Protective Effects of Sphingosine 1-phosphate and FTY720 on Rat Pulmonary Microvascular Endothelial Cells Stimulated by TNFα

Objective In this study, the effects of sphingosine 1-phosphate （S1P） and its analogue FTY720 on lactate dehydrogenase （LDH）, prostacyclin and PGE2 release, monolayer paracellular permeability, and F-actin structural changes of cultured rat pulmonary microvascular endothelial cells stimulated by TNFα were examined. Methods Rat pulmonary microvascular endothelial cells（rPMECs） were isolated, cultured and identified by immunofluorescent stain of yon Willebrand-associatedan-antigen （vWF）. Incubation with TNFα at a dose of 150 ng/ml for 12 hours induced a significant increase in paracellular permeability determined by measurement of a paracellular solute flux, 3-kDa FD-3. Results PGE2, PGF2α and LDH productions by cell cultures exposed to TNFα for 12 hours increased significantly compared with control cells. Also, the 12-hour exposure to TNFα resulted in reorganization and retraction of the rat pulmonary microvascular endothelial cells that was visible both by phase contrast microscopy and cytoskeletal F-actin stain. Paracellular permeability of the monolayers, PGE2, PGFα and LDH productions were significantly reduced following pretreatment with S 1P or FTY-720. Moreover, S 1P or FTY-720 pretreatment prevented the changes of morphologic and cytoskeletal F-actin induced by TNFa. Conclusion S 1P and FTY-720 can inhibited the LDH, PGF2α and PGE2 release by strengthening the cytoskeleton and protecting the barrier function, and may represent a novel therapeutic method for pulmonary vascular barrier damage.     

MiR-503 regulates cisplatin resistance of human gastric cancer cell lines by targeting IGF1R and BCL2

Background Studies have shown that the drug resistance of gastric cancer cells can be modulated by abnormal expression of microRNAs （miRNAs）.We investigated the role of miR-503 in the development of cisplatin resistance in human gastric cancer cell lines.Methods MiR-503 expression was measured by quantitative real-time PCR.MTT （3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide） and clonogenic assays were used to examine changes in cell viability and the drug resistance phenotype of cancer cells associated with upregulation or downregulation of the miRNA.A dual-luciferase activity assay was used to verify target genes of miR-503.Immunohistochemistry,Western blotting analysis,and a flow cytometric apoptosis assay were used to elucidate the mechanism by which miR-503 modulates drug resistance in cancer cells.Results MiR-503 was significantly downregulated in gastric cancer tissues and several gastric cancer cell lines.Additionally,downregulation of miR-503 in the cisplatin （DDP）-resistant gastric cancer cell line SGC7901/DDP was concurrent with the upregulation of insulin-like growth factor-1 receptor （IGF1R） and B-cell lymphoma 2 （BCL2） expression compared with the parental SGC7901 cell line.An in vitro drug sensitivity assay showed that overexpression of miR-503 sensitized SGC7901/DDP cells to cisplatin.The luciferase activity of reporters driven by IGF1R and BCL2 3＇-untranslated regions in SGC7901/DDP cells suggested that IGF1R and BCL2 were both direct target genes of miR-503.Enforced miR-503 expression in SGC7901/DDP cells reduced expression of the target proteins,inhibited proliferation,and sensitized the cells to DDP-induced apoptosis.Conclusion Our findings suggest that hsa-miR-503 modulates cisplatin resistance of human gastric cancer cells at least in part by targeting IGF1R and BCL2.     

Effects of Bone Marrow-derived Mesenchymal Stem Cells on TGF-β and MCP-1 in Injured Lung of Rats

The primary objective of the present study is to investigate the therapeutic effect of bone marrow-derived mesenchymal stem cells （MSCs） on bleomycin （BLM）-induced lung injury of rats and the effect on transforming growth factor-β （TGF-β） and monocyte chemoattractant protein-1 （MCP-1）. MSCs were isolated from SD rats. The recipients rats were divided randomly into four groups： lung injury group, MSC treatment group, MSC control group and normal control group. Rats of lung injury group and MSC treatment group were perfused with BLM of 5mg/kg （0.2-0.3ml） intratracheally, others were perfused with normal saline. After twelve hours, rats of MSC treatment group and MSC control group were injected MSCs of 0.5×10^6per rat into tail vein. Haematoxylin-eosin staining was used to observe the morphology in lung tissue. ELISA was used to detect the contents of TGF-β and MCP-1 in serum and bronchoalveolar lavage fluid （BALF）. Collagen content of the lung tissue was assessed by hydroxyproline （HYP） concentration. It was found that the thickness of alveolar wall and lung interstitium were significantly reduced in the rats of MSC treatment group compared with the lung injury group. HYP content in lung interstitium, TGF-β and MCP-1 in serum and BALF were increased significantly in rats of lung injury group two weeks after BLM perfusion, but they were reduced significantly in the rats of MSC treatment group compared with the injured rats. These observations provide evidence that MSCs engraftment could alleviate bleomycin-induced lung injury and fibrosis in rats and the therapeutic effects might relate with the decrease of TGF-β and MCP-1.     

Glycogen synthase kinase-3: a key kinase in retinal neuron apoptosis in early diabetic retinopathy

Background Diabetes-related pathogenic factors can cause retinal ganglion cell （RGC） apoptosis, but the specific mechanism is not very clear. The aim of this study is to investigate the correlation between glycogen synthase kinase-3 （GSK-3） activation and retinal neuron apoptosis. Methods In an in vitro experiment, the number of apoptotic RGC-5 cells differentiated by staurosporine was evaluated via flow cytometry and nuclei staining using Hoechst 33258. GSK-3 phosphorylation and caspase-3 activation in RGC-5 cells after serum deprivation were determined using Western blotting. Mitochondrial membrane potential was detected using the dye 5,5＇,6,6＇-tetrachloro-l,l＇,3,3＇-tetrethyl benzimidalyl carbocyanine iodide, and reactive oxygen species （ROS） levels were measured with dihydroethidium. In an in vivo experiment, the number of apoptotic retinal neurons was evaluated via terminal transferase dUTP nick-end labeling （TUNEL）, and GSK-3 phosphorylation was determined using Western blotting, in the retinal nerve epithelial tissue of rats in which diabetes was induced by intravenous tail-vein injection of streptozotocin for 4 weeks. Results The levels of phosphorylated Ser21/9 in GSK-3α/13 and p-T308/S473-AKT were lower and the cleaved caspase-3 levels were higher in the serum-deprived model （P 〈0.05）. Lithium chloride treatment was associated with a slower rate of apoptosis, increased mitochondrial membrane potential, and decreased ROS levels in differentiated RGC-5 cells （P 〈0.05）. The level of blood glucose and the number of TUNEL-positive cells in the whole-mounted retinas were higher （P 〈0.01）, and the levels of phosphorylated Ser21/9 in GSK-3α/13 and body weight were lower （P 〈0.05）. However, the thickness of the retinal nerve epithelial layer was not significantly less in diabetic rats compared with control group. Lithium chloride intravitreal injection increased the levels of phosphorylated Ser21/9 in GSK-3α/13 and decreased TUNEL-positive cells in the whole-moun     

支气管哮喘患者白细胞介素12、免疫球蛋白E及内皮素1的水平测定及意义

支气管哮喘是临床常见的呼吸道疾病，是呼吸道慢性变态反应性炎症性疾病。气道慢性变态反应性由多种炎性细胞、炎性介质和细胞因子共同参与，检测哮喘患者自细胞介素（interleukin，IL）-12、免疫球蛋白E（immunoglobulin—E，Ig—E）及内皮素1（endothelin-1，ET-1）水平，以探讨三者在支气管哮喘不同病程中的作用。     

Association between interferon gamma receptor 1 -56C/T gene polymorphism and tuberculosis susceptibility: a meta-analysis

Background Genetic variations in the interferon-gamma （IFN-γ） receptor 1 gene （IFNGR1） may contribute to tuberculosis （TB） risk in different populations.Many studies have investigated the relationship between IFNGR1 56C/T polymorphism and the susceptibility to TB,but have yielded conflicting results.A comprehensive meta-analysis is needed to provide a more accurate estimation of the relationship between them.Methods A literature search based on a combination of manual and computer-based methods was conducted on four English databases （PubMed,Science Direct,SpringerLink,and EBSCO） and three Chinese databases （Wanfang,CQVIP,and Chinese National Knowledge Infrastructure databases）.Pooled odds ratios （ORs） and 95％ confidence intervals （95％ Cls） were calculated using either the fixed-effects model or the random-effects model for different genetic models based on the heterogeneity examination.Results A total of six studies comprising 1 497 confirmed TB cases and 1 802 controls were included in this meta-analysis.Overall,no significant association was observed between IFNGR1-56C/T polymorphism and TB susceptibility （C vs.T,OR=0.90,95％ Cl 0.69-1.17; CC vs.TT,OR=0.87,95％ Cl 0.65-1.18; TC vs.TT,OR=-1.031,95％ Cl 0.872-1.219; CC＋TC vs.TT,OR=0.89,95％ Cl 0.64-1.26; CC vs.TC＋TT,OR=0.92,95％ Cl 0.66-1.29）.In subgroup analysis,a significant association was found in the dominant model （CC＋TC vs.TT,OR=1.24,95％ Cl 1.02-1.51） in Africans,but not in Asians or Caucasians.Conclusions Our meta-analysis did not provide enough powerful evidence to identify a significant association between IFNGR1-56C/T polymorphism and TB susceptibility in the overall population.In subgroup analysis,it indicates that IFNGR1-56C/T is possibly associated with increased TB risk in Africans,but not in Asians or Caucasians.However,larger sample size and better-designed case-control studies are needed to validate these findings.     

Aberrant expression of Wnt antagonist SFRP1 in pancreatic cancer

Pancreatic cancer is one of the malignant tumor with a very poor prognosis. Both genetic and epigenetic alterations are involved in the pathogenetic mechanisms of pancreatic cancer. Hypermethylation and subsequent loss of expression of some tumor suppressor genes and tumor-related genes occur frequently in pancreatic cancer, such as loss of expression of p16, ^1RASSF1A,^2 SOCS-1,^3 and hMLH1^4 genes were repoted.     

Effects of Rosiglitazone on Adiponectin Expression in 3T3- L1 Adipocytes at High Levels of Both Testosterone and Insulin In Vitro Culture

Objective To evaluate effects of rosiglitazone （RSG） on the expression of adiponectin in mature adipocytes at high levels of both testosterone （T） and insulin in vitro culture. Methods Mouse 3T3-L1 preadipocytes were induced to be mature adipocytes and used in this study. According to RSG concentrations, the cells added with T （10^-5 mol/L） and insulin （10^-4 mol/L） were divided into 4 groups： free-RSG group （0 mol/L RSG, FR-TI）, low-dose group （10^-9 mol/L RSG, LR-TI）, middle-dose group （lO-7mol/L RSG, MR-T1） and high-dose group （10^-6 mol/L RSG, HR-TI）. Besides, the cells added with RSG without T and insulin were also divided into 4 groups： FR, LR, MR, and HR. These 8 groups were incubated for 42 h. Cell viability was determined by MTT assay. Expression of adiponectin was detected by Western blotting. Results The maximum viability in FR-TI group was observed at point of 42 h. The growth of the adipocytes was significantly inhibited in MR-TI group compared with FR-TI （P〈0.01）. The level of adiponectin in MR-TI group was higher than that in LR- TI group （P〈0.01）. However, with RSG increasing; HR-TI group showed the lowest level of adiponectin among three treatment groups （P〈0.01）. In addition, adiponectin expression in MR-TI group was significantly higher than that in MR group （P〈0.01）. Conclusion RSG could increase the expression of adiponectin in 3T3-L1 adipocytes under high levels of both T and insulin, but it acts in a narrow concentration range.     

Comparative study of mutation spectrums of MT-RNR1 m.1555A>G, GJB2, and SLC26A4 between familial and sporadic patients with nonsyndromic sensorineural hearing loss in Chinese Han

Background The mutation frequencies of three common deafness genes （MT-RNR1 m.1555A〉G,GJB2,and SLC26A4） among patients with nonsyndromic sensorineural hearing loss （NSHL） were different in previous studies.Inconsistent selection criteria for recruiting patients could have led to differences in estimating the frequencies of genetic mutations thus resulting in different mutation frequencies among these studies.The aim of this study was to reveal the differences in the mutation spectrums of the three common genes between familial and sporadic Chinese Han patients.Methods Totally,301 familial probands and 703 sporadic patients with NSHL were enrolled in this study.Three genes,MT-RNR1 m.1555A〉G,GJB2,and SLC26A4,were screened for mutation in our study cohort.A X2 test was performed to compare the mutation frequencies between the two groups.Results The study showed that the disease-causing mutation frequencies of MT-RNR1 m.1555A〉G,GJB2,and SLC26A4 were 12.29%,14.62%,and 18.27% in familial probands and 3.56%,18.63%,and 18.92% in sporadic patients,respectively.The mutation frequency of MT-RNR1 m.1555A〉G in familial probands was significantly higher than in sporadic patients （X2 test,P=0.000）,while there were no significant differences in the mutation frequencies of GJB2 and SLC26A4 between the familial and sporadic groups （X2 test,P 〉0.05）.Conclusions It is necessary to reveal the differences in gene mutation frequencies between patients of different sources or characteristics by comparative studies in order to avoid selection bias.The mutations of GJB2,SLC26A4,and MTRNR1 m.1555A〉G are the most important etiological factors in Chinese Han patients,among which SLC26A4 might be the most frequent.     

Glutamate Transporter 1-mediated Antidepressant-like Effect in a Rat Model of Chronic Unpredictable Stress

In recent years, more attention has been paid to the role of the glutamate transporter 1 （GLT-1, EAAT2） in major depressive disorder （MDD）. However, experimental data on brain GLT-1 levels are, to some extent, inconsistent in human postmortem and animal studies, These discrepancies imply that the role of GLT-1 in the pathophysiology of MDD and the action of antidepressants remain obscure. This work was designed to study the impact of chronic unpredictable stress （CUS） for 2 ses- sions per day for 35 days and four weeks of fluoxetine （FLX） on depressive-like behaviors in rats, as well as the concomitant expression of the GLT-1 protein in the hippocampus. Behavioral changes were assessed by the sucrose preference and open field tests. GLT-1 levels were detected by immunohisto- chemistry and Western blot analysis. Our study demonstrated that the animals exposed to CUS showed depressive-like behaviors and exhibited a significant decrease in GLT-1 expression in the hippocampus. Chronic FLX treatment reversed the behavioral deficits and the CUS-induced decrease in GLT-1 levels. Taken together, our results support the reduction of GLT-1 in human postmortem studies in MDD and suggest that GLT-1 may be involved in the antidepressant activity of FLX. Our studies further support the notion that GLT-1 is an attractive candidate molecule associated with the fundamental processes of MDD and may be a potential, and novel pharmacological target for the treatment of MDD.     

Effect of behavior training on learning,memory and the expression of NR2B,GluR1 in hippocampus of rats offspring with fetal growth restriction

Objective： To study effects of behavior training on learning, memory and the expression of NR2B, GluR1 in hippocampus of rat＇ s offspring with fetal growth restriction（FGR）. Methods： The rat model of FGR was established by passive smoking method. The rats offspring were divided into the FGR group and the control group, then randomly divided into the trained and untrained group, respectively. Morris water maze test was proceeded on postnatal month（PM2/4） as a behavior training method, then the learning-memory of rats was detected through dark-avoidance and step-down tests. The expressions of NR2B and GluR1 subunits in hippocampal CA1 and CA3 areas were detected by immunohistochemical method. Results： In the dark-avoidance and step-down tests, the performance record of rats with FGR was worse than that of control rats, and the behavior-trained rats was better than the untrained rats, when the FGR model and training factors were analyzed singly. The model factor and training factor had significant interaction（P 〈 0.05）. The expressions of NR2B and GluR1 subunits in hippocampal CA1 and CA3 areas of rats with FGR reduced. In contrast, the expressions of GluR1 and NR2B subunits in CA1 area of behavior-trained rats increased, when the FGR model and training factors were analyzed singly. Conclusion： These findings suggested that the effect of behavior training on the expressions of NR2B and GluR1 subunits in CA1 area should be the mechanistic basis for the training-induced improvement in learning-memory abilities.     

Significant association between Nijmegen breakage syndrome 1 657de15 polymorphism and breast cancer risk

Many studies were published to evaluate the association between Nijmegen breakage syndrome 1 （NBS1） 657de15 polymorphism and breast cancer risk, but the results remained inconsistent. To derive a more precise estimation on the possible association,we performed a meta-analysis of previous published studies. Case-control studies on the association between NBS1 657de15 polymorphisms and breast cancer risk were included into this meta-analysis. We used the odds ratio （OR） with 95% confidence interval （95% CI） to assess the strength of the association. Ten studies with a total of 25,365 subjects were identified and included into this meta-analysis. Meta-analysis of those ten studies showed that there was a significant association between NBS1 657de15 polymorphisms and breast cancer risk （pooled OR = 2.66,95 % CI 1.82 - 3.90, P 〈 0.001 ）. The cumulative meta-analyses showed a trend of a more significant association between NBS1 657de15 polymorphisms and breast cancer risk as data accumulated by publication year. Thus, our meta-analysis suggests that there was a significant association between NBS1 657de15 polymorphisms and breast cancer risk, and NBS1 657de15 polymorphism results in an increased risk of breast cancer.     

Expression Patterns of Sarcomeric α-Actin, α-Actinin and UCP2 in the Myocardium of Kunming Mice after Exposure to C-Terminal Polypeptide of Cardiotrophin-1

Cardiotrophin-1 （CT-1） activates a distinct form of cardiac muscle cell hypertrophy in which the sarcomeric units are assembled in series. The aim of the study was to determine the expres- sion pattern of sarcomeric contractile protein α-actin, specialized eytoskeletal protein α-actinin and mitochondrial uncoupling protein-2 （UCP2） in myocardial remodeling induced by chronic exposure to CT-1. Kunming mice were intraperitoneally injected with carboxy-terminal polypeptide （CP） of CT-1 （CT-1-CP, 500 μg·kg-1·day^-1） for 1, 2, 3 and 4 week （s）, respectively （4 groups obtained according to the injection time, n=10 each, with 5 males and 5 females in each group）, Those injected with physiological saline for 4 weeks served as controls （n=10, with 5 males and 5 females）. The heart tissues of mice were harvested at 1, 2, 3 or 4 week （s）. Immunohistochemistry （IHC） and Western blotting （WB） were used to detect the distribution and expression of sarcomeric α-actin, α-aetinin and mitoehondrial UCP2 in myocardial tissues. IHC showed that α-actin was mainly distributed around the nuclei of cardiomyo- cytes, α-actinin concentrated around the striae and UCP2 scattered rather evenly in the plasma. The ex- pression of α-actin was slightly greater than that of α-actinin and UCP2 in the control group （IHC： χ^2=6.125; WB： F=0.249, P〉0.05） and it gradually decreased after exposure to CT-1-CP. There was no significant difference in the expression of α-actin between the control group and the CT-1-CP-treated groups （χ^2=7.386, P〉0.05）. But Western blotting revealed significant difference in the expression of α-actin between the control group and the 4-week CT-1-CP-treated group （F=2.912; q=4.203, P〈0.05）. Moreover, it was found that the expression of α-actinin increased stepwise with the exposure time in CT-1-CP-treated groups and differed significantly between CT-1-CP-treated groups and the control group （ICH： χ^2=21.977; WB： F=50.388; P〈0.01）. The e     

Over-expression of heme oxygenase-1 in peripheral blood predicts the progression and relapse risk of chronic myeloid leukemia

Background There are limited eligible clinical markers at present to monitor the progress of chronic myeloid leukemia （CML）.Heme oxygenase-1 （HO-1）,as one of the most important oxidation-regulating enzymes in vivo,suggests the onset and progression of cancer when highly expressed.Furthermore,HO-1 level is related with the occurrence and development of hematological diseases.But the relationship between HO-1 expression and progression/relapse of CML has seldom been studied hitherto.This study aimed to investigate the relationship between them to find out a new molecular marker for prediction.Methods A total of 60 peripheral blood and bone marrow （BM） samples from 25 CML patients in different phases were collected respectively to detect the expressions of HO-1 and bcr/abl using real-time PCR.Routine blood test was performed to detect the changes of leukocyte and platelet counts.The proportion of primitive cells in BM was detected by flow cytometry.The relationship between high HO-1 expression and CML progression and relapse was explored by the analysis of variance by Wilcoxon test and linear regression analysis.The diagnostic accuracy and cutoff values were determined by receiver operating characteristic curve.Results Relative expression of HO-1 mRNA in CML patients peripheral blood was significantly higher than that of donors （P 〈0.0001）,which were 0.57±3.78 and （1.417±1.125）×10-6,respectively.HO-1 expression level in CML patients was 0.061 5±0.062 4,which decreased to 0.009 4±0.006 7 upon CMoR,and remained remarkably higher 0.016 3±0.017 5 than that of normal donors （1.417±1.125）× 10-6,P 〈0.001.When relapse occurred,HO-1 expression significantly increased from 0.020 6±0.021 0 to 3.852±10.285 in CMoR stage and undergoing relapse.According to progression of CML,HO-1 expression level in CML patients increased from CP （0.009 5±0.017 6） to AP （0.028 0±0.055 7） and then to BP （0.276 7± 0.447 0）.And there was a linear correlation between HO-1 expression and proportion     

Heparin inhibits burn-induced spleen cell apoptosis by suppressing interleukin-1 expression

Background Epidermal burn injury may trigger significant apoptosis of the spleen cells,which might be caused by a burninduced systemic inflammatory reaction.Heparin has been shown to possess anti-inflammatory properties.Interleukin 1 （IL-1） is centrally important among pro-inflammatory cytokines.We hypothesized that heparin might inhibit burn-induced apoptosis in the spleen via suppression of the IL-1 pathway.Methods Burn injury was performed on IL-1 R＋/＋ （IL-1 receptor wild-type mouse） and IL-1 R-/-（IL-1 receptor knockout mouse） mice,and they were then treated with heparin,saline or IL-1 receptor antagonist IL-Ra.Apoptosis,IL-1α and IL-1β expression were assessed in the spleens and serum.Survival curve analysis was further applied to elucidate the mechanism of heparin＇s protective properties.Results Burn induced significant apoptosis （sham：3.6％±2.1％ vs.burn：28.8％±5.9％; P ＜0.001）and remarkable expression o IL-1α and IL-1β in the mouse spleens and serum.Heparin reduced the burn-induced apoptosis in the spleens （heparin treated：8.6％±3.4％,P ＜0.005）,which could be blocked by IL-1Ra.Heparin markedly decreased both IL-1α and IL-1β expression in the spleens and serum of burned mica.IL-1 R-/-mice demonstrated considerably less apoptosis in the spleens and had a higher survival rate after burns.Heparin did not significantly decrease apoptosis in the spleen and the mortality rate in IL-1 R-/-mice after burns.Conclusion Heparin inhibits burn-induced apoptosis of the spleen cells by suppressing IL-1 expression in mice.