreased in DADLE treatment group, with significantly reduced lung congestion, the degree of lung injury, and the infiltration of neutrophils. Compared with I/R group, the PaO2 and oxygenation index in DADLE treatment group were increased. CONCLUSION：Various degrees of pulmonary injury were observed in acute global cerebral ischemia reperfusion model. DADLE might have a protective effect on lung tissues of ALI in rats.     

Effects of CYP2J3/EETs on myocardial apoptosis under the condition of hypoxic postconditioning

AIM: To investigate the effects and underlying mechanism of endogenous cytochrome P450 2J3/epoxyeicosatrienoic acids (CYP2J3/EETs) system on myocardial apoptosis under the condition of hypoxic postconditioning (HPost).METHODS: Primary myocardial cells from neonatal Wistar rats (12 h-24 h) were cultured and divided into 7 groups as follows: control group, hypoxia/reoxygenation (H/R) group, HPost group, CYP2J3 transfection group, empty vector group, 6-(2-propargyloxyphenyl)hexanoic acid (PPOH, an inhibitor of CYP2J3) group and dimethyl sulfoxide (DMSO, as solvent) group. The H/R treatment was conducted in all the groups except control group. The cell viability was tested by MTT method. The concentration of 11,12-EET in the cell culture medium was measured by high-perfor-mance liquid chromatography (HPLC). The expression of caspase-3 was detected by Western blotting and its activity was determined by caspase-3 activity assay kit.RESULTS: Compared with H/R group, the cell viability and the 11,12-EET concentration were significantly elevated in HPost group (P<0.01). The two indexes in CYP2J3 transfection group were higher than those in HPost group (P<0.01), but they were lowered in PPOH group than those in HPost group (P<0.01). The comparisons of the expression and activity of caspase-3 among groups were as follows: H/R group > HPost group (P<0.01), PPOH group > HPost group (P<0.01) and empty vector group > CYP2J3 transfection group (P<0.05). Additionally, the caspase-3 expression in CYP2J3 transfection group was lower than that in HPost group (P<0.01).CONCLUSION: The CYP2J3/EETs system protects myocardial cells through inhibiting caspase-3-induced apoptosis.     

Effect of intestinal lymphatic pathway on apoptosis of lung tissue in rats by two-hit

AIM: To observe the effects of mesenteric lymph duct ligation on apoptosis of lung tissue,correlated gene expression with apoptosis and TNF-α,IL-6 contents in rats by two-hit.METHODS: 45 Wistar rats were divided into three groups: the ligation group,the non-ligation group and sham group,and the two-hit model was established by means of hemorrhage and LPS treatments.Ligating mesenteric lymph duct was conducted in ligation group.After 24 hours,the pathological sections of lung tissue were prepared for determining the apoptosis rate by TUNEL method and expressions of Bcl-2 and Bax were observed by immunohistochemical test.The lung homogenate was also prepared for determining the contents of TNF-α and IL-6 by ELISA.RESULTS: After two-hit,the apoptosis rate,Bax expression in lung tissue and contents of TNF-α and IL-6 in serum and lung homogenate in non-ligation group were increased and Bcl-2 expression was lower than that in sham group and ligation group significantly (P<0.01,P<0.05).Apoptosis rate in ligation group was no statistics difference with sham group (P>0.05),and the expression of Bcl-2 protein was increased and Bax was lower than that of sham group (P<0.01,P<0.05).CONCLUSION: Blockage of intestinal lymphatic pathway reduces the apoptosis of lung in two-hit rats,and its mechanism might relate to reduced the levels of TNF-α and IL-6 and improved the expression of Bcl-2 protein in lung by the ligation of mesenteric lymph duct.The mesenteric lymph of two-hit might play an important role in the pathogenesis of acute lung injury (ALI).     

Effect of NG-nitro-L-arginine on mitochondria in acute lung injury induced by LPS in rats

AIM: To examine the effect of nonselective nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NA), on mitochondria from acute lung injury induced by lipopolysaccharides(LPS) in rats. METHODS: The rats were randomly divided into control group, LPS injury group and L-NA treatment group. The model of acute lung injury was prepared with injection of LPS in rats. L-NA was respectively administrated through intraperitoneal injection at 3 h after injury induced by LPS. The rats were killed and the mitochondria in lung tissues were isolated by differential centrifugation. The activities of T-NOS, iNOS, ATPase, SOD and GSH-Px, and the contents of NO and MDA from mitochondria were respectively measured. The changes of ultrastructure in lung mitochondria were examined by electronic microscope after injury and L-NA treatment. RESULTS: The activities of T-NOS and iNOS were significantly increased, the activities of ATPase, SOD and GSH-Px were significantly decreased, the contents of NO and MDA were increased after acute lung injury. L-NA significantly enhanced the activities of ATPase, SOD and GSH-Px, and markedly decreased the contents of NO and MDA and the activities of T-NOS and iNOS. CONCLUSION: L-NA inhibits the activity of NOS in mitochondria, decreases the production of NO, improves mitochondria energy pump, ameliorates oxidative injury, and effectively protects lung tissue against acute lung injury induced by LPS.     

Effects of lipopolysaccharide on aquaporin-1 expression and function in rat lung microvessel endothelial cells

AIM: To determine if aquaporin-1 (AQP-1) expression and function is influenced after lipopolysaccharide (LPS) stimulation in rat lung microvessel endothelial cells and to investigate the mechanisms of lung fluid abnormal metabolism in acute lung injury. METHODS: LPS at different concentrations (100 μg/L, 1 mg/L or 10 mg/L) was used to stimulate cultivated rat lung microvessel endothelial cells in vitro at different stimulatory times (4 h, 12 h or 24 h), respectively. Tritium water permeation was conducted for determining the intracellular signal intensity in rat lung microvessel endothelial cells. The RT-PCR technique was applied for the assay of the expression of AQP-1 mRNA. RESULTS: The signal intensity of intracellular tritium water in the LPS stimulation group was less than that in normal control significantly. Compared with the normal control group (P<0.01), there was obvious decrease in the expression of AQP-1 mRNA in LPS stimulation groups. CONCLUSION: The decreased expression of AQP-1 mRNA and the weaken function of transporting water suggest that AQP-1 may play a major role in abnormal fluid transportation in acute injured lung.     

Effect of nitric oxide and peroxynitrite on lung injury induced by ischemia-reperfusion of hind limbs in rats

AIM:To observe the changes in nitric oxide(NO) and peroxynitrite anion (ONOO^- ) in the injuried lung following the ischemia-reperfusion of hind limbs and evaluate the contribution of NO and ONOO^- to tissue injury.METHODS:A model of hind limbs ischemia was made by clamping infrarenal aorta with a microvascular clip and lung injury occurring after reperfusion.Lung t issue was obtained from the animals received sham operation(group 1),4 hours ischemia without reperfusion(group2),1 hour reperfusion following 4 hours ischemia(group3)and 4 hours reperfusion fol owing 4 hours ischemia(group4).The contents of MDA,NO₂^-/NO₃^- and the activities of SOD in the lung were examined.Immunohistochemical echnique was used to determine the immunoreactivity to iNOS and nitrotyrosine(NT)-a specific "footprint" of peroxynitrite.RESULTS:Compared with group1 and group2,the contents of MDA and NO₂^-/NO₃^- increased significantly (P<0.05) and the activities of SOD decreased markedly(P＜0.05) in group3 and group4.Immunohistochemical examination demonstrated intense staining for iNOS and NT throughout the lung in group3 and group4.CONCLUSION:NO and ONOO^- are involved in oxidant-mediated lung injury following reperfusion of ischemic hind limbs.