OBJECTIVETo examine the effects of continuous and intermittent exercises on obesity and fatty liver in rats fed with high-fat diet.

METHODSWistar rats were randomly assigned into routine diet (R) and high-fat diet (H) groups, and each group were subdivided into sedentary group (S), continuous exercise (CE) group, and intermittent exercise (IE) group (n=8). In the CE group, the rats were forced to swim continuously for 90 min once daily, and those in the IE group swam for 30 min for 3 times (at a 4-h interval) daily. Both the CE and IE groups exercised for 5 days a week for 8 consecutive weeks. After the experiment, the retroperitoneal, epididymal, and visceral white and brown adipose tissues, the liver, and the gastrocnemius muscle of the rats were weighed. The lipogenesis rate was determined by incorporation of (3)H(2)0 into saponified lipids, and the blood lipid profiles were analyzed. The body weight and food intake of the rats were recorded daily.

RESULTSIE appeared to be more efficient than CE in reducing the adverse effects of high-fat diet and sedentarism. Compared with CE, IE resulted in an improved lipid profile with reduced food intake, body weight gain, visceral and central adiposity, and fatty liver. The effect of high-fat diet and different exercises on weight gain, adiposity, fatty liver, and lipid profile in rats was associated to the manner of exercise, time of each session, age, gender, and length of observation period.

CONCLUSIONIntermittent exercise is an important nonpharmacological strategy to control obesity and the related complications.

Animals ; Diet, High-Fat ; Fatty Liver ; etiology ; therapy ; Male ; Obesity ; etiology ; therapy ; Physical Conditioning, Animal ; methods ; Rats ; Rats, Wistar

OBJECTIVETo prepare stable chicken red blood cells for the calibration of flow cytometry.

METHODSThe traditional isolation method of chicken red blood cells was modified by incorporating gelatin technique, Ca2+-free HBSS treatment and low-speed centrifugation. The effect of fluorescence staining of the cells was improved by the addition of TritonX-100 to enhance the membrane permeability and Rnase enzymes to disintegrate RNA tiles. The modified method was compared with the traditional method for viability of the freshly isolated cells and the DNA content coefficient of variation (CV) of the fixed cells.

RESULTSChicken red blood cells obtained by the modified method showed a significantly higher viability than those obtained by the traditional method [(98.5∓3.5)% vs (93.5∓2.7)%, P<0.05]. After glutaraldehyde fixation, the isolated cells with the modified method were stable during the 90-day preservation with a significantly lower CV than the cells obtained by the traditional method [(6.0∓0.3)% to 6.2∓0.4% vs (8.6∓0.5)% to (13.1∓1.4)%, P<0.01].

CONCLUSIONThe chicken red blood cells isolated using the modified method can be applicable for calibration of flow cytometry.

Animals ; Calibration ; Chickens ; Erythrocytes ; cytology ; Flow Cytometry ; instrumentation ; methods

OBJECTIVETo establish a rapid and sensitive high-performance liquid chromatography (HPLC) method for detecting pazufloxacin concentrations in the saliva, gingival crevicular fluid and serum of healthy adults.

METHODSSamples of saliva, gingival crevicular fluid and serum were obtained from healthy adults receiving intravenous infusion of pazufloxacin. The concentrations of pazufloxacin in the samples were quantified by HPLC equipped with a reversed-phase column (Agilent Zorbax SB-C18 5 µm, 250 mm×4.6 mm). The mobile phase for pazufloxacin was a mixture of acetonitrile and 0.5% phosphoric acid containing 1% triethylamine (155:850), and 20 µl of the resulting solution was injected into the HPLC system at a flow rate of 1.0 ml/min. The detection wavelength was set at 245 nm. The samples were first deproteinized by precipitation with methanol followed by supernatant drying; the residue was reconstituted with the mobile phase and centrifuged, and the supernatants were directly injected into the HPLC system.

RESULTSPazufloxacin in the samples were totally separated without interference by any endogenous substances. The calibration curves showed a good linear regression (r>0.999). The detection limit was 10 ng/ml with within-day and between-day coefficients of variation performance all below 5% and recovery rates all above 91%.

CONCLUSIONHPLC is both sensitive and selective for quantification of pazufloxacin in saliva, gingival crevicular fluid and serum.

Adult ; Chromatography, High Pressure Liquid ; methods ; Female ; Fluoroquinolones ; analysis ; blood ; Gingival Crevicular Fluid ; chemistry ; Humans ; Male ; Oxazines ; analysis ; blood ; Saliva ; chemistry ; Sensitivity and Specificity ; Young Adult

OBJECTIVETo investigate the feasibility of constructing a capsular poly L-lactic acid (PLLA) ureteral stent seeded with autologous urothelial cells using tissue engineering methods.

METHODSThe capsular ureteral stent was constructed by subcutaneously embedding PLLA ureteral stent in the back of beagles for 3 weeks to induce the formation of connective tissue on the surfaces. After decellularization of the stent, the expanded autologous urothelial cells were seeded on the stent. The surface structure and cell adhesion of the stent were observed using HE staining, scanning electron microscope (SEM) and immunocytochemical staining. MTT assay was used to evaluate urothelial cell proliferation on the capsular PLLA ureteral stent and on circumferential small intestinal submucosa graft.

RESULTSHE staining and VIII factor immunohistochemistry revealed numerous capillaries in the connective tissue encapsulating the stent without obvious local inflammatory response. The results of SEM and immunocytochemical staining showed that the capsule contained rich collagenic fibers forming three-dimensional structures, and the seeded autologous urothelial cells could adhere and well aligned on the surface. MTT assay showed normal growth of the cells on the stent as compared with the cells grown on circumferential small intestinal submucosa graft.

CONCLUSIONThe capsular PLLA ureteral stent allows adhesion and proliferation of autologous urothelial cells and shows a potential in applications of constructing tissue-engineered ureter.

Animals ; Bacterial Adhesion ; Cell Proliferation ; Dogs ; Epithelial Cells ; cytology ; transplantation ; Female ; Lactic Acid ; Polyesters ; Polymers ; Stents ; Tissue Engineering ; methods ; Transplantation, Autologous ; Ureter ; surgery ; Urothelium ; cytology

OBJECTIVETo investigate the effect of small interfering RNA-mediated glucose-regulated protein 78 (GRP78) knockdown on the chemosensitivity of breast cancer cells to cisplatin.

METHODSHuman breast cancer cell line MDA-MB-231 was exposed to different doses of cisplatin (0, 1, 2, 4, 8, and 16 µmol/L), and the changes in cell viability were detected using MTT assay. PI/Annexin V staining was used to observe the apoptosis of the cells in response to transfection with a small interfering RNA targeting GRP78 (pSH1Si-GRP78). Western blotting was employed to detect GRP78 expression in pSH1Si- GRP78-transfected cells after exposure to 8 µmol/L cisplatin for 24, 48 and 72 h.

RESULTSExposure of the cells to 8 µmol/L cisplatin for 24, 48 and 72 h resulted in a cell survival rate of 83.13%, 54.22% and 35.79%, respectively, but the cell apoptosis rate was only 10.8% at 24 h. Transfection of MDA-MB-231 cells with pSH1Si-GRP78 caused a cell apoptosis rate of 24.6%, which increased to 48.9% in cells with both pSH1Si-GRP78 transfection and cisplatin exposure. Cisplatin exposure caused an initial up-regulation followed then by a down-regulation of GRP78 expression in MDA-MB-231 cells, while pSH1Si-GRP78 transfection produced an obvious down-regulation of GRP78 expression.

CONCLUSIONSInhibition of GRP78 expression increases the apoptosis and enhance cisplatin chemosensitivity of breast cancer cells in vitro, suggesting the value of GRP78 as a potential therapeutic target in the clinical treatment of breast cancer.

Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Survival ; Cisplatin ; pharmacology ; Female ; Gene Knockdown Techniques ; Heat-Shock Proteins ; metabolism ; Humans ; RNA, Small Interfering ; Transfection

OBJECTIVETo investigate the accuracy of ultrasound monitoring during pulsed high intensity focused ultrasound (PHIFU) treatment and improve the sensitivity of ultrasound monitoring of tissue necrosis caused by PHIFU treatment.

METHODSBovine liver ex vivo was dot-exposed with HIFU at the therapeutic doses of 2000 J (group A) and 1440 J (group B). The two groups were further divided into groups A1 (power 100 W, duty cycle 100%, irradiate 20 s) A2 (power 100 W, duty cycle 50%, irradiate 40 s), A3 (power 100 W, duty cycle 40%, irradiate 50 s), B1 (power 120 W, duty cycle 100%, irradiate 12 s), B2 (power 100 W, duty cycle 50%, irradiate 24 s), and B3 (power 100 W, duty cycle 40%, irradiate 30 s). The gray scale changes in the ultrasonic images after the exposures were observed, and the correlation function of the image was calculated before and after the exposure. The accuracy of evaluations based on the correlation function and gray-scale changes was compared.

RESULTSThe correct rate of gray scale-based evaluation of tissue necrosis caused by PHIFU was only 51%, while that by correlation function-based evaluation reached 85%.

CONCLUSIONMonitoring of tissue necrosis caused by PHIFU treatment can not rely solely on evaluation of gray-scale change of the ultrasound images, and the correlation function-based evaluation can be more accurate and sensitive for that purpose.

Animals ; Cattle ; High-Intensity Focused Ultrasound Ablation ; methods ; In Vitro Techniques ; Likelihood Functions ; Liver ; diagnostic imaging ; Ultrasonography

OBJECTIVETo assess a minipig model of acute myocardial infarction (AMI) established by percutaneous balloon occlusion using delayed-enhanced magnetic resonance imaging (DE-MRI).

METHODSA minipig model of AMI was established by placement of a 2.0 mm×15.0 mm percutaneous transluminal coronary angioplasty balloon in the middle left anterior descending artery (LAD) through a percutaneous femoral puncture in the right inguinal region. The left anterior descending coronary artery (LAD) was occluded for 90 min, followed by assessment of the infarct size and cardiac function with DE-MRI, and the results were confirmed by pathological examination.

RESULTSDE-MRI showed a mean infarcts size of 10.2∓2.9 cm3 in the minipig models. Compared to the control group, the minipigs with AMI had significantly increased end-diastolic and end-systolic volumes (P<0.05) with a decreased stroke volume, ejection fraction and cardiac output (P<0.001). These DE-MRI values were matched with the microsphere values obtained from short-axis slices in pathological examination.

CONCLUSIONWe have established a feasible approach for evaluating minipig models of AMI as a platform for assessing the therapeutic effect of stem cell transplantation for AMI.

Angioplasty, Balloon, Coronary ; Animals ; Disease Models, Animal ; Magnetic Resonance Imaging ; Myocardial Infarction ; pathology ; Swine ; Swine, Miniature

OBJECTIVETo investigate the effect of Biejiajian Pills on Wnt signal pathway and its inhibitory gene (DKK-1 and FrpHe) expressions and explore the mechanism underlying the action of Biejiajian Pills to suppress the invasiveness of hepatocellular carcinoma.

METHODSTwenty-four Wistar rats were randomized equally into 3 groups for gavage of normal saline and Biejiajian Pills at 20- and 10-fold clinical doses for 3 days. Blood samples were then collected from the rats, and the serum was separated and added in HepG2 cell cultures. After 48 h of culture, the cells were collected to determine the cellular content of β-catenin protein using flow cytometry and detect DKK-1 and FrpHe mRNA expressions using qRT-PCR.

RESULTSHepG2 cells cultured in the presence of sera from rats fed with Biejiajian Pills showed significantly lowered β-catenin protein expression and obvious down-regulation of DKK-1 mRNA expression, and the effect was correlated with the doses of the drug administered. The expression of FrpHe mRNA showed no significant differences between the 3 groups.

CONCLUSIONSBiejiajian Pills can effectively inhibit the invasiveness and migration of hepatocellular carcinoma cells, which is closely related to decreased expressions of β-catenin and DKK-1 to cause block of the Wnt/β-catenin signal pathway.

Animals ; Carcinoma, Hepatocellular ; genetics ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Hep G2 Cells ; Humans ; Intercellular Signaling Peptides and Proteins ; metabolism ; Liver Neoplasms ; genetics ; metabolism ; pathology ; Male ; Proto-Oncogene Proteins ; metabolism ; Rats ; Rats, Wistar ; Wnt Proteins ; metabolism ; Wnt Signaling Pathway ; drug effects ; beta Catenin ; metabolism

OBJECTIVETo examine whether calcineurin/NFAT signaling pathway mediates endothelin-1 (ET-1)-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) by regulating phosphodiesterase-5 (PDE5) and the effect of the selective calcineurin inhibitor cyclosporine A and PDE5 inhibitor sildenafil on ET-1-induced PASMC proliferation.

METHODSPASMCs were treated with ET-1 to stimulate their proliferation with or without prior treatment of the cells with CsA or sildenafil. Calcineurin activity in the cells was measured using a calcineurin activity assay kit, PDE5 expression examined using immunoblotting, and cGMP level detected using a cGMP direct immunoassay kit. PASMC proliferation following the treatments was determined using [(3)H]thymidine incorporation assay.

RESULTSET-1 caused a 2.05-fold increase in the cellular calcineurin activity, a 1.80-fold increase in PDE5 expression, and a 3.20-fold increase in the DNA synthesis rate, and reduced the cGMP level by 67%. Pretreatment of the cells with Cyclosporine blocked the effects of ET-1, and PDE5 inhibition by sildenafil pretreatment also abolished ET-1-induced reduction of cGMP level in the cells. Both Cyclosporine and sildenafil suppressed ET-1-stimulated PASMC proliferation.

CONCLUSIONActivation of calcineurin/NFAT signaling pathway mediates ET-1-induced PASMC proliferation by stimulating PDE5 expression, which further degrades cGMP. Both Cyclosporine and sildenafil can suppress ET-1-stimulated PASMC proliferation in vitro.

Animals ; Calcineurin ; metabolism ; Cell Proliferation ; drug effects ; Cells, Cultured ; Cyclic GMP ; metabolism ; Cyclic Nucleotide Phosphodiesterases, Type 5 ; metabolism ; Cyclosporine ; DNA ; biosynthesis ; Endothelin-1 ; pharmacology ; Muscle, Smooth, Vascular ; cytology ; Myocytes, Smooth Muscle ; cytology ; enzymology ; NFATC Transcription Factors ; metabolism ; Piperazines ; Pulmonary Artery ; cytology ; Purines ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Sildenafil Citrate ; Sulfones

OBJECTIVETo investigate the cytotoxic effect of γδ T cells from osteosarcoma patients against interferon-γ (IFN-γ)-treated osteosarcoma cells in vitro.

METHODSHuman γδ T cells were amplified by zoledronate from peripheral blood cells of osteosarcoma patients. The expression of Fas on the osteosarcoma cells were measured by flow cytometry and quantitative real-time PCR analysis before and after IFN-γ treatment. The cytotoxicity of γδ T cells against osteosarcoma cells was evaluated with LDH assay.

RESULTSIFN-γ significantly enhanced the susceptibility of the osteosarcoma cell lines HOS and U2OS to the cytotoxicity of γDelta; T cells from osteosarcoma patients (P<0.01). IFN-γ obviously up-regulated the expression of Fas in HOS and U2OS cells (P<0.01). Anti-FasL mAb failed to inhibit the cytotoxicity of γδ T cells in untreated osteosarcoma targets (P>0.05), but significantly impaired γδ T cell cytotoxicity in IFN-γ pre-treated osteosarcoma targets (P<0.01).

CONCLUSIONIFN-γ can enhance the cytotoxic effect of human γδ T cells from osteosarcoma patients against osteosarcoma cells in vitro.

Bone Neoplasms ; metabolism ; Cell Line, Tumor ; Cytotoxicity, Immunologic ; Humans ; Interferon-gamma ; pharmacology ; Osteosarcoma ; immunology ; metabolism ; Receptors, Antigen, T-Cell, gamma-delta ; immunology ; T-Lymphocytes, Cytotoxic ; cytology ; drug effects ; immunology ; fas Receptor ; metabolism