Beneficial effects and related mechanism of hawthorn leaves flavonoids on cardiac function in rats with acute myocardial infarction / 中华心血管病杂志

Objective: To explore the effect and related regulatory mechanism of hawthorn leaf flavonoids (FHL) on cardiac function in rats with acute myocardial infarction (AMI). Methods: Sixty SPF male Sprague-Dawley rats (9-week-old, weighing 300-350 g) were used in this study. Ten rats were assigned to sham operation group, and the remaining 50 rats were used to establish the AMI model with coronary artery ligation method, AMI was successfully established in 36 rats. AMI rats were randomly divided into AMI group and FHL low-, medium-, and high-dose groups (n=9 for each group). Rats received intraperitoneal injection (10 ml·kg-1·day-1) with physiological saline and FHL solution with concentrations of 0.3, 0.6, and 1.2 mg/ml, respectively for 4 consecutive weeks. Echocardiography was performed at the end of experiments. Left ventricular end diastolic diameter (LVEDD), left ventricular end diastolic anterior wall thickness (LAWD), left ventricular ejection fraction (LVEF) and left ventricular end diastolic pressure (LVEDP) were measured. Then the rats were sacrificed under deep anesthesia, and the left ventricular anterior wall tissue was used for pathological examinations by hematoxylin-eosin (HE) staining. Other myocardial tissue was used for in situ terminal transferase labeling (TUNEL) staining, and the apoptosis rate of cardiomyocytes was calculated. The myocardial cell apoptosis rate, the mRNA, and protein expressions of phosphatidylinositol 3β-kinase (PI3K), protein kinase B (Akt), glycogen synthetase kinase-3 (GSK3β), cyclin D1 and the protein expressions of p-Akt and p-GSK3β were detected by real-time fluorescent quantitative PCR (RT-qPCR) and Western blot respectively. Results: Compared with sham operation group, the LVEDD and LVEDP of the rats in AMI group and FHL low-, medium-and high-dose groups were increased, and the LAWD and LVEF were reduced (all P<0.05). Compared with AMI group, LVEDD and LVEDP were reduced, and LAWD and LVEF were increased in FHL low-, medium-and high-dose groups (all P<0.05). LVEDD and LVEDP decreased, and LAWD and LVEF increased in proportion to the increase of FHL dose (all P<0.05). LVEDD and LAWD values were similar between FHL low-dose and medium-dose groups (both P>0.05). HE staining results evidenced necrotic myocardial tissue, together with disordered arrangement of myocardial fibers, and a large number of inflammatory cells infiltrated in the myocardial tissue in AMI group. The myocardial damage of rats in FHL low-, medium-, and high-dose groups was less than that of AMI group. The myocardial fibers were arranged neatly, but there were still partial breaks and a small amount of inflammatory cell infiltration in the myocardial tissue and there were scattered islands of normal myocardial tissue in the infarct area of these groups. Among them, myocardial damage was the least in FHL high-dose group. The results of TUNEL staining showed that compared with AMI group, the apoptosis rate of myocardial cells was significantly reduced in FHL low-, medium-, and high-dose groups (all P<0.001), but was still higher than that in sham operation group (all P<0.001). Myocardial cell apoptosis rate decreased in proportion with increasing FHL dose (P<0.05). The RT-qPCR results showed that compared with AMI group, the expression levels of PI3K and cyclin D1 mRNA were significantly upregulated in the myocardial tissue of rats in FHL low-, medium-, and high-dose groups, but still lower than those in sham operation group (all P<0.05), and PI3K and cyclin D1 mRNA expression levels increased with the increase dose of FHL (P<0.05). Western blot results showed that compared with AMI group, the expression levels of PI3K, p-Akt, p-GSK3β, and cyclin D1 were significantly upregulated in the myocardial tissue of rats in FHL low-, medium-, and high-dose groups, but still lower than those in sham operation group (all P<0.05), and the protein expression levels of PI3K, p-Akt, p-GSK3β, and cyclin D1 increased in proportion with the increase dose of FHL (all P<0.05). Conclusion: FHL can effectively improve cardiac function in rats with AMI, and the beneficial effects may be partly mediated through activating PI3K/GSK3β/cyclin D1 signaling pathway.

Role of poly (ADP-ribose) polymerase 1 on DNA methylation variation induced by B(a)P in human bronchial epithelial cell / 中华预防医学杂志

<p><b>OBJECTIVE</b>To investigate DNA methylation variation in human cells induces by B(a)P, and to explore the role of PARP1 during this process.</p><p><b>METHODS</b>The changes of DNA methylation of 16HBE and its PARP1-deficient cells exposed to B(a)P (1.0, 2.0, 5.0, 10.0, 15.0, 30.0 µmol/L) were investigated by immunofluorescence and high performance capillary electrophoresis, and simultaneously, the expression level of PARP 1 and DNMT 1 were monitored dynamically.</p><p><b>RESULTS</b>The percentage of methylated DNA of overall genome (mCpG%) in 16HBE and 16HBE-shPARP1 cells were separately (4.04 ± 0.08)% and (9.69 ± 0.50)%. After being treated by 5-DAC for 72 hours, mCpG% decreased to (3.15 ± 0.14)% and (6.07 ± 0.54)%. After both being exposed to B(a)P for 72 hours, the mCpG% in 16HBE group (ascending rank) were separately (5.10 ± 0.13), (4.25 ± 0.10), (3.91 ± 0.10), (4.23 ± 0.27), (3.70 ± 0.15), (3.08 ± 0.07); while the figures in 16HBE-shPARP1 group (ascending rank) were respectively (10.63 ± 0.60), (13.08 ± 0.68), (9.75 ± 0.55), (7.32 ± 0.67), (6.90 ± 0.49) and (6.27 ± 0.21). The difference of the results was statistically significant (F values were 61.67 and 60.91, P < 0.01). For 16HBE group, expression of PARP 1 and DNMT 1 were 141.0%, 158.0%, 167.0%, 239.0%, 149.0%, 82.9% and 108.0%, 117.0%, 125.0%, 162.0%, 275.0%, 233.0% comparing with the control group, whose difference also has statistical significance (t values were 11.45, 17.32, 32.24, 33.44, 20.21 and 9.87, P < 0.01). For 16HBE-shPARP1 group, expression of PARP 1 and DNMT 1 were 169.0%, 217.0%, 259.0%, 323.0%, 321.0%, 256.0% and 86.0%, 135.0%, 151.0%, 180.0%, 229.0%, 186.0% comparing with the control group, with statistical significance (t values were 9.06, 15.92, 22.68, 26.23, 37.19 and 21.15, P < 0.01). When the dose of B(a)P reached 5.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE group (ascending rank) were 125.0%, 162.0%, 275.0%, 233.0% times of it in control group, with statistical significance (t values were 12.74, 24.92, 55.11, 59.07, P < 0.01); while the dose of B(a)P reached 2.0 µmol/L, the mRNA expression of DNMT 1 in 16HBE-shPARP1 group were 135.0%, 151.0%, 180.0%, 229.0%, 186.0% of the results in control group, and the differences were statistically significant (t values were 23.82, 40.17, 32.69, 74.85, 46.76, P < 0.01).</p><p><b>CONCLUSION</b>The hypomethylation of 16HBE cells induced by B(a)P might be one important molecular phenomenon in its malignant transformation process. It suggests that PARP1 could regulate DNA methylation by inhibiting the enzyme activity of DNMT1, and this effect could be alleviated by PARP1-deficiency.</p>

Effect of ZGDHu-1 on proliferation and apoptosis of A549 cells in vitro and antitumor activity in vivo / 药学学报

This study is to explore the mechanism and effect of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation and apoptosis of A549 cells in vitro and on A549 xenograft tumor in nude mice. With different concentrations of ZGDHu-1 at different times were used to treat A549 cells in vitro. The proliferation was determined by living cell count, SRB assay and Brdu-ELISA. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin V/PI and Hoechst 33258 labeling method. The nude mice model of A549 xenograft tumor was established by subcutaneous inoculation. The suppression activity of ZGDHu-1 by intraperitoneal injection on xenograft mice model was detected. The expressions of bcl-2, bax and p53 gene and protein were analyzed by RT-PCR and flow cytometry. ZGDHu-1 can inhibit A549 cell proliferation viability within a certain range of treating time and does, and a majority of A549 cells were arrested in G2-M phase. The A549 cells apoptosis was confirmed by typical cell morphology, DNA fragment, Sub G1 phase, Hoechst 33258 and Annexin V/PI labeling method with a time and dose related manner. When the xenograft tumor mice model were treated with 10, 20 and 40 mg x kg(-1) ZGDHu-1 for 14 days, the tumor growth inhibition rate were 43.7%, 56.9% and 60.0%, respectively. The expression of bax, bax/bcl-2 and p53 gene and protein increased significantly and bcl-2 decreased slightly by the treatment of ZGDHu-1. ZGDHu-1 can significantly suppress the growth of A549 xenograft tumor in vivo and inhibited proliferation by inducing tumor cell apoptosis in vitro. The mechanism may associate with its up-regulation of bax and p53 during the apoptosis process.

ZGDHu-1-inducing apoptosis of SHI-1 leukemia cells and its molecular mechanism / 中国实验血液学杂志

The aim of study was to investigate the mechanism of N, N'-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) inducing apoptosis in SHI-1 human leukemia cell line. Different concentrations of ZGDHu-1 and different times of culture were used to treat SHI-1 cells; the apoptosis of SHI-1 cells was analyzed by morphology, DNA agarose gel electrophoresis, DNA content detection, Annexin-V/PI and Hoechst33258 labeling method, the mitochondrial transmembrane potential (Delta Psi m) were measured by dihydrorhodamin 123, and expressions of bcl-2, bax, Fas, p53 and mitochondrial membrane protein were analyzed by flow cytometry, while the bcl-2, bax and p53 gene were analyzed by RT-PCR. The transcriptional level of hTERT-mRNA was measured by real-time fluorescence quantitative RT-PCR. The results showed that after exposure to ZGDHu-1, SHI-1 cells were induced to apoptosis in a time-and does-dependent manner. SHI-1 cell apoptosis was confirmed by typical cell morphology, DNA fragmentation, sub-G(1) phase, Hoechst33258 and Annexin-V/PI labeling etc. The expression of bax, bax/bcl-2, p53 and Fas gene significantly increased and bcl-2 slightly decreased. ZGDHu-1 could increased the expression of mitochondrial membrane protein in a dose-dependent manner while Delta Psi m reduced. The expression of hTERT-mRNA significantly decreased. It is concluded that ZGDHu-1 can up-regulate the expression of p53, bax and bax/bcl-2. The mitochondrial pathway mediated by descent of mitochondrial transmembrane potential may be one of the mechanisms inducing apoptosis by ZGDHu-1, in which Fas gene also participates. Telomerase may be an effective gene target for anti-tumour effect of ZGDHu-1.

Effects of N, N'-Di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide on proliferation, apoptosis and differentiation of NB4 leukemia cells in vitro / 中国实验血液学杂志

The purpose of this study was to explore the effect of N, N'-di-(m-methylphenyi)-3, 6-dimethyl-1, 4-dihydro-1, 2, 4, 5-tetrazine-1, 4-dicarboamide (ZGDHu-1) on proliferation, differentiation and apoptosis in NB4 human leukemia cell line and its possible mechanism. Different concentrations of ZGDHu-1 and the different time of cultivation were used to treat NB4 cells. The proliferation inhibition of NB4 cells was analysed by cell counting, alive cell count, MTT assay. Cell apoptosis was determined by cell morphology, DNA agarose gel electrophoresis, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. The analysis of cell morphological change, expression of CD11b, CD13 and NBT reduction were performed to evaluate the differentiation of NB4 cells. The expressions of bcl-2, bax and phosphorylated p38MAPK or STAT3 were detected by flow cytometry. While the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that ZGDHu-1 could inhibit NB4 cell proliferation viability within a certain range of treating time and does, IC(50) values at 48 and 72 hours were 450 ng/ml and 200 ng/ml respectively. A majority of NB4 cells were arrested in G(2/M) phase and a progressive decline of cells was seen in G(0/1). The NB4 cells apoptosis was confirmed by cell typical cell morphology, DNA fragments and sub-G(1) phase peak as well as Hoechst33258 and Annexin-V/PI labeling method with a time-dose-related manner. The morphology of NB4 cells cultured in the presence of 2 - 100 ng/ml ZGDHu-1 for three days was more mature with higher NBT positivity and expressions of CD11b and CD13 than those in control. The expression of phosphor-p38MAPK and bax was increased while phosphor-STAT3 and bcl-2 were unchanged by the treatment of ZGDHu-1. ZGDHu-1 could decrease the expression of hTERT-mRNA in a dose-dependent manner. It is concluded that ZGDHu-1 can inhibit proliferation, induce differentiation and apoptosis of NB4 cells. The mechanism may be associated with up-regulation of bax expression, enhancement of phosphor-p38MAPK activation and inhibition of hTERT-mRNA.

Effects of N, N-di-(m-methylphenyl)-3, 6-dimethyl-1, 4-dihydro-1,2,4, 5-tetrazine-1,4-dicarboxamide (ZGDhu-1) on SHI-1 leukemia cells in vitro / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of ZGDHu-1 on proliferation, differentiation and apoptosis in SHI-1 human leukemia cell line and explore its possible mechanism. Methods SHI-1 cells were cultured with different concentration of ZGDHu-1 and for different time. The cell proliferation was analysed by cell counting, alive cell count, MTT assay and Brdu-ELISA. Cell apoptosis was analysed by morphology, DNA content, Annexin-V/PI and Hoechst 33258 labeling method. Cell differentiation were assayed by morphology,expression of CD11b,CD14 and CD64 and NBT reduction. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry.</p><p><b>RESULTS</b>ZGDHu-1 inhibited SHI-1 cell proliferation in a time and dose dependent manner, the IC50- 48 h and IC50- 72 h were 250 ng/ml and 85 ng/ml, respectively. The majority of SHI-1 cells were arrested in G2/M phase. 48h after treated with 200 ng/ml ZGDHu-1, and those in G2/M phase accounted for (48.4 +/- 2.1)%. The SHI-1 cells apoptosis was increased with a time- and does-dependent manner. The morphology of SHI-1 cells cultured with 2-50 ng/ml ZGDHu-1 for three days become more mature with higher NBT positivity and up-regulated expressions of CD11b,CD14 and CD64. The expression of phosphor-p38MAPK was increased and phosphor-STAT3 down-regulated by the treatment of ZGDHu-1.</p><p><b>CONCLUSION</b>ZGDHu-1 can inhibit SHI-1 cell proliferation and induce the cell differentiation and apoptosis. The mechanism may associate with its up-regulation of phosphor-p38MAPK and down-regulation phosphor-STAT3.</p>

Effects of sodium nitroprusside on P38MAPK/STAT3 activation and telomerase reverse transcriptase mRNA expression in inducing apoptosis of K562 cell line / 中国实验血液学杂志

This study was aimed to investigate the activation of P38MAPK/STAT3 and expression of telomerase reverse transcriptase during sodium nitroprusside (SNP) inducing apoptosis of human leukemia cell line K562 and to explore the molecular mechanisms of SNP-inducing apoptosis in K562 cells. The K562 cell were treated with different concentrations of SNP and were cultured for different time. Cell apoptosis was analysed by cell morphology, DNA agarose gel electrophoresis, DNA content, and Annexin-V/PI labeling method. The TdT-mediated dUTP nick end labeling (TUNEL) assay was used to quantitate the in situ cell apoptosis. The expressions of phosphorylated p38MAPK or STAT3 were analysed by flow cytometry, while the expression of hTERT mRNA in transcriptional level was measured by fluorescence quantitative RT-PCR. The results showed that SNP inhibited K562 cell growth. The K562 cell apoptosis was confirmed by typical cell morphology and DNA fragment, peak of sub-G1 phase, TUNEL and Annexin-V/PI labeling. A majority of K562 cells were arrested in G0/G1 phase. After treatment with SNP at 0.5-3.0 mmol/L, the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 increased first and decreased afterwards. Incubation of K562 cell with SNP (2 mmol/L) could increase the expression of phosphorylated-P38MAPK and phosphorylated-STAT3 at 12 hours and 24 hours respectively, and down-regulated at 72 hours and 48 hours. SNP could decrease the expression of hTERT-mRNA in time-and dose-dependent manner. It is concluded that SNP can significantly induce K562 cells apoptosis, its mechanism may be related to the activation of P38MAPK and suppression of phosphorylated-STAT3 and hTRET-mRNA.

Study on Immune Efficacy of Single and Double Fusion DNA Vaccine from Mycobacterium bovis / 中国生物工程杂志

The DNA fragments of ag85b、esat-6、hsp65、mpb64 and ag85b-esat-6、hsp65-esat-6、mpb64-esat-6 were amplified by PCR and SOE technique.These seven fragments were inserted into pCDNA3.1(+)vector to construct recombinant plasmids pCA、pCE6、pCH、pCM、pCAE、pCHE and pCME.The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of target genes.BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vector pCDNA3.1(+)and PBS respectively.The serum antibodies and the spleen lymphocyte proliferation(SLP)and secreted IFN～? of spleen were tested.The results of indirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantly higher than the two control groups(P

Isolation and Resistance of Acinetobacter calcoaceticus-baumannii in Hospital: A 7-Year Surveillance Study / 中华医院感染学杂志

OBJECTIVE To study the isolation and resistance tendency of Acinetobacter calcoaceticus-baumannii to antimicrobial agents from 1998 to 2004 to provide valuable data for infection prevention and therapy. METHODS We reviewed the isolation rates,distribution in clinical specimens and wards,and the resistance rates of(A.calcoaceticus-baumannii)to 14 kinds of antimicrobial agents from 1998 to 2004. RESULTS There was an increasing tendency of isolation rates of A.calcoaceticus-baumannii every year,which was 0.18% in 1998 but 1.48% in 2004.In the seven years,there was the highest isolation rate of 70.58% in specimens from respiratory tract,the next was from the urine(9.42%),and blood(4.63%).Concerning the wards distribution,ICU had the highest rate of 47.28%.In 1998,A.calcoaceticus-baumannii had resistance rates more than 50% only to one kind of antimicrobial agents(aztreonam),but in 2004,it had increased to thirteen kinds(except cefoperazone/sulbactam).About the fourteen kinds of antimicrobial agents we inspected,that were increased in their resistance rate.The highest increasing of resistance rate was ceftazidime from 11.1% in 1998 to 88.9% in 2004,the imipenem was second for 0.0% to 64.8%,and the third was sulfamethoxazole/trimethoprim form 0.0% to 64.0%,while there still was an increasing resistance tendency to them. CONCLUSIONS The clinical isolation rate of A.calcoaceticus-baumannii is increasing,and it has higher resistance rates to many antimicrobial agents as well as an increasing resistance tendency to relatively susceptive antimicrobial agents every year.So physicians should prescribe on the basis of antimicrobial agents susceptibility tests in vitro.

Comparison of long- term efficacy between preoperative radiotherapy or radiochemotherapy followed by lower-anterior resection and Miles abdominoperineal resection in patients with low rectal cancer / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To compare the long-term efficiency between preoperative radiotherapy or radiochemotherapy followed by lower-anterior resection and abdominoperineal resection (APR) for lower and locally advanced rectal cancer.</p><p><b>METHODS</b>From January 1983 to December 2000, 157 consecutive patients suffering from lower rectal cancer were enrolled in the study, which included 69 cases of clinical stage II and 88 cases of stage III respectively. All patients were divided in to three groups. Patients in group A (n=52) received preoperative radiotherapy with a total dose of 35-45 Gy within 4-5 weeks plus preoperative chemotherapy with 5-fluorouracil (5- FU) followed by lower-anterior resection; patients in group B (n=51) received radiotherapy followed by lower-anterior resection; patients in group C (n=54) received APR only. Clinical data of all patients were reviewed retrospectively.</p><p><b>RESULTS</b>The follow-up rate was 91.7%. The 5- year survival rate was higher in group A (71.1%) than those in group B (47.1%) and group C (42.6%)(P< 0.05). The tumor- free survival rate was higher in group A (61.5%) than those in group B (37.3%) and group C (35.2%)(P< 0.05). The local recurrence rate was 13.5%, 15.7% and 11.1% in group A, B and C respectively, there was no significant difference in recurrence rate among three groups (P> 0.05). The distant metastasis rate was lower in group A (23.1%) than those in group B (49.0%) and group C (46.3%)(P< 0.05), but there was no significant difference in distant metastasis rate between group B and group C.</p><p><b>CONCLUSIONS</b>The combined preoperative radiochemotherapy followed by lower-anterior resection can improve the 5-year survival rate and tumor-free survival rate, and decrease distal metastasis rate.</p>

Change of Bcl-2, Bax proteins and mitochondrial membrane protein on nitric oxide induced apoptosis in HL-60 cells / 中国实验血液学杂志

To investigate the possible mechanisms of nitric oxide (NO)-induced apoptosis in leukemia cell line HL-60, HL-60 cells in vitro were incubated with sodium nitroprusside (SNP), the in situ cell apoptosis quantitatively was assayed by TdT-mediated dUTP nick end labeling (TUNEL), the cell cycle DNA and proteins expression of Bcl-2, Bax, mitochondrial membrane protein (APO2.7) were analyzed by flow cytometry. The results showed that SNP induced HL-60 cell apoptosis in a dosage- and time-dependent manner. After exposure to SNP at the concentration of 1.0 mmol/L for 48 hours, the percentage of apoptosis HL-60 was (42.2 +/- 3.5)% for subG1 and (52.5 +/- 7.6)% for TUNEL respectively, and they are significantly higher than those in control and potassium ferricyanide (PFC) groups as same concentration. During the apoptosis process, it showed a decrease of Bcl-2 protein and an increase of Bax protein and mitochondrial membrane protein in HL-60 cell, proteins of Bcl-2, Bax and mitochondrial membrane were expressed in a dosage- and time-dependent manner too. In conclusion, during the process of SNP induced apoptosis in HL-60 cell, the expression of mitochondrial membrane protein was increased, Bcl-2 and Bax proteins may be important regulators.