OBJECTIVETo explore the liver-toxic fraction in Rhigoma of Dioscorea bulbifera.

METHODThe rats were randomized into four groups: control group (20% PVP-water), T001(10% total methanol extraction), F002(5% chloroform fraction) and F003(5% methanol fraction). Direct bilirubin (DBil) and Glutamic-pyruvic transaminase (GPT) were examined, and liver index was measured. The histological and morphological observations were performed with optical and electrical microscope.

RESULTT001 and F002 showed significant liver toxicity.

CONCLUSIONThe chloroform fraction was the liver-toxic fraction of D. bulbifera.

Alanine Transaminase ; blood ; Animals ; Bilirubin ; blood ; Chemical and Drug Induced Liver Injury ; blood ; etiology ; pathology ; Dioscorea ; chemistry ; Drugs, Chinese Herbal ; toxicity ; Female ; Liver ; pathology ; ultrastructure ; Male ; Plants, Medicinal ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley

Objective To explore the dosage and injection method of concanavalin A(Con A) for inducing Wistar rats into the acute hepatic injury model. Methods (1)According to the dosage of Con A, 42 Wistar rats were randomly divided into groups A, B, C, D, E, N, 7 rats in each group. Group N was given tail intravenous injection of normal saline as normal control group. Groups A, B, C, D, E were given intravenous injection of 4, 8, 16, 30, 40 mg/kg of Con A respectively. At the 8th hour after modeling, the levels of alanine transaminase(ALT), aspartate aminotransferase(AST), albumin(ALB), interleukin(IL)-2 , IL-10, interferon (IFN)-γ, and tumor necrosis factor(TNF)-αwere detected. And HE staining was used to observe the pathological feature of hepatic tissue. (2)According to the injection method of Con A, 21 Wistar rats were randomly divided into normal control group, intraperitoneal injection group and tail intravenous injection group, 7 rats in each group. The dosage of Con A for the rats in intraperitoneal injection group and tail intravenous injection group was 16 mg/kg. At the 8th hour after modeling, the levels of serum ALT, AST, and ALB were determined. Results The number of abnormal deaths in various dose Con A groups at the end of each experiment was 0 in groups A, B, C, and 2 in group D, and 7 in group E. A small amount of spotty necrosis, inflammatory cell infiltration, and hepatic lobule with almost integrity of structure were found in groups A, B, while obvious bridging-like necrosis was seen in groups C, D. Serum ALT, AST, and ALB levels in intraperitoneal injection group had no statistically significant difference as compared with the normal control group. Conclusion Tail intravenous injection of 16 mg/kg of Con A can be used to induce an acute immunological liver injury rat model successfully.

OBJECTIVETo observe the gene silencing and disruption of WNT pathway mediated by the specific shRNA targeted against beta-catenin and its effect on cell proliferation of the human colon cancer cell line Colo205.

METHODSThe shRNA plasmid vectors against beta-catenin were constructed and transfected into Colo205 cells with Lipofectamine 2000. The expression of beta-catenin was detected by RT-PCR and Western blot. Immunofluorescence staining was also performed to detect the beta-catenin protein expression in cells. The cell proliferation inhibition was determined by MTT assay and soft agar colony formation assay.

RESULTSThe shRNA vectors targeted against beta-catenin were successfully constructed and efficiently suppressed the expression of beta-catenin mRNA and protein(P<0.05). The expression inhibition rates were 47.89% and 45.26% at the mRNA and protein level respectively. Immunofluorescence microscopy also demonstrated the inhibition of beta-catenin protein induced by these specific shRNAs. The MTT assay indicated that the specific shRNA resulted in significant inhibition of cell growth on the culture plates in time-dependent manner. At 72 h post-transfection, the cell viability of CAT group was 48.5%, which was significantly different as compared with that of blank control group's 91.3%(P<0.05). In the soft agar, there were 9, 46, 43 cell colonies in the CAT, blank, and negative control groups respectively, which were significantly different(P<0.05).

CONCLUSIONSThe specific shRNAs targeted against beta-catenin has a gene silencing effect and blocks the WNT signaling pathway, which can inhibit the growth of Colo205 cells.

Cell Line, Tumor ; Cell Proliferation ; Colonic Neoplasms ; genetics ; metabolism ; Humans ; In Vitro Techniques ; RNA Interference ; Signal Transduction ; Transfection ; Wnt Proteins ; metabolism ; beta Catenin ; genetics

OBJECTIVE: To investigate the effect of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, on the activation of hepatic stellate cells (HSCs) and its mechanism. METHODS: Primary HSCs isolated from SD rats were cultured and treated with different concentrations (1, 3 or 10micromol/L) of ADMA for various periods (12 approximately 48h). Expression of alpha-smooth muscle actin (alpha-SMA) and synthesis of type-I collagens in HSC were determined. Messenger RNA levels of the transforming growth factor-beta1 (TGF-beta(1)) in the HSCs were determined using RT-PCR. Intracellular reactive oxidant species (ROS) production was measured using oxidant-sensitive fluorescent indicator. Activation of nuclear factor-kappaB (NF-kappaB) was detected by electrophoretic mobility shift assay (EMSA). RESULTS: ADMA could increase alpha-SMA-positive cells ratio and Type I collagens production of HSCs in a concentration- and time-dependent manner, concomitant with the increase of the TGF-beta(1) mRNA level. Treatment with ADMA (10micromol/L) significantly increased the intracellular ROS production and activated NF-kappaB. Such effects of ADMA on the level of TGF-beta(1) mRNA could be markedly attenuated by pretreatment with antioxidant pyrrolidine dithiocarbamate (25micromol/L). CONCLUSION ADMA can induce the HSC activation by increasing TGF-beta(1) expression through ROS-NF-kappaB-dependent pathway. Therefore, ADMA should be a novel and endogenous activator of HSC, which may be involved in the development of liver fibrosis.
Actins ; biosynthesis ; Animals ; Arginine ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Collagen Type I ; metabolism ; Dose-Response Relationship, Drug ; Gene Expression ; drug effects ; Hepatocytes ; cytology ; drug effects ; metabolism ; Male ; NF-kappa B ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Transforming Growth Factor beta ; genetics

Objective To explore the relationship between gene polymorphism of neuropeptide Y (NPY) promoter and cerebral stroke subtypes according to TOAST (Trail of ORG 10172 in Acute Stroke Treatment) criteria in Chinese Han population. Methods The gene polymorphisms at position of-399T/C, -883Tgins/del and -602G/T in NPY promoter were detected by PCR method and gene sequencing in 190 cases of large-artery atherosclerosis stroke (LAA), 260 cases of small-artery occlusion (SAO), 60 cases of cardioembolism stroke (CE), 29 cases of stroke of other demonstrated etiology (ODE),10 cases of stroke of other undemonstrated etiology (UE) and 423 healthy control subjects. The PCR products were directly sequenced. Multivariate logistic regression was performed to analyze the relationship between gene polymorphism of NPY promoter and cerebral stroke subtypes according to TOAST by removing the confounding variables. Results Significant differences in the frequency of genotype CC and allele C at position of-399T/C were noted between the patients with SAO and controls (P=0.046, P=0.010). Compared with the control group, patients with LAA and SAO were more likely having high level of uric acid, hypertension, diabetes mellitus and heard disease (P＜0.05). No statistic differences in the frequency of genotype DD and allele D at position of-883Tgins/del were noted between patients with SAO and controls (P=0.0605, P=0.155). Gene polymorphisms of-399T/C,-883Tgins/del and -602G/T did not associate with an increased risk of having LAA, CE, ODE and UE.Conclusions The gene polymorphisms of promoter in position of-399T/C gene maybe associate with the happening of SAO; allele C at the position of-399T/C may raise the risk of the disease. There is no relationship between the gene polymorphisms of promoter at position of-399T/C, -883Tgins/del, -602G/T and the patients with LAA, CE, ODE and UE. High level of uric acid, hypertension, diabetes mellitus and heard disease history are the risk factors of LAA and SAO.

Objective To investigate the role of interleukin-18 (IL-18) gene promoter polymorphisms in the susceptibility of sporadic Alzheimer's disease (SAD). Methods A total of 109 patients with SAD and 109 healthy controls matched for age and gender were enrolled in our study with case-control method. Polymerase chain reaction with sequence specific primer was used to detect the single nucleotide polymorphisms (C/A) of gene promoter of IL-18 at position -607. Results Significant differences in the C/A genotype and allele distributions of IL-18 promoter at position -607 were noted between patients with SAD and controls (P=0.041 and P=0.021, respectively). The CC genotype frequency of gene promoter of IL-18 at position -607 in patients with SAD was significantly higher than that in healthy controls (x2=4.109, P=0.043); and the people with this genotype had a 1.90-fold increased risk as compared with the people with other genotypes (OR=1.90, 95%CI: 1.017-3.550).Conclusion The C/A polymorphisms of IL-18 gene promoter at position -607 are correlated with SAD in Han Chinese population. People with -607CC in IL-18 gene promoter have an increased risk of SAD.

Objective To investigate the effects of different dialysates on expression of protein kinase C-? (PKC?) and apoptosis of U937 cell line. Methods Different dialysates were added into culture fluid with U937 cell line at exponential phase of growth, and groups were divided: fluid A+fluid B group (dialysate A+dialysate B), fluid A+fluid B+rottlerin (PKC? specific inhibitor)group, fluid A+powder B group (dialysate A+powder B) and fluid A+powder B + rottlerin group. Besides, blank control group and normal control group were established. Cells were harvested 24 h and 48 h after treatment, morphological changes were observed by Hoechst33258 fluorescence staining, cell apoptosis was measured by Annexin-V-FITC/PI double staining, and expression of PKC? mRNA and protein was detected by RT-PCR and Western blotting, respectively. Results Cell apoptosis significantly increased in fluid A+powder B group, with typical morphology of apoptosis. After treatment for 24 h and 48 h, cell apoptosis rates in fluid A+powder B group were significantly higher than those at corresponding time points in blank control group, normal control group and fluid A+powder B+rottlerin group (P0.05). Conclusion Fluid A+powder B can significantly increase apoptosis of U937 cell line, the mechanism of which may be associated with the up-regulation of expression of PKC?. Compared with fluid A+powder B, fluid A+fluid B is superior in reducing apoptosis of peripheral blood monouclear cells.

OBJECTIVETo investigate the effects of progesterone and progestin on the expressions of regulated on activation, normal T cell expressed and secreted (RANTES) in eutopic endometrium from patients with endometriosis.

METHODSWe collected the samples of endometrium from patients with endometriosis before operation or after insertion of levenorgestrel releasing intrauterine system (LNG-IUS), administration of oral medroxyprogesterone (MPA), or injection of gonadotrophic hormone releasing hormone agonist (GnRHa). Reverse transcription-polymerase chain raction was used to assay the expression of RANTES mRNA. On the other hand, progesterone (Po) and tumor necrosis factor-alpha (TNFalpha) of different concentrations and different manners were used to treat cultured cells in vitro. RANTES secretion was evaluated in the culture medium using ELISA. In order to evaluate the effect of Po on the secretion of RANTES under stimulation of TNFalpha, the cells were cultured in medium containing 100 U/ml TNFalpha and Po of different concentrations for 24 hours. After the pretreatment of Po for 48 hours at different concentrations, TNFalpha (100 U/ml, 16 h) was added to observe whether Po inhibits RANTES or not.

RESULTSThe expression of RANTES mRNA in eutopic endometrium of patients with endometriosis was significantly higher than in control group (28.0 +/- 9.0 vs. 22.0 +/- 5.6, P < 0.05). Following the exposures to LNG-IUS (24.0 +/- 4.2 vs. 25.9 +/- 4.2, P > 0.05) or GnRHa (23.0 +/- 12.9 vs. 26.9 +/- 5.2, P > 0.05), the expression of RANTES mRNA had no change. MPA significantly increased the expression of RANTES mRNA (42.6 +/- 3.1 vs. 24.3 +/- 5.7, P < 0.05). Po itself had no significant effect on the secretion of RANTES. Stimulated by Po and TNFalpha at the same time, the secretion of RANTES significantly increased. After pretreatment with Po for 48 hours, the reaction of RANTES to the stimulating effect of TNFalpha was down-regulated.

CONCLUSIONThe eutopic endometrium of patients with endometriosis has high chemotactic activity. It may be feasible to prevent and treat endometriosis with progestins.

Cells, Cultured ; Chemokine CCL5 ; biosynthesis ; Endometriosis ; drug therapy ; metabolism ; Endometrium ; drug effects ; metabolism ; Female ; Gonadotropin-Releasing Hormone ; agonists ; Humans ; Intrauterine Devices, Medicated ; Levonorgestrel ; therapeutic use ; Medroxyprogesterone ; therapeutic use ; Progesterone ; pharmacology ; therapeutic use ; Progestins ; therapeutic use ; Transforming Growth Factor alpha ; pharmacology

OBJECTIVETo investigate the apoptosis-related mechanisms of levenorgestrel-releasing intrauterine system (LNG-IUS), oral medroxyprogesterone (MPA), and injective gonadotrophic hormone releasing hormone agonist (GnRHa) on eutopic endometrium of patients with endometriosis. Methods We collected the samples of endometrium from patients with endometriosis before operation and after insertion of LNG-IUS, administration of oral MPA, or injection of GnRHa. The ultrastructure of endometria was observed and compared by electron microscopy. Apoptotic cells were assessed by the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick-end labeling (TUNEL) assay, and the expressions of Bax, Fas, and Fas-L mRNA were determined by semi-quantitative reverse transcription-polymerase chain raction. Results After have been exposured to LNG-IUS, the apoptotic rate of endometrial epithelial cells and stromal cells increased from (24. 4 +/- 35.0)% to (51.0 +/- 37.8)% (P = 0.027) and (35.3 +/- 30.2)% to (76.4 +/- 11.2)% (P = 0.008), respectively. The degree of apoptosis under transmission electron microscopy was in an order of GnRHa > LNG-IUS > MPA. The expression of Fas-L mRNA in eutopic endometrium of patients with endometriosis was significantly higher than that of the normal control (P < 0.05). The expressions of three apoptosis-related proteins had no significant difference.

CONCLUSIONMedical treatments can increase the apoptosis of eutopic endometrial cells, and such effect was strongest in GnRHa and relatively weaker in LNG-IUS and MPA.

Apoptosis ; Endometriosis ; drug therapy ; pathology ; Endometrium ; drug effects ; pathology ; ultrastructure ; Female ; Gonadotropin-Releasing Hormone ; agonists ; Humans ; Intrauterine Devices, Medicated ; Levonorgestrel ; therapeutic use ; Medroxyprogesterone ; therapeutic use

Objective The role of this essay is to explore the relationship of BIM deletion polymorphism and paclitaxel intrinsic resistance in breast cancer. Methods Human breast cancer cell lines were screened by techniques of PCR and gene sequencing to detect BIM deletion polymorphism.MTS was used to detect the cytotoxic effects of paclitaxel in different cell lines. Meanwhile,levels of BIM mRNA and protein were detected by rtPCR and Western Blot. Results Comparing with the wild type cell line(MCF7),T47D was a deletion cell line with BIM deletion polymorphism and resistant to paclitaxel(T47D vs.MCF-7,IC50>30le vs.IC50=0.16 vs.02 μmol/L). Moreover,the ratio of BIM EXON3 to EXON4 was significantly increased and the level of functional BIM protein was down-regulated in T47D compared with MCF7(P < 0.05). Conclusion BIM deletion polymorphism might initiate intrinsic resistance to paclitaxel therapy in breast cancer.