Gene Expression and Correlation of Pten and Fabp4 in Liver, Muscle, and Adipose Tissues of Type 2 Diabetes Rats.

BACKGROUND: The aim of this work was to study the Fabp4 and Pten gene expression and correlation in the liver, muscle, and adipose tissues of type 2 diabetes mellitus (T2DM) rats. MATERIAL AND METHODS: Male Wistar rats (8 weeks old) were randomly divided into 2 groups (n=12/group): a control group fed a normal diet for 8 weeks and an experimental group fed a high-fat, high-sugar diet for 8 weeks and that received 25 mg/kg streptozotocin by intraperitoneal injection to induce T2DM. The random blood glucose, fasting blood glucose, and fasting insulin levels were measured. The expression of Pten and Fabp4 in the liver, muscle, and epididymal adipose tissues was estimated by real-time quantitative PCR. Pearson correlation coefficient analysis was used to investigate the expression correlation between Pten and Fabp4 in T2DM rats. RESULTS: The gene expressions of Pten and Fabp4 in the liver, muscle, and adipose tissues of T2DM rats were all significantly higher than those in the control group (P<0.05). Pten was highly expressed in the muscles and Fabp4 was highly expressed in muscle and adipose tissues. Furthermore, expressions of Fabp4 and Pten in the muscle and adipose tissues of T2DM rats were positively correlated (P<0.05), but not in the liver. CONCLUSIONS: The increased expression of PTEN and FABP4 in the adipose and muscles of T2DM rats may play an important role in the insulin resistance of T2DM. However, the mechanism by which these 2 genes function in T2DM needs further study.

Increased plasma levels of FABP4 and PTEN is associated with more severe insulin resistance in women with gestational diabetes mellitus.

BACKGROUND: The aim of this study was to investigate the relationship between plasma fatty acid binding protein 4 (FABP4), phosphatase and tensin homolog (PTEN), and insulin resistance in patients with gestational diabetes mellitus (GDM). MATERIAL AND METHODS: Plasma FABP4 and PTEN were determined by ELISA in GDM patients (GDM group, n=30) and in euglycemic pregnant women (control group, n=30). The clinical features, body mass index (BMI), homeostasis model assessment of insulin resistance (HOMA-IR), and lipid profiles were compared between the 2 groups. The influence of risk factors on insulin resistance, including BMI, lipid profiles, FABP4, and PTEN, were further investigated by multiple-factor stepwise regression analysis. RESULTS: Higher levels of BMI, ΔBMI, triglyceride (TG), fasting plasma glucose (FPG), 2-hour plasma glucose (2hPG), fasting insulin, HOMA-IR, FABP4, PTEN, and lower level of high-density lipoprotein cholesterol (HDL-C) were found in the GDM patients than in the controls (all P<0.005). The plasma FABP4 was 1.47±0.25 vs. 0.20±0.07 ng/ml in the GDM and control group, respectively (P<0.0001). Plasma PTEN was 6.46±1.57 vs. 4.72±0.82 ng/ml in the GDM and control group, respectively (P<0.0001). There was a positive relation between plasma FABP4 and PTEN when all blood samples, including GDM and control groups, were analyzed (P<0.05). The multiple-factor regression analysis revealed that plasma FABP4, TG, and PTEN were independent risk factors for increased insulin resistance. CONCLUSIONS: GDM patients have more severe insulin resistance compared to euglycemic pregnant women. Higher levels of plasma FABP4 and PTEN are associated with increased insulin resistance and may participate in the pathogenesis of insulin resistance during gestation.

[Involvement of heme oxygenase in PM2.5-toxicity in human umbilical vein endothelial cells].

OBJECTIVE: To investigate the involvement of heme oxygenase (HO-1) in PM2.5 induced toxic responses in human umbilical vein endothelial cells (HUVECs). METHODS: The experiment groups are as follows: (1) control group; (2) PM2.5 groups: the cells were cultured with various concentrations of PM2.5 (200, 400, 800 µg/ml) for 24 h and 400 µg/ml was chosen for the main study; (3) PM2.5+Trion group: the cells were pre-treated by 10 µmol/L Trion [a scavenger of reactive oxygen species(ROS)] for 1 h before PM2.5 (400 µg/ml) treatment for 24 h; (4) PM2.5+ZnPP group: the cells were pretreated by HO-1 inhibitor ZnPP (10 µmol/L) for 1 h before treatment with PM2.5 (400 µg/ml) for 24 h. MTT assay was used to detect cell viability. Reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence assay were used to determine the mRNA and protein expressions of HO-1. Fluorescence labeling probe method was used to measure intracellular ROS level and flow cytometry was used for cell apoptosis. Colorimetric assay was used to detect intracellular caspase-3 activity. RESULTS: Compared with control, PM2.5 significantly decreased cell viability, increased intracellular ROS, cell apoptosis and caspase-3 activity (all P < 0.05), these effects were significantly attenuated in PM2.5+Tiron group while enhanced in PM2.5+ZnPP group (all P < 0.05 vs. PM2.5 group). PM2.5 upregulated HO-1 mRNA and protein expressions in HUVECs which was downregulated in both PM2.5+Tiron group and PM2.5+ZnPP group. CONCLUSION: PM2.5 could induce oxidative injury through increasing ROS production via modulating HO-1 mRNA and protein expressions, the injury could be aggravated with inhibition of the activity of HO-1 suggesting a potential protective role of HO-1 against PM2.5 induced oxidative stress in HUVECs.

[Study on the effect of extracts from the leaves of Phyllanthus emblica on immune function of mice].

OBJECTIVE: To explore the effect of extracts from the leaves of Phyllanthus emblica (PLFs) on the immune function of mice. METHODS: 70 Kunming mice were choosed to conduct the acute toxicity test of PLFs. The mice were randomly divided into four groups: PLFs high-dosage group, mid-dosage group, low-dosage group and control group. The high,mid,low-dosage groups were treated with PLFs 1.982, 0.991 and 0.496 g/kg respectively per day. The same volume of double distilled water was given to the control group. All by intragastric administration for 7 d. The animals were killed and indexes of thymus and spleen were calculated. The expurgation index K and phagocyte index a were detected after the mice being injected with a dilute India ink through caudal vein. In addition, prepared spleen cells conventionally,the activity of Natural Killer cells was measured and the proliferation of T and B cells were detected. The effect of the extracts on serum hemolysin was detected after the SRBC was injected into the enterocoelia. RESULTS: The LD50 of PLFs was 9. 911 g/kg. Compared with the control group, the indexes of thymus and spleen in the treatment groups had no markedly difference (P > 0.05). The high- and mid-dosage groups could obviously improve the expurgation index K (P < 0.05), phagocyte index alpha (P < 0.05) and NK cell activity (P < 0.05). CONCLUSION: The extracts from Phyllanthus emblica leaves can promote nonspecific immunity immune function in mice.

Ginsenoside Rg1 reduces toxicity of PM(2.5) on human umbilical vein endothelial cells by upregulating intracellular antioxidative state.

Ambient airborne particulate matter (PM) is an important environmental pollutant responsible for many human diseases. Oxidative stress is suggested to be involved in PM-induced cell injury. The present study is designed to study unsalutary effects of the organic extracts of PM with an aerodynamic diameter of less than 2.5µm (PM(2.5)) and protective effect of Ginsenoside Rg1 (Rg1) against PM(2.5) on human umbilical vein endothelial cells (HUVECs) in vitro. Cytotoxic effects of the organic extract PM(2.5) on HUVECs were measured by means of HUVEC cell viability and the generation of intracellular reactive oxygen species (ROS). Expression of heme oxygenase-1(HO-1) and Nuclear factor-erythroid 2-related factor 2 (Nrf2) and Nrf2 cytoplasm-nucleus location were assayed. The present results showed that PM(2.5) (50-800µg/ml) decreased HUVEC viability and increased intracellular generation of ROS and malondialdehyde (MDA) in a concentration dependent manner, but increased HO-1 expression without concentration dependence. Rg1 (10 and 40µg/ml) diminished PM(2.5)-induced HUVEC viability, decrease ROS and MDA generation, increased HO-1 and Nrf2 expression and promoted Nrf2 translocation to nucleus in a concentration dependent manner. These results suggested that organic extracts of PM(2.5) increase oxidative stress and decrease cell viability; Rg1 antagonize PM(2.5)-induced excess oxidative stress; HO-1 expression increase and Nrf2 translocation to nucleus may be involved in the effects of both PM(2.5) and Rg1 on HUVECs.

Discovery of PG545: a highly potent and simultaneous inhibitor of angiogenesis, tumor growth, and metastasis.

Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3ß-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-ß-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.

[Effect of ginsenoside on fine particulate matter induced oxidative injury in human endothelial cells].

OBJECTIVE: To explore the mechanism of fine particulate matter (PM(2.5)) induced endothelial injury and the efficacy and mechanism of ginsenoside Rg1 on the inhibition of endothelium injuries in human endothelial cells exposure to PM(2.5). METHODS: Human umbilical vein endothelial cells (HUVECs) were stimulated with various concentrations PM(2.5) (0.1, 0.2, 0.4, 0.8 mg/ml) and PM(2.5) at concentration 0.8 mg/ml induced significant endothelial injury and was chosen for the main study in the presence or absence of Rg1 (0.04 mg/ml). After 24 h treatment, cell growth A value was detected through MTT, intracellular reactive oxygen species (ROS) level through fluorescence labeling probe method and HO-1, Nrf2 mRNA expression was detected by RT-PCR. RESULTS: The cell A value was significantly lower while the ROS fluorescence gray value and the average optical density ratio of HO-1 were significantly higher in PM(2.5) group than in the control group (all P < 0.05). The average optical density ratio of Nrf2 was similar between PM(2.5) group and control group (P > 0.05). The A value and the average optical density ratio of HO-1 were significantly higher while the ROS fluorescence gray value was significantly lower in co-treated PM(2.5) (0.8 mg/ml) + Rg1 (0.04 mg/ml) group than in the PM(2.5) (0.8 mg/ml) group (all P < 0.05). CONCLUSION: PM(2.5) could induce human endothelial cells injury by increasing oxidative stress which could be attenuated by ginsenoside Rg1.

Synthesis and biological evaluation of polysulfated oligosaccharide glycosides as inhibitors of angiogenesis and tumor growth.

A series of polysulfated penta- and tetrasaccharide glycosides containing alpha(1-->3)/alpha(1-->2)-linked mannose residues were synthesized as heparan sulfate (HS) mimetics and evaluated for their ability to inhibit angiogenesis. The compounds bound tightly to angiogenic growth factors (FGF-1, FGF-2, and VEGF) and strongly inhibited heparanase activity. In addition, the compounds exhibited potent activity in cell-based and ex vivo assays indicative of angiogenesis, with tetrasaccharides exhibiting activity comparable to that of pentasaccharides. Selected compounds also showed good antitumor activity in vivo in a mouse melanoma (solid tumor) model resistant to the phase III HS mimetic 1 (muparfostat, formerly known as PI-88). The lipophilic modifications also resulted in reduced anticoagulant activity, a common side effect of HS mimetics, and conferred a reasonable pharmacokinetic profile in the rat, as exemplified by the sulfated octyl tetrasaccharide 5. The data support the further investigation of this class of compounds as potential antiangiogenic, anticancer therapeutics.

Development of a colorimetric assay for heparanase activity suitable for kinetic analysis and inhibitor screening.

The role that heparanase plays during metastasis and angiogenesis in tumors makes it an attractive target for cancer therapeutics. Despite this enzyme's significance, most of the assays developed to measure its activity are complex. Moreover, they usually rely on labeling variable preparations of the natural substrate heparan sulfate, making comparisons across studies precarious. To overcome these problems, we have developed a convenient assay based on the cleavage of the synthetic heparin oligosaccharide fondaparinux. The assay measures the appearance of the disaccharide product of heparanase-catalyzed fondaparinux cleavage colorimetrically using the tetrazolium salt WST-1. Because this assay has a homogeneous substrate with a single point of cleavage, the kinetics of the enzyme can be reliably characterized, giving a K(m) of 46 microM and a k(cat) of 3.5 s(-1) with fondaparinux as substrate. The inhibition of heparanase by the published inhibitor, PI-88, was also studied, and a K(i) of 7.9 nM was determined. The simplicity and robustness of this method, should, not only greatly assist routine assay of heparanase activity but also could be adapted for high-throughput screening of compound libraries, with the data generated being directly comparable across studies.

A surface plasmon resonance-based solution affinity assay for heparan sulfate-binding proteins.

A surface plasmon resonance-based solution affinity assay is described for measuring the K(d) of binding of heparin/heparan sulfate-binding proteins with a variety of ligands. The assay involves the passage of a pre-equilibrated solution of protein and ligand over a sensor chip onto which heparin has been immobilised. Heparin sensor chips prepared by four different methods, including biotin-streptavidin affinity capture and direct covalent attachment to the chip surface, were successfully used in the assay and gave similar K(d) values. The assay is applicable to a wide variety of heparin/HS-binding proteins of diverse structure and function (e.g., FGF-1, FGF-2, VEGF, IL-8, MCP-2, ATIII, PF4) and to ligands of varying molecular weight and degree of sulfation (e.g., heparin, PI-88, sucrose octasulfate, naphthalene trisulfonate) and is thus well suited for the rapid screening of ligands in drug discovery applications.

Synthesis, biological activity, and preliminary pharmacokinetic evaluation of analogues of a phosphosulfomannan angiogenesis inhibitor (PI-88).

The phosphosulfomannan 1 (PI-88) is a mixture of highly sulfated oligosaccharides that is currently undergoing clinical evaluation in cancer patients. As well as its anticancer properties, 1 displays a number of other interesting biological activities. A series of analogues of 1 were synthesized with a single carbon (pentasaccharide) backbone to facilitate structural characterization and interpretation of biological results. In a fashion similar to 1, all compounds were able to inhibit heparanase and to bind tightly to the proangiogenic growth factors FGF-1, FGF-2, and VEGF. The compounds also inhibited the infection of cells and cell-to-cell spread of herpes simplex virus (HSV-1). Preliminary pharmacokinetic data indicated that the compounds displayed different pharmacokinetic behavior compared with 1. Of particular note was the n-octyl derivative, which was cleared 3 times less rapidly than 1 and may provide increased systemic exposure.

An experimental and molecular-modeling study of the binding of linked sulfated tetracyclitols to FGF-1 and FGF-2.

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc2N-R-NCyc2, where Cyc = C6H6(OSO3Na)3 and R = (CH2)n (n = 2-10), p-xylyl or (C2H4)2-Ncyc] for the fibroblast growth factors FGF-1 and FGF-2 have been measured by using a surface plasmon resonance assay. The KD values range from 7.0 nM to 1.1 microM for the alkyl-linked ligands. The binding affinity is independent of the flexibility of the linker, as replacement of the alkyl linker with a rigid p-xylyl group did not affect the KD. Calculations suggest that binding modes for the p-xylyl-linked ligand are similar to those calculated for the flexible alkyl-linked tetracyclitols. The possible formation of cross-linked FGF:cyclitol complexes was examined by determining KD values at increasing protein concentrations. No changes in KD were observed; this suggesting that only 1:1 complexes are formed under these assay conditions. Monte Carlo multiple-minima calculations of low-energy conformers of the FGF-bound ligands showed that all of the sulfated tetracyclitol ligands can bind effectively in the heparan sulfate-binding sites of FGF-1 and FGF-2. Binding affinities of these complexes were estimated by the Linear Interaction Energy (LIE) method to within a root-mean-square deviation of 1 kcal mol(-1) of the observed values. The effect of incorporating cations to balance the overall charge of the complexes during the LIE calculations was also explored.

[Genetic polymorphisms of short tandem repeat loci D3S1358, D13S317, D5S818 from Nongqu Mongolia].

OBJECTIVE: To understand the allele structure and genetic polymorphism at D3S1358, D13S317, D5S818 short tandem repeats (STRs) loci in Nongqu Mongolian of China, and to construct a preliminary database. METHODS: The allele frequencies of the three STRs loci in 291 unrelated individuals from Nongqu Mongolian were analyzed by polymerase chain reaction and polyacrylamide gel electrophoresis. RESULTS: Six, ten, and eight alleles were observed at D3S1358, D13S317, D5S818, respectively, and all 3 loci met Hardy-Weinberg equilibrium. The statistical analysis of 3 STR loci showed the heterozygosity >or=0.7332, the polymorphic information content >or=0.6884; the combined discrimination power and the probabilities of paternity exclusion were 0.9991 and 0.9806 respectively. CONCLUSION: All three of the loci in this study were found to have high heterozygosity and polymorphic information content, so they could provide useful markers for genetic purposes. These results could serve as valuable data to enrich the Mongolian genetic database and play an important role in Chinese population genetic application.