This study aims to explore the effect and mechanism of Danggui Buxue Decoction(DBD)-containing serum in alleviating the H9c2 cell injury caused by the exposure to intermittent low oxygen. H9c2 cells were assigned into five groups: control(CON) group, intermittent low oxygen(IH) group, intermittent low oxygen plus DBD-containing serum(IH+DBD) group, intermittent low oxygen plus the autophagy enhancer rapamycin(IH+RAPA) group, and intermittent low oxygen plus DBD-containing serum and the autophagy inhibitor 3-methyladenine(IH+DBD+3-MA) group. Monodansylcadaverine(MDC) staining was employed to detect the changes of autophagosomes. Cell counting kit-8(CCK-8) assay was employed to determine the activity of myocardial cells, and lactate dehydrogenase(LDH) and creatine kinase(CK) kits were used to measure the LDH and CK levels in the cell culture, which would reflect the degree of cell damage. TdT-mediated dUTP nick-end labeling(TUNEL) staining was used to detect the apoptosis of myocardial cells, and JC-1 fluorescence probe to detect the changes in mitochondrial membrane potential. Western blot was employed to determine the expression levels of the autophagy-related proteins microtubule-associated proteins light chain 3Ⅱ(LC3Ⅱ), microtubule-associated proteins light chain 3Ⅰ(LC3Ⅰ), P62, Parkin and apoptosis related proteins pro caspase-3, caspase-3, B-cell lymphoma-2(Bcl-2), Bcl-2-associated X(Bax). The results showed that compared with the CON group, the IH group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated expression of P62. In addition, the IH group showed decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, and decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. Compared with the IH group, the IH+DBD and IH+RAPA groups showed increased fluorescence intensity of MDC staining, increased LC3Ⅱ/LC3Ⅰ ratio, up-regulated Parkin expression, and down-regulated P62 expression. In addition, the two groups showed increased cell survival rate, reduced content of LDH and CK in the culture medium, decreased number of TUNEL positive cells, and increased pro caspase-3/caspase-3 and Bcl-2/Bax ratios and mitochondrial membrane potential. The IH+DBD+3-MA and IH groups showed no significant differences in the above indicators. Compared with the IH+DBD group, the IH+DBD+3-MA group showed decreased fluorescence intensity of MDC staining, decreased LC3Ⅱ/LC3Ⅰ ratio, down-regulated Parkin expression, and up-regulated P62 expression. In addition, the group had decreased cell survival rate, increased content of LDH and CK in the culture medium, increased number of TUNEL positive cells, decreased pro caspase-3/caspase-3 and Bcl-2/Bax ratios, and declined mitochon-drial membrane potential. To sum up, DBD could promote the mitophagy, inhibit the apoptosis, and alleviated the injury of H9c2 cells exposed to low oxygen.
Oxygen ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/genetics* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; Apoptosis ; Autophagy ; Ubiquitin-Protein Ligases ; Microtubule-Associated Proteins

In order to investigate the role of melatonin in inhibiting the proliferation of murine gastric cancer and the underlying molecular mechanism, we performed an in vivo study by inoculating murine foregastric carcinoma (MFC) cells in mice, and then tumor-bearing mice were treated with different concentrations of melatonin (i.p.). The changes of Bcl-2, Bax, p21 and p53 expressions in tumor tissue were detected by using real-time fluorescence quantitative RT-PCR and Western blot. We found that: (1) melatonin resulted in reductions of tumor's volume and weight in the gastric cancer-bearing mice and thus showed anti-cancer effect; (2) melatonin reduced Bcl-2 expression, but increased the expression of Bax, p53 and p21 in tumor tissue. Our results suggest that melatonin could inhibit the growth of tumors in gastric cancer-bearing mice through accelerating the apoptosis of tumor cells.
Animals ; Apoptosis ; Melatonin ; pharmacology ; Mice ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Stomach Neoplasms ; drug therapy ; metabolism ; Tumor Suppressor Protein p53 ; metabolism ; bcl-2-Associated X Protein ; metabolism

BACKGROUND/AIMS: The aim of this study was to investigate the immunohistochemical expression of bcl-2, bcl-xL, bax, and p53 proteins according to the pathological parameters such as grade of dysplasia, histological type, depth of invasion, lymph node metastasis, and TNM stage in the gastric adenoma and gastric adenocarcinoma. METHODS: Immunohistochemical staining using monoclonal bcl-2, bcl-xL, bax, p53 antibodies were performed on paraffin embedded specimens from forty-one gastric adenomas and 100 gastric adenocarcinomas. RESULTS: The expression rate of bcl-2 was higher in adenomas (34.2%), especially in high grade dysplasia (52.4%), than adenocarcinomas (2.0%). The expression rate of bcl-xL was higher in adenocarcinomas (55.0%) than adenomas (22%). The expression rate of the bax was higher in adenocarcinomas (58.0%) than adenomas (14.6%). In the adenocarcinoma, the bax expression was significantly related with the depth of invasion, lymph node metastasis, and TNM stage. The expression rate of p53 was higher in adenocarcinomas (64.0%) than adenomas (14.6%). CONCLUSIONS: Bcl-2 protein would be related with the development of gastric adenoma, especially with high grade dysplasia. Bcl-xL and p53 proteins would be involved in the development of relatively early stage of gastric adenocarcinoma but not in tumor progression. Bax protein would be involved in the development of gastric adenocarcinoma and related with depth of invasion, lymph node metastasis, and TNM stage.
Adenocarcinoma/*metabolism/pathology ; Adenoma/*metabolism/pathology ; Female ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Proto-Oncogene Proteins c-bcl-2/*metabolism ; Stomach Neoplasms/*metabolism/pathology ; Tumor Suppressor Protein p53/*metabolism ; bcl-2-Associated X Protein/*metabolism ; bcl-X Protein/*metabolism

The aim of this study was to investigate the effect of TIEG1 on K562 cell apoptosis and expression of BCL-2/BAX, PTEN. The different concentration(0, 1, 5, 10, 20 ng/ml) of TIEG1 were used to treat K562 cells, the cell growth inhibition rate was detected by using MTT method. After treating K562 cells with 10.00 ng/ml TIEG1, the cell apoptosis was detected with flow cytometry. The RT-PCR was used to detected the expression levels of BCL-2 /BAX and PTEN. The results showed that TIEG1 displays inhibitory effect on proliferation of K562 cells in time-and dose-dependent manner (r = 0.52, P < 0.05) ; after K562 cells were treated for 6, 12, 24 and 48 h, the IC50 of TIEG1 were 48.19, 18.72, 9.5 and 3.85 ng/ml respectively. After treating K562 cells with 10.00 ng/ml TIEG1 for 0, 6, 12, 24, 48 h, the apoptosis rate were (2.13 ± 0.42)%, (7.79 ± 0.71)%, (11.17 ± 1.37)%, (24.66 ± 0.29)% and (48.60 ± 1.38)% respectively, and there was significant difference between groups(P < 0.05). In process of K562 cell apoptosis, the expression level of BCL-2 gradually decreased (r = 0.48, P < 0.05), meanwhile the expression levels of BAX (r = 0.69, P < 0.05) and PTEN (r = 0.57, P < 0.05) gradually increased. It is concluded that TIEG1 can indue apoptosis of K562 cells and inhibit K562 cell proliferation in time-and dose-dependent manner. In apoptosis process of K562 cells induced by TIEG1, the expression changes of BCL-2/BAX and PTEN associate with the K562 cell apoptosis.
Apoptosis ; Cell Proliferation ; Humans ; K562 Cells ; Kruppel-Like Transcription Factors ; metabolism ; PTEN Phosphohydrolase ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; bcl-2-Associated X Protein ; metabolism

Animals ; Animals, Newborn ; Hippocampus ; metabolism ; Hypoxia, Brain ; metabolism ; Ischemic Preconditioning ; methods ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

OBJECTIVEDuring the critical period of brain development, insufficiency of thyroid hormone results in severe mental retardation and learning deficit. This study was designed to investigate the effects of hypothyroidism on apoptosis and the expression of Bcl-2 and Bax gene in the developing rat hippocampus neurons and to explore the mechanism of brain development regulated by thyroid hormone.

METHODHypothyroidism was induced by administration of propylthiouracil (PTU, 50 mg/d) solution to the dams from gestational day 15 by gavage. Pups from both hypothyroid and control groups were harvested at postnatal day 1 (P1), P5, P10 and P15, respectively. Blood samples were collected at the time of death for the determination of thyroid hormone. Serum free tri-iodothyronine (FT(3)) and free thyroxine (FT(4)) were measured by using chemoluminescence. Hippocampus collected from the control and hypothyroid pups were examined under light and transmissional electron microscopy. Measurement of DNA fragmentation was carried out by agarose gel electrophoresis. The expression of Bcl-2 and Bax protein in the developing rat hippocampus neurons was performed by Western blotting.

RESULTSSignificantly lower circulating FT(4) and FT(3) levels confirmed the hypothyroid status of the experimental pups. The shrunken and contracted degenerations increased in hippocampus neurons of hypothyroid pups under light microscopy. Enhanced apoptotic cells were found in hippocampus neurons of hypothyroid pups under transmission electron microscopy, especially at P10 and P15. Extensive DNA fragmentation was seen throughout development in hippocampus of hypothyroid pups, but not in the euthyroid controls except for basal level at P10. The expression of Bcl-2 in the hippocampus neurons of hypothyroid pups was significantly lower than that of euthyroid controls at all stages of development (P1: 1.95 +/- 0.27 vs. 2.59 +/- 0.19, P < 0.05, P5: 1.86 +/- 0.24 vs. 2.47 +/- 0.17, P < 0.05, P10: 1.29 +/- 0.22 vs. 1.86 +/- 0.28, P < 0.05 and P15: 1.21 +/- 0.27 vs. 2.18 +/- 0.17, P < 0.01, respectively). The relative amount of expression varied significantly with age in the control pups. The level of Bcl-2 was high in hippocampus neurons of euthyroid at P1, P5, and decreased significantly at P10, and showed a trend of recovery at P15. Similar age-related variation in the expression of Bcl-2 gene was observed in the hypothyroid group at P1, P5 and P10, but the level was maintained low at P15. The expression of Bax in the hippocampus neurons of hypothyroid pups was significantly higher than that of control pups at all stages of development (P1: 1.69 +/- 0.14 vs. 1.24 +/- 0.23, P < 0.05, P5: 1.78 +/- 0.16 vs. 1.29 +/- 0.17, P < 0.05, P10: 1.92 +/- 0.18 vs. 1.45 +/- 0.14, P < 0.05 and P15: 1.86 +/- 0.14 vs. 1.51 +/- 0.12, P < 0.05, respectively). The ratio of Bcl-2/Bax in hippocampus neurons of hypothyroid pups was lower than that of age-matched controls (P1: 1.16 +/- 0.17 vs. 2.12 +/- 0.35, P < 0.05, P5: 1.05 +/- 0.16 vs. 1.94 +/- 0.36, P < 0.05, P10: 0.68 +/- 0.17 vs. 1.29 +/- 0.16, P < 0.05 and P15: 0.67 +/- 0.19 vs. 1.45 +/- 0.22, P < 0.01, respectively).

CONCLUSIONThyroid hormone significantly prevents apoptosis of hippocampus neurons. Congenital hypothyroidism increases not only the extent but also the duration of apoptosis by down-regulation of the anti-apoptotic gene Bcl-2 and maintaining a high level of the pro-apoptotic gene Bax.

Animals ; Animals, Newborn ; Apoptosis ; physiology ; Down-Regulation ; Hippocampus ; metabolism ; Hypothyroidism ; physiopathology ; Neurons ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; bcl-2-Associated X Protein ; metabolism

Animals ; Captopril ; pharmacology ; Heart Failure ; metabolism ; Male ; Myocytes, Cardiac ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; metabolism

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

OBJECTIVETo observe the apoptosis of spermatogenic cells and the expressions of Bcl-2 and Bax proteins after burying the testis in the inguinal pocket, and to investigate their relationship.

METHODSWe randomly divided 36 healthy male New Zealand white rabbits into an experimental group (n = 18) and a control group (n = 18). Models were established by burying testes in the inguinal pocket in the experimental group, while the controls were left untreated. At the end of the 8th week after surgery, 6 animals were randomly taken from each group for measurement of the testis surface temperature and testicular biopsy. The apoptosis of spermatogenic cells in the testis tissues was detected by TUNEL assay, and the expressions of Bcl-2 and Bax proteins determined by immunohistochemistry and imaging analysis.

RESULTSAt 8 weeks after burying the testis in the inguinal pocket, the testicular surface temperature was significantly higher in the experimental group than in the control ([ 38.02 +/- 0.36] degrees C vs [36.15 +/- 0.64 ] degrees C, P < 0.05), and so was the apoptosis index (AI) of spermatogenic cells ([89.69 +/- 3.76] % vs [7.73 +/- 4.95 ] %, P < 0.05). The expression of the Bax protein in the testis was significantly increased, while that of the Bcl-2 protein remarkably decreased in the experimental group as compared with the control group (P < 0.05). The apoptotic cells were mostly primary spermatocytes and round spermatids.

CONCLUSIONElevated local temperature of the testis buried in the inguinal pocket increases the apoptosis of spermatogenic cells, and the spermatogenic cell apoptosis is highly correlated with the decreased expression of Bcl-2 and increased expression of Bax. The changes in the expressions of Bcl-2 and Bax proteins were a main mechanism behind the temperature elevation-induced apoptosis of spermatogenic cells.

Animals ; Apoptosis ; Groin ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rabbits ; Spermatids ; metabolism ; Temperature ; Testis ; metabolism ; pathology ; bcl-2-Associated X Protein ; metabolism

OBJECTIVE: To investigate the effectiveness of cisplatin on the expressions of Bcl-2 and Bax in cochlea and spiral ganglion cells (SGC) of guinea pigs. METHOD: Twenty guinea pigs were randomly divided into cisplatin (n = 10) and control groups (n = 10). Cisplatin group were administrated with a dose of intraperitoneal injection of 16 mg/kg, while the control group were received intraperitoneal injection of normal saline as placebo. Before and 7 days following injections, the ototoxic effect was measured with distortion product otoacoustic emission (DPOAE). Bcl-2, Bax in cochlea were detected by Western Blot. Immunohistochemical staining was used to detect the protein levels of Bcl-2 and Bax in spiral ganglion cells. RESULT: In control and cisplatin group, Bcl-2 protein levels were 0.727 8 ± 0.016 9 and 0.467 6 ± 0.020 1, Bax protein levels were 0.384 8 ± 0. 0217 and 0.735 6 ± 0.022 3 in cochlea respectively, both P < 0.01. In Control and cisplatin group, the grey values of Bcl-2 in SGC were 99.00 ± 2.42 and 149.80 ± 2.37 respectively, the grey values of Bax were 154.50 ± 2.80 and 104.50 ± 3.09 respectively, both P < 0.05. CONCLUSION Decreased expression of Bcl-2 and increased expression of Bax may be involved in cisplatin-induced apoptosis in cochlea and SGC of guinea pigs.
Animals ; Apoptosis ; Cisplatin ; pharmacology ; Cochlea ; metabolism ; Guinea Pigs ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Spiral Ganglion ; drug effects ; metabolism ; bcl-2-Associated X Protein ; metabolism