Cell Apoptosis In Lung Tissues Research Articles

Effect of different doses of dexmedetomidine on lung function and tissue cell apoptosis in a rat model of hyperoxic acute lung injury

Purpose: To study the effect of different doses of dexmedetomidine on lung function and lung tissue cell apoptosis in a rat model of hyperoxic acute lung injury.
 Methods: Five groups of healthy male Sprague-Dawley rats were used: normal rats, untreated hyperoxic rats, and hyperoxic rats given 3 different doses of dexmedetomidine, with 20 rats in each group. The levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were determined usingenzyme-linked immunosorbent assay (ELISA). Parietal paraffin cuts were taken from the right upper lobe for measurement of apoptosis using in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and the apoptosis index was calculated.
 Results: At 24 and 48 h, the levels of IL-6 and TNF-α in the hyperoxia model group were significantly higher than those in the normal control group, and their levels in the middle- and high-dose groups were markedly lowered, relative to untreated hyperoxia rats (p < 0.05). Apoptosis index in the hyperoxia model rats significantly increased, relative to normal rats (p < 0.05). The apoptosis index in the mediumand high-dose groups decreased significantly (p < 0.05).
 Conclusion: Dexmedetomidine inhibits inflammatory responses caused by high concentration of oxygen inhalation, minimizes lung injury, improves lung function and inhibits lung apoptosis.
 Keywords: Dexmedetomidine, Hyperoxia, Acute lung injury, Lung function, Apoptosis

Caffeine prevents hyperoxia-induced lung injury in neonatal mice through NLRP3 inflammasome and NF-\u03baB pathway

BackgroundBronchopulmonary dysplasia (BPD) is a common chronic lung disease in premature infants and hyperoxia exposure is a major cause. In hyperoxic lung injury animal model, alveolar simplification and pro-inflammatory cells infiltration are the main pathophysiologic changes. Caffeine is a drug used to treat apnea in premature infants. Early use of caffeine can decrease the rate and the severity of BPD while the mechanisms are still unclear. The purpose of this study was to evaluate the effects of caffeine on inflammation and lung development in neonatal mice with hyperoxic lung injury and to explore the possible mechanism.MethodsFollowing 14 d of 75% oxygen exposure in newborn mouse, the BPD model was established. Caffeine at a dose of 1 g/L was added in drinking water to nursing mouse. We measured the concentration of caffeine in serum and oxidative stress in lung by commercially available kits. Adenosine 2A receptor (A2AR) expression and lung inflammation were measured by Immunohistochemistry and western blotting. Apoptosis and surfactant protein-C (SFTPC) levels were measured by immunofluorescence. The inflammasome and NF-κB pathway proteins were assessed by western blotting.ResultsWe found that the caffeine concentration in plasma at present dose significantly decreased the expression of A2AR protein in mice lung. Caffeine treatment significantly reduced oxidative stress, improved weight gain, promoted alveolar development, attenuated inflammatory infiltration and lung injury in hyperoxia-induced lung injury mice. Moreover, caffeine decreased the cell apoptosis in lung tissues, especially the Type II alveolar epithelial cell. The expression of NLRP3 inflammasome protein and NF-κB pathway were significantly inhibited by caffeine treatment.ConclusionCaffeine treatment can protect hyperoxia-induced mice lung from oxidative injury by inhibiting NLRP3 inflammasome and NF-κB pathway.

Ibudilast, a Phosphodiesterase-4 Inhibitor, Ameliorates Acute Respiratory Distress Syndrome in Neonatal Mice by Alleviating Inflammation and Apoptosis.

BackgroundAcute respiratory distress syndrome (ARDS) is a sudden and serious disease with increasing morbidity and mortality rates. Phosphodiesterase 4 (PDE4) is a novel target for inflammatory disease, and ibudilast (IBU), a PDE4 inhibitor, inhibits inflammatory response. Our study investigated the effect of IBU on the pathogenesis of neonatal ARDS and the underlying mechanism related to it.Material/MethodsWestern blotting was performed to analyze the expression levels of PDE4, CXCR4, SDF-1, CXCR5, CXCL1, inflammatory cytokines, and proteins related to cell apoptosis. Hematoxylin-eosin staining was performed to observe the pathological morphology of lung tissue. Pulmonary edema score was used to assess the degree of lung water accumulation after pulmonary injury. Enzyme-linked immunosorbent assay (ELISA) was used to assess levels of inflammatory factors (TNF-α, IL-1β, IL-6, and MCP-1) in serum. TUNEL assay was used to detect apoptotic cells.ResultsIncreased expression of PDE4 was observed in an LPS-induced neonatal ARDS mouse model, and IBU ameliorated LPS-induced pathological manifestations and pulmonary edema in lung tissue. In addition, IBU attenuated the secretion of inflammatory cytokines by inactivating the chemokine axis in the LPS-induced neonatal ARDS mouse model. Finally, IBU significantly reduced LPS-induced cell apoptosis in lung tissue.ConclusionsIBU, a PDE4 inhibitor, protected against ARDS by interfering with pulmonary inflammation and apoptosis. Our findings provide a novel and promising strategy to regulate pulmonary inflammation in ARDS.

Dexmedetomidine Attenuates Lipopolysaccharide-Induced Acute Lung Injury by Regulation of microRNA-223-3p and HDAC4

Objective: Acute lung injury (ALI) mainly causes acute respiratory distress syndrome, pneumonia, and acute respiratory failure. Due to the high lethality of ALI, this study was for the investigation of the mechanism by which dexmedetomidine (DEX) alleviated lipopolysaccharide (LPS)-induced ALI by regulating histone deacetylase 4 (HDAC4) and microRNA-223-3p (miR-223-3p). Methods: After LPS induction of ALI, the mice were treated with DEX (low, medium and high doses). Then the mice treated with LPS-induced ALI and high dose of DEX were transfected with miR-223-3p mimics or sh-HDAC4 to figure their roles in inflammation, and apoptosis in lung tissue of mice with ALI. Mouse monocyte RAW264.7 was treated with LPS-induced ALI and different doses of DEX and transfected with miR-223-3p mimics or sh-HDAC4 to explore their roles in proliferation, cycle distribution, inflammation, and apoptosis of RAW264.7 cells. RT-qPCR and western blot analysis were functioned to detect the expression of miR-223-3p, HDAC4, and TLR4/MyD88/NF-κB signaling pathway-related factors in lung tissue and RAW264.7 cells of mice with ALI. Results: Lowly expressed miR-223-3p and over-expressed HDAC4 were detected in lung tissue of mice with ALI and RAW264.7 cells. MiR-223-3p mimics or sh-HDAC4 decreased TLR4/MyD88/NF-κB signaling pathway-related factors in lung tissue of mice and RAW264.7 cells. MiR-223-3p mimics or sh-HDAC4 inhibited inflammation and cell apoptosis in lung tissue of mice with ALI, as well as facilitated proliferation, cycle entry and inhibited inflammation and apoptosis of RAW264.7 cells. Conclusion: This study suggests that DEX can alleviate LPS-induced ALI by mediating miR-223-3p and HDAC4 expression. This finding has key implications for developing the mechanism of ALI. Funding Statement: This work was supported by Science and Technology Department of Jilin Province (No. 20190701068GH). Declaration of Interests: The authors declare that they have no conflicts of interest. Ethics Approval Statement: This study was approved and supervised by the animal ethics committee of China-Japan Union Hospital of Jilin University. The treatment of animals in all experiments conforms to the ethical standards of experimental animals.

Mitochondrial Coenzyme Q Protects Sepsis-Induced Acute Lung Injury by Activating PI3K/Akt/GSK-3β/mTOR Pathway in Rats.

The aim of our study was to assess the effects of mitochondrial coenzyme Q (MitoQ) on sepsis-induced acute lung injury (ALI) and investigate its possible mechanisms. The cecal ligation and puncture (CLP) method was used to establish a septic ALI model. Rats were randomly divided into Con group, CLP group, MitoQ group, and MitoQ + LY294002 group. The survival rate of the rats was recorded, and the survival rate curve was plotted. Moreover, the ratio of wet/dry weight (W/D) in lung tissue was measured. The activity of myeloperoxidase (MPO) was measured by using the MPO colorimetric activity assay kit. The levels of high-mobility group box 1 (HMGB1) and interleukin-6 (IL-6), macrophage inflammatory protein 2 (MIP2), and keratinocyte chemoattractant (KC) were analyzed by ELISA. The histopathological changes were measured by HE staining, and the lung injury was scored. TUNEL assay was applied to detect the apoptotic cells in lung tissue. The protein expressions were detected by western blot. MitoQ increased the survival rate and alleviated pulmonary edema in septic ALI rats. In addition, MitoQ inhibited the MPO activity and decreased the levels of HMGB1 and IL-6. After treatment with MitoQ, alveolar wall edema, inflammatory cell infiltration, and red blood cell exudation were relieved. MitoQ inhibited cell apoptosis in lung tissue of septic ALI rats. Meanwhile, MitoQ treatment remarkedly increased the expression of p-Akt, p-GSK-3β, and p-mTOR but decreased Bax, caspase-3, caspase-9, Beclin-1, and LC-3II/LC-3I. The effects of MitoQ were significantly reversed by the PI3K inhibitor (LY294002). Our study demonstrated that MitoQ could protect sepsis-induced acute lung injury by activating the PI3K/Akt/GSK-3β/mTOR pathway in rats.

MicroRNA-124 alleviates the lung injury in mice with septic shock through inhibiting the activation of the MAPK signaling pathway by downregulating MAPK14

ObjectiveAcute lung injury (ALI) is a severe lung disease with high mortality rate. Research has highlighted that the immune response to ALI is associated with significant changes in the expression of several microRNAs (miRNAs) in the lungs. In our research, we speculated that miR-124 moderated the severity of ALI through comprehensive suppression of the mitogen-activated protein kinase (MAPK) signaling pathway activation by targeting MAPK14. MethodsA mouse model of ALI was established by array of experiments. The expression of MAPK14 and miR-124 was assessed in the tissues of ALI mice and the expression of inflammatory cytokines in ALI mice was determined. The expression of the related kinases in the MAPK signaling pathway and key cytokines in the pro-inflammatory response were assessed by a series of experiments. Immunohistochemistry and TUNEL staining were adopted to detect lung tissue cell proliferation and apoptosis in mice with ALI. ResultsMiR-124 was poorly expressed and MAPK14 was highly expressed in tissues of ALI mice. Overexpression of miR-124 or silence of MAPK14 alleviated the symptoms of ALI by down-regulating inflammatory cytokines expression, which could intrinsically suppress the expression of associated proteins in the MAPK signaling pathway and the downstream pro-inflammatory response factors, promote proliferation and inhibit apoptosis of lung tissue cells. Overexpression of MAPK14 inverted the phenotypic changes induced by overexpressing miR-124. ConclusionThese results indicated that miR-124 could alleviate the symptoms of ALI by inhibiting the activation of MAPK signaling pathway via subsequent targeting of MAPK14. Additionally, miR-124 may serve as a useful biomarker to alleviate the severity of septic shock-induced lung injury.

Heat-killed Salmonella typhimurium mitigated radiation-induced lung injury.

Radiation-induced lung injury (RILI) is a serious complication in thoracic tumour radiotherapy. It often occurs in clinical chest radiotherapy and acute whole-body irradiation (WBI) caused by nuclear accidents or nuclear weapon attack. Some radioprotective agents have been reported to exert protective effects when given prior to radiation exposure, however, there is no treatment strategy available for preventing RILI. In this study, we demonstrated that heat-killed Salmonella typhimurium (HKST), a co-agonist of Toll-like receptors 2 (TLR2), Toll-like receptors 4 (TLR4) and Toll-like receptors 5 (TLR5), mitigated radiation-induced lung injury through the transforming growth factor-β (TGF-β) signalling pathway. We found that HKST alleviated lung hyperaemia and pathological damage after irradiation, indicated that HKST inhibits the early inflammatory reaction of radiation-induced lung injury. Then, for the first time, we observed HKST reduced collagen deposit induced by irradiation in the later phase (7-14week) of RILI, and we found that HKST inhibited radiation-induced cell apoptosis in lung tissues. We found that HKST reduced the level of TGF-β and regulated its downstream signalling pathway. Finally, it was found that HKST inhibited radiation-induced epithelial-mesenchymal transition (EMT) in lung tissues. In conclusion, our data showed that HKST effectively mitigated RILI through regulating TGF-β, provide novel treatment strategy for RILI in whole-body irradiation and radiotherapy.

Role and related mechanism of resolvin D1 in lung ischemia reperfusion injury in rats

Objective: To investigate the role and related mechanism of resolvin D1 (RvD1) in lung ischemia-reperfusion injury (LIRI) in rats. Methods: Forty male Sprague-Dawley rats, 7-8 weeks, weighing 220-280 g, were divided into 4 groups using a random number table method: sham operation group, lung ischemia reperfusion control group, normal saline group, and RvD1 group. The rat model of LIRI was produced by 45 min of occlusion of the left hilum of lungs followed by 150 min reperfusion. In sham group, no blocking of the left hilum of lung after thoracotomy; Normal saline 2 ml/kg and RvD1 100 μg/kg were injected respectively at 10 min of reperfusion in normal saline group and RvD1 group. Blood samples were collected from the femoral vein for determination of interleukin (IL)-6, tumor necrosis factor (TNF)-α, soluble inter-cell adhesion molecules (sICAM-1) concentrations at 150 min of reperfusion. The rats were sacrificed after collection of blood samples and then lung tissues were taken for observation of the pathological changes and for measurement of lung wet/dry weight ratio (W/D). The the contents of malondialdehyde (MDA), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-2 and the activity of myeloperoxidase (MPO) in lung tissues were determined. The protein relative expression of nuclear factor (NF)-κB in lung tissues was detected by Western blot. Lung tissue cell apoptosis was detected with TUNEL method. Results: The plasma level of IL-6, TNF-α, sICAM-1 in normal saline group and RvD1 group were significantly higher than those in the Sham group [(110±7), (100±4) vs (72±3) ng/L, (151±8), (153±6) vs (104±5) ng/L, (2 690±133), (2 760±167) vs (1 953±125) ng/L]. Besides, NF-κB protein relative expression level of lung tissues up-regulated [(0.681±0.033), (0.664±0.024) vs (0.292±0.011)] (all P<0.05). The W/D, apoptosis index, MDA, MCP-1, MIP-2 contents and MPO activity in lung ischemia reperfusion control group, normal saline group and RvD1 group were significantly higher than those in the Sham group [(5.92±0.31), (5.85±0.24), (5.06±0.08) vs (4.14±0.10), (32.9±1.5)%, (31.9±1.3)%, (17.7±1.8)% vs (8.1±0.6)%, (72.1±2.3), (66.7±3.7), (34.0±1.4) vs (22.0±0.8) nmol/mg, (3.99±0.28), (3.86±0.25), (2.66±0.16) vs (1.47±0.17) pg/mg, (9.45±0.53), (9.68±0.62), (7.62±0.22) vs (4.70±0.41) pg/mg, (3.01±0.18), (2.92±0.19), (1.58±0.11) vs (0.98±0.07) U/g] (all P<0.05). The plasma levels of the cytokines mentioned above, the W/D, the apoptosis index, MDA, MCP-1, MIP-2 contents and MPO activity in RvD1 group were significantly lower than those in the lung ischemia reperfusion control group [(63±4) vs (110±7) ng/L, (90±8) vs (151±8) ng/L, (1 835±182) vs (2 690±133) ng/L, (5.06±0.08) vs (5.92±0.31), (17.7±1.8)% vs (32.9±1.5)%, (34.0±1.4) vs (72.1±2.3) nmol/mg, (2.66±0.16) vs (3.99±0.28) pg/mg, (7.62±0.22) vs (9.45±0.53) pg/mg, (1.58±0.11) vs (3.01±0.18) U/g]. Besides, NF-κB protein relative expression level of lung tissues down-regulated [(0.313±0.012) vs (0.681±0.033)] (all P<0.05). Inflammatory cell infiltration in LIRI groups increased significantly, while it was significantly reduced in RvD1 group. Conclusion: RvD1 can effectively alleviate the tissue damage caused by lung ischemia-reperfusion through down-regulating NF-κB expression, relieving inflammatory reaction and oxidative stress, reducing apoptosis in rats.

Effect of sevoflurane on unfolded protein response-related cell apoptosis during acute lung injury in rats undergoing cardiopulmonary bypass

Objective To evaluate the effect of sevoflurane on unfolded protein response (UPR)-related cell apoptosis during acute lung injury in rats undergoing cardiopulmonary bypass (CPB). Methods Forty-eight clean-grade healthy adult male Sprague-Dawley rats, aged 6-8 weeks, weighing 250-300 g, were allocated into 3 groups (n=16 each) using a random number table method: sham operation group (Sham group), CPB group and sevoflurane group (Sev group). Left common carotid artery and right internal jugular vein were only cannulated in group Sham.After establishing CPB, the flow rate was gradually adjusted to the maximum (100 ml·kg-1·min-1) and maintained for 60 min in group CPB.Two percent sevoflurane was inhaled for 30 min, and 15 min later the model of CPB was established in Sev group.Rats were sacrificed at 1 h after the end of CPB, lungs were removed and lung tissues were obtained.The pathological changes and ultrastructure of lung tissues were examined with a light microscope and with an electron microscope, respectively.The wet to dry weight ratio (W/D ratio), apoptosis in lung cells (by TUNEL assay), expression of glucose-regulated protein 78 (GRP78), CCAAT/enhancer-binding protein homologous protein (CHOP), c-Jun N-terminal kinase (JNK) and caspase-12 mRNA was determined by real-time polymerase chain reaction.The expression of GRP78, CHOP, phosphorylated JNK (p-JNK) and caspase-12 was detected by Western blot.The index of quantitative assessment of histologic lung injury (IQA) was measured, and apoptotic index (AI) was calculated. Results Compared with Sham group, the W/D ratio, IQA and AI were significantly increased, the expression of GRP78, CHOP, JNK and caspase-12 was up-regulated (P<0.05), and the pathological changes of lung tissues were accentuated in CPB group.Compared with CPB group, the W/D ratio, IQA and AI were significantly decreased, the expression of GRP78, CHOP, JNK and caspase-12 was down-regulated (P<0.05), and the pathological changes of lung tissues were significantly attenuated in Sev group. Conclusion The mechanism by which sevoflurane mitigates acute lung injury induced by CPB is related to inhibiting UPR-related cell apoptosis in lung tissues of rats. Key words: Anesthetics, Inhalation; Extracorporeal circulation; Lung injury; Apoptosis

Protein S is Protective in Acute Lung Injury by Inhibiting Cell Apoptosis.

Acute lung injury is a fatal disease characterized by inflammatory cell infiltration, alveolar-capillary barrier disruption, protein-rich edema, and impairment of gas exchange. Protein S is a vitamin K-dependent glycoprotein that exerts anticoagulant, immunomodulatory, anti-inflammatory, anti-apoptotic, and neuroprotective effects. The aim of this study was to evaluate whether human protein S inhibits cell apoptosis in acute lung injury. Acute lung injury in human protein S transgenic and wild-type mice was induced by intratracheal instillation of lipopolysaccharide. The effect of human protein S on apoptosis of lung tissue cells was evaluated by Western blotting. Inflammatory cell infiltration, alveolar wall thickening, myeloperoxidase activity, and the expression of inflammatory cytokines were reduced in human protein S transgenic mice compared to the wild-type mice after lipopolysaccharide instillation. Apoptotic cells and caspase-3 activity were reduced while phosphorylation of extracellular signal-regulated kinase was enhanced in the lung tissue from human protein S transgenic mice compared to wild-type mice after lipopolysaccharide instillation. The results of this study suggest that human protein S is protective in lipopolysaccharide-induced acute lung injury by inhibiting apoptosis of lung cells.

Protective effect of Rhubarb on newborn rats with bronchopulmonary dysplasia

Objective To investigate the effect of Rhubarb on bronchopulmonary dysplasia(BPD) and its potential mechanism. Methods Neonatal Sprague-Dawley rats (postnatal day 4) were exposed to 600 mL/L O2 to induce BPD.The experimental rats were randomly divided into 4 groups with 16 in each group: air+ saline group, air+ Rhubarb(600 mg/kg)group, and hyperoxia+ saline group, and hyperoxia+ Rhubarb group.The rats were sacrificed and lung tissues were obtained on day 14 and 21 after birth.Hematoxylin-easin staining was used to detect the pathomorphology of the lungs.Apoptosis of the lung tissue was detected by means of TdT-mediated dUTP nick-end labeling(TUNEL). The expression of Fas was detected by adopting Western blot.The activity of Caspase-8 and Caspase-3 was detected by using spectrophotometer. Results The lung structure of rats was markedly abnormal (decreased, enlarged and simplified alveoli) after being exposed to hyperoxia at any time point.The apoptosis indexes (39.91±1.91 vs.10.11±1.64, 48.80±4.51 vs.12.90±3.18), the expression levels of Fas (0.47±0.02 vs.0.21±0.01, 0.55±0.02 vs.0.22±0.01) and the activities of Caspase-8 (52.59±1.23 vs.40.74±1.08, 60.20±3.48 vs.40.39±2.47) and Caspsase 3 (57.17±1.88 vs.42.00±1.19, 64.57±2.79 vs.41.54±1.27) were all increased in the hyperoxia+ saline group compared with those in the air+ saline group on day 14 and 21, and the differences were statistically significant(all P<0.001). However, compared with the hyperoxia+ saline group, Rhubarb administration dramatically decreased pulmonary apoptosis index (26.49±2.65 vs.22.97±3.66), Fas protein level (0.27±0.03 vs.0.31±0.01) and the activity of Caspase-8 (32.70±2.69 vs.30.66±4.48) and Caspase-3 (44.94±1.60 vs.44.59±1.66), and the differences were statistically significant(all P<0.001). Meanwhile, lung damage after hyperoxia was significantly attenuated in the hyperoxia+ Rhubarb group. Conclusion Rhubarb can reduce the hyperoxic lung injury of BPD by reducing the apoptosis of newborn rat lung tissue cells, and the mechanism may involve the apoptosis pathway of Fas. Key words: Bronchopulmonary dysplasia; Rhubarb; Apoptosis

Effect of exogenous hydrogen sulfide on mitochondrion-dependent apoptosis in lung tissues of rats with endotoxemia

Objective To evaluate the effect of exogenous hydrogen sulfide (H2S) on mitochondrion-dependent apoptosis in lung tissues of rats with endotoxemia. Methods Forty healthy male Sprague-Dawley rats, aged 2-3 months, weighing 250-310 g, were divided into 5 groups (n=8 each) using a random number table: control group (group C), lipopolysaccharide (LPS) group, low dose sodium hydrosulphide (NaHS) group (group L-NaHS), moderate dose NaHS group (group M-NaHS) and high dose NaHS group (group H-NaHS). Endotoxemia was induced by IV LPS 5 mg/kg in chloral hydrate-anesthetized rats.The equal volume of 0.9% sodium chloride solution was intravenously injected in group C. NaHS (an exogenous donor of H2S) 0.78, 1.56 and 3.12 mg/kg were intraperitoneally injected at 3 h after LPS injection in L-NaHS, M-NaHS and H-NaHS groups, respectively.The rats were sacrificed at 6 h after injection of LPS or 0.9% sodium chloride solution, and lungs were removed for examination of the mitochondrial ultrastructure of lung tissues and for determination of apoptosis in lung cells (by flow cytometry) and expression of caspase-3, caspase-9, Bcl-2 and Bax protein and mRNA in lung tissues (by Western blot or real-time polymerase chain reaction). The apoptosis rate and ratio of Bcl-2 expression to Bax expression (Bcl-2/Bax ratio) were calculated.The expression of cytochrome c (Cyt c) in cytoplasm and mitochondria of lung tissues was detected by Western blot. Results The apoptosis rate was significantly increased, the expression of caspase-3, caspase-9, Bcl-2 and Bax protein and mRNA was up-regulated, Bcl-2/Bax ratio was decreased, the expression of Cyt c in cytoplasm was up-regulated, and the expression of Cyt c in mitochondria was down-regulated in group LPS (P 0.05), and the damage to mitochondrial ultrastructure was significantly mitigated in M-NaHS and H-NaHS groups. Conclusion The mechanism by which exogenous H2S inhibits cell apoptosis in lung tissues may be related to inhibition of mitochondrion-dependent apoptosis in rats with endotoxemia. Key words: Hydrogen sulfide; Endotoxemia; Lung; Apoptosis; Mitochondria

Erythropoietin attenuates hyperoxia-induced lung injury by upregulating epidermal growth factor-like domain 7 in newborn rats.

The aim of the present study was to observe the effects of recombinant human erythropoietin (rhEPO) on the expression of epidermal growth factor-like domain 7 (EGFL7) and cell apoptosis in lung tissue following hyperoxic lung injury in newborn rats. The 96 Sprague-Dawley newborn rats were randomly divided into 4 groups (n=24) as follows: Room air-exposed control group, room air-exposed rhEPO-treated group, hyperoxia-exposed group and the hyperoxia-exposed rhEPO-treated group. Pups (n=8) from each group were sacrificed on postnatal days 3, 7 and 14. The pulmonary morphometric and microvessel density changes were observed. In addition, the mRNA and protein expression levels of EGFL7, B-cell lymphoma 2 (Bcl-2) and Bcl-2-like protein 4 (Bax) in lung tissue samples were measured. The rats in the hyperoxia-exposed group exhibited alveolar and pulmonary vascular dysplasia, as well as low mRNA and protein expression levels of EGFL7 and Bcl-2, in addition to high level of Bax in the lung tissue samples when compared with the room air-exposed control group (P<0.05). However, in the hyperoxia-exposed rhEPO-treated group the lung histopathology was improved, and the protein and mRNA expression levels of EGFL7 and Bcl-2 were increased compared with the hyperoxia-exposed group (P<0.05). Furthermore, the expression level of Bax was lower than that of the hyperoxia-exposed group (P<0.05). The present study demonstrated that rhEPO promotes alveolar development and increases pulmonary vascular density by upregulating the expression level of EGFL7 in hyperoxia-induced lung injury of newborn rats.

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.

Preventing cleavage of Mer promotes efferocytosis and suppresses acute lung injury in bleomycin treated mice

Mer receptor tyrosine kinase (Mer) regulates macrophage activation and promotes apoptotic cell clearance. Mer activation is regulated through proteolytic cleavage of the extracellular domain. To determine if membrane-bound Mer is cleaved during bleomycin-induced lung injury, and, if so, how preventing the cleavage of Mer enhances apoptotic cell uptake and down-regulates pulmonary immune responses. During bleomycin-induced acute lung injury in mice, membrane-bound Mer expression decreased, but production of soluble Mer and activity as well as expression of disintegrin and metalloproteinase 17 (ADAM17) were enhanced . Treatment with the ADAM inhibitor TAPI-0 restored Mer expression and diminished soluble Mer production. Furthermore, TAPI-0 increased Mer activation in alveolar macrophages and lung tissue resulting in enhanced apoptotic cell clearance in vivo and ex vivo by alveolar macrophages. Suppression of bleomycin-induced pro-inflammatory mediators, but enhancement of hepatocyte growth factor induction were seen after TAPI-0 treatment. Additional bleomycin-induced inflammatory responses reduced by TAPI-0 treatment included inflammatory cell recruitment into the lungs, levels of total protein and lactate dehydrogenase activity in bronchoalveolar lavage fluid, as well as caspase-3 and caspase-9 activity and alveolar epithelial cell apoptosis in lung tissue. Importantly, the effects of TAPI-0 on bleomycin-induced inflammation and apoptosis were reversed by coadministration of specific Mer-neutralizing antibodies. These findings suggest that restored membrane-bound Mer expression by TAPI-0 treatment may help resolve lung inflammation and apoptosis after bleomycin treatment.

Seawater induces apoptosis in alveolar epithelial cells via the Fas/FasL-mediated pathway

Our previous study showed that seawater can cause lung tissue cell apoptosis; in the present study, the immunohistochemistry and Western blot analysis results demonstrated that Fas, FasL, and cleaved caspase-8 and caspase-3 were up-regulated in the rat lungs exposed to seawater. We found that seawater-induced human lung alveolar epithelial A549 cell apoptosis was concentration and time dependent. Moreover, seawater increased the expression of Fas, FasL, and cleaved caspase-8 and caspase-3 in A549 cells. The incubation of A549 cells in the presence of FasL-neutralising antibody (NOK-2) or caspase-8 inhibitor (Z-IETD-FMK) resulted in a decrease of seawater-induced cell apoptosis. NOK-2 inhibited Fas/FasL interaction and reduced the cleavage of caspase-8 and caspase-3, and Z-IETD-FMK blocked caspase-8 and caspase-3 activation. Seawater similarly produced a significant increase in rat alveolar type II cell apoptosis and expression of Fas and cleaved caspase-8. In summary, the Fas/FasL pathway involved in alveolar epithelial cell (AEC) apoptosis could be important in the pathogenesis of seawater-induced acute lung injury (SW-ALI).

RETRACTED: Ischemia postconditioning and mesenchymal stem cells engraftment synergistically attenuate ischemia reperfusion-induced lung injury in rats

BackgroundIt has been reported that ischemic postconditioning (IPO) or mesenchymal stem cell (MSC) engraftment could protect organs from ischemia/reperfusion (I/R) injury. We investigated the synergetic effects of combined treatment on lung injury induced by I/R. MethodsAdult Sprague-Dawley rats were randomly assigned to one of the following groups: sham-operated control, I/R, IPO, MSC engraftment, and IPO plus MSC engraftment. Lung injury was assessed by arterial blood gas analysis, the wet/dry lung weight ratio, superoxide dismutase level, malondialdehyde content, myeloperoxidase activity, and tissue histologic changes. Cytokine expression was detected using real-time polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay. Cell apoptosis was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end assay and annexin V staining. ResultsMSC engraftment or IPO alone markedly attenuated the lung wet/dry weight ratio, malondialdehyde and myeloperoxidase production, and lung pathologic injury and enhanced arterial partial oxygen pressure, superoxide dismutase content, inhibited pro-inflammatory cytokine levels, and decreased cell apoptosis in lung tissue, compared with the I/R group. In contrast, IPO pretreatment enhanced the protective effects of MSC on I/R-induced lung injury compared with treatment alone. Moreover, in the combined treatment group, the number of MSC engraftments in the lung tissue was increased, associated with enhanced survival of MSCs compared with MSC treatment alone. Additional investigation showed that IPO treatment increased expression of vascular endothelial growth factor and stromal cell-derived factor-1 in I/R lung tissue. ConclusionsIPO might contribute to the homing and survival of transplanted MSCs and enhance their therapeutic effects through improvement of the microenvironment of I/R injury.

Intestinal lymph drainage reduces lung tissue cell apoptosis of hemorrhagic shock rats

Objective To explore the effect of the intestinal lymph drainage on lung tissue cell apoptosis in rats with hemorrhagic shock after resuscitation,rich ALI intestinal lymphatic pathway theory.Methods TUNEL method was used to determine the apoptosis of lung tissue cells,the brown nuclei were apoptotic cells.SABC was used to determine Bcl-2 and Bax protein expression.Results The shock group and shock + drainage group lung tissue cell apoptosis rate were significantly higher than those in the sham operation group,but the shock + drainage group,the apoptosis of lung tissue cells was significantly lower than the shock group.Sham operation group showed Bcl-2,Bax protein of expression; In shock group,lung tissue cell Bcl-2 expression was significantly lower than the sham operation group,the Bax expression was significantly higher than that in the sham operation group; In shock + drainage group,lung tissue cells shored enhanced expression of Bcl-2,Bax expression was reduced,and the shock + drainage group lung tissue cell Bcl-2 expression was significantly higher than that in the shock group,the expression of Bax was significantly lower than the shock group.Conclusion The excessive apoptosis of lung tissue cells was one of the mechanisms of lung injury after shock.Intestinal lymph drainage could reduce lung tissue cell apoptosis,the mechanism invdved the regulation of Bcl-2/Bax protein expression. Key words: Shock, hemorrhagic; Lung injury, acute; Mesenteric lymph duct; Apoptosis

Effects of mechanical ventilation on cell apoptosis in lung tissue of rat with acute lung injury

Objective To investigate the effects of different tidal volumes and positive end expiratory pressures on cell apoptosis in lung tissue of rats with acute lung injury.Methods Forty healthy male Sprague-Dawley rats were randomly(random number)divided into five groups,namely low tidal volume group(LV,VT 8 mL/kg),high tidal volume group(HV,VT 30 mL/kg),low tidal volume group with PEEP 2cmH2O(LV2P,VT 8 mL/kg,PEEP 2 cmH2O),low tidal volume group and PEEP 5cmH2O (LV5P,VT 8 mL/kg,PEEP 5 cmH2O)and low tidal volume group and PEEP 8 cmH2O(LV8P,VT 8 mL/kg,PEEP 8 cmH2O).After intravenous administration of oleic acid(OA,0.1 mL/kg),the rat model of acute lung injury was made.Mechanical ventilation was employed in rats of the experiment groups.Rats were sacrificed and their whole lungs were taken after mechanical ventilation for 2 hours.The transferase d-UTP end labeling assay(TUNEL)was used to define the extent and distribution of apoptotic cells in bronchus and lung tissues.The level of caspase-3 protein was determined by immunohistochemistry.Results The apoptotic cells on both alveolar septum and bronchial epithelium obviously increased with high level of caaspase-3 protein in HV group.The number of apoptotic cells obviously decreased with decrease in caspase-3 protein after PEEP ventilation.These changes were more significant in LV5P than those in other groups(P ＜ 0.01).Conclusions The mechanical ventilation with low tidal volume and PEEP produces protective effects on lung from injury.The cell apoptosis plays an important role in the course of VILI. Key words: Acute lung injury; Ventilation; Apoptosis; Caspase-3 protein

Effects of naloxone on pneumocyte apoptosis during pulmonary ischemia reperfusion injury

Objective To investigate the effects of naloxone (Na) on pneumocyte apoptosis and expression of heme oxygenase-1 (HO-1) during ischemia reperfusion injury of lung in rats. Method Forty-two male Sprague-Dawley rats were made models of ischemia reperfusion injury of unilateral lung, and were randomly( random number) divided into three groups: sham operation group (Sh group), ischemia reperfusion group (IR group) and naloxone group (Na group). The hilus of lung was clamped for 45 minutes and the clamp was taken off to build the I/R model. After 3-6 hours reperfusion, naloxone in dose of 1 mg/kg was injected intra-peritoneally in rats of Na group. The rate of cell apoptosis in lung tissue was detected with the way of Annexin-V-PI in flow cy-tometer. The wet to dry weight ratio (W/D) of lung tissue was measured. The expression of HO-1 in lung was measured by using RT-PCR and the ultra-structure change of lung tissue was observed under electron microscope. Results The rate of pneumocyte apoptosis and W/D ratio of lung tissue were significantly higher in IR group than in Sh group (P < 0.01), and the rate of pneumocyte apoptosis and W/D ratio of lung tissue were negatively correlated with the expression of HO-1 mRNA in lung tissue. Compared with IR group, the rate of cell apoptosis and W/D ratio were lower and the expression of HO-1 mRNA was higher in Na group (P < 0.01). The ultra- structure changes of lung tissue were lessened in Na group than in IR group. Conclusions During early period of lung IR injury, HO-1 induced by naloxone can inhibit the cellular apoptosis and protect the lung tissue. Key words: Rat; Lung; Reperfusion injury; Naloxone; HO-1; Apoptosis; RT-PCR; Annexin-V-PI