OBJECTIVETo observe the effects of panax quinquefolius saponin (PQS) of stem and leaf on glucose-lipid metabolism and insulin signal transduction in the insulin resistant model of adipocytes.

METHODSThe insulin resistant model of differentiated 3T3-L1 adipocytes was established in vitro with free fatty acid. After induction of insulin resistance, cells were treated with metformin or PQS for 2 days. The glucose consumption in culture fluid was detected by glucose oxidase method; the effects of PQS on the lipolysis induced by tumor necrosis factor (TNF-alpha) was observed using colorimetry; and the phospholation of signal proteins was detected by Western-blot.

RESULTSThe amount of glucose consumption (mmol/L) in the model group (5.250 +/- 2. 671) was significantly lower than that in the normal control group (14.133 +/- 1.305, P < 0.01), it increased in the meformin treated group (11.807 +/- 1.358), and the groups treated with high-, middle- and low-dose PQS dose-dependently (10.784 +/- 2.373, 10.217 +/- 1.237 and 9.984 +/- 2.006, respectively), significantly higher than that in the model group (P < 0.01). Upon TNF-alpha treatment, the concentration of free fatty acid (FFA) (nmol/ microg) in culture medium was 2.479 +/- 0.597, predominantly higher than that in the control group (1.320 +/- 0.538, P < 0.01), while it was 1.210 +/- 0.566 in the metformin group, 1.105 +/- 0.631 in high-dose PQS group, 1.108 +/- 0.260 in the middle-dose PQS group, 1.201 +/- 0.593 in the low-dose PQS group, all were lower than that in the TNF-alpha group (P < 0.05 or P < 0.01), and a dose-dependent tendency of PQS's action was seen. The tyrosine phosphorylation of insulin receptor and IRS-1 as well as Ser473 phosphorylation of PKB were lower in the model group than in the control group; they were insignificantly changed in the low-dose PQS group, but did show significant difference in comparing with those in the high-and middle-dose PQS groups or metformin group.

CONCLUSIONPQS can accelerate the glucose utilization and depress the lipolysis in adipocytes induced by TNF-alpha, which may be correlated with its promoting insulin signal transduction and improving insulin resistance in adipocytes.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Glucose ; metabolism ; Insulin ; metabolism ; Insulin Resistance ; Lipid Metabolism ; drug effects ; Mice ; Saponins ; pharmacology ; Signal Transduction ; drug effects

AIM AND METHODSUsing in vivo myocardial injury model induced by Isoproterenol (ISO) and the model induced by in vitro global heart ischemia/reperfusion (I/R) in rats, we observed the effects of myocardial ischemia and ischemia/reperfusion on bio-membranes of mitochondria and sarcolemma injury.

RESULTSISO (5 mg/kg, s.c.) and I/R (20 min/20 min) damaged myocardial bio-membranes of rat, in which the lipid peroxidation of mitochondria was significantly increased, the activity of phospholipase A2 was activated and the contents of phospholipid and free fat acid were decreased and increased significantly, respectively. The membrane lipid fluidity and the activities of Ca(2+) -ATPase of mitochondria and Na+, K(+) -ATPase of sarcolemma were significantly lowered. In addition, the respiration function of mitochondria was declined and the oxidative phosphorylation of respiratory chain was uncoupled, which resulted in the decrease in the production of high energy phosphoric acid compounds.

CONCLUSIONMyocardial injuries caused by ISO or I/R injured the structure and function of rat myocardial mitochondria and sarcolemma.

Animals ; Disease Models, Animal ; Male ; Mitochondria, Heart ; pathology ; ultrastructure ; Mitochondrial Membranes ; pathology ; ultrastructure ; Myocardial Reperfusion Injury ; pathology ; Rats ; Rats, Wistar ; Sarcolemma ; pathology ; ultrastructure

Limonin existed in citrus fruits has been shown to have anti-bacterial, anti-viral, anti-feedant, anti-nociceptive, anti-inflammatory activities and anti-carcinogenic activities. But the clinical use is limited by its low bioavailability. The aim of this study is to observe the absorption and secretion transport mechanisms of limonin in intestine which can pave the way for the further study and clinical use. The transport characteristics and mechanisms of limonin in rat were studied by in situ intestine perfusion and in vitro Caco-2 cells method. The intestinal absorption of limonin was probably via a facilitated diffusion pathway which was poor and without segment-selection. Verapamil and ketoconazole improved the absorption remarkably according to the result of in vitro Caco-2 cells study; however, probenecid had no significant effect on the absorption. The P-gp efflux and CYP3A4 metabolism were involved in the poor intestinal absorption and low bioavailability of limonin. The exploration of the intestinal absorption mechanism is crucial to the design of dosage form and clinical use of limonin.
ATP-Binding Cassette, Sub-Family B, Member 1 ; metabolism ; Animals ; Biological Availability ; Biological Transport ; drug effects ; Caco-2 Cells ; Cytochrome P-450 CYP3A ; metabolism ; Dose-Response Relationship, Drug ; Humans ; Intestinal Absorption ; drug effects ; Ketoconazole ; pharmacology ; Limonins ; administration & dosage ; pharmacokinetics ; Male ; Perfusion ; Probenecid ; pharmacology ; Rats ; Verapamil ; pharmacology

Objective To discuss the molecular mechanism of 18F-fluorodeoxyglucose (FDG) uptake in tumor and to assess its value to identify pathologic type and cancer staging in patients with earlystage nasopharyngeal carcinoma.Methods Forty patients with nasopharyngeal carcinoma of early-stage,including 12 cases with T1 stage and 28 cases with T2 stage, underwent FDG PET imaging.The maximum standardized uptake value ( SUVmax ) and mean standardized uptake value ( SUVmean ) of FDG uptake of each patient were measured and compared between T1 and T2 stage by t-test.The expression of glucose transport protein 1 ( Glut1 ) and hexokinase- Ⅱ ( HK- Ⅱ ) of each case was measured in paraffin sections by streptavidin-perosidase (SP) immunohistochemistry.The positive expression rate of Glut1 and HK- Ⅱ was calculated and compared between T1 and T2 by x2 test.Meanwhile, the correlation between the expression of Glut1 or HK-Ⅱ and the SUVmax was tested by Pearson analysis.Results The SUVmax and SUVmean in 40 patients were 9.45 ± 1.87 and 6.04 ± 1.09, respectively.The SUVmax of patients with T1 stage (8.95 ± 1.91 ) was significantly lower (t =4.46, P＜0.001 ) than that of patients with T2 stage (11.55 ± 1.70), and the SUVmean of patients with T1 stage (5.61 ± 1.08) was significantly lower ( t = 6.76, P ＜ 0.001 ) than that of patients with T2 stage (7.98 ± 1.10) too.Among 40 patients, all patients showed positive expression of Glut1 and HK-Ⅱ , and the positive expression rate of Glut1 and HK-Ⅱ was ( 45.2 ± 10.9 )% and ( 68.3 ±9.5)%, respectively.The positive expression rate of Glut1 was (38.4 ±8.1)% in T1 stage and (49.7 ±12.6)% in T2 stage, which displayed no difference (x2 =40.58, P＞0.05), but the HK-Ⅱ positive expression rate showed significant difference (x2 =58.71, P＜0.05) between T1 stage (60.1 ±11.1)% and T2 stage (77.9 ± 14.7 )%.The correlation analysis indicated that there was low-degree positive correlation (r =0.369, P=0.019) between the SUVmax and Glut1 expression, and there was medium-degree positive correlation (r = 0.549, P = 0.001 ) between the SUVmax and HK-Ⅱ expression.Conclusion Expression of Glut1 and HK-Ⅱ was positively correlated with FDG uptake in patients with early-stage nasopharyngeal carcinoma.

OBJECTIVETo amplify the PCR with the internal transcribed spacerl regions measure the base sequence of the amplified products of DNA, and to set up an identified standard on the level of molecule.

METHODDNA from the seeds of G. dahurica was extracted by conventional method, and composed peculiar primer was used to amplify with the internal transcribed spacerl regions of the rRNA gene, and the base sequence of the amplified products by stopping the circle of the end of double deoxidation of four color fluorescent mark was measured.

RESULTIt was proved by agar sugar gel electrophoresis that the PCR amplified products of the internal transcribed spacerl regions of the rRNA gene existed. The base sequence of the seeds of G. dahurica's internal transcribed spacerl regions of the rRNA gene was measured.

CONCLUSIONTo measure the base sequence of internal transcribed spacerl regions of the rRNA gene in the seeds of G. dahurica's is a method to identify vegetal Chinese traditional medicine on the level of molecule.

Base Sequence ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Gentiana ; genetics ; Molecular Sequence Data ; Plants, Medicinal ; genetics ; Polymerase Chain Reaction ; RNA, Ribosomal ; genetics ; Seeds ; genetics

OBJECTIVETo establish a method for the simultaneous determination of 15 pesticides residues in Radix Paeoniae Alba by large volume injection-gas chromatography-mass spectrometry(LVI-GC-MS).

METHODSThe pesticides, including organochlorine pesticides, organophosphorus pesticides and pyrethroid insecticides, were analyzed by LVI-GC-MS using DB-5MS capillary column (30 m X 250 μm, 0.25 μm). The column temperature programming: initial temperature 40 degree for 1 min, with the increasing rate of 20 degree/min to 210 degree for 2 min, then with the increasing rate of 5 degree/min to 280 degree for 22 min. The flow of carrier gas was 1.0 ml/min with the injection volume of 15 μl.

RESULTSThe calibration curves of the pesticides were linear in the specified concentration ranges with correlation coefficients of 0.9937-0.9995. The average recoveries of the pesticides in Radix Paeoniae Alba spiked at two spiked levels ranged from 60.4 % to 106.8 % (for pendimethalin and 4, 4'-DDE those were 53.1 % and 45.2 %) with relative standard deviation(RSD) of 3.6 % to 18.6 % and the detection limits (S/N=3) were in the range of 0.16 μg/kg to 3.59 μg/kg.

CONCLUSIONThe established method for determination of multi-pesticide residue in Radix Paeoniae Alba is rapid, convenient and accurate with high sensitivity and low-cost.

Gas Chromatography-Mass Spectrometry ; methods ; Paeonia ; chemistry ; Pesticide Residues ; analysis

OBJECTIVETo investigate the expression and gene mutation of cluster of differentiation 9 (CD9) in the pathway of the mineral powder induced malignant transformation in immortalized human bronchial epithelial cells (BEAS-2B) in Gejiu.

METHODSBEAS-2B cells served as the control group and its malignant transformation cells induced by mineral powder in Gejiu were considered as experiment group. The expression of CD9 protein in 20 bottles of BEAS-2B cells and 20 bottles of malignant transformation cells was evaluated by immunocytochemistry. The mRNA expression of CD9 in 10 bottles of BEAS-2B cells and 10 bottles of malignant transformation cells was examined by reverse transcriptase polymerase chain reaction (RT-PCR). Gene mutation was detected in the products of RT-PCR by DNA sequencing.

RESULTSThere was significant difference between the expression of CD9 protein in BEAS-2B cells (100%, 20/20) and that in its malignant transformation cells (35%, 7/20 P < 0.01). The expression of CD9 mRNA in BEAS-2B cells 0.91 +/- 0.09 was significantly higher than that in its malignant transformation cells (0.34 +/- 0.14) (P < 0.01). Two point mutation of CD9 gene was detected in the malignant transformation cells of BEAS-2B by DNA sequencing. The change of G-->T in the base of 231 led to the change of Gln-->His in the amino acids of 40. The change of T-->A in the base of 119 led to the change of Val-->Asp in the amino acids of 3.

CONCLUSIONThe absence or down-regulation of CD9 expression and point mutation in the malignant transformation cells of BEAS-2B may play a considerable role in the pathway of the malignant transformation in the BEAS-2B cells induced by mineral powder in Gejiu.

Bronchi ; pathology ; Cell Line ; Cell Transformation, Neoplastic ; drug effects ; genetics ; Dust ; Epithelial Cells ; drug effects ; metabolism ; pathology ; Gene Expression Regulation ; drug effects ; Humans ; Lung Neoplasms ; chemically induced ; genetics ; metabolism ; pathology ; Mining ; Mutation ; drug effects ; Tetraspanin-29 ; genetics ; metabolism

OBJECTIVETo evaluate the estrogenic activity of several kinds of Chinese herbal medicines.

METHODUse zoopery and reporter gene technique to study the estrogenic activity of five Chinese herbal medicines. Zoopery: weanling female Kunming mice weight 9-12 g were administrated botanical extracts of Selaginella tamariscina, Pinus Massoniana, Corallodiscus flabellate, Dryopteris sublaeta and Leonurus heterophyllus, the positive control group with Nilestriol tablets and control group with water, respectiely. On the eighth day, the animals were sacrificed and the uteri were separated solely and weighed. Reporter gene technique: Induce the expression of reporter gene controlled by ERE and measure the activity of luciferase on cell's clear supernatant.

RESULTThe botanical extracts of S. tamariscina can increase weights of mice (P < 0.01); In the expression of reporter gene controlled by ERE, Either ERalpha or ERbeta's has estrogenic activity (P < 0.01). Follow in the zoopery we find the water part and the n-butanol part of S. tamariscina are the two active parts.

CONCLUSIONS. tamariscina and it's water part and n-butanol part have estrogenic activities, effect on ERbeta is greater than ERalpha.

Animals ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Estrogen Receptor alpha ; metabolism ; Estrogen Receptor beta ; metabolism ; Female ; Leonurus ; chemistry ; Mice ; Organ Size ; drug effects ; Phytoestrogens ; isolation & purification ; pharmacology ; Pinus ; chemistry ; Plants, Medicinal ; chemistry ; Selaginellaceae ; chemistry ; Uterus ; anatomy & histology

AIM To investigate the effect of Wendan Decoction on nerve injury in a mouse model of sleep disorders and its mechanism.METHODS A mouse model of insomnia was established by the modified multiple platform sleep deprivation method.After successful modeling,the mice were randomly divided into the model group,the estazolam tablet group(0.15 mg/kg)and the low-dose and high-dose Wendan Decoction groups(12.5,50 g/kg),with 6 mice in each group,in contrast to the 6 mice of the control group.After 7 days of drug intervention,the mice had their changes of cerebral cortex,hippocampal CA1 area and hypothalamus observed by HE staining;their neuronal damage observed by Nissl staining;their levels of neurofilament light chain(NEFL),neuron-specific enolase(NSE),S100 calcium-binding protein B(S100B),tumor necrosis factor(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in brain tissue and serum detected by ELISA;their cerebral expression of glial fibrillary acidic protein(GFAP)detected by immunohistochemical method;and their cerebral expressions of GFAP,phosphorylated IκB kinase β(p-IKKβ)and phosphorylated nuclear transcription factor-κB(p-NF-κB)detected by Western blot.RESULTS Compared with the model group,the high-dose Wendan Decoction group displayed increased number of neurons,complete and neatly arranged structure;decreased number of neurons with nuclear shrinkage and deformation;increased Nissl bodies,decreased levels of NEFL,NSE,S100B,TNF-α,IL-6 and IL-1β in serum and brain tissue(P＜0.01);decreased cerebral expression of GFAP(P＜0.01);and decreased phosphorylation levels of cerebral p-IKKβ and p-NF-κB(P＜0.01).CONCLUSION Wendan Decoction can reduce the nerve damage and the expression of proinflammatory mediator in sleep disorders mice,and the mechanism may be related to the inhibited activation of IKKβ/NF-κB pathway.

OBJECTIVEThis study is aimed at developing a simple and easy way to generate dendritic cells (DCs) from human peripheral blood monocytes (PBMCs) in vitro.

METHODSPBMCs were isolated directly from white blood cell rather than whole blood and purified by patching methods (collecting the attached cell and removing the suspension cell). DCs were then generated by culturing PBMCs for six days with 30 ng/ml recombinant human granulocyte-macrophage stimulating factor (rhGM-CSF) and 20 ng/ml recombinant human interleukin-4 (rhIL-4) in vitro. On the sixth day, TNF-alpha (TNFalpha) 30 ng/ml was added into some DC cultures, which were then incubated for two additional days. The morphology was monitored by light microscopy and transmission electronic microscopy, and the phenotypes were determined by flow cytometry. Autologous mixed leukocyte reactions (MLR) were used to characterize DC function after TNFalpha or lipopolysaccharide (LPS) stimulations for 24 h.

RESULTSAfter six days of culture, the monocytes developed significant dendritic morphology and a portion of cells expressed CD1a, CD80 and CD86, features of DCs. TNFalpha treatment induced DCs maturation and up-regulation of CD80, CD86 and CD83. Autologous MLR demonstrated that these DCs possess potent T-cell stimulatory capacity.

CONCLUSIONThis study developed a simple and easy way to generate DCs from PBMCs exposed to rhGM-CSF and rhIL-4. The DCs produced by this method acquired morphologic and antigenic characteristics of DCs.

Cell Culture Techniques ; methods ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; physiology ; Granulocyte-Macrophage Colony-Stimulating Factor ; pharmacology ; Humans ; Interleukin-4 ; pharmacology ; Leukocytes, Mononuclear ; cytology ; drug effects ; physiology ; Phenotype ; Recombinant Proteins ; pharmacology ; Tumor Necrosis Factor-alpha ; pharmacology