Objective: To investigate the apoptosis-inducing effect of matrine on murine hepatocarcinoma cell line H22 in vivo and in vitro and explore the underlying mechanisms. Methods: The H22 cell apoptosis induced by matrine at the early stage was detected with Annexin V-FITC/PI double staining assay, The expressions of Bcl-2 and Bax proteins in H22 cells as well as in the BALB/c H22 xenograft tumor tissues were detected using immunohistochemical method. Transmission electron microscopy (TEM) was used to observe the ultramicro-structure alterations of H22 xenograft tumor cells in BALB/c mice. The effect of matrine on the kinetics of tumor growth after subcutaneous injection of H22 cells in BALB/c mice was observed and the tumor inhibition rate was calculated. Results: Annexin V staining detected early apoptosis of H22 cells after treatment with matrine 1.0 mg/mL and 1.5 mg/mL for 48 h. The apoptotic rates were 11.71% and 17.86%, respectively, both of which higher than that of control groups (P <0.05). The tumor inhibition rate was above 60% after matrine treatment. Immunohistochemistry analysis revealed that matrine increased Bax protein expression and reduced Bcl-2 protein expression in both H22 cells in vitro and xenograft tumor tissues in vivo. TEM demonstrated the existence of apoptotic cells and apoptotic bodies in H22 xenograft tumor tissues after matrine treatment. Conclusion: Matrine significantly suppresses tumor growth and induced apoptosis both in vitro and in vivo. The apoptosis-inducing effects is related with up-regulation of Bax protein and down-regulation of Bcl-2 protein.

Objective To observe the changing levels of serum sFas before and after intravenous i mmunoglobulin (IVIG) treatment of incomplete Kawasaki disease (IKD),to explore the roles of sFas in the pathogenesis of IKD and IVIG treatment mechanism.Methods Thirty eight cases of IKD children were selected as experimental group and 20 examples of the same age of children as the control group.The IKD children were treated by IVIG in combination with aspirin (ASP) ; and blood test was performed before treatment,3 days after treatment,and 14 days after treatment,respectively.Dual-resistant sandwich enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of serum sFas,plasma Fibrinogen (PT-D),d-dimer (D-D),and c-reactive protein (CRP).Results The levels of serum sFas,PT-D,D-D,and CRP were significantly higher than the control group for IKD children before treatment[(0.55 ± 0.14)ng/L vs (0.24 ±0.04) ng/L,(552.3 ± 147.2) mg/dl vs (277.3 ±82.5)mg/dl,(649.0 ±201.6) μg/L vs (315.4 ±91.8)μg/L,and(72.2 ±28.7)mg/L vs (7.2 ±2.9)mg/L; t' =12.41,9.11,8.64,13.82;All P ＜ 0.05] ;3 days after treatment,compared with those before treatment and control group,the sFas level of IKD children at the third day after treatment was significantly decreased compared to that before treatment and control groups,respectively [(0.43 ± 0.09) ng/L vs (0.55 ± 0.14) ng/L,(0.24 ± 0.04) ng/L,F =47.624,All P ＜0.05] ;For the level of sFas at the 14th day after treatment,no statistical significance was found between IKD children and the control group[(0.24 ±0.05) ng/L vs (0.24 ±0.04) ng/L,t =0.596,P ＞ 0.05].Conclusions The abnormally increased serum sFas level before IVIG treatment suggests that dysfunction of apoptosis be involved in the pathogenesis of the IKD.Intravenous immunoglobulin treatment may be involved in the apoptosis process.

Objectives To explore the change of interleukin-17 (IL-17) in Kawasaki disease (KD). Methods Fourty KD pediatric patients, among them 12 patients with echocardiographic abnormalities in acute phase, 25 age-matched non-KD patients were enrolled. The level of serum IL-17 was measured by enzyme linked immunosorbent assay in acute and convalescent phase of KD patients and non-KD patients. At the same time, C-reactive protein (CRP), globulin, albumin were detected. Results In acute phase of KD patients, the level of serum IL-17 were signiifcantly higher than that in convalescent phase of KD patients and non-KD patients (P<0.05). The level of serum IL-17 was no signiifcant differences in convalescent phase of KD patients and non-KD patients (P>0.05). In acute phase of KD patients with echocardiography abnormalities, the level of serum IL-17 was signiifcantly higher than that with non-echocardiography abnormalities (P<0.05). The level of serum IL-17 in acute phase of KD patients were positively correlated with CRP and globulin (r=0.750, 0.750, P<0.05), and negatively correlated with albumin (r=-0.779, P<0.05). Conclusions IL-17 may be involved in KD immune pathogenesis. Serum IL-17 is one of the activity index of KD, which associ-ated with cardiovascular damages.

Objective To study the expression levels of N-terminal pro-brain natriuretic peptide (NT-proBNP),serum albumin of Kawasaki' s disease (KD),incomplete Kawasaki' s disease (IKD),and children whose fever were unexplained and to explore the clinical significance of the levels of NT-proBNP and serum albumin in the early diagnosis of IKD.Methods The levels of NT-proBNP of 246 cases of KD (KD group),61 cases of IKD (IKD group) and 301 cases of children with unexplained fever (fever group)were measured by the enzyme-linked fluorescence analysis (ELFA) at the day of admission,meanwhile,the levels of albumin were tested in KD,and IKD children were underwent ECG and echocardiography.Based on the test results,patients were further divided into the group with cardiovascular damage and the group without cardiovascular damage.SPSS 19.0 was used for statistical analysis.The t test was used to compare the parameters between each group,the variance analysis and association analysis were carried out with Pearson's correlation analysis.The ROC curve analysis was done to identify the cardiovascular damage threshold.Results ① The level of plasma NT-proBNP of the KD group,the IKD group was significantly h igher than the fever group [(789.1±4.7) ng/L,(824.8±4.4) ng/L vs (92.5±2.3) ng/L,F=230.736,all P＜0.05];② The level of albumin of the KD group and the IKD group was significantly lower than that of the fever group [(33.9±2.8) g/L,(33.8±3.1) g/L vs (40.8±3.6) g/L,F=355.648,all P＜0.05]; ③ The levels of NT-proBNPs between the cardiovascular damage group and the groups without cardiovascular damage among the KD group,and those of the IKD groups were compared.In the KD group,the NT-proBNPs level of the two subgroups was (2948±3) g/L (n=103) vs (305±3) g/L,n=143; while in the IKD group,the NT-proBNPs of the two subgroups was (1454±4) g/L (n=38) vs (323±4) g/L (n=23).The dif-ferences were statistically significant (t=16.464,4.356,all P＜0.05).④ The plasma NT-proBNP level higher than 933.5 ng/L was identify as the indicator for cardiovascular damage in both KD and IKD children.Its sensi-tivity was 88.1％,and its specificity was 89％.⑤ When the level of NT-proBNP was higher than 250 ng/L,the sensitivity for diagnosis in the KD,the IKD was 80.9％,85.2％ respec-tively,and the specificity was 85.7％.When the level of NT-proBNP was higher than 250 ng/L and that of albumin was lower than 35 g/L,the sensitivity for diagnosis of KD,IKD was 67.5％,70.5％ respectively,the specificity was 99.7％.Conclusion The level of plasma NT-proBNP (＞250 ng/L) accompanied by decreased albumin (＜35 g/L) may be specific markers for early diagnosis of IKD.In addition,the level of NT-proBNP ≥933.5 ng/L can be used as a diagnostic threshold,which has good sensitivity and specificity for identifica-tion of cardiovascular damage in the KD and IKD in children.

ObjectiveTo diagnose and classify 249 patients with myelodysplastic syndrome (MDS) according to the WHO standards.MethodsAccording to the WHO standards,cell morphology,cytogenetics,immune phenotype and bone marrow pathologic biopsy in 249 cases of MDS were analyzed.ResultsGreat shape and oval cell of mature erythrocyte could be observed in all MDS patients peripheral blood. The incidence of immature erythrocyte,immature granulocyte,pelger-like abnormal nucleus and neutrophils cells without granular increased with subtypes progressing.These abnormal characteristics and proportion tended to more apparent with MDS subtypes progressing.With the dynamic follow-up,we found the rate of MDS transition to AL increased with subtypes progressing(P＜0.05 ).The immune phenotype analysis of 148 patients was undertook and found that the trend to express myeloid specific antigen (CD33) increased gradually with subtypes progressing The chromosome inspection in 138 patients was undertook and found that 53 patients (38.7 ％ with abnormal karyotype,mainly in 20q- and +8;16 cases with complex abnormal karyotype (28 ％), two patients in 5q-. 180 patients were underwent bone marrow biopsy at the same time and found that 19 patients with abnormal morphology;42 patients with bone marrow fibrosis.ConclusionsCombining with multiple index to detect the MDS contributes to the classification and diagnosis more accuratcly and long-term follow-up helps to judgment the prognosis.

A highly sensitive, rapid and selective liquid chromatography-tandem mass spectrometry(LC-MS/MS) method for the quantitative determination of retinamido-ester in rat plasma was developed and validated. A simplified protein precipitation with acetonitrile was employed for the sample preparation.The separation was carried out on an Agilent TC C18 column ( 150 mm×4. 6 mm ID, 5 μm particle size)with the mobile phase consisted of methanol-water-formic acid (93 : 7 : 0.1). Simvastatin was used as internal standard. The detection was performed on a trap-quadrupele tandem mass spectrometer by selected reaction monitoring (SRM) scan mode via atmospheric pressure chemical ionization (APCI). The range of and inter-day precision values were between 95.97% and 104. 43%, and RSD was between 4. 63% and 10. 69%, respectively. This method was applied to determine the pharmacokinetic parameters. The main pharmacokinetic parameters of retinamido-ester after oral administration via gastric gavage of 2. 5, 5, 10mg·kg-1 were as follows,T1/2;(11.28±7.23),(8.90±3.82),(8.01±5.56)h; AUC0-∞:(103.41±61.46),(190.23±74.99),(421.66±299.20)ng·h·mL-1;MRT:(6.31±0.75),(5.98±0.71), (6.18±0.97) h; CL/F: (30. 10 ± 13.67), (29.58±10.59), (31.18 ±17.51)L·h-1·kg-1;Vd/F:(414.94±159.82),(356.16±139.85),(369.28±322.73)L·kg-1,respectively.

An HPLC method was established for the determination of the related substance in erdosteine. Waters ODS-SunFire (250 mm x 4.6 mm ID, 5 microm) column was used, the mobile phase was composed of methanol-acetonitrile-0.01 mol x L(-1) citric acid (20:4:76, the pH value was adjusted by triethylamine to 2.5). The flow rate was 1 mL x min(-1). The detection wavelength was 254 nm. The related substances in the sample of erdosteine taken were calculated by self control with or without the response factor of impurity relative to that of erdosteine. Under the chromatographic condition developed, the impurities in erdosteine were isolated well. The detection limit was 0.2 microg x mL(-1) (signal/noise = 3) by principal component calculated. The method can be adopted to control the related substances in erdosteine.
Chromatography, High Pressure Liquid ; Drug Contamination ; Expectorants ; chemistry ; Limit of Detection ; Thioglycolates ; chemistry ; Thiophenes ; chemistry

This study was aimed to investigate the change of tissue factor pathway (TFP) ratio during the attack of acute myocardial infarction (AMI) and its clinical significance. Plasma recalcification time was assayed by manual operation. Plasma tissue factor (TF), TF pathway inhibitor (TFPI) antigen, FVII:Ag, activated FVII (FVIIa) and D-Dimer were measured by enzyme linked immunoabsorbent assay (ELISA). TF activity was determined by chromogenic assay, plasma FVII coagulation activity (FVII:C) was detected by one-stage system. Blood samples were taken from 59 patients with AMI and 84 healthy volunteers. The results indicated that (1) plasma recalcification time was significantly shorter in the AMI group than that in the control; (2) compared with the control, TF activity in AMI patients showed no significant change (p > 0.05); the antigen levels of TF and TFPI in patients with AMI were remarkably increased (p < 0.05), and the increment degree of TF was remarkably higher than that of TFPI, therefore the TF/TFPI ratio was enlarged; total TFPI (t-TFPI) and full-length TFPI (fl-TFPI) were significantly higher (p < 0.01), truncated TFPI (tr-TFPI) was significantly lower (p < 0.01); the TF/t-TFPI ratio was higher than that in normal group, the TF/t-TFPI ratio was lower than that in normal group (p < 0.01), but the TF/tr-TFPI and fl-TFPI/t-TFPI ratios in AMI group were more remarkably higher than that in control group (p < 0.01), the tr-TFPI/t-TFPI and tr-TFPI/fl-TFPI ratios were significantly lower (p < 0.01). (3) compared with the control, the levels of plasma FVIIa and FVII:C in AMI group were higher (p < 0.05), FVII:Ag did not significatly change; FVIIa/FVII: Ag ratio was more remarkably higher (p < 0.01), but the elevation of FVIIa/FVII:C and FVII:C/FVII:Ag ratios showed no significant change (p > 0.05); (4) plasma D-dimer was significantly higher, compared with the normal control (p < 0.01). It is concluded that TFP is initiated during the attack of AMI, suggesting the circulating blood in AMI patients is in hypercoagulable status, therefore the simultaneous detection of multiple coagulation factors is necessary for evaluating risk factors in AMI patients, and the use of ratio for reflecting hypercoagulable status and risk factors is more reliable to detect each of them separately.
Aged ; Blood Coagulation ; Case-Control Studies ; Factor VII ; metabolism ; Female ; Fibrin Fibrinogen Degradation Products ; metabolism ; Humans ; Male ; Middle Aged ; Myocardial Infarction ; blood ; Thromboplastin ; metabolism

OBJECTIVETo compare gene expression profile in K562 cells induced by matrine, so as to screen for differentiation related genes.

METHODSK562 cells were exposed to 0.1 mg/ml matrine for 3 hours, and gene expression profiles in matrine-treated and non-treated cells were studied by cDNA microarray.

RESULTSFrom 8465 screened genes, 30 differentially expressed genes were found. Among them 18 showed decreased expression including oncogene and proto-oncogene, signal transduction gene, DNA binding and transcription gene etc, 12 showed increased expression including cell receptor gene, immune-associated gene, metabolism-associated gene etc in matrine-treated K562 cells as compared with that in non-treated cell.

CONCLUSIONThe genes, that are closely associated with differentiation of can-cer cell, could be the potential targets for cancer treatment.

Alkaloids ; pharmacology ; Cell Differentiation ; drug effects ; genetics ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; K562 Cells ; cytology ; drug effects ; metabolism ; Oligonucleotide Array Sequence Analysis ; Quinolizines ; pharmacology

OBJECTIVETo investigate NK cell cytotoxicity to leukemic cell by NKG2D receptors and NKG2D ligands interaction upregulated by hydroxyurea (HU).

METHODSLeukemic cell lines OUN-1 and primary leukemic cells were cultured for 24 hours in the presence of HU, then the NKG2D ligands expressions were analyzed by flow cytometry (FCM). Isolated NK cells from healthy individual cultured for 72 hours in presence of IL-2 were used as effect cell, and leukemic cell line OUN-1 treated with HU was used as target cell, NK cell cytotoxicity against leukemic cell line was assessed using chromium-51 release assay.

RESULTSLeukemic cell lines showed upregulation of MIC A/B (MFI: 8.9 ± 0.9 vs 23.5 ± 3.4, P = 0.01) and ULBP2 (MFI: 14.5 ± 0.6 vs 33.5 ± 4.8, P = 0.03) following incubation with HU. HU also upregulated the NKG2DLs on primary leukemia cells from patients with acute myeloid leukemia. Treatment of OUN-1 with HU significantly increased the cytotoxicity of NK cells isolated from healthy individual \[(62.0 ± 5.6)% vs (76.0 ± 5.3)%, P = 0.02\], and the enhancing effect of HU was partly blocked by anti-NKG2D Abs \[(76.0 ± 5.3)% vs (46.0 ± 4.5)%, P = 0.00\].

CONCLUSIONHU selectively upregulated NKG2D ligand expression on leukemic cell lines, and enhanced NK cell cytotoxicity against leukemic cells through NKG2D receptors and NKG2D ligands interaction.

Cell Line, Tumor ; Humans ; Hydroxyurea ; pharmacology ; Killer Cells, Natural ; drug effects ; immunology ; Leukemia ; immunology ; Ligands ; NK Cell Lectin-Like Receptor Subfamily K ; immunology