Clinicopathological significance of pEZRThr567 expression in lung squamous carcinoma

AIM: To investigate the clinicopathological significance of the protein expression of phosphorylated ezrin at threonine 567 (pEZR^Thr567) in lung squamous cell carcinoma, adjacent tissues and normal tissues. METHODS: pEZR^Thr567 protein was detected in lung squamous carcinoma, adjacent and normal tissues by the method of immunohistochemistry. The correlation of pEZR^Thr567 expression with clinicopathological parameters of lung squamous carcinomas was also analyzed. The localization of pEZR^Thr567 was detected by immunofluorescence staining in lung squamous cell line EBC-1. RESULTS: The protein expression of pEZR^Thr567 in lung squamous carcinoma was significantly higher than that in the adjacent and normal lung tissues (P<0.01). pEZR^Thr567 mainly localized on the cell membrane, and its over-expression signi-ficantly correlated with the differentiation, clinical stage and lymph node metastasis in lung squamous carcinoma. CONCLUSION: pEZR^Thr567 may be an effective biomarker for prediction of malignant potential and poor prognosis of lung cancer.     

Inhibitory effect of cholecystokinin octapeptide on expression of CD14 on rat pulmonary interstitial macrophages induced by lipopolysaccharide in vitro

AIM:To study the effect of cholecystokinin octapeptide(CCK-8) on lipopolysaccharide (LPS)-stimulated pulmonary interstitial macrophage(IM)in vitro.METHODS:Pulmonary IM were isolated and cultured in the presence of LPS, CCK-8, proglumide(the antagonist of CCK receptors) and vehicle alone or together. The expression of mCD14 protein was assayed by flow cytometry, and sCD14 in the supernatant was analyzed semi-quantitatively by Western blot, and TNF-α in the supernatant was detected with ELISA.RESULTS:CCK-8, at concentrations from 10^-7mol/L to 10^-6mol/L inhibited significantly the expression of mCD14, the release of sCD14 and TNF-α to the supernatant up-regulated by LPS(1mg/L). The effect of CCK-8 was inhibited by proglumide. CONCLUSION:CCK-8 modulated negatively several functions of LPS-stimulated pulmonary IM through CCK receptors, which may be one of the mechanisms for CCK-8 to alleviate the inflammation in lung tissues during endotoxemia.     

Clinical significance of KLF15 expression in human lung adenocarcinoma

AIM: To explore the clinical significance of Krüpple-like factor 15 (KLF15) protein expression in the patients with lung adenocarcinoma for exploring the therapeutic and prognositic biomarkers of lung cancer. METHODS: Four cases of lung adenocarcinoma tissues and matched adjacent tissues were collected from our hospital, and the expression of KLF15 protein in these tissues was analyzed by Western blot. At the same time, 72 cases of archived paraffin-embedded samples and clinical data of the patients with lung adenocarcinoma were also collected. The KLF15 protein expression in the archived paraffin-embedded lung adenocarcinoma samples was detected by immunohistochemical staining. The correlations between KLF15 protein expression and clinical characteristics of the patients including prognosis were also analyzed. In addition, the KLF15 protein was up-regulated in A549 cells, and then the effects of KLF15 protein on the viability of the cells were measured by CCK-8 assay. RESULTS: The protein expression of KLF15 in the 4 cases of lung adenocarcinoma tissues was significantly lower than that in matched paracancerous tissues. Fifty-three cases of lung adenocarcinoma specimens showed low expression or no expression of KLF15 protein in total 72 cases (73.6%). The 5-year survival rate of the patients with high expression of KLF15 protein in their specimens was higher than that of the patients with the low expression of KLF15 protein (P<0.01), and the expression of KLF15 protein was significantly correlated with the pathological staging (P<0.01) and T stage (P<0.01) of the patients with lung adenocarcinoma. Furthermore, the low expression of KLF15 protein was an important poor prognostic indicator of the patients. Up-regulation of KLF15 protein in the A549 cells significantly inhibited the growth of the cells. CONCLUSION: KLF15 inhibits the growth of lung adenocarcinoma cells. It could be used as a therapeutic target and a prognostic biomarker for the patients with lung adenocarcinoma.     

Aquaporins in lung

Aquaporins (AQPs) is a novel family of membrane protein whichis in charge of the transportation of water across the membranes. It has been testified that AQPs may be related to many physiologicalfunctions and pathophysiological disorders. In lung, 6 subtypes of AQPs were found, 4 of which have been located in certain tissue. AQP1, AQP3, AQP4 and AQP5 are located in upper respiratory tract and may contribute to the mucus production, bronchial moisturizing and gland secretion. AQP1 and AQP5 are located in lower respiratory tract and may participate in directing the water movement across the blood-gas barrier. AQPs in natal lung may be an important factor to accelerate the reabsorption of the lung liquid. In adult, some diseases such as asthma, pulmonary edema, adult respiratory distress syndrome, which have been considered unbalanced water movement, may be also concerned with AQPs. The relationship between lung edema and AQPs have not been elucidated in details. It should be verified how AQPs work in more organs and tissues as well as in some diseases.     

Application of multiple gene methylations in plasma for diagnosis of lung cancer

AIM: To determine the aberrant methylation status in the gene promoter regions of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3by detecting the plasma specimens and the value of their combined detection for diagnosis of lung cancers. METHODS: Nest methylation specific PCR (nMSP) was used to detect the promoter methylation status of the 5 genes in the plasma from 106 normal controls， lung cancer tissues, lung benign tissues and the plasma from 106 patients with lung cancers. Multiple displacement amplification (MDA) was used to amplify modified genomic DNA to solve the problem of insufficient of plasma DNA template. RESULTS: The positive rates of promoter methylation of CDH13, RASSF1A, DLEC1, SEPT9and RUNX3in the lung cancer tissues were 51.9%, 44.3%, 54.7%, 36.8%, 24.5%, respectively, and those in the plasma were 46.2%, 41.5%, 50.9%, 31.1%, 19.8%, respectively. The results of the Kappa consistency check showed that the lung cancer tissues and the plasma had obviously coherence in the methylation status of the 5 gene promoter regions. Combination of DLEC1, CDH13, RASSF1A, and SEPT9 had a higher diagnostic efficiency than the others, as their ACC value was 0.8208 and youden index was 0.6415 (with the sensitivity of 81.13% and the specificity of 83.02%). CONCLUSION: Combination detection of promoter methylation of lung cancer-related genes in the plasma is expected to apply to the early diagnosis of lung cancer.     

Expression of DNA repair gene ERCC1 and its relationship with PAH-DNA adducts in lung cancer tissues

AIM: To investigate the expression of nucleotide excision repair gene ERCC1 and its relationship with PAH (polycyclic aromatic hydrocarbons)-DNA adducts in lung cancer tissues. METHODS: ERCC1 mRNA expression and the PAH-induced DNA adducts were detected in 150 lung cancer tissues, 120 adjacent lung tissues without cancer cells, 40 benign lung lesions and 40 normal lung tissues. The effects of some exposure factors on the expression of ERCC1 gene and the connection between ERCC1 and PAH-DNA adduct was analyzed. RESULTS: Reduced expression levels of ERCC1 were observed in 46 of 150 (30.7%) lung cancer specimens and 1 of 40 (2.5%) normal lung tissues. Smoking may suppress the expression of ERCC1 gene. The level of PAH-DNA adduct was negatively correlated with the expression of ERCC1 gene, the Spearman coefficient was -0.648, P<0.01. CONCLUSION: ERCC1 is an important nucleotide excision repair gene and may participate in the repair of DNA damage, such as PAH-DNA adduct. Low expression of ERCC1 may play an important role in the development of human lung cancer.     

Protein expression of EMMPRIN,MMP-2 and P53 detected by technique of quantum dots immunofluorescence in human lung cancer

AIM: To investigate the expression of extracellular matrix metalloproteinase inducer (EMMPRIN/CD147), matrix metalloproteinase2 (MMP-2) and P53 proteins in human lung cancer tissues and to explore the relationship between EMMPRIN protein and malignant biological behaviour of lung cancer.METHODS: Fluorescent semiconductor nanocrystals were applied, which were nanometer-sized light-emitting particles and were emerging as a new class of fluorescent probes for cancer detection due to the unique optical and electronic properties. The technique of QDs immunofluorescence histochemistry (QDs-IHC) was used to detect the protein expression of EMMPRIN, P53 and MMP-2 in the human lung cancer microarray, and co-expression of EMMPRIN/P53 proteins was also simultaneously detected by double-labeling immunofluorescence.RESULTS: Compared with non-cancerous lung tissues, the positive rates of EMMPRIN, P53 and MMP-2 proteins in the lung cancer tissues were 70.00%, 77.14% and 72.86%, respectively, and the differences were all significant (P<0.05). The positive rates of EMMPRIN, P53 and MMP-2 proteins were all significantly related with tumor staging(TNM stage) and lymph node metastasis (P<0.05). The positive correlation between EMMPRIN expression and protein levels of MMP-2 and P53 was observed (P<0.01).CONCLUSION: The protein expression of EMMPRIN, P53 and MMP-2 is correlated with the development of lung cancer. Malignant progression of lung cancer promoted by EMMPRIN may be closely related with the expression of MMP-2 and P53.     

Curcumin attenuates rat lung ischemia/reperfusion injury by interfering with autophagy

AIM To investigate the role of curcumin （CUR） in lung ischemia/reperfusion （I/R） injury （LIRI） and its relationship with autophagy. METHODS 40 SD rats were randomly divided into sham operation （sham） group, I/R group, solvent （DMSO） group, CUR group and CUR+rapamycin （CUR-Rap） group. The rats were intraperitoneally injected with normal saline, DMSO, CUR or CUR+Rap before operation. After the rat LIRI model was established, the lung tissues were taken to measure W/D, TLW, IAR, and the contents of SOD and MDA were also measured to indicate the oxidative stress level. Light and electron microscopes were used to observed the morphology and ultrastrucure of lung tissues. The expression levels of autophagy-related proteins were determined by Western blot to evaluate autophagy levels. RESULTS Compared with sham group, wet weight/dry weight （W/D）, total lung water （TLW）, injured alveoli rate（IAR） and malondialdehyde （MDA） content in all other groups were increased, superoxide dismutase （SOD） activity was decreased, the levels of autophagy were increased （P<0.05）, and lung tissue injury and cell ultrastructural damage were aggravated in CUR group. Compared with DMSO group, W/D, TLW and IAR and MDA content were decreased, SOD activity was decreased, autophagy levels were also decreased （P<0.05）, and lung tissue and cell ultrastructural damage were attenuated. Compared with CUR group, W/D, TLW, IAR and MDA content were increased, SOD activity declined, the autophagy levels were increased （P<0.05）, and damage of lung tissues and cells were more serious in CUR-Rap group. CONCLUSION Curcumin attenuates the lung I/R injury in rats, and its mechanism may be related to the reduction of oxidative stress and the inhibition of autophagy.     

Protective effect of emodin on lung injury induced by hepatic fibrosis in rats

AIM:To explore the protective effect of emodin on lung injury induced by hepatic fibrosis in rats. METHODS:The hepatic fibrosis rat model was established with multiple pathogenic factors (CCl 4, ethanol, high fat, high cholesterol and low choline) and treated with different doses (20 mg/kg and 40 mg/kg) of emodin for 4 weeks. The hepatic index was measured. The biochemical indexes, endotoxin, homocysteine, albumin, aspartate aminotransferase,alanine aminotransferase, total bilirubin, total cholesterol and triglyceride, and hepatic fibrosis indexes, hyaluronic acid, laminin, collagen IV and procollagen Ⅲ, were detected. The histopathological changes of the liver were observed. The pulmonary index was determined. The histopathological changes of the lungs were observed. The levels of tumor necrosis factor α (TNF-α), malondialdehyde (MDA), nitric oxide (NO) and peroxynitrite (ONOO-) in the lung tissues were analyzed. RESULTS:The rat hepatic fibrosis model was successfully established. In model group, lung edema and inflammation occurred, and the pulmonary index and the levels of TNF-α, MDA, NO and ONOO- in the lung tissues were increased significantly. In emodin treatment groups, the pulmonary indexes were lower than that in model group. The pathological injury of the lung tissues was alleviated. The levels of TNF-α, MDA, NO and ONOO- in the lung tissues were decreased. CONCLUSION:Emodin has a protective effect on lung injury induced by hepatic fibrosis in rats.     

Effects of propofol on lung injury and PI3K/Akt pathway in rats after liver ischemia and reperfusion

AIM：To study the effect of propofol on phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway in the model of rat lung injury after hepatic ischemia and reperfusion (IR). METHODS：Sixty-six SD rats were randomly divided into 4 groups：sham operation group (n=6), IR group (n=24), propofol group (n=24) and propofol+wortmannin group (n=12). The rats in IR group and propofol group were further divided into 4 subgroups according to the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The rats in propofol+wortmannin group were also divided into 2 subgroups according to the time points of 3 h and 6 h after reperfusion. The rats in sham group were only dissected porta without ligation. The ligation of hepatic pedicle in the rats in IR group was performed to induce liver ischemia for 30 min and then reperfusion was conduced. The rats in propofol group were given slow injection of propofol (20 mg/kg) from the caudal vein 10 min before ischemia, and then propofol was continuously pumped at dose of 20 mg·kg-1·h-1 until rats' death. Other procedures were the same as the rats in IR group. The rats in propofol+wortmannin group were injected with wortmannin, a blocker of PI3K, at dose of 15 μg/kg before ischemia, and also given the injection of propofol as the rats in propofol group. The lung tissues of the rats were collected at the time points of 1 h, 3 h, 6 h and 12 h after reperfusion. The protein levels of total Akt (t-Akt), phosphorylated Akt (p-Akt) and Bcl-2 in the lung tissues were detected by Western blotting. The apoptotic rate was analyzed by flow cytometry with annexin V-FITC/PI staining. RESULTS：Compared with sham group, the protein levels of p-Akt and Bcl-2, and the cell apoptotic rate in the lung tissues in IR group, propofol group and propofol+wortmannin group increased. Compared with IR group, the protein levels of p-Akt and Bcl-2 increased, and cell apoptotic rate of the lung tissues decreased in propofol group. Compared with propofol group, the protein levels of p-Akt and Bcl-2 decreased, and cell apoptotic rate in the lung tissues increased in propofol+wortmannin group. CONCLUSION：Propofol reduces the lung injury in rats induced by liver ischemia and reperfusion, and its mechanism may be involved in PI3K/Akt signaling pathway.     

Effects of early liquid resuscitation combined with hydrogen inhalation on lung injury in septic shock rats induced by lipopolysaccharides

AIM：To study the effects of fluid resuscitation combined with 2% hydrogen inhalation on lung injury in septic shock rats induced by lipopolysaccharides (LPS). METHODS：Sixty male Wistar rats were randomized to 4 groups： normal control group, septic shock control group, fluid resuscitation group and fluid resuscitation plus hydrogen inhalation group. Liquid resuscitation was applied to the rats in fluid resuscitation group and fluid resuscitation plus hydrogen inhalation group. The rats in fluid resuscitation plus hydrogen inhalation group also received 2% hydrogen inhalation and the rats in other groups inhaled air for control. The pathological changes of the lung were observed under light microscope. The levels of inflammatory mediators in bronchoalveolar lavage fluid (BALF) were measured by ELISA. The expression of Fas and Bcl-2 was detected by Western blotting. RESULTS：Compared with fluid resuscitation group, the levels of inflammatory mediators in BALF and the expression of Fas in lung tissues were decreased, and the expression of Bcl-2 in lung tissue was increased in fluid resuscitation plus hydrogen inhalation group. CONCLUSION：Fluid resuscitation with 2% hydrogen inhalation exhibits a protective effect on lung injury induced by LPS.     

Effect of ischemic post-conditioning on Egr-1 and IL-1β expression in ischemia-reperfusion injured lung in rats

AIM: To investigate the protective effects of ischemic post-conditioning on the expression of early growth response factor 1 (Egr-1) and interleukin-1β(IL-1β) in ischemia-reperfusion injured lung in rats. METHODS: The model of lung ischemia-reperfusion injury was established in 24 rats and the rats were randomly allocated to 3 different groups (n=8 in each group): (1) sham group: only sham operation (thoracotomy) and no ischemia for 3 h; (2)ischemia-reperfusion group (I/R group): interruption of pulmonary perfusion and ventilation for 1 h followed by reperfusion for 2 h; (3) ischemic post-conditioning group (IPostC group): ischemic post-conditioning (5 min of reperfusion and 5 min of ischemia for 3 times) between the end of ischemia and the beginning of the reperfusion followed by reperfusion for 1.5 h. The lung tissues (prepared to small pieces of about 20 mg) were collected and homogenized at the end of the experiment. The concentration of myeloperoxidase (MPO) in the homogenate was determined. The wet to dry weight ratio (W/D) of the lung tissues was also measured at the end of reperfusion. The pathological changes of the lung tissues were observed under light microscope after reperfusion. The mRNA expression of Egr-1 and IL-1β in the lung tissues was detected by RT-PCR. RESULTS: Compared with sham group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D were significantly increased in I/R group (P<0.05). The inflammatory responses of the lungs in I/R group were significantly severer than those in sham group. Compared with I/R group, the mRNA expression of Egr-1 and IL-1β, the levels of MPO and W/D in IPostC group were significantly decreased (P<0.05). The inflammatory responses of the lungs in IPostC group were also significantly attenuated. CONCLUSION: Ischemic post-conditioning significantly reduces ischemic reperfusion injury of the lung by inhibiting the expression of Egr-1 and IL-1β.     

Knock-down of NEDD1 expression inhibits lung adenocarcinoma cell proliferation and migration

AIM: To explore the role of neural precursor cell expression developmentally down-regulated protein 1 (NEDD1) in the development and progression of lung cancer. METHODS: The differences of NEDD1 expression levels between lung cancer tissues and tumor-adjacent tissues were analyzed by the method of immunohistochemistry and TCGA database. Kaplan-Meier curve was used to analyze the correlation between lung cancer prognosis and the expression level of NEDD1. The proliferation of A549 cells was tested by plate colony formation experiment after knock-down of NEDD1 expression. The apoptosis rate and cell cycle distribution were examined by flow cytometry. The migration ability of the A549 cells was detected by Transwell assay. The protein levels of cell cycle-related molecules were determined by Western blot. Database analysis was performed to evaluate the relationship between the expression of NEDD1 and cyclin-dependent kinases 2 (CDK2). RESULTS: Compared with the tumor-adjacent tissues, the expression level of NEDD1 in the lung cancer tissues was increased, so as the database analysis, and the higher expression of NEDD1 showed a poorer prognosis. Under light microscope, the A549 cells showed a low proliferation rate after silencing the NEDD1 expression, and the colony formation ability of the cells was also reduced; knock-down of NEDD1 expression induced the apoptosis and inhibited the cell migration; knock-down of NEDD1 expression blocked the cells in G₁/S phase, and the protein levels of p-Rb and cyclinD1 were decreased, while the protein levels of p-Chk1, p-Chk2 and p-p53 were increased (P<0.05). A positive correlation between the expression of NEDD1 and CDK2 was noted by database analysis. CONCLUSION: NEDD1 plays an crucial role in promoting cell proliferation via inhibiting apoptosis and accelerating cell cycle, high expression of NEDD1 in lung adenocarcinoma tissue is related to poor prognosis, thus NEDD1 may be used as a candidate marker molecule for the diagnosis and prognosis of lung cancer.     

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.     

Protective role of endogenous hydrogen sulfide in cholecystokinin octapeptid against acute lung injury induced by lipopolysaccharide

AIM: To explore the role of endogenous hydrogen sulfide (H₂S) in the mechanism of cholecystokinin octapeptide (CCK-8) to alleviate acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Eighty-four Sprague-Dawley rats were randomly divided into seven groups: control, LPS (instilled intratracheally to reproduce the model of ALI), NaHS (H₂S donor) +LPS, propargylglycine ［inhibitor of cysathionine-γ-lyase (CSE), PPG］+LPS, CCK-8+LPS, PPG+CCK-8+LPS and CCK-8 group. Animals were sacrificed at 4 h and 8 h after agent instillation. The wet and dry ratio (W/D) of the lung weight was measured and calculated. Morphological changes of lung tissues were observed. H₂S concentration in plasma, malondialdehyde (MDA) content, myeloperoxidase (MPO) and CSE activities in the lung were determined. Furthermore, the level of P-selectin of lung tissue was measured by radioimmunoassay, the CSE mRNA expression in the lung was detected by RT-PCR, and the protein content in bronchoalveolar lavage fluid (BALF) was detected. RESULTS: Compared with control, severe injury of lung tissues and increase in W/D, protein content in BALF, MDA content, MPO activity and P-selectin level in the lung were observed in rats treated with LPS. LPS also lead to a drop in plasma H₂S concentration, lung CSE activity and CSE mRNA expression. Administration of NaHS before LPS could attenuate the changes induced by LPS, while H₂S concentration, CSE activity and CSE mRNA expression were higher than those in LPS group. However, pre-treatment with PPG exacerbated the lung injury induced by LPS, H₂S concentration, CSE activity and CSE mRNA expression were lower than those in LPS and CCK-8 +LPS group, respectively. CONCLUSION: CCK-8 attenuates LPS-induced acute lung injury by means of anti-oxidation and inhibition of PMN adhesion and aggregation, both of which are mediated by endogenous H₂S.