To observe the effect of ginsenoside Re on cardiomyocyte apoptosis and Bcl-2/Bax gene expression after ischemia (30 min) and reperfusion (6 h) in rats and to elucidate the possible mechanisms of ginsenoside Re on inhibition of cardiomyocyte apoptosis, the ischemia/reperfusion heart model was established by ligating the left anterior descending branch of coronary artery in Wistar rats. The apoptotic cardiomyocytes were confirmed by transmission electron microscopy and counted by in situ nick end labeling (TUNEL) method and light microscopy. The mRNA and protein expression of Bcl-2 and Bax genes were studied by in situ hybridization and immunohistochemical staining. Mean optical density (OD) value of the positive fields of mRNA and protein expression was quantitatively examined by image analysis system. The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischemia/reperfusion group and weren't observed in the sham-operation group by transmission electron microscopy; (2) The numbers of the apoptotic cells were 134.45 +/- 45.61/field in the ischemia/reperfusion group, and 90.66 +/- 19.22/field in the ginsenoside Re-treated group. The differences was significant between two groups (P < 0.01); (3) Gene expression of Bcl-2 and Bax were increased significantly in the ischemia/reperfusion group and ginsenoside Re-treated group when compared with the sham-operation group. There was no significant difference in the gene expression of Bcl-2 between the ginsenoside Re-treated group and ischemia/reperfusion group (P > 0.05), but gene expression of Bax was decreased significantly in the ginsenoside Re-treated group as compared with the ischemia/reperfusion group (P < 0.01). The ratio of Bcl-2/Bax was increased significantly in the ginsenoside Re-treated group when compared with the ischemia/reperfusion group and sham-operation group. These findings suggest that myocardial ischemia-reperfusion can induce cardiomyocyte apoptosis, and ginsenoside Re can significantly inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion in rats. It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by inhibiting expression of pro-apoptotic Bax gene and raising the ratio of Bcl-2/Bax.
Animals ; Apoptosis ; drug effects ; Female ; Ginsenosides ; pharmacology ; Male ; Myocardial Reperfusion Injury ; genetics ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Panax ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; bcl-2-Associated X Protein

OBJECTIVETo study the relationship between the expression of Bax, Bcl-2 proteins, and apoptosis in radiation compound wound healing of rats.

METHODSApoptosis, Bax and Bcl-2 proteins were estimated by in situ terminal labeling (TUNEL) and immunohistochemical methods.

RESULTS(1) Changes of the apoptosis in wound healing showed three typical characteristics: early occurrence, high frequency and delayed disappearance after radiation to rats when compared with those of simple wound group, which might be an important reason for radiation-induced delayed wound healing. (2) The expression of Bax protein increased evidently with the increment of apoptosis and showed a good corresponding relationship with the apoptotic frequency in the process of wound healing. While the expression of Bcl-2 protein decreased obviously as the apoptosis reached a maximum and showed increasing tendency up to normal level when the apoptosis decreased distinctively.

CONCLUSIONSBax and Bcl-2 proteins play an important role in the apoptotic regulation of radiation compound wound healing in rats.

Animals ; Apoptosis ; radiation effects ; Female ; Gamma Rays ; Immunohistochemistry ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Skin ; pathology ; radiation effects ; Wound Healing ; genetics ; radiation effects ; bcl-2-Associated X Protein

OBJECTIVESTo investigate the relationship between germ cell apoptosis and expression of Bcl-2 and Bax in experimental torsed/detorsed testes of the adult male rats.

METHODSThirty healthy male Sprague-Dawley rats were divided into torsion group (n = 15) and control group (n = 15) randomly. Animals were submitted to unilateral 720 testicular torsion, then detorsion were done in two hours. Three days later, the evidence of germ cell apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) technique. The expression of Bcl-2 and Bax were detected by immunohistochemical method.

RESULTSIn the torsed testes, the apoptosis index of germ cell and Bax expression significantly increased compared with that in the control group (P < 0.01) while Bcl-2 expression obviously decreased (P < 0.01). The apoptotic cells were mostly pechytene spermatocytes and round spermatides.

CONCLUSIONSThe germ cell apoptosis is highly associated with expression of Bcl-2 and Bax in experimental testicular torsion. Bcl-2/Bax plays an important role in germ cell apoptosis.

Animals ; Apoptosis ; Gene Expression ; Germ Cells ; pathology ; Male ; Proto-Oncogene Proteins ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; Rats ; Rats, Sprague-Dawley ; Spermatic Cord Torsion ; metabolism ; pathology ; bcl-2-Associated X Protein

To investigate whether the Bcl-2 gene family is involved in modulating mechanism of apoptosis and change of cell cycle protein induced by curcumin in acute myeloid leukemia HL-60 cell line and primary acute myelogenous leukemic cells, the Bcl-2 family member Mcl-1, Bax and Bak and cell cycle proteins including P27kipl, P21wafl, cyclin D3 and pRbp- were selected and their expression detected by SABC immuno-histochemical stain method. The attitude of sub-G1 peak in DNA histogram was determined by FCM. The TUNEL positive cell percentage was identified by terminal deoxynucleotidyl transferase (TdT)-mediated Biotin dUNP end labeling technique. It was found that when HL-60 cells were treated with 25 mumol/L curcumin for 24 h, the expression level of Mcl-1 was down-regulated, but that of Bax and Bak up-regulated time-dependently. There was significant difference in the expression level of Mcl-1, Bax and Bak between the curcumin-treated groups and control group (P < 0.05-0.01). At the same time, curcumin had no effect on progress of cell cycle in primaty acute myelogenous leukemia at newly diagnosis, but could increase the peak of Sub-G1 (P < 0.05), and down-regulate the expression of Mcl-1 and up-regulate the expression of Bax and Bak with the difference being statistically significant. The expression of P27kipl, P21wafl and pRbp- were elevated and that of cyclin D3 decreased in the presence of curcumin. These findings suggested that the Bcl-2 gene family indeed participated in the regulatory process of apoptosis induced by curcumin in HL-60 cells and AML cells. Curcumin can induce apoptosis of primary acute myelogenous leukemic cells and disturb cell cycle progression of HL-60 cells. The mechanism appeared to be mediated by perturbing G0/G1 phases checkpoints which associated with up-regulation of P27kipl, P21wafl and pRbp- expression, and down-regulation of cyclin D3.
Antineoplastic Agents, Phytogenic/pharmacology ; Apoptosis/*drug effects ; Cell Cycle Proteins/*metabolism ; Curcumin/*pharmacology ; Genes, bcl-2/genetics ; HL-60 Cells ; Leukemia, Myelocytic, Acute/pathology ; Neoplasm Proteins/biosynthesis ; Neoplasm Proteins/genetics ; Proto-Oncogene Proteins/biosynthesis ; Proto-Oncogene Proteins/genetics ; Proto-Oncogene Proteins c-bcl-2/*metabolism ; Tumor Cells, Cultured ; bcl-2-Associated X Protein

The study was purposed to explore the effect of adriamycin (ADM) on K562 cells in vitro and the mechanism of expression changes of relevant apoptotic genes and oncogene Gfi-1. The apoptosis was assayed by flow cytometry (FCM) and the DNA electrophoresis; the expression changes of Gfi-1, Bcl-2, bax mRNA and protein were detected by RT-PCR and FCM after K562 cells were treated with different concentrations of ADM for 24 hours. The results showed that when K562 cells were treated with 0 - 2.0 mg/L ADM for 24 hours, the typical apoptotic DNA electrophoresis band of K562 cells were observed with the dose increasing. When concentration of ADM was 0.5 and 2.0 mg/L, the expression of Gfi-1 decreased and the expression of bax increased; when concentration of ADM was 0.5 - 2.0 mg/L, the expression of Bcl-2 was not found to be significantly changed, the levels of Bcl-2 mRNA and protein were of no statistical difference. When dose of ADM was higher than 2.0 mg/L, the percentage of apoptotic K562 cells decreased with cell necrosis. It is concluded that at certain range of concentration, apoptosis or necrosis of K562 cells can be induced by ADM, the percentage of apoptosis, the changes of expression of Bcl-2, bax and Gfi-1 depend on the dose of ADM. The mechanism of apoptosis in K562 cells induced by ADM may be related to suppression of Gfi-1 oncogene and activation of expression of bax gene.
Antibiotics, Antineoplastic ; pharmacology ; Apoptosis ; drug effects ; DNA-Binding Proteins ; biosynthesis ; genetics ; Doxorubicin ; pharmacology ; Humans ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; biosynthesis ; genetics ; Transcription Factors ; biosynthesis ; genetics ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the effects of long-term estrogen replacement treatment (ERT) on the expression of bcl-2 and H-ras in rat endometrium. METHODS: Thirty 5-month-old SD female rats were randomly divided into 3 groups: Control group ( sham operated and vehicle injected, n 10) , OVX group (OVX operated and vehicle injected, n = 10) , and ERT group (OVX operated and 17 beta-estradiol injected, n = 10). The rats were killed in the 13th week and the uteri were isolated and weighed, pathologically analyzed, and we measured the thickness of the endometrium. Immunochemistry and in situ hybridization analysis were used to examine the changes of bcl-2 and H-ras mRNA and Bcl and H-ras protein expression in the endometrium of the rats. RESULTS: Uterine weight and endometrial thickness of OVX decreased much more than those of the control (P <0.01 ) and ERT rats (P < 0.01). One simple hyperplasia and one squamous metaplasia of endometrium were found in ERT rats. Quantitatively, bcl-2 and H-ras mRNA and Bcl and H-ras protein level of ERT were higher than those of OVX rats (P < 0.01 ), and there were no statistical significances between the ERT group and the control rats. CONCLUSION Long-term estrogen replacement can keep the endometrium from atrophy, and lead to the genesis of simple hyperplasia and squamous metaplasia of the endometriun, which can increase the risk of endometrial carcinomas. Estrogen may inhibit apoptosis and promote the proliferation of endometrial cells through increasing the expression of bcl-2 and H-ras mRNA and Bcl-H-ras proteins.
Animals ; Endometrium ; metabolism ; Estradiol ; pharmacology ; Estrogen Replacement Therapy ; Female ; Genes, bcl-2 ; Genes, ras ; Ovariectomy ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Time Factors ; ras Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate the toxicological mechanism of microcystin-LR (MCLR) on L-02 cells.

METHODSL-02 cells was treated with MCLR at different concentrations and the subsequent changes such as cell proliferation (MTT assay), morphology, lactate dehydrogenase (LDH) leakage, apoptosis rate and apoptosis-related gene expression were examined.

RESULTSMTT assay showed that MCLR mildly inhibited the cell growth within the initial 24 h of treatment but enhanced the cell viability after that till 60 h in a time- and dose-dependent manner. LDH leakage underwent no marked changes in response to 48-hour MCLR treatment but increased upon prolonged treatment for 60 h, indicating the presence of oxidative damage. After a 48-h treatment with MCLR at 50 microg/ml, obvious apoptosis of L-02 cells occurred as manifested by cell rounding, detachment from the substrate, cell shrinkage and membrane blebbing. The apoptosis rates were rather low (between 22% and 29%) after treatment with MCLR at different concentrations for 36 h, and increased to as much as 80% after a 60-h treatment with 50 microg/ml MCLR. The expressions of p53 and bcl-2 increased in the cells after treatment with high-concentration MCLR, suggesting that MCLR up-regulated the expression levels of the two proteins.

CONCLUSIONMCLR can induce apoptosis and up-regulate p53 and bcl-2 expressions in human normal liver cell line L-02.

Apoptosis ; drug effects ; Cell Line ; Hepatocytes ; cytology ; Humans ; Microcystins ; toxicity ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics

OBJECTIVESTo investigate the cause of spermatogenic cells decreasing after spinal cord injury.

METHODSImmunohistochemical S-P method was used to detect the expression of iNOS and Bcl-2 protein in the testis of spinal cord injured and sham-operated adult rats in second week and fourth week after operation.

RESULTSSecond week and fourth week after operation, the expression of iNOS of operated rats increased significantly (P < 0.05). Fourth week after operation, the number of Bcl-2 positive cells in spinal cord injured group decreased significantly (P < 0.05).

CONCLUSIONSThe expression veition of iNOS and Bcl-2 is the cause that inducing the decrease of spermatogenic cells in testis of spinal cord injured rats.

Animals ; Male ; Nitric Oxide Synthase ; biosynthesis ; genetics ; Nitric Oxide Synthase Type II ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; metabolism

The modulatory role of bcl-2 gene in hepatocellular apoptosis of rats with glycochenodeoxycholate (GCDC)-induced obstructive jaundice was investigated. The hepatocytes in normal rats and those with bile duct-ligation for 7 days, 14 days and 21 days were isolated and obtained by in situ collagenase perfusion and primary culture. The expression of bcl-2 mRNA in the hepatocytes was detected by RT-PCR. Primary culture was performed on the hepatocytes from normal rats and those with bile duct-ligation for 14 days. 100 mumol/L GCDC was added to the hepatocytes for incubation for 24 h. The hepatocellular apoptotic ratio was measured by using FCM and hepatocellular apoptosis detected in situ by using TUNEL technique. Results showed that the expression of bcl-2 mRNA was not detectable in the hepatocytes of normal rats by RT-PCR technique, while detectable in the hepatocytes of those with bile duct ligation (BDL) for 7, 14 and 21 days. Hepatocellular apoptosis in the BDL group was obviously decreased as compared with normal control group after addition of 100 mumol/L GCDC to the cells for 24 h. It was concluded that the hepatocytes in the BDL rats expressed bcl-2. During obstructive jaundice, expression of bcl-2 from the hepatocytes can inhibit the bile salt-induced hepatocellular apoptosis.
Animals ; Apoptosis ; Cholestasis, Extrahepatic ; chemically induced ; metabolism ; pathology ; Glycochenodeoxycholic Acid ; Hepatocytes ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar

Aldehydes ; pharmacology ; Animals ; Anthracenes ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Hepatocytes ; cytology ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Rats