Aim To investigate the anti-neuroinflam-matory activities of dehydromiltirone and the underlying mechanisms in LPS-stimulated microglial cell line BV2 cells. Methods BV2 cells were pre-treated with de-hydromiltirone, then stimulated by LPS. The levels of nitric oxide( NO) were measured by Griess assay, and the concentrations of pro-inflammatory cytokines were measured by ELISA assay. Confocal fluorescence mi-croscopy was used to measure the expression of MAC-1, the biomarker of activated BV2 cells. The levels of-inducible nitric oxide synthase ( iNOS ) , cyclooxygen-ase-2 ( COX-2 ) , NF-κB and PI3 K/Akt were deter-mined by Western blot analysis. Results The treat-ment of dehydromiltirone significantly inhibited the pro-duction of NO, TNF-α and IL-6, attenuated the ex-pression of iNOS and COX-2 protein, and dampened the microglial activation in LPS-stimulated BV2 cells. The mechanistic study revealed that dehydromiltirone inhibited the phosphorylation of PI3 K and Akt in LPS-stimulated BV2 cells, and decreased NF-κB activation by suppressing the degradation of IκB. Conclusion dehydromiltirone shows significant anti-neuroinflamma-tory effects through inhibiting PI3 K/Akt phosphoryla-tion and then inhibiting NF-κB signaling pathway.

A study on the microbial transformation of glycyrrhetinic acid (GA) was conducted by a fungus, Cunninghamella blakesleeana CGMCC 3.970 systematically. After incubation with the cell cultures of C. blakesleeana CGMCC 3.970 at 25 degrees C for 7 days on a rotary shaker operating at 135 r x min(-1), GA was converted into one major product and five minor products. The products were extracted and purified by solvent extraction, macroporous adsorbent resin, silica gel column chromatography, and semi-preparative RP-HPLC chromatography. Their structures were identified as 3-oxo-15α-hydroxy-18β-glycyrrhetinic acid(1), 3-oxo-15β-hydroxy-18β-glycyrrhetinic acid (2), 7β,15α-dihydroxy-18β-glycyrrhetinic acid (3), 3-oxo-7β, 15α-dihydroxy-18β-glycyrrhetinic acid (4), 7β-hydroxy-18β-glycyrrhetinic acid(5) and 15α-hydroxy-18β-glycyrrhetinic acid(6) by the analyses of MS, 1H-NMR and 13C-NMR spectroscopic data respectively. Among them, 2 was a new compound. These results suggest that C. blakesleeana CGMCC 3.970 has the capability of selective ketonization and hydroxylation for GA. [Key words] glycyrrhetinic acid; Cunninghamella blakesleeana CGMCC 3. 970; microbial transformation
Biotransformation ; Cunninghamella ; metabolism ; Glycyrrhetinic Acid ; analogs & derivatives ; chemistry ; metabolism ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization

Objective To investigate insulin sensitivity and beta cell function in female systemic lupus erythematosus (SLE) patients with different glucose tolerances. Methods Insulin sensitivity and beta cell function were compared between SLE patients and non-SLE subjects in the states of normal glucose tolerance (NGT), impaired glucose tolerance (IGT)and diabetes mellitus (DM) respectively.Furthermore, risk factors for insulin sensitivity and beta cell function in SLE patients were analysed by linear regression. Results In NGT state, insulin sensitivity and beta cell function of newly diagnosed SLE patients without glucocorticoids treatment were not significantly different from those of normal control group ( P ＜0. 05). Compared with newly diagnosed SLE patients without glucocorticoids treatment and normal control group, HOMA insulin resistance index (HOMA-IR) , In (HOMA-β), In (early phase insulin secretion index, EISI ) and In ( late phase insulin secretion index, LISI ) of SLE patients with glucocorticoids treatment were significantly higher( 1.91 ± 1.04 vs 0. 81 ±0. 75,0. 94 ±0. 27;5.05 ±0. 65 vs 4. 01 ±0. 63,4. 23 ±0.47;3. 14±0.81 vs 2.42 ±0.39,2.50±0.65;2.30 ±0.55 vs 1.62 ±0.57,1.56 ±0.43;P ＜0.05),while In ( Matsuda index, MI ) was significantly lower ( 4. 53 ± 0. 54 vs 5. 27 ± 0. 68,5. 18 ± 0. 38; P ＜0. 05). In IGT and DM state, HOMA-IR (2. 84 ± 1. 87 vs 1.82 ± 1.22, 3. 18 ±2. 29 vs 2. 94 ±2. 26) and In (HOMA-β) (5. 18 ±0. 93 vs 4. 06 ±0. 58, 3. 99 ± 1.04 vs 3.43 ±0. 83) were significantly higher in SLE patients with glucocorticoids treatment than those of non-SLE subjects ( P ＜ 0. 05 ) respectively. BMI and In (daily glucocorticords doses) were independent risk factors for insulin sensitivity, and age, the SLE disease activity index(SLEDAI) and In(daily glucocorticords doses) were related factors beta cell function.Conclusion In NGT, IGT and DM state,SLE female patients with glucocorticoids treatment have reduced insulin sensitivity and increased beta cell function, these changes are related to the use of glucocorticoids.

This study was aimed to dynamically observe the expression level of lactate dehydrogenase (LDH) in MDS patients and to explore the significance of LDH level for prognostic judgement of MDS patients. The expression level of LDH in 163 confirmedly diagnosed patients from 2001 to 2009 years in our hospital, the changes of LDH level in follow-up patients and relation of the LDH changes to prognosis, survival time and MDS progression, as well as the relation of LDH level to blood cell count, ratio and karyotype of blast cells in bone marrow were analyzed retrospectively. The results showed that the median LDH level in 163 MDS patients at diagnosis was 214 U/L (range 102 - 865 U/L), the median survival time of patients with increased LDH (> 240 U/L) was 25.6 months which was significantly shorter than that of patients with normal LDH level (56.8 months)(p < 0.05). When MDS patients were classified according to IPSS, the increased LDH level in MDS patients was observed in high risk and intermediate II groups (337.20 ± 298.00 U/L and 234.07 ± 216.00 U/L, respectively) which was significantly higher than that in low risk group (154.94 ± 46.08 U/L) (p < 0.05). The LDH level in patients with MDS progression was obviously enhanced while LDH level in patients without progression was not enhanced, mainly maintained in stable level as compared with LDH level at diagnosis and before progression (p < 0.005). By multivariate analysis, the increase of LDH level was found to be an independent prognostic factor. It is concluded that the LDH level may be used as indicator for judging prognosis of MDS patients, which is helpful to early recognition of MDS progression and risk stratification of disease, as well as selection of rational therapy.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; L-Lactate Dehydrogenase ; blood ; Male ; Middle Aged ; Myelodysplastic Syndromes ; blood ; diagnosis ; Prognosis ; Retrospective Studies ; Young Adult

OBJECTIVETo investigate the effects and the mechanisms of cell growth inhibition in hepatocellular carcinoma cells after induction with antisense survivin-liposome (LIP) complex, and to provide evidence in treatment for hepatocellular carcinoma and tumors expressing survivin.

METHODSSurvivin ODNs was transfected into HepG2 cells mediated by LiP reagent. The expression of survivin mRNA and protein was detected by RT-PCR and Western blot. MTT assay was applied to determine cell proliferation in HepG2 cells. Active caspase-3 and apoptosis rate were evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Cell cycle was analyzed by flow cytometry in the cell cycle-synchronized hepatocellular carcinoma cells treated with the antisense compound.

RESULTSAntisense compound efficiently down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC(50) of 250 nmol/L. Its maximal effect was achieved at a concentration of 600 nmol/L, when expression levels were down-regulated by 80%, as revealed by gradually increase of caspase-3-like protease activity and apoptosis rate in a time-dependent manner. Morphological apoptotic changes such as membrane blebbing, loss of microvilli, cytoplasmic vasculization, condensation of cytoplasm and nucleus, chromatin fragmentation, and apoptosis and cell growth inhibition were observed. In the cell cycle-synchronized hepatocellular carcinoma cells, antisense compound induced cell cycle arrest followed by apoptosis. After treated with low concentration of compound, the cell cycle was arrested at S phase or G2/M phase; while at high concentration, the cell cycle was mainly arrested at S phase. Apoptosis was obviously observed and the rate of apoptosis was increased in a time and concentration-dependent manner.

CONCLUSIONAntisense survivin has significant inhibitory effect on growth of hepatocellular carcinoma cells in vitro. This is associated with cell cycle arrest and apoptosis.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Caspase 3 ; Caspases ; metabolism ; Cell Cycle ; drug effects ; Cell Proliferation ; drug effects ; Humans ; Inhibitor of Apoptosis Proteins ; Liposomes ; Liver Neoplasms ; pathology ; Microtubule-Associated Proteins ; metabolism ; pharmacology ; Neoplasm Proteins ; metabolism ; pharmacology ; Oligonucleotides, Antisense ; pharmacology

OBJECTIVETo assess the characteristics of enhanced magnetic resonance image with ultrasmall superparamagnetic iron oxide (USPIO) in the inflammatory and tumor metastatic rabbit model, and explore its relevance with histologic ultrastructural findings.

METHODSTotally 36 New Zealand white rabbits were randomly divided into lymphadenitis group and metastatic group. Complete Freund's adjuvant was injected into the bilateral dorsal footpads of 18 rabbits to set up ipsilateral lymphadenitis model. The other 18 rabbits received a subcutaneous implantation of VX2 tumor cell suspension (1.5 x 10(7) cells/ml) in both thighs to set up metastatic lymph node model. Magnetic resonance scan were performed 24 hours before and after USPIO (90 micromol Fe/kg) injection. T2 values of each lymph node were measured and lymph node T2 enhancement rate was calculated as well. HE staining, Prussian blue staining, and electronic microscopy were performed to observe the pathological microstructure changes and the distribution of the iron particle in lymph node. Relationship between lymph nodes USPIO enhancement and its microstructures were further analyzed. Results Thirty-six lymph nodes in lymphadenitis group showed different degrees of reactive hyperplasia. Twenty-six lymph nodes in metastatic group were invaded by tumor cell. Non-enhanced scan showed mild difference between T2 signal intensity of the two pathological lymph node types. After USPIO enhancement, inflammatory lymph nodes showed distinct T2 signal reduction at the center, and metastatic lymph nodes showed homogenous and faint T2 signal reduction. Enhancement rate of benign and malignant lymph nodes were 57.39% and 29.45% respectively (P < 0.01). HE staining and Prussian blue staining indicated USPIO particles located mainly in the macrophages at inflammatory lymphatic medulla, while paracortical area and cortical area contained relatively much less USPIO particles due to less macrophages distribution. MRI findings were correlated with the pathological results. Electronic microscopy also verified that the majority of USPIO particles were located in the numerous cytophagic bubbles of macrophages. Lymph nodes metastasis including 4 lymph nodes with completed structure destruction due to entire tumor infiltration, 19 lymph nodes with partially lymph node structure destruction but reduced USPIO-contained macrophage numbers or reduced USPIO particles in macrophages, and 3 lymph nodes with only localized foci tumor metastasis at subcapsular area. Conclusions USPIO enhancement pattern of different lymph nodes is closely related to distribution and functional status of the intra-node macrophages. It may affect the accuracy of the lymph node property diagnosis based on USPIO enhanced image.

Animals ; Dextrans ; metabolism ; Female ; Image Enhancement ; methods ; Lymph Nodes ; ultrastructure ; Lymphadenitis ; diagnosis ; pathology ; Lymphatic Metastasis ; diagnosis ; ultrastructure ; Magnetic Resonance Imaging ; methods ; Magnetics ; Magnetite Nanoparticles ; Male ; Nanoparticles ; Rabbits ; Random Allocation

OBJECTIVETo investigate the anti-hyperglycemic effect and its mechanism of ethanol extraction from Calamintha chinensis (EJCT).

METHODFasting serum glucose (FSG) in normal mice was determined after oral administration of EJCT. Effects of EJCT on hyperglycemia mice induced by adrenaline were investigated by observing the contents of FSG and liver glucogen. Effect of EJCT on the diabetic mice induced by alloxan was investigated by observing the contents of FSG and the injured degree of pancreatic islet. The antilipid-peroxidation of EJCT on liver homogenate was measured by determination of malondiadehyde (MDA) induced by Fe2+/Cys.

RESULTEJCT showed no obvious effect on FSG in normal mice. However, EJCT 300, 600 mg x kg(-1) could remarkably decrease the contents of FSG and increase liver glucogen in hyperglycemia mice induced by adrenaline. In diabetic mice induced by alloxan, EJCT 150, 300, 600 mg x kg(-1) could remarkably decrease the contents of FSG. The damage of pancreatic islet induced by alloxan was also significantly attenuated by EJCT. Furthermore, EJCT 30, 60, 90, 120 mg x L(-1) inhibited lipid peroxidation initiated by Fe2+/Cys in liver homogenate.

CONCLUSIONThese results suggest that EJCT can significantly attenuate hyperglycemia in diabetic mice, which is probably due to decreasing the decomposition of liver glucogen, increasing the synthesis of liver glucogen, antioxidation and amelioration of damaged pancreatic islet.

Animals ; Blood Glucose ; metabolism ; Diabetes Mellitus, Experimental ; blood ; drug therapy ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fasting ; Hypoglycemic Agents ; pharmacology ; therapeutic use ; Islets of Langerhans ; drug effects ; Lamiaceae ; chemistry ; Lipid Peroxidation ; drug effects ; Liver ; drug effects ; metabolism ; Male ; Mice ; Mice, Inbred ICR

OBJECTIVEThis study was to investigate the growth and proliferation characteristics of rat bone mesenchymal stem cells (BMSCs) isolated by the method of whole bone marrow culture and to explore the effect of cell inoculation density and incubation period on cell proliferation, with an aim to provide multipotential seed cells for preventing from degenerative disease.

METHODSBone mesenchymal stem cells were isolated by the method of whole bone marrow culture and then cultured in vitro. The cell morphologic features were observed by inverted microscope. The cell surface antigens were identified by flow cytometry. The effect of cell inoculation density and culture period on cell growth and proliferation was explored by analyzing the characteristics of a ten-day cell growth curve in 96-well plates.

RESULTSFlow cytometry results showed the detection rates for CD29, CD34 and CD45 were 97.68% (7607/7788), 7.93% (661/8340) and 2.76% (215/7788) respectively, which was consistent with the expression characteristics of BMSCs surface antigens. BMSCs became uniform after three cell passages, existing in a typical shape of whirlpool or radial colony. The senescent cells started to appear at 7(th) passage, and more senescent cells were found at 10(th) passage. The growth curve for moderate inoculation density was typically S-shaped. Lag phase was found during the first two days, and logarithm growth phase was in the following three days. Plateau phase started from the 6(th) day and cell numbers decreased slightly from the 8(th) day.

CONCLUSIONThe whole bone marrow culture is an effective way to obtain BMSCs. A moderate inoculation density was more advantageous to cell proliferation, by which more seed cells could be obtained.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Flow Cytometry ; Male ; Mesenchymal Stromal Cells ; cytology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the lipid peroxidation induced by deltamethrin (DM) in the cerebral cortex and hippocampus of rat.

METHODSWistar male rats were administrated with DM (daily dose was 3.125, 12.500 mg/kg respectively). The content of malondialdehyde (MDA) and the activity of total-superoxide dismutase (T-SOD, including Mn-SOD and CuZn-SOD), catalase (CAT), glutathione-S-transferase (GST), glutathione peroxidase (GSH-Px) and glutathione reductase (GR) in cerebral cortex and hippocampus tissue were determined. The reduced glutathione (GSH) content and gamma-glutamylcysteine synthetase (gamma-GCS) activity in cytosolic fraction of cerebral cortex and hippocampus tissue was determined by reversed-phase high performance liquid chromatographic assay with o-phthalaldehyde pre-column derivation.

RESULTS(1) MDA content in cerebral cortex of the high dose group was significantly higher than those in the low dose group, and MDA content in hippocampus tissue of the high dose group was significantly higher than those in both the control and the low dose group after 5 d of DM exposure. (2) The activity of T-SOD and CuZn-SOD in cerebral cortex of both high and low dose group were significantly lower than that in the control group, and there was no effect on CAT activity in cerebral cortex (P < 0.01 or P < 0.05). (3) GSH content in cerebral cortex of the high dose group was significantly higher than that in control group (P < 0.05), and that in hippocampus tissue of high dose was significantly lower than that in both control and low dose group (P < 0.05). GR activity of low dose group in cerebral cortex was significantly lower than that in both control and high group [(11.80 +/- 5.15) vs (18.98 +/- 3.68), (17.35 +/- 2.47) U/mg pro] (P < 0.01). Gamma-GCS activity in hippocampus tissue of the high dose group was significantly lower than that in both control and low dose group [(1.75 +/- 0.60) vs (3.17 +/- 0.79), (2.72 +/- 0.75) nmol x mg pro(-1) x min(-1)] (P < 0.01). GR activity in hippocampus tissue of both high and low dose group was significantly lower than that in the control group [(21.63 +/- 4.92), (21.46 +/- 8.89) vs (31.22 +/- 6.97) U/mg pro] (P < 0.05).

CONCLUSIONThe oxidative stress in nerve tissue, which could be resulted from effect of DM on the activity of SOD, gamma-GCS and GR and GSH content, is one of the mechanisms of neuro-toxicity induced by DM; The decreased activity of gamma-GCS and GR may be the primary cause of DM-induced decrease in that GSH content in hippocampus tissue.

Animals ; Cerebral Cortex ; drug effects ; metabolism ; Dose-Response Relationship, Drug ; Hippocampus ; drug effects ; metabolism ; Insecticides ; toxicity ; Lipid Peroxidation ; drug effects ; Male ; Malondialdehyde ; metabolism ; Nitriles ; toxicity ; Oxidative Stress ; drug effects ; Oxidoreductases ; metabolism ; Pyrethrins ; toxicity ; Rats ; Rats, Wistar

The column chromatography on silica gel, Sephadex LH-20 and semi-preparative HPLC were used to separate and purify the compounds from the EtOAc extract of medium and MeOH extract of cell cultures of Morus alba. Eight compounds were isolated. Based on physico-chemical properties and spectroscopic data, their structures were identified as isobavachalcone (1), genistein (2), norartocarpetin (3), albanin A (4), guangsangon E (5), mulberrofuran F (6), chalcomoracin (7), kuwanon J (8). Compounds 3-6 were isolated from the cell cultures of M. alba for the first time.
Benzofurans ; isolation & purification ; Cell Culture Techniques ; methods ; Chalcones ; isolation & purification ; Chromatography, Gel ; methods ; Chromatography, High Pressure Liquid ; Dextrans ; Genistein ; isolation & purification ; Morus ; chemistry ; cytology ; Plant Leaves ; chemistry ; cytology ; Plants, Medicinal ; chemistry ; cytology ; Silica Gel ; Terpenes ; isolation & purification