OBJECTIVETo observe the effect of total flavones of Epimedium leptorrhizum (YYH-C) on osteoporosis in ovariectomized rats.

METHODOvariectomized female rats were randomly divided into the model group, YYH-C lower, middle and high dose (0.7, 1.4, 2.8 g x kg(-1)) groups, the positive drug Bujiale (0.15 mg x kg(-1)) group, and the sham group. The rats were orally ad-ministrated with drugs for three months. Parathyroid hormone (PTH), procollagen I N-terminal peptide (PINP), alkaline phosphatase (ALP), calcium (Ca) and phosphrous (P) in serum were detected. Femur bones and vertebrae bones of left side were collected to determined bone metrological indexes, including bone mineral density (BMD), bone Ca, and bone ash weight/dry weight percentage. Femur bones of right side were collected to for a morphological observation of bone.

RESULTCompared with the sham group, the model group showed significantly higher PTH and ALP content but obviously lower PINP and Ca content. The three YYH-C 3 groups could resist the decrease of PINP. Specifically, low and middle dose groups could remarkably inhibit the increase of PTH, and the high dose group could increase the Ca content in serum, but without significant effect on the rise in ALP. There was no significant difference in P content in serum in each group. BMD, ash weight/dry weight percentage, Ca and P content of the model group were significantly lower than those in the sham group. The high dose YYH-C group could significantly increase BMD. All of the three YYH-C groups could notably increase ash weight/dry weight percentage and Ca, P content in femur bones and vertebrae bones. YYH-C could significantly increase average thickness, area, area percentage of bony trabeculae, cortical bone area percentage of femoral shaft and the number of osteoblasts on the surface of bony trabeculae, and decrease the number of osteoclasts.

CONCLUSIONYYH-C can effectively control the bone mass loss of rats with ovariectomy-induced osteoporosis, prevent the changes in bone microstructure, and inhibit bone absorption, so as to resist high turn-over osteoporosis after ovariectomy. [Key words] total flavones of Epimedium leptorrhizum; ovariectomized rat; osteoporosis

Alkaline Phosphatase ; metabolism ; Animals ; Bone Density ; drug effects ; Calcium ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; Epimedium ; chemistry ; Female ; Flavones ; administration & dosage ; Humans ; Osteoporosis, Postmenopausal ; drug therapy ; metabolism ; physiopathology ; Ovariectomy ; Parathyroid Hormone ; metabolism ; Rats ; Rats, Sprague-Dawley

The present study was aimed to investigate the role and relationship between N-methyl-D-aspartic acid (NMDA) receptor and neuropeptide Y (NPY) in depression induced by chronic unpredictable mild stress (CUMS). CUMS-induced depression model was established in Sprague-Dawley rats. Intrahippocampal injections of NMDA, non-competitive NMDA receptor antagonist MK-801 and NPY-Y1 receptor antagonist GR231118 were respectively adopted by rat brain stereotaxic coordinates. The behavioral observations were conducted by sucrose consumption test, open field test and forced swimming test. The expression of NPY in hippocampus was detected by immunohistochemistry. The results showed that compared with the control group, rats receiving CUMS for 21 days or intrahippocampal injection of GR231118 or NMDA showed depression-like behavioral changes, including a reduction in sucrose preference, body weight, locomotor activity, rearing and grooming in open field test, and increased duration of immobility in forced swimming test. Intrahippocampal injection of NMDA decreased the expression of NPY in hippocampal CA3 region and dentate gyrus (DG) region. Intrahippocampal injection of MK-801 improved the depression-like behavioral changes induced by CUMS, and increased the expression of NPY in hippocampal CA3 region and DG region. Co-injection of GR231118 and MK-801showed that GR231118 suppressed the antidepressant effect of MK-801. These data suggest that CUMS might induce depression through excessive release of glutamate (Glu), over-activation of NMDA receptors, and downregulation of NPY. Antidepressant effect of NPY was mainly mediated via NPY-Y1 receptor.
Animals ; Depression ; etiology ; physiopathology ; Dizocilpine Maleate ; pharmacology ; Hippocampus ; metabolism ; physiology ; Male ; Neuropeptide Y ; antagonists & inhibitors ; physiology ; Peptides, Cyclic ; pharmacology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; antagonists & inhibitors ; physiology ; Stress, Physiological

This study was aimed to apply the electronic nose (E-nose) in the research of traditional Chinese medicine (TCM). The discussion was made on difficulties of using E-nose. The solution plan was proposed and the discrimination model was established. It provided a simple, rapid and effective analysi method in the identification of TCM. It also provided new ideas for the research and application of gas sensor arrays. E-nose was used in the ex-traction of TCM scent characteristics. Based on ion mobility spectrometry of MOS sensor, the fingerprint of TCM scent was established. The maximum response value of the sensor was used as analysis index. According to the diffi-culties of identification, two solution plans were proposed. Firstly, different detectors were employed to complete the classification. Secondly, radial basis function (RBF) and random forests (RF) were combined and then a cascade classifier was constructed in order to achieve the maximum of information obtained in conditions where the number of measurements, metal oxide semiconductor sensors in E-nose was limited. The results showed that both plans were accurate and practical with relatively high upper correct judge rate and better cross-validation (The highest upper correct judge rates were 95% and 100%, 96% and 80%, respectively). It was concluded that this study firstly ap-plied cascade classifier in the establishment of TCM identification by E-nose. With limited amount of sensors, the maximum information was received through data mining. Using E-nose in the identification of TCM was rapid and accurate. The established pattern recognition method was maneuverable with accurate identification rate and stability compared to conventional sensory identification method. It provided a simple and rapid analysis method for the iden-tification of TCM.

Objective To study the expression of co-stimulatory molecules, GL50-ICOS, in thyroid tissue of patients with Graves' disease (GD) and to explore their relationship with the immune pathogenesis of GD.Methods RT-PCR, Western blot, immunohistochemistry were applied to detect the expression of GL50-ICOS in thyroid of GD. Thyrocytes were cultured in the absence or presence of pro-inflammatory cytokines. The expression of GL50 on thyroid follicular cells (TFC) was further measured by flow cytometry. Results (1) In GD patients,the percentage of CD4+ CD28- T cells was significantly increased as compared with the control healthy individuals. The expression of co-stimulatory molecule ICOS was up-regulated. (2) The mRNA level of ICOS was significantly increased in GD patients than that in nontoxic goiter(NTG) patients(P＜0.01). (3)Compared with NTG control group, the GL50 protein expression was much higher in thyroid tissues of GD patients (P ＜0.01). (4)The results of immunohistochemistry showed that GL50 expression was observed in all GD thyroid tissues, while no expression of GL50 was detected in NTG thyroid tissues(P＜0. 01). (5) The expression of GL50on primary cultured thyroid follicular cells was significantly increased under the stimulatation of pro-inflammatory cytokines in vitro. Conclusion GL50-ICOS is expressed abnormally in thyroid tissue of patients with GD.

OBJECTIVETo investigate the association between the expression of turnor necrosis factor alpha (TNF-alpha) mRNA in fat tissue of intrauterine growth retarded (IUGR) rats and insulin resistance, and the long-term effects of early different nutritional diet.

METHODSThe IUGR rat model was established by food restriction of pregnant rats. A total of 32 newborn IUGR rats were randomly divided into 4 groups: IUGR model (S/N) group, IUGR high caloric diet (A) group, IUGR high caloric and high protein diet (B) group, IUGR high protein diet (C) group. Only the mother rats were given those different diets individually, and all IUGR newborn pups were lactated for 3 weeks. From the beginning of the 4(th) week, all IUGR pups were weaned and fed with normal diet till the end of the experiment. Eight normal birth weight newborn rats were used as the control group fed with the normal diet. Weight, perirenal fat weight, fasting glucose and insulin concentration and quantified TNF-alpha mRNA expression in adipose cell were measured at the 48(th) week. The insulin sensitive index (ISI) and the relation index between TNF-alpha mRNA and fat weight, fat weight/body weight (fw/bw) ratio and ISI were calculated.

RESULTSISI of IUGR model group, IUGR A and B groups was lower than normal control group, while perirenal fat weight, fw/bw and the expression of TNF-alpha mRNA in adipose cells were all significantly higher (P < 0.05 or 0.01). There were no significant differences in these indexes between IUGR C group and normal control groups (P > 0.05). A positive correlation was found between TNF-alpha mRNA and fat weight and fw/bw (r(1) = 0.755, r(2) = 0.782, P = 0.000). Significant inverse associations between ISI and TNF-alpha mRNA (r = -0.556, P = 0.000) and fw/bw (r = -0.513, P = 0.02) were also found.

CONCLUSIONThe occurrence of insulin resistance in IUGR rats is possibly associated with central obesity and accumulation of the abdominal fat and adipose cell over-expression of TNF-alpha. The adipose TNF-alpha may be an important pathogenic factor of insulin resistance of IUGR. High protein diet is a reasonable nutritional intervention. Because it promotes the skeleton muscle catch-up growth but not fat catch-up growth, it can avoid the occurrence of central obesity and insulin resistance in IUGR rats.

Adipose Tissue ; metabolism ; Animals ; Diet ; Female ; Fetal Growth Retardation ; Insulin Resistance ; Nutritional Status ; Pregnancy ; Random Allocation ; Rats ; Tumor Necrosis Factor-alpha ; metabolism

Cadherins ; metabolism ; Female ; Humans ; MCF-7 Cells ; Neoplastic Stem Cells ; metabolism ; Receptor, Notch1 ; metabolism ; Receptors, CXCR4 ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Vascular Endothelial Growth Factor C ; metabolism

Drug allergy and pseudoallergic reactions are main adverse drug reactions. Allergy is mainly induced by the immunogenicity of drug, drug metabolic products or drug additive. Pseudoallergic reactions may result from the irritation or activation of inflammatory material release. Pre-clinical evaluation of drug allergy and pseudoallergic reactions is included in immunotoxicity evaluation. Now there is no in vivo or in vitro method that could predict all kinds of allergy or pseudoallergic reactions due to the different mechanisms. In the past few years, FDA, SFDA OECD, ICH and WHO have published several guidelines on per-clinical immunotoxicity evaluation, however, no agreement has been reached on allergy and pseudoallergic reactions evaluation. This article reviews the requirements of allergy and pseudoallergic reactions in pre-clinical evaluation.
Drug Hypersensitivity ; diagnosis ; Humans ; Immune System ; drug effects ; Practice Guidelines as Topic

Serum used widely in mammalian cell culture is also a potential source of bacterial, mycoplasmal and viral contaminations. In addition, the complex biological components in serum make harder the subsequent product recovery process. High cost, high batch variation and potential source limitation are among the other shortcomings. So serum-free or even protein-free medium are preferable for recombinant protein production. However, without serum to provide essential components such as hormones, growth factors and binding proteins, cells are easy to die. In this study, CHO-dhfr- cells were genetically engineered to make them adapted to IMEM, a protein-free medium, and resistant to apoptosis. The genes in choice are insulin-like factor (Igf-1), Bcl-2 and cyclin E. Bcl-2 is a mitochondrial membrane-integrated protein. It can block the release of cytochrome c by maintaining the integrity of mitochondrial membrane, and thus inhibit apoptosis. Igf-1 is similar both in structure and function to insulin, a growth factor added to serum-free medium to promote cell growth and is the only protein component in many currently used serum-free media. cyclin E is a cell cycle protein expressed continuously in G1 phase. When cyclin E accumulates to certain amount, cell cycle was driven to S phase. So cyclin E is a proliferation-promoting protein. By co-express Igf-1/Bcl-2 or Bcl-2/ cyclin E in CHO-dhfr- cells with a dicistronic expression vector, we constructed two cell lines: CHO-IB and CHO-BC. The high expression of each protein was confirmed by Western blot and flow cytometry. Apoptosis was analyzed by flow cytometry and DNA ladder detection, and the two cell lines were both found much more resistant to apoptosis induced by withdrawal of serum or addition of actinomycin D than the CHO-dhfr- parent cell. Cell proliferation assay by MTT method showed that the two cell lines proliferated much faster than CHO-dhfr- in IMDM medium without serum. Continuously culture assay proved that the two cell lines grow very well in IMEM protein-free medium supplemented with fibronectin and vitronectin to ease adherence. When compared to CHO-dhfr-, the two cell lines exhibited much more viable cell numbers and faster growth rate.
Animals ; Apoptosis ; CHO Cells ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Culture Media ; Cyclin E ; genetics ; Genes, bcl-2 ; Insulin-Like Growth Factor I ; genetics

Mammalian cells are prone to apoptosis when cultured in large scale for production of biopharmaceuticals. And this will reduce production duration and result in high cost of production. Apoptosis is triggered by various factors, and delicately regulated by a set of genes. Bcl-2, a component integrated in mitochondria membrane, is an important member of these genes. By maintaining the integrity of mitochondria membrane, Bcl-2 keeps cytochrome C from releasing into cytoplasm, and thus blocks the activation of caspases, and subsequent onset of apoptosis. Over-expression of Bcl-2 has proven to be useful in blocking apoptosis in various cell lines, including CHO, hybridoma, myeloma, lymphoma and insect cells. Ammonia, a metabolite of cultured cells, however, showed apparent pro-apoptosis activity. In living cells, ammonia can be utilized by glutamine synthetase (GS) to synthesize glutamine, and thus lower the concentration of ammonia in medium, and its negative effects. Glutamine is essential to living cells. If not added into medium, glutamine can only be synthesized by GS, which makes GS a qualified selection marker. This marker can be used for gene amplification by adding into medium increased concentration of MSX, an inhibitor of GS. In this study, we over-expressed Bcl-2 using GS amplification in a recombinant CHO cell line stably expressing human interferon-beta. The modified cell line, with higher expression of Bcl-2 and lower production of ammonia, exhibited good anti-apoptosis quality and higher interferon-beta production in continuous culture.
Animals ; Apoptosis ; genetics ; physiology ; Biopharmaceutics ; CHO Cells ; cytology ; metabolism ; Cricetinae ; Cricetulus ; Glutamate-Ammonia Ligase ; genetics ; metabolism ; Interferon-beta ; metabolism ; Models, Genetic ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism

OBJECTIVETo compare the differences of expressions of adenylyl cyclase (AC) and phosphodiesterase (PDE) in ejaculated spermatozoa between healthy volunteers and the patients with asthenospermia.

METHODSEjaculated spermatozoa were collected from healthy volunteers and the patients with asthenospermia. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect mRNA expression of AC and PDE subtypes in human spermatozoa. The concentrations of cAMP and cGMP in the samples were detected by enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with healthy volunteers, expression of sAC mRNA and concentration of cAMP were significantly decreased in the patients with asthenospermia (P < 0.01) , while the expression of PDE4C mRNA was significantly increased at the same time (P <0.01). There were no marked differences in the expression of ACIII mRNA and concentration of cGMP between the two groups.

CONCLUSIONThe sAC down-regulation and PDE4C up-regulation are possible reasons for asthenospermia.

Adenylyl Cyclases ; biosynthesis ; Asthenozoospermia ; metabolism ; Cyclic AMP ; metabolism ; Humans ; Male ; Phosphoric Diester Hydrolases ; biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Spermatozoa ; metabolism