Objective To estimate the effect of glycyrrhetinic acid on epidermal growth factor (EGF)-induced proliferation of HaCaT cells,and to investigate its possible mechanism.Methods Methyl thiazolyl tetrazolium (MTT) assay was used to evaluate the proliferation of HaCaT cells treated with different concentrations of EGF (0,1,5,10,25,50,100 μg/L) and glycyrrhetinic acid (0,0.1,1.0,10,25,50,100μmol/L) alone,or the combination of 25 μg/L EGF with 25 μ mol/L glycyrrhetinic acid or 10 μ mol/L U0126 (an inhibitor of MEK1/2).Western blot was carried out to measure the protein expression of proliferating cell nuclear antigen (PCNA),Notch-1,ERK 1/2 and phosphorylated ERK 1/2 in HaCaT cells treated with 25 μg/L EGF,10 μmol/L U0126,25μmol/L glycyrrhetinic acid alone or in combination.Data were statistically analyzed by using t test,analysis of variance and correlation analysis with SPSS 17.0 software.Results EGF of 0-100 μg/L promoted the proliferation of HaCaT cells in a dose-dependent manner (r =0.798,P ＜ 0.05),and there was a linear correlation between the effect and concentration within the concentration range 0-50 μg/L (r =0.859,P ＜ 0.05).However,glycyrrhetinic acid of 10-100 μmol/L inhibited the proliferation of HaCaT cells in a dose-dependent manner (r =-0.945,P ＜0.01),and 10 μmol/L glycyrrhetinic acid could suppress the EGF (25 μg/L)-induced proliferation and phosphorylation of ERK1/2 in HaCaT cells.Also,both 25 μmol/L glycyrrhetinic acid and 10 μmol/L U0126 could attenuate the increase in PCNA and Notch-1 expression in HaCaT cells induced by 25 μg/L EGF.Conclusion Glycyrrhetinic acid can inhibit the EGF-induced proliferation of HaCaT cells,likely by suppressing the activation of ERK1/2 signaling pathway.

Objective To evaluate the effect of curcumin on the proliferation of and apoptosis in HaCaT cells induced by tumor necrosis factor α (TNF-α).Methods HaCaT cells were cultured with the presence of different concentrations (0,1,5,10,25,50,100 ng/ml) of recombinant TNF-α,curcumin of 20 μmol/L,or the combination of recombinant TNF-α (25 ng/ml) and curcumin (20 μmol/L),for 24 hours followed by the determination of cell proliferation with methyl thiazolyl tetrazolium (MTT) assay.Western blot was conducted to measure the protein expression of proliferating cell nuclear antigen (PCNA) and Notch-1 in HaCaT cells treated with recombinant TNF-α (25 ng/ml) and curcumin (20 μ mol/L) alone or in combination for 24 hours.Flow cytometry using annexin-V/propidium iodine (PI) was performed to assess the early apoptosis in HaCaT cells incubated with recombinant TNF-α of 25 ng/ml and curcumin of 20 μmol/L alone or in combination for 12 hours.Statistical analysis was carried out with one-way analysis of variance.Results Recombinant TNF-α promoted the proliferation of HaCaT cells in a dose-dependent manner,with the maximum proliferation activity observed in HaCaT cells treated with TNF-α of 25 ng/ml,while curcumin of 20 μmol/L effectively inhibited the proliferation of HaCaT cells induced by TNF-α of 25 ng/ml (P ＜ 0.01).TNF-α of 25 ng/ml had no obvious effect on cell apoptosis,while curcumin of 20 μ mol/L markedly induced the apoptosis in HaCaT cells,and there was a synergy between TNF-α of 25 ng/ml and curcumin of 20 μmol/L in the induction of apoptosis in HaCaT cells,with the apoptosis rate being 2.3％,3.4％,11.6％ and 16.8％ respectively in untreated cells,cells treated with TNF-α,curcumin,and the combination of TNF-α and curcumin,respectively.Conclusions Curcumin could enhance the inductive effect of TNF-α on the apoptosis in,but suppress the promotive effect of TNF-α on the proliferation of,HaCaT cells.

OBJECTIVETo determine the content of 7 anthraquinones in Semen Cassiae.

METHODA HPLC method was developed, with Inertsil ODS-3 column, acetonitrile and 0.1% H3PO4 solution as mobile phases in gradient elution. The detection wavelength wasset at 278 nm, and the flow rate was 0.8 mL x min(-1).

RESULTRecoveries of all 7 anthraquinones were between 95%-105%. The content of the anthraquinones in crude drug produced in different habitation were different.

CONCLUSIONThe method is convenient and accurate, which provides the foundation for the research of Semen Cassiae.

Anthraquinones ; analysis ; Cassia ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; classification ; isolation & purification

Objective To study the mechanism of marcosomia by investigating insulin-like growth factor 2(IGF_2)imprinting status,expression level and the promoter usage in the placenta of macrosomia. Methods We selected heterozygous cases for Apa Ⅰ polymorphism in exon 9 of IGF_2 gene and then analyzed its imprinting status in 168 placentas of macrosomia and normal pregnancies.IGF_2 transcription levels and promoter usages in macrosomic and normal placenta were evaluated by using semi-quantitative RT- PCR assay.Results Thirty specimens of macrosomic placenta and 30 of normal placenta were identified as heterozygous for IGF_2.All of the heterozygous specimens showed maintenance of imprinting.The expression of placental IGF_2 mRNA(2.2?1.2)was significantly higher in macrosomia than that of normal weight group (1.6?0.6,P 0.05).Conclusion It is possible that over expression of IGF_2 in placenta contributes to macrosomia while the promoter usage and imprinting status are not associated with macrosomia.

OBJECTIVETo study the imprinting status of IGF2 and phenotypes of loss of imprinting (LOI) in cord blood of neonates of Chinese Han population and to investigate relative factors to LOI.

METHODSCord blood of 1010 Chinese Han newborns were collected and the imprinting status of IGF2 was detected by reverse transcription-PCR(RT-PCR) and restriction fragment length polymorphism.The relationships between LOI and fetal growth indices, features of parents and grandparents, clinical characteristics were analyzed.

RESULTSOf all cases, 42.8% (432/1010) were heterozygous for a polymorphism of Apa I site in exon 9 of IGF2, while 21.6%(66/306) displayed IGF2 LOI. Maternal factors including average age, gestational age, BMI pre-pregnancy, weight gain during pregnancy and the level of HB, HCT, and other indices of biochemistry in their second and third trimester were not correlated with LOI expression. However in newborns with fathers older than 35 yrs, 31.7%(19/60) displayed LOI, which was significantly more common than that in newborns with younger fathers (P< 0.05, chi square is 4.69). There were no difference in birth weight (BW) between normal imprinting and LOI groups. But if the newborn's weights were in 2500-2999 g, LOI was 6.25%(2/32), which was significantly lower than that in 3000 g group (P< 0.05, chi square is 4.89). In groups with BW being less than 2500 g and more than/equal to 4000 g, the LOI newborn's blood glucose was decreased significantly after 2 hrs (P< 0.01, t is 7.47 and 10.9).

CONCLUSIONIn newborns of Chinese Han population, 21.6% showed IGF2 LOI in cord blood. IGF2 LOI may have some influences on fetal growth. Paternal age is associated with LOI.

Asian Continental Ancestry Group ; genetics ; China ; Female ; Fetal Blood ; metabolism ; Genomic Imprinting ; genetics ; Humans ; Infant, Newborn ; Insulin-Like Growth Factor II ; genetics ; Pregnancy ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo evaluate the value of gray-scale ultrasound, contrast-enhanced ultrasound and multislice spiral CT in early and differential imaging diagnosis of small hepatocellular carcinoma (SHCC).

METHODSThis study included 35 patients with space-occupying lesions in the liver identified by routine ultrasound examination. The hemodynamics of the patients was recorded during the arterial, portal and lag phases using contrast-enhanced ultrasound. The enhancement features of the 3 phases were observed using multislice spiral CT. All the cases were confirmed by pathological examinations.

RESULTSFor SHCC diagnosis, gray-scale ultrasound, contrast-enhanced ultrasound and multislice spiral CT showed a sensitivity of 77.8%, 94.4%, and 100%, specificity of 88.2%, 100%, and 94.1%, positive predictive value of 87.5%, 100%, and 94.7%, negative predictive values 78.9%, 94.4%, and 100%, concordance rate of 82.9%, 97.1%, and 97.1% and Younden index of 0.66, 0.94, and 0.94, respectively.

CONCLUSIONSContrast-enhanced ultrasound and multislice spiral CT have significantly greater diagnostic efficacy than gray-scale ultrasound in early and differential diagnosis of SHCC. But in some atypical cases, gray-scale ultrasound, contrast-enhanced ultrasound and multislice CT have to be combined to establish a diagnosis.

Carcinoma, Hepatocellular ; diagnosis ; Contrast Media ; administration & dosage ; Female ; Humans ; Image Enhancement ; methods ; Liver ; diagnostic imaging ; Liver Neoplasms ; diagnosis ; Male ; Reproducibility of Results ; Sensitivity and Specificity ; Tomography, Spiral Computed ; methods ; Ultrasonography, Doppler, Color ; methods

OBJECTIVETo investigate the function of abnormal fibrinogen in two inherited dysfibrinogenemia pedigrees.

METHODSRoutine coagulation tests were conducted in the probands and related family members. The antigen and activity levels of fibrinogen were detected by immunoturbidimetry assay and clauss assay, respectively. All the exons and exon-intron boundaries of the three fibrinogen genes and antithrombin gene（AT3）were analyzed by PCR amplification and direct sequencing. Routine thrombelastography (TEG) test and functional fibrinogen TEG test were both used to make a comprehensive evaluation of coagulation status and functional fibrinogen level in patients. The molecular weights of the three peptides from fibrinogen were measured by Western blot. The function of abnormal fibrinogen was assessed by fibrinogen dynamic polymerization and fibrinolysis velocity.

RESULTSThe coagulation routine tests were normal in two probands except for prolonged thrombin time (TT) and reptilase time (RT), as well as reduced activity levels of 0.5 g/L and 0.6 g/L fibrinogen, respectively. The antigen levels of fibrinogen were 2.32 g/L and 2.66 g/L in two probands, which were in the normal reference range. The genotype analysis showed that Arg275His in fibrinogen γ chain (γ Arg275His) existed in both probands and patients in these two pedigrees. Meanwhile, proband B's grandfather and aunt also carried heterozygote g.5876T>C (Ser116Pro) mutation in AT3. The results of routine TEG test demonstrated that the α values of proband B and his father were close to and lower than the lower limit of reference range, respectively, while the MA values were normal in both of them. However, functional fibrinogen TEG test revealed obviously reduced MA value. All the probands and patients demonstrated prolonged lag-off time and reduced peak value in fibrinogen dynamic polymerization tests. Meanwhile, most of fibrin formed from the patients' plasma could not be dissolved completely by plasminogen (PLG) and urokinase-typeplasminogenactivator (u-PA) at a certain time.

CONCLUSIONWe first reported cases of inherited dysgibrinogenemia associated with inherited AT deficiency. γArg275His mutation caused the abnormal fibrinogen in terms of fibrin mono polymerization and possibly in fibrinolysis. Combined use of routine TEG test and functional fibrinogen TEG test with comprehensive analyses of the parameters in both tests could better evaluate the level of functional fibrinogen and predict the risk of hemorrhage and thrombosis in patients with inherited dysfibrinogenemia.

Adult ; Afibrinogenemia ; genetics ; Child ; Female ; Fibrinogen ; genetics ; Fibrinogens, Abnormal ; genetics ; physiology ; Genotype ; Humans ; Male ; Mutation ; Pedigree ; Phenotype

OBJECTIVETo observe the therapeutic effect of Yishen Qingre Huashi Recipe (YQHR) in treating proteinuria of chronic glomerular disease patients with Pi-Shen deficiency complicated damp-heat syndrome (PSDCDHS).

METHODSTotally 121 stage 1 -2 primary chronic glomerular disease patients with PSDCDHS were randomly assigned to the treated group (85 cases) and the control group (36 cases) according to 2:1. All patients received conventional and symptomatic treatment. Patients in the treated group took YQHR additionally, while those in the control group took Losartan Potassium Tablet (50 mg each time, once per day) additionally. The therapeutic course for all was 6 months. Changes of 24 h urine protein, blood urea nitrogen (BUN), serum creatinine(SCr), and estimated glomerular filtration rate (eGFR) were observed at different time points. And the difference in therapeutic effects were compared between the two groups.

RESULTSCompared with the control group after 6 months of treatment, 24 h urine protein obviously decreased in the treated group (P <0. 05). There was no statistical difference in SCr, BUN, or eGFR between the two groups after 6 months of treatment (P >0. 05). The total effective rate after 2, 4, and 6 months of treatment in the treated group was 77. 6% (66/85 cases), 82. 4% (70/85 cases), and 89. 4% (76/85 cases), respectively. They were 47. 2% (17/36 cases), 55. 6% (20/36 cases), and 61. 1% (22/36 cases) in the control group, respectively. Compared with before treatment in the treated group, the total effective effect after 6 months of treatment was higher than that after 2 months of treatment (χ2=4. 28, P <0. 01). Compared with the control group at the same time points, the total effective rate in the treated group after 2, 4, and 6 months of treatment was higher (χ2=10. 87, 9. 53, 13.16, P <0. 01).

CONCLUSIONYQHR could significantly lower proteinuria in chronic glomerular disease patients with PSDCDHS, improve the clinical effect, thereby providing clinical evidence for treating chronic glomerular disease proteinuria from resolving dampness and clearing heat.

Blood Urea Nitrogen ; Drugs, Chinese Herbal ; therapeutic use ; Hot Temperature ; Humans ; Kidney Diseases ; complications ; therapy ; Kidney Glomerulus ; pathology ; Losartan ; Medicine, Chinese Traditional ; Phytotherapy ; Proteinuria ; etiology ; therapy ; Syndrome ; Tablets

OBJECTIVETo modify the structure of psoralidin using in vitro enzymatic glycosylation to improve its water solubility and stability.

METHODSA new psoralidin glucoside (1) was obtained by enzymatic glycosylation using a UDP- glycosyltransferase. The chemical structure of compound 1 was elucidated by HR-ESI-MS and nuclear magnetic resonance (NMR) analysis. The high-performance liquid chromatography (HPLC) peaks were integrated and sample solution concentrations were calculated. MTT assay was used to detect the cytotoxicity of the compounds against 3 cancer cell lines in vitro. Results Based on the spectroscopic data, the new psoralidin glucoside was identified as psoralidin-6',7-di-O-β-D- glucopyranoside (1), whose water solubility was 32.6-fold higher than that of the substrate. Analyses of pH and temperature stability demonstrated that compound 1 was more stable than psoralidin at pH 8.8 and at high temperatures. Only psoralidin exhibited a moderate cytotoxicity against 3 human cancer cell lines. Conclusion In vitro enzymatic glycosylation is a powerful approach for structural modification and improving water solubility and stability of compounds.

Antineoplastic Agents ; metabolism ; Benzofurans ; metabolism ; Cell Line, Tumor ; Chromatography, High Pressure Liquid ; Coumarins ; metabolism ; Glucosides ; biosynthesis ; Glycosylation ; Glycosyltransferases ; metabolism ; Humans ; Magnetic Resonance Spectroscopy ; Solubility

In order to study the influence of trichosanthin (TCS) on apoptosis and growth inhibition of human NB4 cells in vitro, the expression of annexin V and the change of DeltaPsim of NB4 cells induced by TCS was analyzed by FACS, and MTT assay was adopted to measure the growth inhibition ratio of NB4 cells treated with TCS. Apoptosis was assayed by agarose gel electrophoresis. The results showed the higher concentration of TCS and the longer the acting time, the stronger growth inhibition of NB4 cells. The expression of annexin V was positive, and the positive ratio was greatly enhanced with prolongation of acting time. DeltaPsim reduced gradually while the apoptosis cells increasing. DNA agarose gel electrophoresis showed a gradient, which confirmed that TCS could induce NB4 cells apoptosis. In conclusion, taken together, data show that TCS can inhibit NB4 growth in vitro, and induce apoptosis. Experiment provides an important evidence for application of TCS in clinical treatment of acute promyelocytic leukemia.
Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; DNA Fragmentation ; drug effects ; Dose-Response Relationship, Drug ; Flow Cytometry ; Humans ; Trichosanthin ; pharmacology