The effects of interleukin-1β in modulating osteoclast-conditioned medium's influence on gelatinases in chondrocytes through mitogen-activated protein kinases

Osteoarthritis is recognised to be an interactive pathological process involving the cartilage, subchondral bone and synovium. The signals from the synovium play an important role in cartilage metabolism, but little is known regarding the influence of the signalling from bone. Additionally, the collagenases and stromelysin-1 are involved in cartilage catabolism through mitogen-activated protein kinase （MAPK） signalling, but the role of the gelatinases has not been elucidated. Here, we studied the influence of osteoclastic signals on chondrocytes by characterising the expression of interleukin-1β （IL-1β）-induced gelatinases through MAPK signalling. We found that osteoclast-conditioned media attenuated the gelatinase activity in chondrocytes. However, IL-1β induced increased levels of gelatinase activity in the conditioned media group relative to the mono-cultured chondrocyte group. More specifically, IL-1β restored high levels of gelatinase activity in c-Jun N-terminal kinase inhibitor-pretreated chondrocytes in the conditioned media group and led to lower levels of gelatinase activity in extracellular signal-regulated kinase or p38 inhibitor-pretreated chondrocytes. Gene expression generally correlated with protein expression. Taken together, these results show for the first time that signals from osteoclasts can influence gelatinase activity in chondrocytes. Furthermore, these data show that IL-11~ restores gelatinase activity through MAPK inhibitors; this information can help to increase the understanding of the gelatinase modulation in articular cartilage.

Osteoglycin Expression Influences Gelatinases Activity of Murine Hepatocarcinoma Cells Cultured with Extract of Lymph Node

Objective： To investigate the possible correlation between osteoglycin expression and gelatinases activity of tumor cells. Methods： Eukaryotic expression plasmid and antisense expression plasmid of osteoglycin were constructed, and transfected into mouse hepatocarcinoma Hca-F cells and Hca-P cells, respectively. RT-PCR and Western blot were employed to analyze osteoglycin expression in tumor cells. Zymographic analysis was used to observe the possible correlation between osteoglycin expression and gelatinases activity of tumor cells cultured under different conditions. Results： High expression of osteoglycin after transfection of osteoglycin gene attenuated secretion of gelatinases in Hca-F cells cultured with extract of lymph node （P〈0.05）. However, transfection of osteoglycin gene into Hca-F cells failed to influence gelatinases activity of tumor cells cultured with extracts of liver and spleen or in DMEM medium. Effective suppression of osteoglycin expression in Hca-P cells by osteoglycin shRNA resulted in enhanced activity of gelatinases in Hca-P cells cultured with extract of lymph node （P〈0.05）. However, inhibition of osteoglycin expression in Hca-P cells failed to influence gelatinases activity of tumor cells cultured with extracts of liver and spleen or in DMEM medium. Conclusion： Osteoglycin expression influences gelatinases activity of murine hepatocarcinoma cells cultured with extract of lymph node. Regulation of gelatinases activity might be one of the mechanisms by which osteoglycin contribute to lymphatic metastasis suppression.

Relationship of gelatinases-tight junction proteins and blood-brain barrier permeability in the early stage of cerebral ischemia and reperfusion

Gelatinases matrix metalloproteinase-2 and matrix metalloproteinase-9 have been shown to mediate claudin-5 and occludin degradation, and play an important regulatory role in blood-brain barrier permeability. This study established a rat model of 1.5-hour middle cerebral artery occlusion with reperfusion. Protein expression levels of claudin-5 and occludin gradually decreased in the early stage of reperfusion, which corresponded to the increase of the gelatinolytic activity of matrix metalloproteinase-2 and matrix metalloproteinase-9. In addition, rats that received treatment with matrix metalloproteinase inhibitor N-[（2R）-2-（hydroxamidocarbonylmethyl）-4-methylpenthanoyl]-L- tryptophan methylamide （GM6001） showed a significant reduction in Evans blue leakage and an inhibition of claudin-5 and occludin protein degradation in striatal tissue. These data indicate that matrix metalloproteinase-2 and matrix metalloproteinase-9-mediated claudin-5 and occludin degradation is an important reason for blood-brain barrier leakage in the early stage of reperfusion. The leakage of the blood-brain barrier was present due to gelatinases-mediated degradation of claudin-5 and occludin proteins. We hypothesized that the timely closure of the structural component of the blood-brain barrier （tight junction proteins） is of importance.

Study of Lipocalin-2 Associated with Neutrophilic Gelatinases (uNGAL) in the Urine in Children with the Microbial Inflammatory Diseases of Kidneys and Urinary Tract

Purpose of the study: Research of the clinical and diagnostic significance of determination of Lipocalin-2 associated with neutrophilic gelatinases (uNGAL) in the urine of children with urinary tract infection (UTI) and pyelonephritis. Materials and methods: We examined 30 children with acute pyelonephritis and UTI aged 1 to 16 years (average age 7.32 ± 4.52) including 26 girls and 4 boys. Verification of the diagnosis was conducted on the basis of clinical and laboratory data, medical history and instrumental examination of patients. All children were divided into 2 groups: 1st group—15 children with acute pyelonephritis, 2nd group—15 children with urinary tract infection. uNGAL was measured in the urine by enzyme-linked immunosorbent assay (EISA) (BioVendor Laboratoty Medicine). Results: It is found, that the urine level of NGAL depends on the damage degree of renal parenchyma. The correlation of medium strength was found between the excretion level of uNGAL during the acute period of pyelonephritis and the detection of renal scars according to the DMSA-nephroscintigraphy data. In the group of children with the acute pyelonephritis the direct correlation of medium strength was found between the excretion level of uNGAL/creatinine and leukocytosis value and also with the CRP blood level. Conclusion: The results allow us to recommend the determination of the excretion level of uNGAL/creatinine as an additional non-invasive marker for the early detection of renal parenchyma injury.

Expression of gelatinases and tissue inhibitors of metallo-proteinases in the rhesus monkey(Macaca mulatta) corpus luteum

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are believed to play important roles in the formation and regression of corpus luteum (CL). This study is to investigate the expression of gelatinases (MMP-2, -9) and TIMPs in the rhesus monkey CL in both early and late luteal phases and during the early stages of pregnancy. Ovaries were collected from regularly cycling rhesus monkey at D5 and D15 following ovulation and at D12, D18 and D26 of pregnancy. In situ hybridization revealed that in the CL MMP-2 niRNA was expressed during both formation and regression, while MMP-9 mRNA was mainly localized in the late luteal phase. Reduction of MMP-2, -9 transcripts in the CL was observed during pregnancy. MMP-2 mRNA in the CL reduced to an undetectable level at D26 of pregnancy. TIMP-1 mRNA was highly expressed in the CL in both early and late luteal phases and persisted throughout the early stages of pregnancy. Strong signal for TIMP-2 mRNA was also detected in both luteal phases, and the

Biofilm Formation and Virulence Genes in Clinical Isolates of Enterococcus faecalis

Enterococcus faecalis is a Gram-positive bacterium commonly found in the gastrointestinal tract that can cause serious infections. Many enterococci have broad resistance to antibiotics including penicillin, cephalosporins, aminoglycosides and glycopeptides. There are several adaptation mechanisms that bacteria can undergo to become more resistant, among them is the formation of biofilm. Several genes have been linked to the increase in the capacity of biofilm formation by bacteria such as gelE, esp and asa1. The aim of this research was to evaluate the biofilm formation of 12 E. faecalis isolates collected in hospitals and a standard strain, as well as to evaluate the hydrophobicity of its membrane and the presence of virulence genes. All the isolates formed biofilm and the characteristics of their membrane were variable. In addition, the presence of at least one virulence gene was found in all the 12 isolates, and none of the genes in the standard strain, indicating the acquisition of these genes in the hospital environment. With this, we can conclude that there is a close relationship between biofilm formation, acquisition of antibiotic resistance and the presence of virulence genes.

MMP-2和MMP-9在声带息肉发病机制中的作用

有研究表明,声带息肉基底膜（basilar membrane,BM）存在质的改变,并认为这种改变可能是声带息肉形成的重要原因。MMP-2和MMP-9是胶原降解的关键酶,其特异性底物是Ⅳ型胶原纤维，而Ⅳ型胶原纤维是基底膜主要骨架，所以胶原酶在启动基底膜降解中起主导作用。本文拟通过分析基质金属蛋白酶2（matrix metalloproteinase-2，MMP-2）和MMP-9在声带息肉中的表达，同时观察影响其表达及活性调控的转化生长因子β1（transforming growth factor beta 1，TGF-β1），探讨其在声带息肉形成中的作用。

NGAL基因克隆载体的构建与鉴定

目的构建中性粒细胞明胶酶相关脂质运载蛋白(neutrophil gelatinase-associated lipocalin,NGAL)基因克隆载体。方法RT-PCR扩增宫颈癌Hela细胞NGAL cDNA片断,将纯化后PCR产物插入pGEM-T载体,构建重组质粒,重组子通过琼脂糖电泳、特异性内切酶切割及测序予以鉴定。结果成功构建了NGAL基因克隆载体2个。结论为NGAL基因的定量检测提供了基本实验条件。

High expression of osteoglycin decreases gelatinase activity of murine hepatocarcinoma Hca-F cells

AIM:To investigate the possible correlation between osteoglycin expression and gelatinase activity of mouse hepatocarcinoma Hca-F cells.METHODS:A eukaryotic expression plasmid pIRE Spuro3 osteoglycin(+)was constructed and transfected into Hca-F cells to investigate the possible correlation between osteoglycin expression and gelatinase activity of Hca-F cells cultured with extract of lymph node,liver,spleen or in DMEM medium.The activity of gelatinases was examined through zymographic analysis.RESULTS:High expression of osteoglycin attenuated the gelatinase activity of Hca-F cells cultured with extract of lymph node,and at the same time,decreased the metastatic potential of Hca-F cells to peripheral lymph nodes in vivo.CONCLUSION:High expression of osteoglycin decreases the gelatinase activity of Hca-F cells cultured with extract of lymph node;regulation of gelatinase activity might be one of mechanisms that osteoglycin contributes to lymphatic metastasis suppression.

ID7 Isolated from Bauhinia variegata Stem Inhibits Tumor Progression and Metastatic Mechanisms of Triple Negative Breast Cancer in Vivo

Background:The breast cancer has been the most common form of cancer among women.The triple negative subtype represents 20%of all breast cancer cases in the world and is standing out by affecting young women and being aggressive.The main cause of death of patients with cancer is due to metastasis,which can reach the liver and lungs.Objective:The activities of ID7 fraction of the stems of Bauhinia variegata L.on breast cancer,lung metastasis and liver inflammatory process were evaluated.Method:ID7 was characterized by mass-spectrometry.The viability of murine mammary cells(4T1)treated with ID7 was assessed by MTT,trypan blue and fluorescence assay and viability of BT-20,MDA-MB-231 and MCF-7 human breast cancer tumor lines by MTS.The cell migration,invasion using matrigel and adhesion were performed.The expression of cell death proteins was quanitified by western blot and the gelatinases by zimogram.The ID7 activity of the tumor(4T1)and metastatic progession in vivo was evaluated.Results:ID7 reduced the 4T1 and MDA-MB-231viability and increased the late apoptosis,inhibited the 4T1 migration and invasion,increased the 4T1 adhesion and decreased the secreted active gelatinases.ID7 also increased the expression of PARP,caspase-7 and caspase-8,RIP and TNF-R1.In vivo,the ID7 decreased the volume and weight of the tumors and decreased lung metastasis and inflammation in the liver.The characterization showed mainly the presence of oleic acid,myricetin,quercetin and kaempferol in ID7.Conclusion:Thus,it was found that ID7 fraction exhibits selective antitumor and on the mechanisms of breast cancer metastasis activity,preventing lung metastasis and inflammation in the liver.It is suggested that fatty acids and flavonoids are correlated with such activities.

Effects of Plasmodium falciparum—infected erythrocytes on matrix metalloproteinase-9 regulation in human microvascular endothelial cells

Objective:To investigate the regulation of matrix metalloproteinases(MMPs) and tissue inhibitors of melalloproleinases(TIMPs) in human microvascular endothelium(HMEC-1) exposed to erythrocytes infected by different strains of Plasmodium falciparum(P.falciparum).Methods: HMEC—1 eells were co—incubated for 72 h with erythrocytes infected by late stage trophozoite of D10(chloroquine-sensilive) or W 2(chloroquine-resistant) P.falciparum strains.Cell supernatants were then collected and the levels of pro- or active gelatinases MMP-9 and MMP-2 were evaluated by gelatin zymograpln and densitometry.The release of pro-MMP-9,MMP-3.MMP-1 and TIMP-1 proteins was analyzed by western blotting and densitometry.Results:Infected erythrocytes induced de novo proMMP-9 and MMP-9 release.Neither basal levels of proMMP-2 were altered,nor active MMP-2 was found.MMP-3 and MMP-1 secretion was significant!) enhanced,whereas basal TIMP-1 was unaffected.All effects were similar for both strains. Conclusions:P.falciparum parasites,either chloroquine-sonsitive or -resistant,induce the release of active MMP-9 protein from human microvascular endothelium,by impairing balances between proMMP-9 and its inhibitor,and by enhancing the levels of its activators.This work provides new evidence on MMP involvement in malaria,pointing at MMP-9 as a possible target in adjuvant therapy.

Molecular mechanism about lymphogenous metastasis of hepatocarcinoma cells in mice

AIM: To investigate the correlation between lymphogenous metastasis and matrix metalloproteinases (MMPs) activity and the expression of Fas ligand of tumor cells in lymph nodes. METHODS: Fifty-six inbred 615-mice were equally divided into 2 groups and inoculated with Hca-F and Hca-P cells. Their lymph node metastatic rates were examined. Growth fraction of lymphocytes in host lymph nodes was detected by flow cytometry. The Hca-F and Hca-P cells were cultured with extract of lymph node, liver or spleen. The quantity of MMPs in these supernatants was examined by zymographic analysis. The expression of Fas ligand, PCNA, Bcl-2 protein of Hca-F and Hca-P cells in the mice were examined by immunohistochemistry. The apoptosis signals of macro-phages in lymph nodes were observed with in situ DNA fragmentation. RESULTS: On the 28th day post-inoculation, the lymph node metastatic rate of HcaF was 80%(16/20), whereas that of Hca-P was 25%(5/20). The growth fraction of lymphocytes was as follows: in the Hca-F cells, the proliferating peak of lymphocytes appeared on the 14th day post inoculation and then decreased rapidly, while in HcaP cells, the peak appeared on the 7th day post inoculation and then kept at a high level. With the extract of lymph node, the quantity of the MMP-9 activity increased (P【0.01) and active MMP-9 and MMP-2 were produced by both Hca-F and Hca-P tumor cells, which did not produce MMPs without the extract of lymph node or with the extracts of the liver and spleen. The expression of Fas Ligand of Hca-F cells was stronger than that of Hca-P cells (P 【0.01). The expressions of PCNA and Bcl-2 protein of Hca-F cells in the tumors of inoculated area were the same as that of Hca-P cells. In situ DNA fragmentation showed that the positive signals of macrophages were around Hca-F cells. CONCLUSION: Secretion of MMPs which was associated with metastatic ability of Hca-F and Hca-P tumor cells depends on the environment of lymph nodes. The increased expression of Fas ligand protein of Hca-F tumor cells with high lymphogenous metastatic potential in lymph nodes may help tumor cells escape from being killed by host lymphocytes.

Alterations in metastatic properties of hepatocellular carcinoma cell following H-ras oncogene transfection

AIM: To demonstrate the relationship between H-ras oncogene and hepatocellular carcinoma (HCC) metastasis. METHODS: Activated H-ras oncogene was transfected into SMMC 7721, a cell line derived from human HCC, by calcium phosphate transfection method. Some metastasis-related parameters were detected in vitro, including adhesion assay, migration assay, expression of collagenase IV(c IV ase) and epidermal growth factor receptor (EGFR). RESULTS: The abilities of H-ras-transfected cell clones in adhesion to laminin (LN) or fibronectin (FN), migration, c IV ase secretion increased markedly, and the expression of EGFR elevated moderately. More importantly, these alterations were consistent positively with the expression of p21, the protein product of H-ras oncogene. CONCLUSION: H-ras oncogene could induce the metastatic phenotype of HCC cell in vitro to raise its metastatic potential.

Increase of TNFα-stimulated Osteoarthritic Chondrocytes Apoptosis and Decrease of Matrix Metalloproteinases 9 by NF-κB Inhibition

Objective To investigate the in vitro effect of caffeic acid phenethyl ester （CAPE）, a NF-KB inhibitor, on the apoptosis of osteoarthritic （OA） chondrocytes and on the regulation of the gelatinases matrix metalloproteinase 2 （MMP-2） and matrix metalloproteinase 9 （MMP-9）. Methods Annexin V-FITC/propidium iodide （PI） labeling and western blotting were used to observe and determine the apoptosis in TNFa-stimulated primary cultured osteoarthritic chondrocytes. Also, gelatin zymography was applied to examine MMP-2 and MMP-9 activities in supernatants. Results it was confirmed by both flow cytometry and western blotting that chondrocytes from OA patients have an apoptotic background. Use of CAPE in combination with 10 ng/mL of TNFa for 24 h facilitated the apoptosis. MMP-9 in the supernatant could be autoactivated （from proMMP-9 to active MMP-9）, and the physiologic calcium concentration （2.5 mmol/L） could delay the autoactivation of MMP-9. The activities of MMP-2 and MMP-9 in the fresh supernatant increased significantly in response to stimulation by 10 ng/mL of TNFa for 24 h. The stimulatory effect of TNFa just on proMMP-9 was counteracted significantly by CAPE. Conclusion NF-KB could prevent chondrocytes apoptosis though its activation was attributed to the increase of proMMP-9 activity induced by TNFa （a pro-apoptotic factor）. Therefore, therapeutic NF-KB inhibitor was a ＇double-edged swords＇ to the apoptosis of chondrocytes and the secretion of MMP-9.

Infection of Schistosomiasis japanicum is likely to enhance proliferation and migration of human breast cancer cells:mechanism of action of differential expression of MMP2 and MMP9

Objective:To study whether the infection of Schistosomiasis japanicum(S.japanicum) is related to enhanced proliferation and migration of cancer cells,and the molecular mechanism pertains to cancer cell metastasis in human host.Methods:The gene of S.japanicum glutathione transferase(sjGST) cloned from 5.japanicum was expressed,purified and applied in a series of assays to explore the effect of sjGST on proliferation and migration of MDA-MB-435S,and the expression of MMP2 and MMP9.Immunofluorescence assay for the binding of sjGST to MDA-MB-435S was also carried out.Results:Results showed that sjGST enhanced proliferation and migration in human breast cancer cell MDA-MB-435S signifycantly at 50-200 nM,but did not enhance them in human lung cancer cell A549.Immunofluorescence assay for the binding of sjGST to MDA-MB-435S and A549 showed that GST was readily hound to the breast cancer cells,but showed almost no binding to human lung cancer cells.The assays for gelatinase activity showed that both MMP2 and MMP9 activities were increased significantly in the presence of sjGST(50-200 nM) in MDA-MB-435S, but they were not significant in A549.Conclusions:Our current results show strongly that S. japanicum GST binds to MDA-MB-435S probably via its i'eceptor,and enhances proliferation and migration of the cancer cells by up-regulatory expression of MMP2 and MMP9.

Effects of hypoxia,hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 in hepatic stellate cells

AIM: To study the effects of hypoxia, hyperoxia on the regulation of expression and activity of matrix metalloproteinase-2 (MMP-2) in hepatic stellate cells (HSC). METHODS: The expressions of MMP-2, tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) and membrane type matrix metalloproteinase-1 (MT1-MMP) in cultured rat HSC were detected by immunocytochemistry (ICC) and in situ hybridization (ISH). The contents of MMP-2 and TIMP-2 in culture supernatant were detected with ELISA and the activity of MMP-2 in supernatant was revealed by zymography. RESULTS: In the situation of hypoxia for 12h, the expression of MMP-2 protein was enhanced (hypoxia group positive indexes: 5.7 +/- 2.0, n=10; control: 3.2 +/- 1.0, n = 7; P【0.05), while TIMP-2 protein was decreased in HSC (hypoxia group positive indexes: 2.5 +/- 0.7, n = 10; control: 3.6 +/- 1.0, n = 7; P 【 0.05), and the activity (total A) of MMP-2 in supernatant declined obviously (hypoxia group: 7.334 +/- 1.922, n = 9; control: 17.277 +/- 7.424, n = 11; P 【 0.01). Compared the varied duration of hypoxia, the changes of expressions including mRNA and protein level as well as activity of MMP-2 were most notable in 6h group. The highest value(A(hypoxia)-A(control)) of the protein and the most intense signal of mRNA were in the period of hypoxia for 6h, along with the lowest activity of MMP-2. In the situation of hyperoxia for 12h, the contents (A(450)) of MMP-2 and TIMP-2 in supernatant were both higher than those in the control, especially the TIMP-2 (hyperoxia group: 0.0499 +/- 0.0144, n = 16; control: 0.0219 +/- 0.0098, n = 14; P 【 0.01), and so was the activity of MMP-2 (hyperoxia group: 5.252 +/- 0.771, n = 14; control: 4.304 +/- 1.083, n = 12; P 【 0.05), and the expression of MT1-MMP was increased. CONCLUSION: HSC is sensitive to the oxygen, hypoxia enhances the expression of MMP-2 and the effect is more marked at the early stage; hyperoxia mainly raises the activity of MMP-2.

Hemozoin triggers tumor necrosis factor alpha-mediated release of lysozyme by human adherent monocytes:new evidences on leukocyte degranulation in P.falciparum malaria

Objective:Avidly phagocytosed hemozoin(malarial pigment) alters several functions of human monocytes and stimulates generation of several cytokines.Recently,we showed that phagocytosis of hemozoin by human monocytes increases expression and activity of matrix metalloproteinase-9,a proteolytic enzyme available in specific gelatinase granules,which contain several enzymes including lysozyme.Present work investigated active lysozyme release after phagocytosis of hemozoin and its dependence on production of tumor necrosis factor alpha. Methods:After phagocytosis of hemozoin,hemozoin-containing trophozoites or control meals(opsonized nonparasitized red blood cells and latex particles),monocyte supematants were monitored for 2 hours,in presence of blocking anti-human tumor necrosis factor alpha antibodies or recombinant human tumor necrosis factor alpha cytokine in selected experiments.Lysozyme release was evaluated by a specific spectrometric assay measuring lysozyme activity after coincubation of cell supematants with suspensions of Mycrococcus Lysodeikticus,while levels of soluble tumor necrosis factor alpha were analyzed by specific enzyme-linked immunodsorbent assay. Results:Levels of lysozyme activity and soluble tumor necrosis factor alpha protein were increased in hemozoin in-or trophozoites-laden monocytes supematants.Phagocytosis per se(control meals) also increased lysozyme release,but levels were significantly lower than those obtained after phagocytosis of hemozoin or trophozoites. Interestingly,all effects on lysozyme release observed after phagocytosis were abrogated by blocking anti-human tumor necrosis factor alpha antibodies,while they were mimicked by recombinant human tumor necrosis factor alpha cytokine.Conclusions:Present work shows that phagocytosis of hemozoin promotes monocyte degranulation and enhances active lysozyme release.The effect requires tumor necrosis factor alpha mediation.

Effect of a nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester on invasion of human colorectal cancer cell line SL-174T

AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.

Lipocalin-2 Test in Distinguishing Acute Lung Injury Cases from Septic Mice Without Acute Lung Injury

Objective To explore whether the amount of lipocalin-2 in the biofluid could reflect the onset of sepsis-induced acute lung injury(ALI) in mice. Methods Lipopolysaccharide(LPS, 10 mg/kg) injection or cecal ligation and puncture(CLP) was performed to induce severe sepsis and ALI in C57 BL/6 male mice randomly divided into 5 groups(n=10 in each group): group A(intraperitoneal LPS injection), group B(intravenous LPS injection via tail vein), group C(CLP with 25% of the cecum ligated), group D(CLP with 75% of the cecum ligated), and the control group(6 sham-operation controls plus 4 saline controls). All the mice received volume resuscitation. Measurements of pulmonary morphological and functional alterations were used to identify the presence of experimental ALI. The expressions of lipocalin-2 and interleukin(IL)-6 in serum, bronchoalveolar lavage fluid(BALF), and lung tissue were quantified at both protein and mRNA levels. The overall abilities of lipocalin-2 and IL-6 tests to diagnose sepsis-induced ALI were evaluated by generating receiver operator characteristic curves(ROC) and computing area under curve(AUC). Results In both group B and group D, most of the "main features" of experimental ALI were reproduced in mice, while group A and group C showed septic syndrome without definite evidence for the presence of ALI. Compared with septic mice without ALI(group A+group C), lipocalin-2 protein expression in septic mice with ALI(group B+group D) was significantly up-regulated in BALF(P<0.01) and in serum(P<0.01), and mRNA expression boosted in lung tissues(all P<0.05). Lipocalin-2 tests performed better than IL-6 tests in recognizing sepsis-induced ALI cases, evidenced by the larger AUC of the former(BALF tests, 0.8800 versus 0.6625; serum tests, 0.8500 versus 0.7000). Using a dual cutoff system to diagnose sepsis-induced ALI, BALF lipocalin-2 test exhibited the highest positive likelihood ratio(13.000) and the lowest negative likelihood ratio(0.077) among the tests of lipocalin-2 and IL-6 in blood and BALF. A statistically significant correlation was found between lipocalin-2 concentration in BALF and that in serum(Spearman r=0.8803,P<0.0001). Conclusions Lipocalin-2 expression is significantly up-regulated in septic ALI mice compared with those without ALI. Lipocalin-2 tests with a dual cutoff system could be an effective tool in distinguishing experimental ALI cases.

Urinary Neutrophil Gelatinase Associated Lipocalin as a Marker of Tubular Damage in Type 2 Diabetic Patients with and without Albuminuria

Background: Neuttrophil gelatinase associated lipocalin (NGAL) was shown to be a good marker for predicting acute kidney injury (AKI). Some recent reports demonstrated that NGAL may be an early biomarker for kidney affection in diabetic patients. The aim of this work is to investigate urinary NGAL (UNGAL) in type 2 diabetic patients with and without albuminuria. Methods: This study included 46 type 2 diabetic patients and 15 healthy age and sex matched individuals as the control group. Diabetic patients were divided into three groups according to urinary albumin excretion (UAE), normoalbuminuria, microalbuminuria and macroalbuminuria. UNGAL was measured in all populations and corrected to urinary creatinine to account for day to day variation in urine volume and transformed log. Comparison between 4 groups (control, normoalbuminuria, microalbuminuria and macroalbuminuria) was done. Results: Log UNGAL/Creatinine ratio showed significant difference when comparing control group (0.70 ± 0.58) versus normoalbuminuria (1.71 ± 1.06), microalbuminuria (1.57 ± 0.72) and macroalbuminuria (1.92 ± 0.63), however, there was no significant difference among diabetic groups. Pearson’s correlation showed that log UNGAL/Creatinine ratio positively correlated with glycated hemoglobin (HbA1c) and inversely with estimated glomerular filtration rate (eGFR). Regression analysis showed that HbA1c, urinary creatinine and eGFR were the independent predictors of log UNGAL/Creatinine ratio. Conclusion: Tubular markers like UNGAL may be early elevated in type 2 diabetic patients even before the incidence of glomerular injury detected by microalbuminuria and it can be used as an early marker for detection of kidney involvement in diabetic patients.