Ubiquitin carboxyl-terminal hydrolase isozyme L1/UCHL1 suppresses epithelial-mesenchymal transition and is under-expressed in cadmium-transformed human bronchial epithelial cells.

Cadmium (Cd), a highly toxic heavy metal, is widespreadly distributed in the environment. Chronic exposure to Cd is associated with the development of several diseases including cancers. Over the decade, many researches have been carried on various models to examine the acute effects of Cd; yet, limited knowledge is known about the long-term Cd exposure, especially in the human lung cells. Previously, we showed that chronic Cd-exposed human bronchial epithelial BEAS-2B cells exhibited transformed cell properties, such as anchorage-independent growth, augmented cell migration, and epithelial-mesenchymal transition (EMT). To study these Cd-transformed cells more comprehensively, here, we further characterized their subproteomes. Overall, a total of 63 differentially expressed proteins between Cd-transformed and passage-matched control cells among the five subcellular fractions (cytoplasmic, membrane, nuclear-soluble, chromatin-bound, and cytoskeletal) were identified by mass spectrometric analysis and database searching. Interestingly, we found that the thiol protease ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1) is one of the severely downregulated proteins in the Cd-transformed cells. Notably, the EMT phenotype of Cd-transformed cells can be suppressed by forced ectopic expression of UCHL1, suggesting UCHL1 as a crucial modulator in the maintenance of the proper differentiation status in lung epithelial cells. Since EMT is considered as a critical step during malignant cell transformation, finding novel cellular targets that can antagonize this transition may lead to more efficient strategies to inhibit cancer development. Our data report for the first time that UCHL1 may play a function in the suppression of EMT in Cd-transformed human lung epithelial cells, indicating that UCHL1 might be a new therapeutic target for chronic Cd-induced carcinogenesis. Graphical abstract.

PTPL1 suppresses lung cancer cell migration via inhibiting TGF-ß1-induced activation of p38 MAPK and Smad 2/3 pathways and EMT.

Epithelial-mesenchymal transition (EMT) enables dissemination of neoplastic cells and onset of distal metastasis of primary tumors. However, the regulatory mechanisms of EMT by microenvironmental factors such as transforming growth factor-ß (TGF-ß) remain largely unresolved. Protein tyrosine phosphatase L1 (PTPL1) is a non-receptor protein tyrosine phosphatase that plays a suppressive role in tumorigenesis of diverse tissues. In this study we investigated the role of PTPL1/PTPN13 in metastasis of lung cancer and the signaling pathways regulated by PTPL1 in terms of EMT of non-small cell lung cancer (NSCLC) cells. We showed that the expression of PTPL1 was significantly downregulated in cancerous tissues of 23 patients with NSCLC compared with adjacent normal tissues. PTPL1 expression was positively correlated with overall survival of NSCLC patients. Then we treated A549 cells in vitro with TGF-ß1 (10 ng/mL) and assessed EMT. We found that knockdown of PTPL1 enhanced the migration and invasion capabilities of A549 cells, through enhancing TGF-ß1-induced EMT. In nude mice bearing A549 cell xenografts, knockdown of PTPL1 significantly promoted homing of cells and formation of tumor loci in the lungs. We further revealed that PTPL1 suppressed TGF-ß-induced EMT by counteracting the activation of canonical Smad2/3 and non-canonical p38 MAPK signaling pathways. Using immunoprecipitation assay we demonstrated that PTPL1 could bind to p38 MAPK, suggesting that p38 MAPK might be a direct substrate of PTPL1. In conclusion, these results unravel novel mechanisms underlying the regulation of TGF-ß signaling pathway, and have implications for prognostic assessment and targeted therapy of metastatic lung cancer.

Protein tyrosine phosphatase L1 represses endothelial-mesenchymal transition by inhibiting IL-1ß/NF-κB/Snail signaling.

Endothelial-mesenchymal transition (EnMT) plays a pivotal role in various diseases, including pulmonary hypertension (PH), and transcription factors like Snail are key regulators of EnMT. In this study we investigated how these factors were regulated by PH risk factors (e.g. inflammation and hypoxia) in human umbilical vein endothelial cells (HUVECs). We showed that treatment with interleukin 1ß (IL-1ß) induced EnMT of HUVECs via activation of NF-κB/Snail pathway, which was further exacerbated by knockdown of protein tyrosine phosphatase L1 (PTPL1). We demonstrated that PTPL1 inhibited NF-κB/Snail through dephosphorylating and stabilizing IκBα. IL-1ß or hypoxia could downregulate PTPL1 expression in HUVECs. The deregulation of PTPL1/NF-κB signaling was validated in a monocrotaline-induced rat PH (MCT-PH) model and clinical PH specimens. Our findings provide novel insights into the regulatory mechanisms of EnMT, and have implications for identifying new therapeutic targets for clinical PH.

Crosstalk between the Akt/mTORC1 and NF-κB signaling pathways promotes hypoxia-induced pulmonary hypertension by increasing DPP4 expression in PASMCs.

Abnormal wound healing by pulmonary artery smooth muscle cells (PASMCs) promotes vascular remodeling in hypoxia-induced pulmonary hypertension (HPH). Increasing evidence shows that both the mammalian target of rapamycin complex 1 (mTORC1) and nuclear factor-kappa B (NF-κB) are involved in the development of HPH. In this study, we explored the crosstalk between mTORC1 and NF-κB in PASMCs cultured under hypoxic condition and in a rat model of hypoxia-induced pulmonary hypertension (HPH). We showed that hypoxia promoted wound healing of PASMCs, which was dose-dependently blocked by the mTORC1 inhibitor rapamycin (5-20 nM). In PASMCs, hypoxia activated mTORC1, which in turn promoted the phosphorylation of NF-κB. Molecular docking revealed that mTOR interacted with IκB kinases (IKKs) and that was validated by immunoprecipitation. In vitro kinase assays and mass spectrometry demonstrated that mTOR phosphorylated IKKα and IKKß separately. Inhibition of mTORC1 decreased the level of phosphorylated IKKα/ß, thus reducing the phosphorylation and transcriptional activity of NF-κB. Bioinformatics study revealed that dipeptidyl peptidase-4 (DPP4) was a target gene of NF-κB; DPP4 inhibitor, sitagliptin (10-500 µM) effectively inhibited the abnormal wound healing of PASMCs under hypoxic condition. In the rat model of HPH, we showed that NF-κB activation (at 3 weeks) was preceded by mTOR signaling activation (after 1 or 2 weeks) in lungs, and administration of sitagliptin (1-5 mg/kg every day, ig) produced preventive effects against the development of HPH. In conclusion, hypoxia activates the crosstalk between mTORC1 and NF-κB, and increased DPP4 expression in PASMCs that leads to vascular remodeling. Sitagliptin, a DPP4 inhibitor, exerts preventive effect against HPH.

Phosphorylation of histone H2B serine 32 is linked to cell transformation.

Various types of post-translational modifications of the histone tails have been revealed, but a few modifications have been found within the histone core sequences. Histone core post-translational modifications have the potential to modulate nucleosome structure and DNA accessibility. Here, we studied the histone H2B core domain and found that phosphorylation of H2B serine 32 occurs in normal cycling and mitogen-stimulated cells. Notably, this phosphorylation is elevated in skin cancer cell lines and tissues compared with normal counterparts. The JB6 Cl41 mouse skin epidermal cell line is a well established model for tumor promoter-induced cell transformation and was used to study the function of H2B during EGF-induced carcinogenesis. Remarkably, cells overexpressing a nonphosphorylatable H2BS32A mutant exhibited suppressed growth and EGF-induced cell transformation, possibly because of decreased activation of activator protein-1, compared with control cells overexpressing wild type H2B. We identified ribosomal S6 kinase 2 (RSK2) as the kinase responsible for H2BS32 phosphorylation. Serum-starved JB6 cells contain very little endogenous H2BS32 phosphorylation, and EGF treatment induced this phosphorylation. The phosphorylation was attenuated in RSK2 knock-out MEFs and RSK2 knockdown JB6 cells. Taken together, our results demonstrate a novel role for H2B phosphorylation in cell transformation and show that H2BS32 phosphorylation is critical for controlling activator protein-1 activity, which is a major driver in cell transformation.

[Determination of trace lead and cadmium in transgenic rice by crosslinked carboxymethyl konjac glucomannan microcolumn preconcentration combined with graphite furnace atomic absorption spectrometry].

A novel method was developed for the determination of trace lead and cadmium in transgenic brown rice based on separation and preconcentration with a micro column packed with crosslinked carboxymethyl konjac glucomannan (CCMKGM) prior to its determination by graphite furnace atomic absorption spectrometry. Variables affecting the separation and preconcentration of lead and cadmium, such as the acidity of the aqueous solution, sample flow rate and volume, and eluent concentration and volume, were optimized. Under optimized condition, detection limits of the method for the determination of trace lead and cadmium in transgenic brown rice were 0.11 and 0.002 microg x L(-1), respectively. The obtained results of lead and cadmium in the certified reference material (GBW10010, GBS1-1) were in good agreement with the certified values. The recoveries were in the range of 90%-103% and 93%-105% for detection of Pb and Cd in transgenic brown rice and the wild-type brown rice samples respectively. This study could provide technical support for determination of trace Pb and Cd in transgenic rice.

Transcriptional upregulation of MAPK15 by NF-κB signaling boosts the efficacy of combination therapy with cisplatin and TNF-α.

The efficacy of cisplatin in treating advanced non-small cell lung cancer is limited mainly because of insensitivity and/or acquired resistance. MAPK15, previously shown by us to enhance the sensitivity of the anti-cancer drug arsenic trioxide, could also enhance the sensitivity of other anti-cancer drugs. Here, we explore the potential role of MAPK15 in chemosensitivity to cisplatin in human lung cancer cells. Our results indicated that the expression level of MAPK15 was positively correlated with cisplatin sensitivity through affecting the DNA repair capacity of cisplatin-treated cells. The expression of MAPK15 was transcriptionally regulated by the TNF-α-activated NF-κB signaling pathway, and TNF-α synergized with cisplatin, in a MAPK15-dependent manner, to exert cytotoxicity in vitro and in vivo. Therefore, levels of TNF-α dictate the responsiveness/sensitivity of lung cancer cells to cisplatin by transcriptionally upregulating MAPK15 to enhance chemosensitivity, suggesting manipulation of MAPK15 as a strategy to improve the therapeutic efficacy of chemotherapeutic drugs.

Retrospective Study of Critically Ill COVID-19 Patients With and Without Extracorporeal Membrane Oxygenation Support in Wuhan, China.

Background: Extracorporeal membrane oxygenation (ECMO) might benefit critically ill COVID-19 patients. But the considerations besides indications guiding ECMO initiation under extreme pressure during the COVID-19 epidemic was not clear. We aimed to analyze the clinical characteristics and in-hospital mortality of severe critically ill COVID-19 patients supported with ECMO and without ECMO, exploring potential parameters for guiding the initiation during the COVID-19 epidemic. Methods: Observational cohort study of all the critically ill patients indicated for ECMO support from January 1 to May 1, 2020, in all 62 authorized hospitals in Wuhan, China. Results: Among the 168 patients enrolled, 74 patients actually received ECMO support and 94 not were analyzed. The in-hospital mortality of the ECMO supported patients was significantly lower than non-ECMO ones (71.6 vs. 85.1%, P = 0.033), but the role of ECMO was affected by patients' age (Logistic regression OR 0.62, P = 0.24). As for the ECMO patients, the median age was 58 (47-66) years old and 62.2% (46/74) were male. The 28-day, 60-day, and 90-day mortality of these ECMO supported patients were 32.4, 68.9, and 74.3% respectively. Patients survived to discharge were younger (49 vs. 62 years, P = 0.042), demonstrated higher lymphocyte count (886 vs. 638 cells/uL, P = 0.022), and better CO2 removal (PaCO2 immediately after ECMO initiation 39.7 vs. 46.9 mmHg, P = 0.041). Age was an independent risk factor for in-hospital mortality of the ECMO supported patients, and a cutoff age of 51 years enabled prediction of in-hospital mortality with a sensitivity of 84.3% and specificity of 55%. The surviving ECMO supported patients had longer ICU and hospital stays (26 vs. 18 days, P = 0.018; 49 vs. 29 days, P = 0.001 respectively), and ECMO procedure was widely carried out after the supplement of medical resources after February 15 (67.6%, 50/74). Conclusions: ECMO might be a benefit for severe critically ill COVID-19 patients at the early stage of epidemic, although the in-hospital mortality was still high. To initiate ECMO therapy under tremendous pressure, patients' age, lymphocyte count, and adequacy of medical resources should be fully considered.

[Crosslinked carboxymethyl konjac glucomannan microcolumn preconcentration of trace lead, cadmium and copper in water samples and determination by graphite furnace atomic absorption spectrometry].

A novel solid phase extraction technique was developed for the determination of trace lead, cadmium and copper in environmental water samples based on separation and preconcentration with a microcolumn packed with crosslinked carboxymethyl konjac glucomannan (CCMKGM)prior to its determination by graphite furnace atomic absorption spectrometry. Various influential factors on the separation and preconcentration of lead, cadmium and copper, such as the acidity of the aqueous solution, sample flow rate and volume, and eluent concentration and volume, were investigated systematically, and the optimized operation conditions were established. The analytes could be quantitatively retained by CCMKGM in the pH range of 5.0-7.0, then eluted completely with 1.0 mL 0.5 mol x L(-1) HCl. The detection limits (3sigma) for analyte ions were 0.038 microg x L(-1) for Pb2+, 0.0005 microg x L(-1) for Cd2+ and 0.014 microg x L(-1) for Cu2+ with an enrichment factor of 50, and the relative standard deviations were 3.5% for Pb2+, 9.2% for Cd2+ and 4.7% for Cu2+ (c(Pb2+) = C(Cu2+) = 1.0 microg x L(-1); c(Cd2+) = 0.1 microg x L(-1), n=11). The proposed method was successfully applied to the determination of trace lead, cadmium and copper in environmental water samples (local tap water and lake water). In order to validate the method, the developed method was applied to the determination of lead, cadmium and copper in environmental water reference materials (ERMs, GSBZ 50009-88, PR China), and the results obtained were in good agreement with the certified values.

Serum and Seminal Plasma Element Concentrations in Relation to Semen Quality in Duroc Boars.

Element concentrations in serum and seminal plasma were studied in Duroc boars with different semen quality characteristics. Based on the utilization rate of 2174 ejaculates from June to August in 2016, a total of 166 Duroc boars were allocated into three groups: low utilization rate group (LG, 0 to 60% utilization rate), medium utilization rate group (MG, 60 to 80%), and high utilization rate group (HG, 80 to 100%). Serum and seminal plasma samples were collected, and element levels were analyzed using inductively coupled plasma mass spectrometry. The results showed that LG boars had higher concentrations of serum copper and seminal plasma cadmium compared with MG and HG boars (P < 0.05), and serum copper and seminal plasma cadmium were negatively correlated with sperm motility, while positively correlated with the abnormal sperm rate. We observed the abnormal sperm rate increased by approximately 4.53% with serum copper increasing from 1.63 to 2.44 mg/L, while sperm motility decreased by approximately 2.85% with seminal plasma cadmium increasing from 0 to 0.82 µg/L. Moreover, serum iron and manganese levels in the LG group were significantly reduced compared with the HG boars (P < 0.05), and the two elements were negatively correlated with the abnormal sperm rate (P < 0.05). In conclusion, excessive copper and absence of iron and manganese in serum as well as higher seminal plasma cadmium may reduce the utilization rate of semen by impairing sperm motility and morphology, indicating the importance of adding and monitoring microelements in boar diet.

[Determination of lead in microemulsified rapeseed oil and bio-diesel oil by GFAAS].

Bio-diesel oil has attracted much attention as a substitutable energy sources for its renewable and eco-friendly property. However, problems of lead contamination in fuel are also emphasized increasingly at present. So it was of quite significance to determine the contents of lead in bio-diesel oil and its raw material rapeseed oil. An effective method was developed for the rapid determination of lead in rapeseed oil and bio-diesel oil by graphite furnace atomic absorption spectrometry (GFAAS) after their stabilization as microemulsions. In this research work, polyethyleneglycol octyl phenyl ether and n-butanol were used for emulsifier and auxiliary emulsifying agent, respectively. For Pb, efficient thermal stabilization was obtained using NH4H2PO4 as matrix modifier. Sample stabilization was necessary because of evident analyte losses that occurred immediately after sampling. Excellent long-term sample stabilization and the influence of the microemulsion composition on the GFAAS response were observed by mixing different organic solvents. The ashing and atomization temperature and ramp rate influenced the sensitivity obtained for Ph. Take this into account, the optimum conditions of the graphite furnace atomic absorption spectrometric determination of Pb in rapeseed oil and bio-diesel oil samples were investigated. The results showed that the microemulsion was quite stable when the value of V(20% polyethyleneglycol octyl phenyl ether), V(n-butanol), V(oil) and V(water) was 0.1: 8.9: 0.5: 0.5, without matrix interference effect. The determination limit of the proposed method was 126.2 microg x L(-1) for Pb, comfortably below the values found in the analyzed samples. The recoveries were from 81.8% to 109.0%, which performed using the addition of different concentrations of lead to bio-diesel oil, rapeseed oil and petrochemical diesel samples. The relative standard deviation of determination was 5.84%. This work showed the great efficiency of the microemulsion, indicating that it is possible to extract lead from the oil phase. The method was applied to the determination of lead in oil samples with satisfactory results.

[Expression of apoptosis related protein in transforming growth factors-beta signaling pathway and its effects on the cell apoptosis in the lung tissues after acute pulmonary embolism].

OBJECTIVE: To study the expression changes in apoptosis related protein in transforming growth factors-beta signaling pathway (ARTS) in the lung tissues in a rat acute pulmonary embolism (APE) model and its effects on cell apoptosis. METHODS: A rat APE model was reproduced. Samples of lung tissues were harvested at time points of 1, 8, 24 and 48 hours after APE. Healthy rats were used as control. The changes in mRNA level of ARTS were identified by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR), and the changes in its protein level, and also Bcl-2, Bcl-xL, XIAP and H2Ax proteins, which were related with ARTS-mediated cell apoptosis, were determined by Western blotting. Immunohistochemical method was employed to study the distribution and expression changes in ARTS in the lung tissue before and after APE. Apoptotic cells in lung tissue sections were identified by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) method. RESULTS: At different time points, the mRNA levels and the protein levels of ARTS significantly increased in the lung tissues of rats with APE at 1 hour and 48 hours (P<0.05 or P<0.01). The immunohistochemical study showed that ARTS had low expression levels and could not be detected in the normal lung tissue, but it was up-regulated obviously at 48 hours after APE, mainly expressed in the bronchial epithelium and the lung alveolar epithelium. Apoptotic cells could be observed in the lung tissue by TUNEL after APE and at the same time when the lung tissue cells exhibited lower levels of the anti-apoptotic proteins Bcl-2, Bcl-xL, and XIAP, as compared with controls. Apoptosis level (as evaluated by H2Ax, apoptotic marker staining) in the lung tissue cells was obviously raised compared with controls (P<0.05 or P<0.01). CONCLUSION: The expression of ARTS is up-regulated after APE, and ARTS-mediated apoptosis plays an important role in the cell apoptosis of lung tissue.

Inhibition of non-small cell lung cancer cell proliferation and tumor growth by vector-based small interfering RNAs targeting HER2/neu.

Amplification and over-expression of HER2/neu oncogene is found in diverse types of human cancers, and is closely related to tumor occurrence, metastasis, angiogenesis and chemotherapy resistance. Therapeutic agents targeting HER2/neu have been intensively addressed over the past decades. In non-small cell lung cancers (NSCLCs), the prevalence of HER2/neu activation, its role in prognosis, and its possible implications as a therapeutic target, are still to be elucidated. Here we show that the abundant or moderate over-expression of HER2/neu could be detected in both pulmonary adenocarcinoma and pulmonary large cell carcinoma cell lines. Stable knockdown of HER2/neu expression in the NSCLC cell line SPC-A-1 was achieved by vector-based small interfering RNAs (siRNAs), which consequently caused significant decrease in cell proliferation and clone forming efficiency, as well as cell cycle arrest at G(1) phase. Compared with the parental NSCLC cells, HER2/neu knockdown cells exhibited attenuated capacities in developing tumors in nude mice, and the growth tumors xenografts derived from these cells were dramatically regressed. These data provided direct evidence that HER2/neu signaling is essential for tumorigenicity of NSCLC cells, and suggested that siRNAs targeted to HER2/neu may provide a novel therapeutic strategy in the treatment of NSCLC, especially when combined with traditional therapeutics or via development of vector-based siRNAs of multiple targets that synergistically contribute to carcinogenesis, e.g. EGFR and HER2/neu.

Comparative proteomic study of acute pulmonary embolism in a rat model.

Pulmonary embolism (PE) is a common, potentially fatal disease, whose blood clots originate from the deep venous system of the lower extremities. PE is of clinical importance because of the considerable mortality and morbidity. In this study, at first we established a rat PE model by injecting 3-4 emboli into the left jugular vein. Before collecting the lung tissues, we perfused them with saline through the right jugular vein and at the same time cut off the right carotid to remove the blood. Then we separated and identified differentially expressed proteins in lung tissues at different time points using the techniques of 2-DE and MS. After image analysis of 2-DE gels, 46 protein spots of interest were excised from the gels and identified by MALDI-TOF-MS. Thirty-two protein spots of them found their corresponding protein candidates in the database. These proteins are associated with distinct aspects of PE such as the contractive function of smooth muscles, metabolism of energy, collagen and toxicant, cellular differentiation, apoptosis and injury, blood pressure adjustment, maintaining of acid-base balance, and so on. Ten of the identified proteins were validated by semiquantitative RT-PCR, and three of them were further validated by Western blot analysis. The differential expression patterns of these proteins suggest the distinct roles they may play in different stages of the rat PE model, and information from this study may be helpful to uncover the pathophysiologic molecular mechanisms involved in PE.

Comparative proteome analysis of serum from acute pulmonary embolism rat model for biomarker discovery.

Pulmonary embolism (PE) is a common, potentially fatal disease and its diagnosis is challenging because clinical signs and symptoms are nonspecific. In this study, to investigate protein alterations of a rat PE model, total serum proteins collected at different time points were separated by two-dimensional electrophoresis (2-DE) and identified using matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Bioinformatics analysis of 24 differentially expressed proteins showed that 20 had corresponding protein candidates in the database. According to their properties and obvious alterations after PE, changes of serum concentrations of Hp, Fn, DBP, RBP, and TTR were selected to be reidentified by western blot analysis. Semiquantitative RT-PCR showed DBP, RBP, and TTR to be down-regulated at mRNA levels in livers but not in lung tissues. The low serum concentrations of DBP, RBP, and TTR resulted in the up-regulation of 25(OH)D3, vitamin A, and FT4 (ligands of DBP, RBP, and TTR) after acute PE in rat models. The serum levels of Hp and Fn were detected in patients with DVT/PE and controls to explore their diagnostic prospects in acute PE because the mRNA levels of Hp and Fn were found to be up-regulated both in lung tissues and in livers after acute PE. Our data suggested that the concentration of serum Fn in controls was 79.42 +/- 31.57 microg/L, whereas that of PE/DVT patients was 554.43 +/- 136.18 microg/L (P < 0.001), and that the concentration of serum Hp in controls was 824.37 +/- 235.24 mg/L, whereas that of PE/DVT patients was 2063.48 +/- 425.38 mg/L (P < 0.001). The experimental PE rat model selected in this study was more similar to the clinical process than the other existing PE animal models, and the findings indicated instant changes of serum proteins within 48 h after acute PE. The exploration of these differentially expressed proteins or their combination with existent markers such as D-dimer may greatly improve the accuracy of the diagnosis of acute PE, but diagnostic tests are still needed to evaluate the sensitivity and specificity of these markers and also the number of false positives and false negatives.

[NF-kappaB expression in lung tissue of acute lung injury rat model and the influence by antioxidant N-acetylcysteine].

AIM: To observe the NF-kappaB expression in the lung tissue of LPS-induced acute lung injury(ALI) rat model and the influence of N-acetylcysteine (NAC) on NF-kappaB expression. METHODS: The expression of NF-kappaB in lung tissue in ALI rat model and the influence of NAC on NF-kappaB expression were detected by immunohistochemical (ABC) staining and Western blot. RESULTS: There were a small amount of sporadic NF-kappaB cells in airway epithelium and interstitium in normal control group. In contrast, nuclear NF-kappaB expression-positive cells increased obviously in airway mucosa, lung interestium, alveolar cavity and vascular wall of ALI rats. NF-kappaB(+) cells were mainly airway mucosa epithelial cells, infiltrating inflammatory cells, alveolar epithelial cells, and vascular endothelial cells. The NF-kappaB expression-positive cells in NAC therapy group notably decreased compared with ALI group and control group(P<0.01). Western blot analysis showed that the expression of NF-kappaB was different at various time points, reaching the peak at 3 h and then decreased (P<0.01) after LPS induced lung injury. CONCLUSION: In LPS induced acute lung injury rat model, the NF-kappaB nuclear expression increased obviously in airway mucosa, lung interestium and alveolar cavity. Most cells in lung tissue participated in the activation of NF-kappaB. NAC could alleviate inflammation by inhibiting activation of NF-kappaB.

[The changes of expression level of matrix metalloprotease 9 and its inhibitor (TIMP-1) in murine pulmonary fibrosis model].

AIM: To evaluate the balance between matrix metalloprotease-9 (MMP-9) and its inhibitor, tissue inhibitor of metalloproteinases-1 (TIMP-1) in bleomycin-induced pulmonary fibrosis in mice. METHODS: Pulmonary fibrosis model was induced by bleomycin in mice. The histological images of lungs were studied by HE staining. Alveolar macrophages(AMs) were separated by anti-CD68 mAbs using immunomagnetic beads(d0.05). The concentration of MMP-9 secreted by AMs was gradually decreased on day 1, 3, 7, 14 and 28, and levels of TIMP-1 gradually increased. CONCLUSION: There was an imbalance between macrophage-derived MMP-9 and TIMP-1 in bleomycin-induced pulmonary fibrosis in mice.

[Purification and characterization of the fusion protein TGF-betaR II/Fc].

AIM: To express the fusion protein TGF-betaR II/Fc in large amounts by using recombinant Bac-TR II baculovirus expression system constructed by our laboratory and to purify and characterize it. Then, to verify whether the fusion protein TGF-betaR II/Fc can be able to block the biological activity of cytokine TGF-beta1. METHODS: The viral titer was determined by plaque forming test. The recombinant baculovirus was amplified by infecting sf9 cells. The fusion protein was purified by FPLC using protein G column. The purified product was analyzed by SDS-PAGE and the amount of target protein calculated by gray scanning. Western blot and sandwich ELISA were used to affirm the expression of the fusion protein. MTT colorimetry was used to test whether the fusion protein can block the inhibition effect of cytokine TGF-beta1 on the growth of L929 cells. It was to verify whether the fusion protein can reduce the fibronectin production in L929 cells accelerated by TGF-beta1 by western blot. RESULTS: The titer of recombinant Bac-TR II baculavirus in the primary culture fluid was 2x10(12) pfu/L. After electrophoresis, gray scanning analysis showed that the target protein accounted for 10 percent of the total protein. Western blot analysis and sandwich ELISA detection proved that the target protein has been expressed. The fusion protein could block the inhibitive effect of cytokine TGF-beta1 on the growth of L929 cells and fibronectin production in L929 cells. CONCLUSION: The fusion protein TGF-betaR II/Fc can inhibit the biological activity of TGF-beta1 in-vitro. This study will be helpful to the mass production of the fusion protein, and will facilitate its further use in the therapy of pulmonary fibrosis.