OBJECTIVETo study the adscription of plasma effective constituents of rat after oral administration of Gegen Qinlian decoction.

METHODThis study was performed by using HPLC-UV to identify the chemical constituents of rat plasma samples obtained after oral administration of the whole and individual herbs of Gegen Qinlian decoction. A reversed phase Globalsil C18 column (4.6 mm x 250 mm, 5 microm) was used. The mobile phase was composed of 20 mmol x L(-1) ammonium acetate (adjusted to pH 4. 5 by acetate acid) and acetonitrile with gradient elution. The flow rate was 1.0 mL x min(-1) and the column maintained at room temperature. The detection wavelength was set at 270 nm.

RESULTThe chromatographic analysis of the rat plasma obtained after oral administration of Gegen Qinlian decoction showed 31 compounds, fifteen of which were the original constituents contained in Gegen Qinlian decoction, thirteen of which were metabolites, while the other three only existed after oral administration of the Decoction. By comparison of the retention times of the unknown peaks with that of standard compounds and, by spiking method with the standard compounds, five peaks were identified as puerarin, baicalin, berberine, jatrorrhizine and palmatine.

CONCLUSIONThis HPLC-UV method is simple and reliable in the analysis of the UV absorbing chemical constituents of rat plasma. The compounds absorbed into blood and its metabolites are the possible effective constituents of Gegen Qinlian decoction because they interact directly within the body. The results obtained in this investigation may provide useful information in the further understanding of the action mechanism of the individual and whole herbs in Gegen Qinlian decoction.

Administration, Oral ; Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; administration & dosage ; pharmacokinetics ; Male ; Rats ; Reproducibility of Results

OBJECTIVETo establish a method for studying efficacious materials of traditional Chinese medicines from an overall perspective.

METHODCarthamus tinctorius was taken the example. Its major components were depleted by preparing liquid chromatography. Afterwards, the samples with major components depleted were evaluated for their antioxidant effect, so as to compare and analyze the major efficacious materials of C. tinctorius with antioxidant activity and the contributions.

RESULTSeven major components were depleted from C. tinctorius samples, and six of them were identified with MS data and control comparison. After all of the samples including depleted materials are compared and evaluated for their antioxidant effect, the findings showed that hydroxysafflor yellow A, anhydrosafflor yellow B and 6-hydroxykaempferol-3, 6-di-O-glucoside-7-O-glucuronide were the major efficacious materials.

CONCLUSIONThis study explored a novel and effective method for studying efficacious materials of traditional Chinese medicines. Through this method, we could explain the direct and indirect contributions of different components to the efficacy of traditional Chinese medicines, and make the efficacious material expression of traditional Chinese medicines clearer.

Alkalies ; chemistry ; Antioxidants ; chemistry ; Carthamus tinctorius ; chemistry ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Glucosides ; chemistry ; Mass Spectrometry

OBJECTIVETo study the effect of Feiji Recipe (FR) intervening indoleamine 2,3-dioxygenase (IDO) induced immune escape on the murine model of Lewis lung carcinoma. Methods Totally 48 C57BL/6 mice inoculated with Lewis lung cancer cells transfected with human (enhanced green fluorescent protein,EGFP)-IDO gene were divided into four groups according to radom digit table, i.e., the model group (administered with normal saline by gastrogavage) , the Chinese medicine group (treated with FR Decoction at the daily dose of 100 mg/g by gastrogavage), the 1-methyl-D-trytaphan (1-MT) group (administered with 1-MT mixed liquor at the daily dose of 100 mg/kg by gastrogavage), and the Paclitaxel group (treated with Paclitaxel at the daily dose of 15 mg/kg by peritoneal injection), 12 in each group. The intervention was started from the 2nd day of modeling. The survival time was observed in 24 of them. Ratios of CD4+ CD25+ FoxP3+ regulatory T cells (Treg) in the spleen were detected in the rest 24 mice by flow cytometry respectively.

RESULTSCompared with the model group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells remarkably decreased in the Chinese medicine group, the 1-MT group, and the Paclitaxel group (P < 0. 01). Compared with the Paclitaxel group, the survival time was significantly prolonged in the Chinese medicine group and the 1-MT group (P < 0.01); ratios of Treg cells decreased significantly in the 1-MT group (P < 0.05).

CONCLUSIONFR could inhibit the proliferation of lung cancer cells and immune eseape, improve the immune function, and prolong the survival of tumor-bearing mice.

Animals ; Antineoplastic Agents ; pharmacology ; therapeutic use ; Carcinoma, Lewis Lung ; drug therapy ; immunology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; Lung Neoplasms ; Mice ; Mice, Inbred C57BL ; Paclitaxel ; T-Lymphocytes, Regulatory

UNLABELLEDOBJECTIVE; To evaluate the clinical value of different CT diagnostic criteria for peripancreatic artery and vein invasion in pancreatic carcinoma through comparison with the findings on surgical exploration.

METHODSOf 72 patients of having suspected pancreatic carcinoma were examined by multiplane spiral CT. Among 43 confirmed by surgical pathology; 15 underwent pancreaticoduodenectomy; 28 were found to have unresectable tumors. The peri-pancreatic major vessels including the superior mesenteric artery, celiac artery, hepatic artery, superior mesenteric vein and portal vein were explored carefully during surgical exploration.

RESULTSThe criteria for peri-pancreatic artery invasion was the presence of one of the following signs: artery embeded in tumor, or more than half of the artery circumference involved by tumor with wall irregularity or stenosis. The sensitivity of the above described criteria was 75.0% (12/16). If the criteria of tumor involvement exceeding half of the vessel circumference were adhered to, the sensitivity was 87.5% (14/16), which was high than the former, but the specificity was lower than that of the former one (90.2% versus 95.1%). The criteria for peri-pancreatic vein invasion was presence of any of the following signs: vein obliteration, more than half of the vein circumference involved by tumor, vein wall irregularity, vein stenosis, tear-drop sign of superior mesenteric artery. The sensitivity of the above described criteria was 92.9% (39/42), higher than that of the criteria that more than half of the vessel circumference was involved by the tumor (69.0%, 29/42), but the specificity of both criteria was the same (97.4%, 37/38).

CONCLUSIONFor assessing peri-pancreatic artery and vein invasion, using the combination of different CT diagnostic criteria has higher accuracy than when using only criteria of more than half of vessel circumference involved by tumor.

Adult ; Aged ; Carcinoma, Pancreatic Ductal ; diagnosis ; surgery ; Celiac Artery ; diagnostic imaging ; Female ; Hepatic Artery ; diagnostic imaging ; Humans ; Male ; Mesenteric Artery, Superior ; diagnostic imaging ; Mesenteric Veins ; diagnostic imaging ; Middle Aged ; Neoplasm Invasiveness ; Pancreatic Neoplasms ; diagnosis ; surgery ; Pancreaticoduodenectomy ; Portal Vein ; diagnostic imaging ; Reproducibility of Results ; Sensitivity and Specificity ; Tomography, Spiral Computed ; methods

OBJECTIVETuberculosis is still a public health problem. Host genetic factors, such as polymorphisms in NRAMP1 gene, may play a role in the development of tuberculosis. To clarify the effect of NRAMP1 gene polymorphisms on the development of childhood tuberculosis, the association of NRAMP1 gene polymorphisms with susceptibility to tuberculosis in the ethnic Han Chinese children was investigated.

METHODSFrom January 2005 to March 2008, 130 ethnic Han children with tuberculosis (TB group) were enrolled. Three hundred and ninety hospitalized ethnic Han children for physical examination in the surgery department were used as the control group. The controls were matched with tuberculosis children by age, sex and area. PCR-RFLP analysis was performed on DNA samples to identify allele genotypes of INT4 and D543N in NRAMP1 gene. Genotype frequency differences between tuberculosis patients and controls were analyzed using x2 test.

RESULTSNo statistical difference was found in the genotype frequency of variants G/C and C/C at the INT4 locus between the TB and the control groups. At the D543N locus, the frequency of genotype variants (G/A and A/A) was significantly higher in the TB group (34/130) than that in the control group (66/390) (x2=5.349, P<0.05; OR=1.74, 95%CI=1.08-2.79). When stratified by sex, differences in the genotype distribution were observed only in females at the D543N locus, which the variant genotypes were higher in the TB group (16/52) than in the control group (21/155) (x2=7.866, P<0.05; OR=2.84, 95%CI=1.34-5.99). For males, there was no difference between the TB and the control groups. At the INT4 locus, no difference was observed between the two groups in boys and girls.

CONCLUSIONSGenotypic variation at the D543N locus in NRAMP1 gene may be associated with susceptibility to tuberculosis in ethnic Han Chinese children. Variant genotypes in NRAMP1 gene (G/A and A/A) may be susceptible genotypes to tuberculosis in ethnic Han Chinese children. Girls with variant genotypes were more susceptible to tuberculosis.

Cation Transport Proteins ; genetics ; Child ; Child, Preschool ; China ; ethnology ; Female ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Humans ; Infant ; Male ; Polymorphism, Genetic ; Sex Factors ; Tuberculosis ; ethnology ; genetics

OBJECTIVETo screen anti-c-Met Fab from a phage antibody library and identify its binding activity.

METHODSThe expression of c-Met of HCC lines was identified by Western blot and immunofluorescence. Antibodies against c-Met were screened with immobilized antigen. After five rounds of panning, 30 randomly selected clones were identified by phage ELISA to select specific clones with high affinity. The positive clones were selected for Fab soluble expression in TOP10F and the binding activities were analysed in HCC lines.

RESULTSc-Met expressed in HCC membrane was confirmed by Western blot and immunofluorescence. A Fab fragment named AM2-26 with fine activity to c-Met was selected. AM2-26 binding specificity was confirmed by IP, FACS and immunofluorescence.

CONCLUSIONThe anti-c-Met Fab binding to c-Met in HCC provides a promising candidate for the biotherapy of hepatoma.

Antibodies ; immunology ; isolation & purification ; Cell Line, Tumor ; Cloning, Molecular ; Gene Library ; Humans ; Immunoglobulin Fab Fragments ; immunology ; Immunoglobulin Variable Region ; immunology ; Peptide Library ; Proto-Oncogene Proteins c-met ; immunology ; Recombinant Fusion Proteins ; immunology

OBJECTIVETo investigate the antitumor effect of natural killer (NK) cells on human colorectal cancer cells HT-29 in vitro by blocking transforming growth factor-β (TGF-β) signaling in NK cells transfected with vector containing dominant negative TGF-β type 2 receptor (DNTβR2).

METHODSTGF-β1 was added at the final concentration of 10 ng/ml for HT-29 cells. Primary NK cells were transfected with recombinant plasmid pIRES2-AcGFP-DNTβR2 and control plasmid pIRES2-AcGFP using Amaxa Nucleofector technology respectively. The cytotoxicity of these two types of NK cells to HT-29 cells was detected and analyzed by cell counting kit-8.

RESULTSThe transfection efficiency of primary NK cells was 18.85% for the plasmid pIRES2-AcGFP-DNTβR2 and 35.28% for the control plasmid pIRES2-AcGFP. The expression of DNTβR2 in NK cells was confirmed by Western blotting and RT-PCR. Primary NK cells displayed significantly lower cytotoxicity against HT-29 cells incubated with TGF-β1 than that without TGF-β1 (effect-target cell ratio 10:1,14.40%∓ 2.00% vs. 26.14% ∓ 2.50%, P > 0.05; effect-target cell ratio 20:1, 19.18% ∓ 2.49% vs. 40.81% ∓ 3.50%, P > 0.05). The cytotoxicity of NK cells transfected with DNTβR2 vector was significantly higher than that with control vector against HT-29 cells cultured with 10 ng/ml TGF-β1 (effect-target cell ratio 10:1, 21.17% ∓ 2.49% vs. 11.48% ∓ 1.11% ,P > 0.05; and effect-target cell ratio 20:1, 35.30% ∓ 3.78% vs. 17.19% ∓ 2.29%, P > 0.05).

CONCLUSIONNK cells transfected with DNTβR2 vector show better antitumor effect, which may provide new method for NK-based adoptive immunotherapy for cancer.

HT29 Cells ; Humans ; Killer Cells, Natural ; immunology ; metabolism ; Plasmids ; genetics ; Receptors, Transforming Growth Factor beta ; genetics ; Transfection ; Transforming Growth Factor beta ; metabolism ; pharmacology

Type B Niemann-Pick disease is an autosomal recessive sphingolipidosis due to mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1). Here we present molecular findings for two sibling patients. One mutation V36A due to c.107T>C in exon 1 is a single nucleotide polymorphism and the other N522S due to c.1565 A>G in exon 6 is a novel missense mutation. This non-fatal missense mutation leads to –20% residual lysosomal acid sphingomyelinase activity in vitro and only results in hepatosplenomegaly without neurologic involvement.
Female ; Humans ; Middle Aged ; Mutation, Missense ; Niemann-Pick Disease, Type B ; genetics ; Polymorphism, Single Nucleotide ; Siblings ; Sphingomyelin Phosphodiesterase ; genetics

OBJECTIVETo establish a reliable neonatal rat model of periventricular leukomalacia (PVL) which is expected to be similar to PVL of human preterm infants pathologically, and to explore the concomitant eye lesions in the PVL model.

METHODSTwo-old-day neonatal rats were randomly divided into a PVL group and a sham-operated group (n=19 each). The PVL model was established by the ligation of bilateral common carotid arteries, followed by a 30-min exposure to 8% oxygen. The cerebral infarction area was assessed with TTC staining 1 day after operation. Cerebral pathology was examined under a light micsrocope 2 and 21 days after operation. The examinations of eyes under a slip lamp and the pathology of eyeballs under a light microscope were performed 21 days after operation.

RESULTSThe TTC staining cerebral slices showed there were extensive white areas of infarction in the brain of the PVL group, with an infarction area of 53.45 +/- 33.90 mm3 and a percentage of infarction of (24.98 +/- 15.44)% . Significant cystic necrosis and apoptosis around the periventricular and subcortical white matter and mild damage in cortical neurons were observed in the PVL group 2 days after operation. The more obvious cystic necrosis around the periventricular area was found in the PVL group 21 days after operation. There were no pathological changes in the brain of the sham-operated group. All of rats in the PVL group had bilateral cataracts, however, no pathological changes were observed in their postbulbar tissues. The sham-operated group did not show eye abnormal.

CONCLUSIONSThe PVL animal model that was similar to PVL of human preterm infants pathologically was successfully established by the ligation of bilateral common carotid arteries, followed by 30-min hypoxia exposure, with a positive effect and a good repeatability. Cataract can also be induced by the method.

Animals ; Animals, Newborn ; Brain ; pathology ; Cataract ; etiology ; pathology ; Disease Models, Animal ; Female ; Humans ; Hypoxia-Ischemia, Brain ; complications ; Infant, Newborn ; Leukomalacia, Periventricular ; etiology ; pathology ; Male ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the brain pathological changes following exdogenous neural stem cells (NSCs) intraventricular transplantation in neonatal rats with periventricular leukomalacia (PVL), and to explore the feasibility of NSCs transplantation for the treatment of PVL in premature infants.

METHODSNSCs were prepared from E14 embryonic rat brain. Two-day-old neonatal rats were randomly divided into six groups: PVL, PVL+culture medium, PVL+NSCs, sham operation, sham operation+culture medium, and sham operation+NSCs (18-21 rats each group). Intraventricular transplantation of exdogenous NSCs was performed 72 hrs after PVL induction or sham operation. The cerebral pathological evaluation was undertaken by light microscopy 7, 14 and 21 days after transplantation.

RESULTSThe pathological changes in the cerebral white matter were gradually improved with the prolonged time after transplantation. After 21 days of transplantation, 50% of the cerebral white matter showed mild pathological changes and 50% of that showed severe pathological changes, with neuronal pathological scores of 1.28+/-0.86, in the untreated PVL group. In the PVL+NSCs group, 30% of normal white matter, 40% of mild and 30% of severe pathological changes in the white matter were observed, with neuronal pathological scores of 0.32+/-0.16, 21 days after transplantation. There were very significant differences in both of pathological changes in the cerebral white matter and neuronal pathological scores between the PVL and PVL+NSCs groups (x2=10.7, P<0.01; F=29.664, P<0.01).

CONCLUSIONSIntraventricular transplantation of exdogenous NSCs can apparently improve cerebral white matter damage. It is suggested that intraventricular transplantation of NSCs is of a great potential feasibility for the treatment of PVL in premature infants.

Animals ; Animals, Newborn ; Brain ; pathology ; Female ; Humans ; Infant, Newborn ; Leukomalacia, Periventricular ; pathology ; therapy ; Neurons ; cytology ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Stem Cell Transplantation