OBJECTIVETo explore the molecule mechanism of Salidroside inducing directional differentiation of mouse mesenchymal stem cells (MSCs) into neuronal cells.

METHODSThe mouse multipotent mesenchymal precursor cell line (D1) was taken as the objective. Cultured MSCs were divided into the negative control group (complete culture solution), the positive control group (containing 1 mmol/L β-mercaptoethanol), the Salidroside induced group (20 mg/L Salidroside), and the blocked group (20 ng/ ml DKK1, a special inhibitor of Wnt/β-catenin signal pathway). All cells were inoculated in a 6-well plate (1 x 10(4) cells/cm2) and grouped for 24 h. The expression of p-catenin was detected by fluorescence Immunochemistry in the negative control group, the positive control group, and the Salidroside induced group. The expression of neuron-specific enolase (NSE), beta 3 class III tubulin (β-tubulin III), nuclear receptor related factor 1 (Nurr1), glial fibrillary acidic protein (GFAP) mRNA, Wnt3a, β-catenin, low-density lipoprotein receptor-related protein6 (LRP6), Axin mRNA were detected using reverse transcrip- tion PCR (RT-PCR). The expression of β-catenin and NSE protein were analyzed by Western blot in the negative control group, the positive control group, and the Salidroside induced group. Ca2+ chelating agents (EGTA), L-type Ca2+ channel blocker (Nifedpine), and IP3Ks special inhibitor (LY294002) were used to block Ca2+ signal pathway respectively. The expression of Wnt3a, LRP-6, Axin, glycogen syn- thase kinase (GSK-3), and β-catenin mRNA were detected by RT-PCR. The β-catenin protein expression was analyzed using Western blot.

RESULTSCompared with the positive control group, β-catenin protein was strong positively expressed; the expression of Wnt3a, β-catenin, LRP6, Axin, NSE, β-tubulin III, Nurr1 mRNA, and NSE protein were obviously up-regulated in the Salidroside induced group (P < 0.01). Compared with the positive control group and the Salidroside induced group, β-catenin, NSE, Nurr1, and β-tubulin III mRNA expression decreased; β-catenin and NSE protein expression were also down-regulated in the blocked group (P < 0.01). Compared with the Salidroside induced group, the expression of Wnt3a, LRP-6, β-catenin, and Axin mRNA were down-regulated in the Ca2+ signal blocked group and the salidroside induced group (P < 0.01, P < 0.05).

CONCLUSIONSalidroside affected directional differentia- tion of MSCs into neuronal cells through Wnt/β-catenin and Ca2+ signal pathway.

Animals ; Cell Differentiation ; drug effects ; Glucosides ; pharmacology ; Glycogen Synthase Kinase 3 ; Lipoproteins, LDL ; Low Density Lipoprotein Receptor-Related Protein-6 ; Mesenchymal Stromal Cells ; physiology ; Mice ; Neurons ; Phenols ; pharmacology ; Phosphopyruvate Hydratase ; RNA, Messenger ; Signal Transduction ; Wnt Signaling Pathway ; physiology ; beta Catenin ; metabolism

A double targets of high throughput screening model for xanthine oxidase inhibitors and superoxide anion scavengers was established. In the reaction system of xanthine oxidase, WST-1 works as the probe for the ultra oxygen anion generation, and product uric acid works as xanthine oxidase activity indicator. By using SpectraMax M5 continuous spectrum enzyme sign reflectoscope reflector, the changes of these indicators' concentration were observed and the influence factors of this reaction system to establish the high throughput screening model were studied. And the model is confirmed by positive drugs. In the reaction system, the final volume of reaction system is 50 μL and the concentrations of xanthine oxidase is 4 mU x mL(-1), xanthine 250 μmol x L(-1) and WST-1 100 μmol x L(-1), separately. The Z'-factor of model for xanthine oxidase inhibitors is 0.537 4, S/N is 47.519 9; the Z'-factor of model for superoxide anion scavengers is 0.507 4, S/N is 5.388 9. This model for xanthine oxidase inhibitors and superoxide anion scavengers has more common characteristics of the good stability, the fewer reagent types and quantity, the good repeatability, and so on. And it can be widely applied in high-throughput screening research.
Enzyme Inhibitors ; pharmacology ; Free Radical Scavengers ; pharmacology ; High-Throughput Screening Assays ; Superoxides ; Uric Acid ; Xanthine ; Xanthine Oxidase ; antagonists & inhibitors

To enhance the quality and efficiency of ozagrel by investigating the differences between the ozagrel polymorphs in bioavailability. Solid ozagrel in different polymorph forms were orally administered to SD rats. An HPLC method was established to determinate plasma level of ozagrel. The bioavailabilities of two polymorph forms were calculated and compared. The pharmacokinetic parameters of ozagrel, were as follows: Cmax was 32.72 ± 17.04 and 34.01 ± 19.13 mg · L(-1), respectively; AUC0-t was 61.14 ± 14.76 and 85.56 ± 18.08 mg · L(-1) · h, respectively; t½ was 1.53 ± 0.51 and 4.73 ± 3.00 h, respectively. There was no significant difference in pharmacokinetic parameters between form I and II polymorphs of ozagrel while the t½ of form II is longer, which indicates that the use of form II polymorph as pharmaceutical product may prolong the effective action time in clinics. This would help the polymorph quality control in drug production.
Animals ; Biological Availability ; Chromatography, High Pressure Liquid ; Methacrylates ; chemistry ; pharmacokinetics ; Rats ; Rats, Sprague-Dawley

The present study aimed to investigate the effect and the mechanisms of Bu-zhong-yi-qi-tang (BZYQ) on Spleen-Qi deficiency rat's model using nuclear magnetic resonance (NMR) metabolomics and multivariate statistical analysis methods. The rat Spleen-Qi deficiency model was established as follows: oral administration of Radix Rhei extract, loaded swimming and starvation for 24 h. The body weight and motor behavior of the rats were measured and recorded once a week. BZYQ could significantly improve body weight and behavioral of Spleen-Qi deficiency model rats compared with the model group (P < 0.05, 0.01). After drug administration, the changes in the levels of endogenous metabolites in the spleen including decreasing lactate, taurine and hypoxanthine, increasing glutamate and scyllo-inositol compared with the model group. The metabolomics approach is an effective tool for the investigation of the pharmacologic mechanism of BZYQ and it is helpful to further research.
Administration, Oral ; Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Medicine, Chinese Traditional ; Metabolomics ; Proton Magnetic Resonance Spectroscopy ; Qi ; Rats ; Spleen

Glioblastoma (GBM) is the most common, malignant, and lethal primary brain tumor in adults.Up to now,there is no effective drug for GBM.Withaferin A(WFA)is mainly derived from Indian Winter cherry.It has been traditionally used in ayurvedic medicine.WFA has wide range of pharmaco-logical activities including cardioprotective,anti-inflammatory,immuno-modulatory properties.Recently, WFA was reported to inhibit the growth of many cancer cells; however, the precise molecular mecha-nisms of its anti-cancer activities in GBM remain unclear. Here, we found that treatment of WFA in U251 and U87-MG glioma cells inhibited the cell proliferation,released the cellular LDH,decreased the DNA synthesis, and inhibited the migration, invasion, and colony formation of cells. WFA also in-creased the apoptotic rate of cells, decreased the mitochondrial membrane potential, arrested cell cy-cle at G2/M,inhibited the activity of caspase 3/7,and increased the protein expression of cleaved-cas-pase 3,cleaved PARP in U251 and U87-MG cells.In addition,cell apoptosis induced by WFA was as-sociated with increasing level of Bim,Bad,P21,P53 and decreasing the level of p-CDK1,cyclin A and B. It was also shown that cell apoptosis induced by WFA was associated with P38 signal pathway. These results demonstrated that WFA induced mitochondrial dependent apoptosis in glioblastoma cells which was associated with arresting the cell cycle at G2/M phase by P38 pathway.Taken together,our findings suggest that WFA might be a promising chemotherapy drug in the treatment of GBM.

Objective To study the risk factors of hyperthyroid heart diseases(HHD) by analyzing clinical features of patients in order to provide a scientific basis for prevention and treatment of HHD. Methods Nine hundred and eighty two cases were selected as objective from in-patient data of Thyroid Disease Treatment Centre of Shandong Province. The cases were divided into hyperthyroidism group and HHD group. The variables of etiology,sex, age, duration of disease, TSH, FT3, FT4 and TRAb were analyzed by comparative analysis. The risk factors were analyzed by logistic regression. Results The prevalence of hyperthyroidism complicated hyperthyroid heart disease was 7.7%(76/982), age, duration of diseases, FT3, TRAb in the HHD group were [(51.4 ± 11.5), (6.3 ±2.1) years, 21.6 pmol/L, 71.6 U/L], in hyperthyroidism group were [(37.9 ± 9.8), (2.6 ± 1.3) years, 14.9pmol/L, 49.6 U/L]. The differences were statistically significant(u = 9.93,15.23, T = 44954,48792.5, P ＜ 0.05)between the two groups. The factors of the older, higher FT3 and TRAb, longer duration, Graves disease (OR =1.751,1.470,1.483,1.445,1.234) increased the risk of HHD. Conclusions Graves disease, longer duration, old age, higher FT3 and TRAb are the risk factors of HHD. Timely prevention and control of risk factors is necessary to reduce the incidence of HHD.

OBJECTIVETo investigate the effect of Jiangtang Yishen Recipe (JTYSR) on high insulin induced cell proliferation of human glomerular mesangial cells (HMCs) and the expression of insulin receptor substrate 1 (IRS-1) and phosphatidylinositol-3-kinase (PI-3K).

METHODSHMCs were divided into 4 groups, i.e., the negative control group, the high insulin model group, the JTYSR group, and the LY294002 group. The concentration of insulin, JTYSR, and LY294002 was respectively confirmed by pre-experiment. Different culture solution was respectively added for different groups. RPMI1640 culture solution was added for HMCs in the negative control group, while HMCs in the rest 3 groups were cultured by 100 nmol/L insulin for 24 h. Meanwhile, HMCs from the JTYSR group and the LY294002 group were exposed to 125 mg/L JTYSR and 80 micromol/L LY294002 respectively for further 48 h. The proliferation of HMCs was detected by MTT and flow cytometry. The protein expression of IRS-1 and PI-3K in HMC was detected by immunohistochemical assay and Western blot. Results The proliferation of HMCs induced by high insulin could be significantly lowered, and the protein expression of IRS-1 and PI-3K could be down-regulated in the JTYSR group and the LY294002 group (P <0.01). Compared with the LY294002 group, the protein expression of IRS-1 and PI-3K could be slightly down-regulated in the JTYSR group (P <0.05).

CONCLUSIONJTYSR could lower high insulin induced proliferation of HMCs, and its mechanism might be related to insulin signaling pathway.

Cell Proliferation ; drug effects ; Chromones ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Insulin Receptor Substrate Proteins ; metabolism ; Mesangial Cells ; physiology ; Morpholines ; Phosphatidylinositol 3-Kinase ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Signal Transduction

This study was conducted to investigate the paclitaxel loaded by hydrazone bonds in poly(ethylene glycol)-poly(caprolactone) micelles (mPEG-PCL-PTX) on proliferation and apoptosis of human lung cancer A549 cells and its possible mechanisms of anti-tumor activity. The cell proliferation was measured with MTT assay. Flow cytometry were used to analyze the cell cycle. The cell apoptosis was analyzed using Hoechst/P staining. The expression levels of apoptotic genes expression in the mitochondrial apoptosis pathway were detected by RT-PCR and Western blotting, respectively. The mPEG-PCL-PTX could inhibit the proliferation of A549 cells and promote the apoptosis. The Bax, caspase-3 protein expression were increased while Bcl-2 protein expression was decreased in A549 cells. Results showed that the polymer containing hydrazone bond is non-toxic in vitro, the mPEG-PCL-PTX micelles can inhibit the proliferation and induce the apoptosis of A549 cells. Key words: paclitaxel; micelle; A549 cell; proliferation; cell cycle; apoptosis
Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Micelles ; Paclitaxel ; pharmacology ; Polyesters ; Polyethylene Glycols ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

The identification of five marine-derived shell traditional Chinese medicine (TCM) recorded in the Chinese Pharmacopoeia were studied. Using near infrared technology (NIR) combined with principal component analysis (PCA) methods, Ostreae Concha, Haliotidis Concha, and Margaritifera Concha could be efficiently distinguished from Meretricis Concha together with Arcae Concha. In the first principal components, Ostreae Concha exhibited obvious differences with high loadings in 4 236, 5 263, 7 142 cm(-1) concerning to the contents of CaCO3 and H2O in the samples. Arcae Concha and Meretricis Concha displayed significant differences with others in the second principal components, which can be illustrated by high loadings in 5 000 -4 430 cm(-1) areas. It is indicated that the second principal components might be related to organics which contained NH and CH groups, for example proteins. Meanwhile, our data showed a correlation between the function of these shell TCM and their distribution in the PCA plot. These results suggested that organic components in marine-derived shell TCM could not be neglected for their quality control.
Animal Shells ; chemistry ; Animals ; Calcium Carbonate ; analysis ; Medicine, Chinese Traditional ; methods ; Mollusca ; chemistry ; classification ; Principal Component Analysis ; Seawater ; Species Specificity ; Spectroscopy, Near-Infrared ; methods

OBJECTIVETo investigate SiO2-induced EMT in human bronchial epithelial cells HBE in vitro.

METHODSHBE cells were cultured and then stimulated with indicated doses of SiO2 (0, 50, 100, 200, 300 µg/ml). The morphological changes were observed by microscope. In addition, Western blot was per-formed to detect the expression of E-cad, α-SMA and Vim. The changes of migration ability were examined by wound-healing assay in vitro.

RESULTS(1) After exposure to SiO2, HBE cells lost contact with their neighbor and displayed a spindle-shape, fibroblast-like morphology. (2) Compared with the control, the E-cad (300 µg/ml group) expression downregulated 2.98 fold (P < 0.05), and the Vim (300 µg/ml group) and α-SMA (200 µg/ml group) expression upregulated 4.46 fold and 3.55 fold (P < 0.05). There were significant differences between 100, 200, 300 µg/ml groups and the control group (P < 0.05). (3) In the test group, the percentage of wound-healing areas/wound areas were larger than those in control group (P < 0.05).

CONCLUSIONSSiO2 could induce EMT in human bronchial epithelial cells.

Bronchi ; cytology ; Cells, Cultured ; Epithelial Cells ; cytology ; drug effects ; Epithelial-Mesenchymal Transition ; drug effects ; Humans ; Silicon Dioxide ; adverse effects ; Stromal Cells ; cytology ; drug effects