OBJECTIVETo explore the in vitro anti-platelet effects of Ginsenoside -2A,a purified extract from Panax notoginseng.

METHODSPlatelet rich plasma (PRP) was prepared routinely from venous blood samples of patients with essential hypertension and normal persons. PRP was incubated with different concentrations of Nifedipine, Ginsenoside-2A ,and SK&F96365. Maximal platelet aggregation rate[ PAG (M) ] induced by 2 micromol/L ADP was taken as the observed index. Five-minute PAG( M) was determined for 5 consecutive times.

RESULTS(1) PAG (M) in essential hypertension group was 0. 89 +/- 0. 06, which was higher than that in the normal group (0. 68 +/-0. 07 ) with significant difference (P <0.01). (2)Nifedipine of two concentrations (10 p.mol/L,20 pVmol/L) had no effect on PAG(M) in either essential hypertension group or normal group(P >0. 05). (3)Different concentrations of SK&F96365 (2.5 micromol/L,5 micromol/L,10 micromol/L and 20 micromol/L) could inhibit the PAG(M) in essential hypertension group; (4) Differen concentrations of Ginsenoside -2A (2. 5 micromol/L, 5 micromol/L, 10 micromol/L and 20 micromol/L) could inhibit PAG ( M) in essential hypertension group; three concentrations of Ginsenoside -2A (5 micromol/L, 10 micromol/L, 20 micromol/L) could inhibit the PAG(M) in the normal group (all P <0.05).

CONCLUSIONPlatelet aggregating function in essential hypertension patients was obviously higher than that in the normal persons and platelets was in the high reactive status. Nifedipine had no inhibitive effect on platelet aggregation. SK&F96365 could inhibit the platelet aggregation. Ginsenoside-2A could inhibit platelet aggregation, and had the definite anti-platelet action.

Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; Ginsenosides ; administration & dosage ; pharmacology ; Humans ; Imidazoles ; administration & dosage ; pharmacology ; Nifedipine ; administration & dosage ; pharmacology ; Platelet Aggregation Inhibitors ; administration & dosage ; pharmacology

OBJECTIVETo explore the effects of chloride channels on the regulation of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG).

METHODSFreshly separated platelets were activated by thrombin. Chloride channel blockers DIDS or NFA and calcium channel blockers SK&F96365 or nifedipine were added to study the effects on platelet [Ca2+]i and PAG by a single reagent or the combination of reagents and find out the interactions among DIDS, NFA, SK&F96365 and nifedipine.

RESULTSBoth DIDS and NFA could inhibit the thrombin (1 U/ml) induced PAG in a dose-dependent manner, whereas had little effect on resting [Ca2+]i. As compared with the control group, DIDS, SK&F96365 and Nifedipine could significantly reduce the PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05). The combination of DIDS and SK&F96365 had greater effects in reducing the PAG, Ca2+ release and Ca2+ influx than either reagent alone (P < 0.05). The combination of DIDS and nifedipine also had greater effect than each alone in reducing Ca2+ release (P < 0.05). The combination of NFA and SK&F96365 weakened each other's effect on Ca2+ release (P < 0.05), while NFA and nifedipine weakened each other's effects on PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05).

CONCLUSIONDIDS and NFA have no effect on the resting [Ca2+]i and the leak calcium influx of platelet. DIDS can inhibit the Ca2+ release, Ca2+ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca2+ release. The chloride channel and calcium channel blockers have interactions in affecting resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; pharmacology ; Adult ; Blood Platelets ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Cytoplasm ; drug effects ; metabolism ; Drug Interactions ; Humans ; Imidazoles ; pharmacology ; Nifedipine ; pharmacology ; Niflumic Acid ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

OBJECTIVETo investigate the effect of valproic acid on the expression of P27(Kip1) and P170 and drug resistance of leukemia HL60/HT cell line and explore its possible mechanisms.

METHODSHL-60/HT cells were derived from HL-60 cells induced by harringtonine (HT) in gradient concentrations. The inhibitory effect of valproic acid on the proliferation of HL-60 and HL-60/HT cells was evaluated by MTT assay, and the P27(Kip1) expression, P170 expression and cell cycle of the cells were analyzed with flow cytometry.

RESULTSThe multidrug-resistant HL-60/HT was acquired, which showed a stable drug-resistant index with increased IC(50) of HT, VCR, DNR and Ara-c by 9.30, 5.20, 4.91 and 3.65 folds, respectively, as compared with those of HL60 cells. The expression of P27(Kip1) in HL-60/HT cells was significantly lower but P170 expression significantly higher than that of HL-60 cells and normal mononuclear cells (P<0.05). The expressions of P27(Kip1) and P170 showed no significant difference between normal mononuclear cells and HL-60 cells. The growth inhibition rate of VPA combined with Ara-C was significantly higher than that of valproic acid or Ara-C alone in HL-60/HT cells and HL-60 cells (q=1.37 and 1.51, respectively). HL-60/HT and HL-60 cells cultured in the presence of VPA resulted in a significant increase in the expression of P27(Kip1) and the G(1)-phase cells (P<0.05), but the expression of P170 underwent no significant changes (P>0.05).

CONCLUSIONHL-60/HT cells have lower P27(Kip1) expression compared with HL-60 cells. Valproic acid can inhibit the growth of HL-60/HT cells and enhance their Ara-C sensitivity possibly by increasing P27(Kip1) expression and causing cell cycle arrest in G(1) phase.

Antineoplastic Agents ; pharmacology ; Cyclin-Dependent Kinase Inhibitor p27 ; genetics ; metabolism ; Cytarabine ; pharmacology ; Drug Resistance, Multiple ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Glycoproteins ; genetics ; metabolism ; HL-60 Cells ; Humans ; Intracellular Signaling Peptides and Proteins ; genetics ; metabolism ; Valproic Acid ; pharmacology

OBJECTIVETo observe the effects of pravastatin on platelet-derived nitric oxide system in hypercholesterolemia (HC) and atherosclerosis (AS) in rabbits, and the relationship between these changes and atherosclerosis courses.

METHODSThirty male New Zealand white rabbits were randomly divided into three groups, 12 in group A, 12 in group B, and 6 in group C. All of them were fed daily with cholesterol-rich food during the first 12 weeks. In addition, in group A, pravastatin (10 mg) was orally administered daily. At the end of the 12th week, 6 in group A and B were killed randomly and their aortas were removed and the pathologic changes were observed. In the following 12 weeks, food enriched with cholesterol was substituted with normal food in all three groups. Pravastatin treatment was continued or started in the remaining members of group A and group B, but not in group C. At the end 24th week, all rabbits were killed and their aortas were examined for the fatty-streaks or atherosclerotic plaques. The expressions of endothelial NOS (eNOS) mRNA and inducible NOS (iNOS ) mRNA, NOS activity, NO production and the level of the serum lipids were measured at 0, 6th, 12th, 18th and 24th week.

RESULTSThe expression levels of platelet-derived NOS mRNA, eNOS mRNA ratio in group A had no difference at above time points, while in group B were reduced significantly at 6th week and 12th week compared with at 0 week (P <0.01), and increased at 18th week and 24th week compared with 12th week (P <0.05). The expression levels of eNOS mRNA in group C were reduced at 6th, 12th and 18th, 24th week compared with 0 week (P <0.05 and P <0.01, respectively), and were reduced in groups B and C compared with group A at 6th ,12th week (P < 0.05) and increased in group A and B compared with group C at 18th, 24th week (P <0.01). The expression levels of iNOS/mRNA among the three groups had no difference. Pathologic finding of the arteries: AS was not found in group A from the 12th to 24th week. While in group B, there were a lot of fatty-streaks on the entire intima of all large arteries at the 12th week. There were also fatty-streaks in the ascending aorta, but were improved at the 24th week. In group C, there were marked plaques in the entire aorta at the 24th week.

CONCLUSIONSThe expressions of platelet-derived eNOS mRNA, NOS activity, NO production are decreased in HC or AS rabbits. Pravastatin can up-regulate expressions of platelet-derived eNOS mRNA, NOS activity, leading to preventing or improving the pathological courses of AS.

Animals ; Atherosclerosis ; blood ; pathology ; Blood Platelets ; metabolism ; Disease Models, Animal ; Male ; Nitric Oxide ; blood ; genetics ; Nitric Oxide Synthase ; blood ; genetics ; Pravastatin ; pharmacology ; RNA, Messenger ; genetics ; Rabbits

This study was aimed to investigate the effect of MK886, a mPGES-1 inhibitor, on apoptosis and drug resistance of leukemia HL-60/A cell line. Expression of mPGES-1 was assayed by QT-PCR and Western blot. The effect of MK886 on HL-60/A cell proliferation was assayed by CCK-8 method, and flow cytometry was used to detect cell apoptosis. The expression of Akt and P-Akt was detected by Western blot. PGE2 was measured by ELISA. Effect of MK886 (10 µmol/L) on the chemotherapeutic sensitivity of HL-60/A cells and expression of mdr-1 mRNA and P170 protein were investigated too. The results indicated the expression of mPGES-1 was higher in HL-60/A cells. MK886 inhibited HL-60/A cell proliferation and induced apoptosis in a time- and concentration-dependent manner. Expression of mPGES-1 and P-Akt and synthesis of PGE2 decreased significantly. MK886 reduced expression of mdr-1 and P170 protein and enhanced the sensitivity of HL-60/A cells to chemotherapeutic drugs. It is concluded that MK886 can inhibit HL-60/A cell proliferation, induce apoptosis and enhance sensitivity to chemotherapeutic drugs, the mechanism of which possibly associates to down-regulation of mPGES-1/PGE2 synthesis, reduction P-Akt expression and decreasing mdr-1 and P170 protein expression.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Drug Resistance, Neoplasm ; drug effects ; Gene Expression Regulation, Leukemic ; HL-60 Cells ; Humans ; Indoles ; pharmacology

This study was aimed to clarify whether valproic acid (VPA) induces apoptosis of leukemia HL-60 cell line and its possible mechanism. The effect of different concentrations and treatment time of VPA on HL-60 cell proliferation was assayed by cytotoxicity test (CCK-8 method) and fluorescence microscopy, and flow cytometry was used to detect cell apoptosis. The expressions of telomerase subunit h-tert mRNA and apoptosis-related protein as well as caspase-3 activity were detected by real time-quantitative PCR, Western blot and ELISA respectively. The results indicated that VPA inhibited proliferation of HL-60 cells and induced cell apoptosis in a dose dependent manner (r = -0.87). The expressions of anti-apoptotic protein BCL-2 and h-tert mRNA were significantly decreased while the pro-apoptotic protein BAX and caspase-3 activity increased after treatment with VPA. The apoptosis rate of HL-60 cell was negatively correlated with expression of h-tert mRNA. It is concluded that VPA can inhibit leukemia HL-60 cell proliferation and induce apoptosis. The VPA displays anti-leukemia activity possibly through reducing h-tert mRNA and BCL-2 protein expression, increasing BAX expression and activity of caspase-3.
Apoptosis ; drug effects ; Caspase 3 ; metabolism ; HL-60 Cells ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Telomerase ; metabolism ; Valproic Acid ; pharmacology ; bcl-2-Associated X Protein ; metabolism

To observe the expression of cyclin D1, hTERT, and telomerase activity in MNC, HL-60, HL-60A and to explore their effects on leukemogenesis and drug-resistance, normal human peripheral blood mononuclear cells, HL-60 cells sensitive to adriamycin and HL-60A cells resistant to adriamycin were investigated. The cell cycle was analyzed by flow cytometry, and the apoptosis was analyzed by Annexin V-FITC(+) PI staining. Expressions of cyclin D1 and hTERT were determined by real-time PCR and Western blot. Telomerase activity was detected by TRAP-ELISA. The results indicated that the percentage of MNC, HL-60 and HL-60A in S phase was (10.21 + 2.11)%, (44.93 + 3.00)%, and (51.38 + 1.10)% respectively; the percentage of apoptosis cells was (16.14 + 2.13)%, (7.53 + 0.92)%, (4.15 + 0.96)% respectively; the expression of mRNA and protein for cyclin D1 and hTERT increased; the telomerase activities of HL-60 and HL-60A were higher (p = 0.000), whereas the difference between HL-60 and HL-60A was no statistically significant (p = 0.232); positive correlation between cyclin D1, hTERT and telomerase activity had been found (p < 0.01). It is concluded that the cells of S phase increased while the apoptotic cells decreased in HL-60 and HL-60A, especially in HL-60A, which may be due to the up-regulation of cyclin D1, hTERT and telomerase activity.
Cell Cycle ; Cyclin D1 ; metabolism ; HL-60 Cells ; Humans ; Leukemia ; metabolism ; Telomerase ; metabolism

OBJECTIVETo explore the effects of 2A-1-1 (purified component from Panax notoginsengs saponins) on the aggregation of and Ca2+ influx into human platelets.

METHODSThe aggregation of platelets was tested by nephelometry, Fura-2 fluorescent technique was used for detecting cell [Ca2+]i. The effects of 2A-1-1, nifedipine and SK&F96365 on Ca(2+) influx into human platelets induced by ADP or CPA were observed separately.

RESULTSNifedipine (< 20 micromol/L) could not inhibit platelet aggregation induced by ADP or the Ca(2+) influx induced by ADP or CPA. SK&F96365 at 20 micromol/L could inhibit the maximal aggregation of platelets induced by ADP with a inhibitory rate of 59.83%, at 15 micromol/L could inhibit the Ca2+ influx induced by CPA or ADP. 2A-1-1 (5, 10 and 20 micromol/L) could inhibit the maximal aggregation of platelets induced by ADP with the inhibitory rates of 47.06%, 53.47% and 71.52%, respectively. 2A-1-1 at 10 and 20 micromol/L could inhibit the Ca2+ influx induced by CPA or ADP.

CONCLUSIONS2A-1-1 can inhibit platelets aggregation, block the ROC (Receptor-dependent Ca2+ channels) and inhibit Ca2+ influx of human platelets.

Adenosine Diphosphate ; pharmacology ; Adult ; Blood Platelets ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; pharmacokinetics ; Calcium Channel Blockers ; pharmacology ; Dose-Response Relationship, Drug ; Female ; Ginsenosides ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Indoles ; pharmacology ; Male ; Nifedipine ; pharmacology ; Platelet Aggregation ; drug effects ; Platelet Aggregation Inhibitors ; pharmacology

To investigate the effect of a microsomal prostaglandin E synthase-1 (mPGES-1) inhibitor MK886 on cell cycle of the human acute myeloid leukemia HL-60 cells. HL-60 cells were treated with different concentration of MK886 (10, 25, 50 µmol/L) for 24 h. Flow cytometry, Western blot and ELISA were used to measure cell cycle, cyclin D1, mPGES-1, PGE(2), Akt, P-Akt and C-MYC. The results indicated that after treated with MK886, the percentage of HL-60 cells decreased in G(0)/G(1) phase and increased in S phase, and expressions of mPGES-1, cyclin D1, P-Akt and C-MYC and synthesis of PGE(2) decreased significantly. It is concluded that MK886 can arrest HL-60 cells in G(0)/G(1) phase, the mechanism of which is possibly associated to inhibition of mPGES-1 expression, reduction of PGE(2) synthesis, suppression of Akt phosphorylation and C-MYC expression, down-regulation of cyclin D1 expression.
Cell Cycle ; drug effects ; HL-60 Cells ; Humans ; Indoles ; pharmacology ; Intramolecular Oxidoreductases ; antagonists & inhibitors ; Leukemia ; metabolism ; pathology ; Prostaglandin-E Synthases

[Objective]Examine the expression of microsomal prostaglandin E synthase-1(mPGES-1)and nuclear transcription factor kappa B p65(NF-κB p65)in diffuse large B cell lymphoma(DLBCL),assessing their correlation with clinical variables,prognosis and potential clinical valve.[Methods]The immunohistochemistry was uesd to investigate the expression of mPGES-1 and NF-κB p65 in 83 DLBCL patiens'tissues.The relationship between these two proteins and the clinical variables and prognosis of these patients was evaluated.[Results]The high expression of mPGES-1 and NF-κB p65 were observed in 65.1%(54/83)and 73.5%(61/83)cases of DLBCL,respectively.The expression level of NF-κB p65 was positively correlated with mPGES-1 expression(P<0.05).The expression of these two proteins was found to be significantly associated with B cell lymphoma/leukemia-2 protein(BCL-2),higher expression of Ki67,higher lactate dehydrogenase (LDH),more extranodal lesions,advanced Ann Arbor stage and higher international prognostic index(IPI)score(P<0.05). In addition,NF-κB p65 was related with multiple myeloma oncogene 1(MUM1),pathological type(P<0.05). Both mPGES-1 and NF-κB p65 overexpression was correlated with worse overall survival(OS)while NF-κB p65 was an in-dependent prognostic factor for OS of DLBCL(P<0.05).[Conclusions]mPGES-1 and NF-κB p65 were highly expressed in DLBCL and closely linked with each other. The overexpression of mPGES-1 and NF-κB p65 was correlated with tumor progression and unfavorable prognosis in patients with DLBCL.