OBJECTIVETo explore the expression of CASZ1 and its relationship with the pulmonary microvascular development in lung tissue of newborn rats exposed to hyperoxia which induced bronchopulmonary dysplasia (BPD).

METHODForty-eight newborn Sprague Dawley(SD) rats (male and female unlimited) were randomly divided into two groups: experimental group and control group according to random digits table with 24 in each.The rats in experimental group were exposed to high oxygen volume fraction of 800 ml/L and the rats in control group were exposed to normal air. Eight rats were randomly selected from each group on day 3 and 7 after oxygen exposure.The sections of lung were stained with HE method in order to assess lung histological changes, the alveolar development was evaluated by the number of radial alveolar count (RAC) and septal wall thickness. CD31 was detected by immunohistochemistry (IHC) method and the capillary density was calculated. The location, distribution and expression of CASZ1 in the lung tissue were detected by the immunohistochemistry, Western blotting, and quantitative PCR (qPCR).

RESULT(1) Stained by HE, lungs of experimental group showed destroyed alveoli, alveoli fusion and increased septal wall thickness, RAC were significantly lower than those in control group(14 d: septal wall thickness (12.69 ± 0.63) μm vs. (6.53 ± 0.16) μm, RAC 5.9 ± 0.4 vs. 8.4 ± 1.0, t = 19.046, 4.760, P both = 0.000). (2) CD31 protein was expressed predominantly in cytoplasm of pulmonary microvascular endothelial cells. The experimental group CD31 average optical density (AIOD) were decreased compared with control group((16.6 ± 1.6) × 10(3) vs.(40.1 ± 2.4) × 10(3), (18.1 ± 1.4) × 10(3) vs.(83.2 ± 5.2) × 10(3), (49.2 ± 5.4) × 10(3) vs.(136.2 ± 28.1) × 10(3), t=16.185, 16.066 and 6.078, P<0.01 for all comparisons). Capillary density in experimental group was also significantly decreased compared with control group ((3.84 ± 0.15)% vs.(6.01 ± 0.22)%, (4.17 ± 0.38)% vs.(6.15 ± 0.24)%, (5.43 ± 0.44)% vs. (9.13 ± 0.25)%, t = 16.124, 8.773 and 14.076, P all < 0.01). (3)RT-qPCR and Western blotting showed that the CASZ1 mRNA significantly increased in experimental group compared with control group(0.56 ± 0.17 vs. 1.00 ± 0.26, 0.32 ± 0.29 vs. 0.58 ± 0.14, 0.14 ± 0.22 vs. 0.56 ± 0.15, t=3.890, 3.303 and 2.388, P < 0.05 for all comparisons), and the protein expression of CASZ1 also significantly increased in experimental group compared with control group (0.65 ± 0.02 vs. 0.78 ± 0.23, 0.46 ± 0.03 vs. 0.75 ± 0.05, 0.34 ± 0.22 vs. 0.75 ± 0.04, t=6.200 and 10.485 and 14.998, P < 0.05 for all comparisons). (4)The protein level of CASZ1 in experimental group was positively correlated with capillary density (r=0.519, P<0.01).

CONCLUSIONCASZ1 is involved in the whole process of newborn rats BPD and may be linked to pulmonary microvascular dysplasia.

Animals ; Animals, Newborn ; Bronchopulmonary Dysplasia ; pathology ; Female ; Hyperoxia ; pathology ; Lung ; blood supply ; pathology ; Male ; Oxygen ; adverse effects ; Pulmonary Alveoli ; RNA, Messenger ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transcription Factors ; metabolism

OBJECTIVETo observe the protective effects of safflor Injection (SI) and extract of Ginkgo biloba (EGB) on lung ischemia-reperfusion injury (LIRI) and investigate its mechanism.

METHODSIn vivo rabbit model of LIRI was reconstructed. Forty rabbits were randomly and equally divided into four groups: sham-operation group (sham group), ischemia-reperfusion group (model group), ischemia-reperfusion plus SI group (safflor group) and ischemia-reperfusion plus EGB injection group (EGB group). Malondialdehyde (MDA) content, superoxide dismutase (SOD) and xanthine oxidase (XO) activity in serum were measured. The wet/dry weight ratio (W/D) of the lung tissue and activity of myeloperoxidase (MPO) were also tested. Ultrastructure change of the lung tissue was observed by the electron microscope. The expression of intercellular adhesion molecule-1 (ICAM-1) was measured by immunohistochemistry (IHC).

RESULTSIn the model group, MDA and XO increased and SOD decreased in serum compared with the sham group (P<0.01). The values of W/D, MPO and ICAM-1 of the model group were higher than those of the sham group (P<0.01), but those of the safflor group and EGB group were significantly lower than those of the model group (P<0.01). The IHC demonstrated that ICAM-1 expression in lung tissue of the model group was significantly higher than those of the safflor group (P<0.01). Compared with safflor group, in the EGB group MDA, XO, MPO decreased, SOD and ICAM-1 expression increased (P<0.05), but the change of W/D was not statistically significant (P>0.05).

CONCLUSIONSSI and EGB may attenuate LIRI through antioxidation, inhibition of neutrophil aggregation and down-regulation of ICAM-1 expression. But EGB had more effect on the antioxidation, while SI did better on regulating ICAM-1 expression.

Animals ; Female ; Ginkgo biloba ; chemistry ; Immunohistochemistry ; Injections ; Intercellular Adhesion Molecule-1 ; metabolism ; Lung ; blood supply ; pathology ; Male ; Malondialdehyde ; metabolism ; Plant Extracts ; administration & dosage ; pharmacology ; therapeutic use ; Protective Agents ; administration & dosage ; pharmacology ; therapeutic use ; Rabbits ; Reperfusion Injury ; blood ; drug therapy ; Safflower Oil ; administration & dosage ; pharmacology ; therapeutic use ; Superoxide Dismutase ; blood ; Xanthine Oxidase ; blood

OBJECTIVETo explore the effect of curcumin (CUR) on cycteinyl aspirate specific protease-12 (Caspase-12) and pneumocyte apoptosis in pulmonary ischemia/reperfusion (I/R) injury mice.

METHODSThe in vivo unilateral in situ pulmonary I/R injury mouse model was established in C57BL/6J mice. Sixty experimental mice were randomly divided into six groups by random digit table, i. e., the sham-operation group (Sham), the I/R group, the I/R + dimethyl sulfoxide group (I/R + DMSO), the I/R + low dose CUR pre-treated group (I/R + CUR-100), the I/R + middle dose CUR pre-treated group (I/R + CUR-150), the I/R + high dose CUR pre-treated group (I/R + CUR-200), 10 in each group. Mice were euthanized and their left lungs were excised. Wet lung weight to dry lung weight (W/D) and the total lung water content (TLW) were tested. The morphological changes of the lung tissue were observed and index of quantitative evaluation for alveolar damage (IQA) detected under light microscope. The ultra-microstructure of the lung tissue was observed under electron microscope. The mRNA and protein expression levels of Caspase-12 and glucose regulated protein (GRP78) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot. Apoptosis index (AI) of the lung tissue was determined by terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) method.

RESULTSCompared with the Sham group, expression levels of Caspase-12, GRP78 mRNA and protein all significantly increased in the I/R group (P < 0.05); W/D, TLW, IQA, and AI were all notably higher (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were notably observed in I/R group. Compared with the I/R + DMSO group, expression levels of GRP78 mRNA and protein were increasingly higher in the I/R + CUR-100 group, the I/R + CUR-150 group, and the I/R +CUR-200 group (P < 0.05), expression levels of Caspase-12 mRNA and protein were lower (P < 0.05); W/D, TLW, IQA, and AI also decreased (P < 0.05, P < 0.01); the morphological and ultrastructural injury of the lung tissue were gradually alleviated in the I/R + CUR groups.

CONCLUSIONCUR had better effect on the lung protection against I/R injury, which might be related to inhibition for pneumocyte apoptosis associated with Caspase-12 in excessive unfolded protein response (UPR).

Animals ; Apoptosis ; drug effects ; Caspase 12 ; metabolism ; Curcumin ; pharmacology ; Heat-Shock Proteins ; metabolism ; Lung ; blood supply ; Male ; Mice ; Mice, Inbred C57BL ; Reperfusion Injury ; metabolism ; pathology ; prevention & control

OBJECTIVETo observe the level of intracellular reactive oxygen species (ROS) in rats with severe burn and pulmonary microvascular endothelial cells (PMVECs) treated with serum of rat with burn injury, and to investigate the relationship between ROS and apoptosis of PMVECs.

METHODS(1) Twenty-four SD rats were divided into sham injury group ( n = 3) and burn group (n = 21) according to the random number table (the same grouping method below). Rats in burn group were inflicted with 30% TBSA full-thickness scald on the back, and rats in sham injury group were sham injured. Blood samples were collected from abdominal aorta at post injury hour 6, 12, 24, 36, 48, 60, 72 respectively from 3 rats of burn group. The serum content of ROS was assayed by ELISA. The same determination was performed in rats of sham injury group. (2) Five rats were subjected to scald injury as above, and burn serum was prepared 24 hours after injury. Another 5 rats without receiving any treatment were used to prepare normal serum. (3) Marginal pulmonary tissue was harvested from 20 SD young rats. Cells were cultured with tissue block method and indentified with immunohistochemical staining. The third passage of PMVECs in logarithmic phase were inoculated in 6-well plates and 12-well plates. PMVECs in both plates were divided into 4 groups: normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, with 3 wells in each group. Cells in the former 2 groups were respectively cultured with special nutrient solution of endothelial cells without serum added with 15% healthy rat serum or 15% burn rat serum. Cells in the latter 2 groups were cultured with the same culture conditions as in the former two groups correspondingly with addition of 100 µmol/L MnTBAP in the nutrient solution. After being cultured for 24 h, the content of ROS in PMVECs in 6-well plates was detected with flow cytometry. The apoptosis of PMVECs in 12-well plates was observed with acridine orange-ethidium bromide staining, and the apoptosis rate was calculated. Data were processed with one-way analysis of variance and LSD-t test.

RESULTS(1) The serum contents of ROS in rats of burn group were respectively (187 ± 21), (235 ± 22), (231 ± 25), (291 ± 20), (315 ±23) nmol/mL at post injury hour 24, 36, 48, 60, 72, which were significantly higher than that in sham injury group [(141 ± 19) nmol/mL, with t values respectively 7. 86, 9. 57, 13. 87, 14.98, 18.40, P values below 0.01]. (2) Primary cells grew slowly and showed a cobblestone appearance. After passages, cells grew with orderly distribution. The positive rate of coagulation factor VIII of cells was (96 ± 5)% , and thus they were identified as PMVECs. (3) In normal serum group, burn serum group, normal serum + MnTBAP group, and burn serum + MnTBAP group, the contents of ROS in PMVECs were respectively 798 ± 40, 1 294 ± 84, 763 ± 59, 926 ± 42 ( F =93.01, P <0.01), and the apoptosis rates of PMVECs were respectively (6.2 ± 1.3)%, (57.3 ± 6. 7)%, (3.7 ± 0. 8)%, (28.7 ± 5. 7)% (F = 224.50, P <0.01) after being cultured for 24 h. Compared with those of normal serum group, the content of ROS and apoptosis rate of PMVECs in burn serum group increased significantly (with t values respectively 10.40 and 49.06, P values below 0.01). The content of ROS and apoptosis rate of PMVECs in burn serum + MnTBAP group were significantly lower than those in burn serum group (with t values respectively 7.48 and 23.94, P values below 0.01).

CONCLUSIONSSerum content of ROS was increased in severely burned rats. Burn rat serum stimulation on PMVECs can lead to the increase of the intracellular ROS and induce apoptosis. However application of MnTBAP can scavenge ROS and reduce the apoptosis induced by burn rat serum.

Animals ; Apoptosis ; Burns ; blood ; therapy ; Endothelial Cells ; pathology ; Enzyme-Linked Immunosorbent Assay ; Lung ; blood supply ; Oxygen ; Rats ; Reactive Oxygen Species ; blood ; Serum ; metabolism

OBJECTIVETo investigate the role of p38 MAPK on ischemic postconditioning (IPO) attenuating pneumocyte apoptosis after lung ischemia/reperfusion injury (LIRI).

METHODSForty adult male SD rats were randomly divided into 5 groups based upon the intervention (n = 8): control group (C), LIR group (I/R), LIR + IPO group (IPO), IPO + solution control group (D), IPO + SB203580 group (SB). Left lung tissue was isolated after the 2 hours of reperfusion, the ratio of wet lung weight to dry lung weight (W/D), and total lung water content (TLW) were measured. The histological structure of the left lung was observed under light and electron transmission microscopes, and scored by alveolar damage index of quantitative assessment (IQA). Apoptosis index (AI) of lung tissue was determined by terminal deoxynuleotidyl transferase mediated dUTP nick end and labeling (TUNEL) method. The mRNA expression and protein levels of and Bax were measured by RT-PCR and quantitative immunohistochemistry (IHC).

RESULTSCompared with C group, W/D, TLW, IQA, AI and the expression of Bax of I/R were significantly increased, the expression of Bcl-2 and Bcl-2/Bax were significantly decreased (P < 0.05, P < 0.01), and was obviously morphological abnormality in lung tissue. Compared with I/R group, all the indexes of IPO except for the expression of Bcl-2 and Bcl-2/ Bax were obviously reduced, the expression of Bcl-2 and Bcl-2/Bax were increased (P < 0.05, P < 0.01). All the indexes between D and IPO were little or not significant( P > 0.05). The expression of Bcl-2 and Bcl-2/Bax of SB were significantly increased and other indexes were reduced than those of IPO (P < 0.05, P < 0.01).

CONCLUSIONIPO may attenuate pneumocyte apoptosis in LIRI by inactivation of p38 MAPK, up-regulating expression of Bcl-2/Bax ratio.

Alveolar Epithelial Cells ; cytology ; Animals ; Apoptosis ; Disease Models, Animal ; Ischemic Postconditioning ; Lung ; blood supply ; enzymology ; pathology ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; enzymology ; pathology ; prevention & control ; bcl-2-Associated X Protein ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism

In order to investigate the expression of nerve growth factor (NGF) and hypoxia inducible factor-1α (HIF-1α) and its correlation with angiogenesis in non-small cell lung cancer (NSCLC), paraffin-embedded tissue blocks from 20 patients with NSCLC were examined. Twenty corresponding para-cancerous lung tissue specimens were obtained to serve as a control. The expression of NGF, HIF-1α, and vascular endothelial growth factor (VEGF) in the NSCLC tissues was detected by using immunohistochemistry. The microvascular density (MVD) was determined by CD31 staining. The results showed that the expression levels of NGF, HIF-1α and VEGF in the NSCLC tissues were remarkably higher than those in the para-cancerous lung tissues (P<0.05). There was significant difference in the MVD between the NSCLC tissues (9.19±1.43) and para-cancerous lung tissues (2.23±1.19) (P<0.05). There were positive correlations between NGF and VEGF, between HIF-1α and VEGF, and between NGF and HIF-1α in NSCLC tissues, with the spearman correlation coefficient being 0.588, 0.519 and 0.588, respectively. In NSCLC tissues, the MVD had a positive correlation with the three factors (P<0.05). Theses results suggest that NGF and HIF-1α are synergically involved in the angiogenesis of NSCLC.
Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; blood supply ; metabolism ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; biosynthesis ; Immunohistochemistry ; Lung ; blood supply ; metabolism ; pathology ; Lung Neoplasms ; blood supply ; metabolism ; Male ; Middle Aged ; Neovascularization, Pathologic ; metabolism ; Nerve Growth Factor ; biosynthesis ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

OBJECTIVETo investigate the correlation between blood flow assessed by CT perfusion imaging and characteristics of microvascular ultrastructure in non-small cell lung cancer (NSCLC).

METHODStwenty-eight patients with non-small cell lung cancer proven surgically and pathologically underwent perfusion CT examination. The patients were divided into a hyper-perfusion group and a hypo-perfusion group by the median value of blood flow, and then the differences of microvascular ultrastructure in the two groups were analyzed.

RESULTSThe median BF value of the 28 patients was 36.40 ml×100 g(-1)×min(-1). Take this median value as the boundary, the group with hypo-perfusion showed a significantly lower BF value than the group with hyper-perfusion [(30.84 ± 4.79) ml×100 g(-1)×min(-1) vs. (49.67 ± 10.89) ml×100 g(-1)×min(-1), t = -5.925, P < 0.001]. The group with lymph node metastasis showed a significantly lower BF value than the group without lymph node metastasis [(30.78 ± 5.24) ml×100 g(-1)×min(-1) vs. (50.73 ± 11.16) ml×100 g(-1)×min(-1), t = 3.490, P = 0.015]. The maturity of microvessels of the hyper-perfusion group was higher than that of the hypo-perfusion group. Under the electron microscope, the microvessels in the hypo-perfusion group showed a more narrow lumen, poorer integrity of basement membrane, a more close relationship between cancer cells and microvascular wall, and cancer cells were more easily seen in the microvascular lumen.

CONCLUSIONThe blood flow value of CT perfusion imaging may be related with the abnormal microvascular ultrastructure, and may be helpful to the prediction of metastasis risk in NSCLC.

Adult ; Aged ; Carcinoma, Non-Small-Cell Lung ; blood supply ; diagnostic imaging ; metabolism ; pathology ; Female ; Follow-Up Studies ; Humans ; Lung Neoplasms ; blood supply ; diagnostic imaging ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Microvessels ; diagnostic imaging ; ultrastructure ; Middle Aged ; Neoplasm Metastasis ; Perfusion Imaging ; Tomography, Spiral Computed ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVE: To observe the effect of intrauterine hypoxia on the development of rat lung after birth under ordinary pressure and normoxia, on the expression of vascular endothelial growth factor (VEGF) in the lung as the age increasing after birth, and to provide experimental basis for the treatment of intrauterine hypoxia after baby was born. METHODS: Intrauterine hypoxia models were established. The rats were divided into an air-control group (the control group) and a hypoxic 6-day group (the hypoxic group). All rats were fed under normal pressure and normoxia after they were born. At postnatal 7, 14, and 21 days, we measured the pulmonary vascular morphometry, detected the expression of VEGF protein with immunohistochemisty, the expression of VEGF mRNA with real-time PCR, and observed the alteration of capillary endothelium in the lung tissues under the electron microscope. RESULTS: The expression of VEGF protein and VEGF mRNA in the 2 groups increased as the rats grew, but the expression increased slower in the hypoxic group than that in the control group. The increase curve of the 2 groups crossed. There was no significant difference between the 2 groups in the pulmonary vascular morphometry at each experiment time point. Hyperplasia of capillary endothelium decreased with age. Cellular edema of capillary endothelium was obvious especially at the 14th day after birth under the electron microscope. CONCLUSION The expression of VEGF protein and VEGF mRNA has slower increase in the intrauterine hypoxic rats than that in the normal control rats. The expression of VEGF may influence the development of lung vessel after rats was born.
Animals ; Animals, Newborn ; Endothelium, Vascular ; pathology ; Hypoxia ; Lung ; blood supply ; metabolism ; RNA, Messenger ; Rats ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVEErythropoietin and erythropoietin receptor (EPO-R) are expressed in many kinds of tumors. The EPO/EPO-R signaling is involved in tumor cell proliferation, invasion and angiogenesis. The aim of this study was to detect the expression of EPO-R in non-small cell lung cancer (NSCLC), and explore its correlation with angiogenesis.

METHODSThe expression patterns of EPO and EPO-R in 31 cases of NSCLC tissues were detected by immunohistochemistry, and that in benign lung lesions of 21 patients as control. To analyze the correlation of EPO/EPO-R expression patterns and clinicopathological factors. CD34 was used to label the vascular endothelial cells and calculate the microvessel density (MVD).

RESULTSThe positive rates of EPO and EPO-R expression in NSCLC were 67.7% and 96.8%, respectively, significantly higher than those in the control ones. The positive rates of EPO and EPO-R expression in adjacent tissues were 19.4% and 35.5%, and in benign lesions were 9.5% and 19.0%, respectively (P < 0.001). The expression patterns of EPO/EPO-R were not related with pTNM stage, histological type, histological grade and lymph node metastasis (P > 0.05). Increased MVD was correlated with poor differentiation, lymph node metastasis, and advanced stage.

CONCLUSIONSHigh expression of EPO/EPO-R in NSCLC patients suggest that they may be involved in tumorigenesis. EPO/EPO-R expression and MVD are closely related, and they might be an endogenous stimulant of angiogenesis during the progression of non-small cell lung cancer. It may provide evidence for clinical diagnosis.

Antigens, CD34 ; metabolism ; Carcinoma, Non-Small-Cell Lung ; blood supply ; metabolism ; pathology ; Erythropoietin ; metabolism ; Humans ; Lung Neoplasms ; blood supply ; metabolism ; pathology ; Lymphatic Metastasis ; Microvessels ; pathology ; Neoplasm Staging ; Neovascularization, Pathologic ; Receptors, Erythropoietin ; metabolism

Maspin belongs to the serine protease inhibitor (serpin) family and has been proven to be a suppressor of tumor growth and metastasis in many types of tumors. The purpose of this study was to investigate the expression of maspin in non-small cell lung cancer (NSCLC) and its relationship to vasculogenic mimicry (VM). A total of 160 specimens of NSCLC were involved in this study and 20 specimens of normal lung tissue served as controls. VM, microvessel density (MVD) and the expression of maspin were detected by using immunohistochemical staining. The results showed that the positive rates of maspin and VM in the NSCLC group were 48.1% (77/160) and 36.9% (59/160), respectively, which were significantly different from those in the control group with the positive rates of maspin and VM being 100% and 0% respectively (P<0.05). VM, MVD and the expression level of maspin were significantly related to tumor differentiation, lymph node metastasis, clinical stages and postoperative survival time (all P<0.05). The maspin expression in patients with squamous cell carcinoma was significantly higher than that in those with adenocarcinoma (P<0.05). The maspin expression was negatively correlated with VM and MVD, and there was a positive correlation between VM and MVD. Maspin-negative expression, VM and high MVD score were negatively related to the 5-year-survival rate. PTNM stages, VM, MVD and maspin expression were independent prognostic factors for NSCLC (P<0.05). It was suggested that the loss of expression of maspin may participate in the invasion and metastasis of NSCLC and it has a positive relationship to VM in NSCLC. Combined detection of maspin, VM and MVD may help predict the progression and prognosis of NSCLC.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Non-Small-Cell Lung ; blood supply ; metabolism ; pathology ; Female ; Humans ; Lung Neoplasms ; blood supply ; metabolism ; pathology ; Male ; Microvessels ; pathology ; Middle Aged ; Neovascularization, Pathologic ; pathology ; Serpins ; metabolism ; Tumor Cells, Cultured ; Young Adult