Objective To detect the effect of osthole on proliferation and apoptosis of HL-60 cells and its molecular mechanism.Methods HL-60 cells proliferation was measured through the CCK8 assay method.The cell morphological changes were observed by Hoechst33342 staining after 8 h of drug effect.Induction of apoptosis was determined by flow cytometry and fluorescent microscopy.Expressions of Bcl-2 and Bax mRNA were evaluated by RT-PCR,and the expressions of cleaved caspase-3,caspase-8,caspase-9,Fas and FasL were evaluated by using western bolt assay.Results Osthole could inhibit the proliferation of HL-60 cells,the maximum inhibiting rate was (90.7 ±4.5)％,F =138.46,P =0.000; the apoptosis rate was 33.6％,F =27.75,P =0.006.The changes of apoptosis of cells and nucleus were shown in cell morphological observation.Osthole affected the decrease of the mRNA levels of Bcl-2 and the increase of the Bax mRNA levels via a dosedependent manner(F =210.12,P =0.000).Western blotting demonstrated that osthole could lead to the increase of the expression levels of cleaved caspase-3,caspase-8,caspase-9,Fas and FasL in the HL-60 cell line via a time-dependent manner.Conclusion Data suggests that osthole inhibits proliferation and induces apoptosis of HL-60 cells through mitochondria-dependent pathway and death-receptor pathway.

Objective To study the molecular regulation mechanism of VEGF in the model of ATRA induced differentiation in HL-60 cells,and to provide new targets for leukemia anti-angiogenic therapy.Methods The morphology was observed by Wright-Gimesa staining; HL-60 cells differentiation was detected by NBT reduction experiment.VEGF,STAT3,c-myc mRNAs were measured by reverse transcription-PCR;VEGF,STAT3 and c-myc proteins were determined by Western blot.Results The proliferation of HL-60 cells was inhibited obviously by ATRA(1 μmol/L) with the induction of differentiation,NBT positive rate was 82.59％ (t =-24.157,P ＜ 0.01) ; VEGF mRNA (t =7.339,P ＜ 0.05),STAT3 mRNA (t =3.667,P ＜0.05) and c-myc mRNA (t =6.858,P ＜ 0.05) were all down-regulated.VEGF protein (t =3.386,P ＜0.05),STAT3 protein(t =4.074,P ＜ 0.05) and c-myc protein (t =3.333,P ＜ 0.05) were all down-regulated.Conclusion VEGF expression level is reduced with the procession of differentiation of HL-60 cells,which may be largely correlated with the down regulation of STAT3 and c-myc.

Objective To explore the effect of vascular endothelial growth factor antisense oligonucleotides(AS-VEGF)on HL60 cell and HL60/VCR multidrug resistance cell and analyze the function of P-gp and the expression of related multidrug resistance genes including Bcl-2,Mcl-1,MDR1,MRP,GST?,TopoⅡ? and TopoⅡ?.Methods A vector AS-VEGF which expressed in eukaryotic cell was established,then transfected the vector into HL60 and HL60/VCR by limposome transfection technology,observed and drew the growth curve by Tapanlan taining,RT-PCR was used to detect the expression of Bcl-2,Mcl-1,MDR1,MRP,GST?,TopoⅡ? and TopoⅡ? in mRNA level after transfected 24 h and 48 h.Western Blot was used to detect the expression of P-gp in proteinum level after transfected 24 h and 48 h.Results The growth of HL60 and HL60/VCR was inhibited by AS-VEGF(1.25 mmol/L).Between HL60 and HL60/VCR,AS-VEGF decreased the expression of MDR1,MRP,GST? and TopoⅡ? but could not influence the expression of Bcl-2,Mcl-1 and TopoⅡ?,and the expression of P-gp was also obviously decreased in 48 h compared with that in 24 h.Conclusions AS-VEGF can inhibite the growth of HL60 and HL60/VCR and reverse multidrug resistance by changing cell microenvironment and the cell membrane correlated protein transportating channel,reduce the cell disintoxicating and the self-repair ability.

Objective To study the molecular mechanism of different sensitivities to apoptosis induced by low concentration of As2O3 in PML-RARα negative HL-60 cells and PML-RARα positive NB4 cells. Meth-ods NB4 and HL-60 cells were cultured with As2O3 for 1 to 4 days; cell proliferation were detected by MTT method; the apoptosis was detected by flow cytometry,Bcl-2,Bax and Fas mRNA were determined by RT-PCR. Results The proliferation of NB4 cells was inhibited obviously by As2O3(1.0 μmol/L)with the induction of apoptosis( P <0.05) ,which was accompanied by the down-regulation of Bcl-2 mRNA expression( P <0.05)and the ratio of Bcl-2/Bax(P <0.05), but there was no obvious variation of Bax and Fas expression( P >0.05). Inhibition of proliferation and apoptosis were not obvious in PML-RARα negative HL-60 cells induced by low concentration As2O3 ( P >0.05), and there was no obvious variation of Bcl-2, Bax, Fas mRNA expres-sion or Bcl/Bax ratio( P >0.05). Conclusion The ratio of Bcl-2/Bax is contributed to the different sensitiv-ities of PML-RARα negative HL-60 cells and positive NB4 cells induced by low concentration of As2O3.

AIMTo investigate the changes in the expression of four kinds of calcium regulatory proteins mRNA on the isolated ischemia/ reperfusion (IR) hearts.

METHODSThe rat hearts were divided into two groups: control group and IR group which received 45 min exposure to Krebs-Henseleit solution after 15 min zero-flow global ischemia. The indexes of left ventricular function, such as LVDP, +dp/dt(max), -dp/dt(max), and an arrhythmia scoring system were compared between the two groups. The messenger ribonucleic acid (mRNA) amount of sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA), phospholamban (PLB), inositol 1,4,5-trisphosphate receptor2 (IP3R2) and ryanodine receptor2 (RyR2) was measured by reverse transcription-polymerase chain reaction (RT-PCR) and normalized to the mRNA levels of beta-actin.

RESULTSIn the IR group, LVDP, +dp/dt(max) and -dp/dt(min) of the isolated hearts were depressed and the high rate of arrhythmias occurred during reperfusion. The levels of SERCA, IP3R2, RyR2 mRNA were lower in the IR isolated hearts group than those in the control group, while there was no difference in the level of phospholamban.

CONCLUSIONThese data suggest that myocardial ischemia/reperfusion can induce the depression of cardiac performance and an increased risk of arrhythmias, concomitant with the decrease in SERCA, IP3R2, RyR2 mRNA steady state levels.

Animals ; Myocardial Reperfusion Injury ; metabolism ; Myocardium ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Sarcoplasmic Reticulum Calcium-Transporting ATPases ; genetics ; metabolism

OBJECTIVETo investigate the effect of Astragalus membranaceus(AM) on vascular circles and the underlying mechanisms.

METHODSThe study was performed with the model of isolate rat thoracic aorta rings in organ bath. When the endothelium of rat thoracic aorta was removed,the effect of accumulated AM on aorta rings in resting tension, or pre-constricted with KCl, or pre-constricted with phenylephrine (PE) was observed. And to explove the mechanism, the aorta rings were incubated with Ca(2+)-free medium alone, or Ca(2+)-free medium plus heparin, or propranolol alone before pre-contraction with PE.

RESULTSAM had no significant effects on aorta rings in resting tension or pre-constricted with KCl. When the concentration of AM was cumulated to 10(-1), 3 x 10(-1),10(0), 3 x 10(0) g/L, it caused concentration-dependent relaxation while aorta rings were pre-constricted with PE(3 x 10(-7)mol/L), compared with the control [(90.4 +/-4.2)% compared with (94.7 +/-2.4)%,(86.1 +/-5.0)% compared with (92.6 +/-3.2)%, (82.3 +/-5.9)% compared with (90.4 +/-3.6) %, (78.3 +/-6.0)% compared with (88.1 +/-4.0)%]. This effect was not inhibited by Ca(2+)-free medium or propranolol alone. However, the effect was attenuated by the co-incubation with heparin and Ca(2+)-free medium [without heparin:(76.2+/-4.3)% compared with (92.3 +/-5.9)%, with heparin: (95.3+/-0.5)% compared with (95.1+/-0.6)%].

CONCLUSIONThe results indicate that AM can relax the rat thoracic aorta rings without endothelium. The mechanism may include the inhibition of intracellular calcium ions release by the 1,4,5-triphosphate inositol-receptor-dependent pathway in vascular smooth muscle cells.

Animals ; Aorta, Thoracic ; cytology ; Astragalus membranaceus ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; In Vitro Techniques ; Male ; Muscle, Smooth, Vascular ; cytology ; Phosphatidylinositols ; metabolism ; Rats ; Rats, Sprague-Dawley ; Vasodilator Agents ; pharmacology

Objective To investigate the clinical efficacy of herbal cake moxibustion plus acupuncture in treating post-stroke urinary incontinence.Methods Fifty patients with post-stroke urinary incontinence were randomized to a treatment group (26 cases) and a control groups (24 cases). The treatment group received herbal cake moxibustion plus acupuncture and the control group, acupuncture alone. Twenty-four-hour urine void number, incontinence severity and the clinical symptom score were recorded in the two groups before and after treatment.Results There were statistically significant pre-/post-treatment differences in twenty-four-hour urine void number and the clinical symptom score in the two groups (P<0.05). There were statistically significant differences in pre-/post-treatment twenty-four- hour urine void number difference value and clinical symptom score difference value between the treatment and control groups (P<0.05). There was a statistically significant pre-/post-treatment difference in incontinence severity in the two groups (P<0.05). There was a statistically significant post-treatment difference in incontinence severity between the treatment and control groups (P<0.05).Conclusion Herbal cake moxibustion plus acupuncture is an effective way to treat post-stroke urinary incontinence.

This paper aims to investigate if the dental restoration of nickel-chromium based alloy (Ni-Cr) leads to the enhanced excretions of Ni and Cr in urine. Seven hundred and ninety-five patients in a dental hospital had single or multiple Ni-Cr alloy restoration recently and 198 controls were recruited to collect information on dental restoration by questionnaire and clinical examination. Urinary concentrations of Ni and Cr from each subject were measure by graphite furnace atomic absorption spectrometry. Compared to the control group, the urinary level of Ni was significantly higher in the patient group of <1 month of the restoration duration, among which higher Ni excretions were found in those with either a higher number of teeth replaced by dental alloys or a higher index of metal crown not covered with the porcelain. Urinary levels of Cr were significantly higher in the three patient groups of <1, 1 to <3 and 3 to <6 months, especially in those with a higher metal crown exposure index. Linear curve estimations showed better relationships between urinary Ni and Cr in patients within 6-month groups. Our data suggested significant increased excretions of urinary Ni and Cr after dental restoration. Potential short- and long-term effects of Ni-Cr alloy restoration need to be investigated.
Adult ; Chromium ; urine ; Chromium Alloys ; chemistry ; Creatinine ; urine ; Crowns ; Dental Porcelain ; chemistry ; Female ; Humans ; Male ; Metal Ceramic Alloys ; chemistry ; Middle Aged ; Nickel ; urine ; Spectrophotometry, Atomic ; Surface Properties ; Time Factors

BACKGROUNDDrug susceptibility assay is very important in tuberculosis therapy. Pyrazinamide is a first line antituberculosis drug and diagnosis of its resistance in Mycobacterium tuberculosis (M. tuberculosis) is difficult and time consuming by conventional methods. In this study, we aimed to evaluate the performance of the microscopic observation drug susceptibility (MODS) assay in the detection of pyrazinamide resistance in M. tuberculosis relative to the conventional Wayne assay and Lowenstein-Jensen (LJ) proportion method.

METHODSM. tuberculosis clinical isolates (n = 132) were tested by the MODS and the Wayne assay: the results were compared with those obtained by the LJ proportion method. Mutations in the gene were identified by direct sequencing of the pncA genes of all isolates in which pyrazinamide resistance was detected by any of the three methods.

RESULTSCompared to the LJ results, the sensitivity and specificity of the MODS assay were 97.8% and 96.5% respectively; the sensitivity and specificity of the Wayne assay were 87.0% and 97.7% respectively. Mutations in the pncA gene were found in 41 of 46 strains that were pyrazinamide resistant (3 tests), in 1 of the 4 strains (LJ only), in 42 of 48 strains (at least 1 test), but no mutations in 1 strain sensitive according to the MODS assay only. The MODS assay, Wayne assay and LJ proportion method provided results in a median time of 6, 7 and 26 days respectively.

CONCLUSIONSMODS assay offers a rapid, simple and reliable method for the detection of pyrazinamide resistance in M. tuberculosis and is an optimal alternative method in resource limited countries.

Antitubercular Agents ; pharmacology ; Microbial Sensitivity Tests ; Microscopy ; methods ; Mycobacterium tuberculosis ; drug effects ; Pyrazinamide ; pharmacology

Severe fever with thrombocytopenia syndrome bunyavirus is a newly emerging virus in China, enveloped with a tripartite, single-stranded RNA genome of negative polarity. The regulatory elements for viral transcription and replication, as well as encapsidation and packaging signals, are thought to be located within these noncoding regions (NCRs). The terminal nucleotides are genus specific and highly conserved. The function of the remaining nucleotides of the NCRs is still not well understood. In this study, we developed the plasmid-driven RNA polymerase I minireplicon system for SFTSV firstly, using reporter genes GFP and luciferase. The function of the noncoding regions of the three Bunyaviridae RNA segments (L, M, S) in transcription was analyzed. Reporter genes are successfully expressed in SFTSV minireplicon system. Our results suggest that the NCRs of SFTSV from all three segments contain the necessary signals to initiate transcription. Quantitative detection of the luciferase expression level shows that promoter activity in the three segments is different.
Bunyaviridae Infections ; virology ; Cloning, Molecular ; Genome, Viral ; Humans ; Phlebovirus ; genetics ; physiology ; Replicon ; Viral Proteins ; genetics ; metabolism ; Virus Replication