The Expression of Apoptosis-Related Genes Bcl-2 and Bax Protein and Apoptosis Positivity in Cervical Carcinoma during Irradiation

To evaluate the apoptosis positivity, the expression of Bcl-2, bax proteinsin 30 patients with squamous cell cervix carcinoma before and after radiotherapy. Methods: By usingimmuno-histochemical and TDT-dUTP nick end labelling techniques, 30 cases of squamous cell cervicalcarcinoma were analyzed. Results: The apoptosis positivity before and after irradiation was 76.7%and 100% respectively, with the difference being significant (P 【 0.05); The positive rates of Bcl-2protein before and after irradiation were 73.3% and 46.7% respectively, with the difference beingsignificant (P 【 0.05); The positive rates of bax protein before and after irradiation were 86% and100% respectively, with the difference being significant (P 【 0.05). Conclusion: bax and Bcl-2protein play an important role in apoptosis induced by fractionated radiation therapy. Apoptosisinduced by irradiation is contributed to upregulation of bax protein or downregulation of Bcl-2protein.

Effects of Ethyl Pyruvate on Myocardial Apoptosis and Expression of Bcl-2 and Bax Proteins after Ischemia-reperfusion in Rats

In order to study the effects of ethyl pyruvate on cardiomyocyte apoptosis following ischemia/reperfusion （I/R） in vitro and the expression of Bcl-2 and Bax proteins, isolated rat hearts were perfused in a Langendorff model. Twenty-four rats were randomly divided into 3 groups （n=8 in each group）： control group was perfused for 120 min. In the I/R group, after 30 min stabilization the injury was induced by 30 min global ischemia followed by 60 min reperfusion. Ethyl pyruvate （EP） group was set up with the same protocol as I/R group except that it was supplied with 2 mmol/L EP 15 rain before ischemia and throughout reperfusion. Myocardial malonaldehyde （MDA） content was measured. Myocardial apoptotic index （AI） was tested by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling （TUNEL） method. The expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in cardiac myocytes was detected by immunohistochemistry. As compared with control group, the content of MDA, myocardial AI and the expression of Bcl-2, Bax proteins were increased significantly in I/R group, but the content of MDA, myocardial AI and the expression of Bax protein were decreased obviously and the expression of Bcl-2 protein was up-regulated in EP group （P〈0.05）. These results demonstrate that EP could inhibit apoptosis of cardiac myocytes possibly via alleviating oxidative stress, up-regulating Bcl-2 and down-regulating Bax proteins.

Effects of genistein on neuronal apoptosis,and expression of Bcl-2 and Bax proteins in the hippocampus of ovariectomized rats

Genistein is one of several isoflavones that has a structure similar to 17β-estradiol, has a strong antioxidant effect, and a high affinity to estrogen receptors. At 15 weeks after ovariectomy, the expression of Bcl-2 in the hippocampus of rats decreased and Bax expression increased, with an obvious upregulation of apoptosis. However, intraperitoneal injection of genistein or 17β-estradiol for 15 consecutive weeks from the second day after operation upregulated Bcl-2 protein expression downregulated Bax protein expression, and attenuated hippocampal neuron apoptosis. Our experimental findings indicate that long-term intervention with genistein can lead to a decrease in apoptosis in hippocampal neurons following ovadectomy, upregulate the expression of Bcl-2, and downregulate the expression of Bax. In addition, genistein and 17β-estradiol play equal anti-apoptotic and neuroprotective roles.

澳洲茄碱通过调控Bcl-2/Bax/caspase-3信号通路促进非小细胞肺癌发生凋亡

目的 探讨龙葵活性成分澳洲茄碱对非小细胞肺癌细胞PC9增殖、凋亡的影响。方法 体外培养PC9细胞,设对照组(0μmol/L)及澳洲茄碱不同剂量组(0、2、5、10、15、20、25μmol/L),CCK-8试剂盒检测澳洲茄碱对PC9细胞的增殖抑制作用;TMRE检测线粒膜电位;caspase3/7活性试剂盒联合GreenNuc?Caspase-3/Annexin V-mCherry染色检测caspase-3活性;Annexin V-FITC/PI双染法检测细胞凋亡率;给药处理或者使用PTEN抑制剂后,Western blot检测细胞中相关蛋白的表达量。结果 与对照组相比,经澳洲茄碱干预24、48、72 h后,PC9细胞的活力均明显降低(P<0.05);经澳洲茄碱干预24 h后,细胞线粒体膜电位明显降低,而细胞凋亡比例明显升高(P<0.05);caspase-3/7活力、活细胞Caspase-3活性及cleaved caspase-3蛋白表达均显著升高(P<0.01);PI3K和Akt磷酸化水平降低(P<0.05);而PTEN、Bax蛋白表达上调(P<0.05);抗凋亡蛋白Bcl-2蛋白的表达下调(P<0.05)。结论 澳洲茄碱可通过调控Bcl-2/Bax/caspase-3通路及其上游蛋白活性而抑制PC9细胞增殖,促进其凋亡。

Brain-derived neurotrophic factor prevents beta-amyloid-induced apoptosis of pheochromocytoma cells by regulating Bax/Bcl-2 expression

Brain-derived neurotrophic factor was utilized in the present study to treat cell injury models induced by aggregated β-amyloid（25 35）. Methylthiazolyldiphenyl-tetrazolium bromide assay and western blot analysis showed that brain-derived neurotrophic factor provided neuroprotection against cellular apoptosis by suppressing the decline in β-amyloid（25 35）-induced cell activity and the increasing ratio of Bax/Bcl-2. After treating pheochromocytoma cells with tyrosine kinase receptor B receptor inhibitor K252a, brain-derived neurotrophic factor reverses the above- mentioned changes. The experimental findings suggested that brain-derived neurotrophic factor prevented β-amyloid peptide-induced cellular apoptosis by modulating Bax/Bcl-2 expression, and this effect was associated with binding to the specific tyrosine kinase receptor B receptor.

Effect of compound preparation Tongqiao Jiannao capsules on neural cell apoptosis and Bcl-2 and Bax protein levels in a rat model of brain ischemia/reperfusion injury

BACKGROUND：Pharmacological studies have demonstrated that compound preparation Tongqiao Jiannao capsules composed of Zexie, Baizhu, Honghua, Danshen, and Shexiang can supplement qi, activate blood circulation, relieve blood stasis, induce resuscitation for alleviating pain, relieve pain, and dilate blood vessels. OBJECTIVE： To observe the effects of Tongqiao Jiannao capsules on the levels of the anti-apoptotic protein Bcl-2 and the proapoptotic protein Bax, and verify the mechanism of action. DESIGN, TIME AND SETTING： Randomized, controlled animal experiment, performed in the Laboratory of Biochemistry and Molecular Biology, Shanxi Medical University between June 2001 and December 2002. MATERIALS： The right middle cerebral arteries of 24 healthy adult Sprague Dawley rats were occluded by the suture method. The primary Chinese herbal medicinal ingredients of Tongqiao Jiannao capsules are Zexie, Baizhu, Honghua, Danshen, and Shexiang, which were purchased from Shanxi Provincial Medicinal Material Company, China, and prepared into condensed granules in the Room for Chinese Herbal Medicine Preparation, Second Hospital, Shanxi Medical University. Bcl-2 and Bax immunohistochemical staining kits, a 3,3-diaminobenzidine（DAB） kit, and an in situ apoptosis detection kit were purchased from Wuhan Boster Bioengineering Co., Ltd., China. METHODS： Twenty-four rats were randomly and evenly divided into three groups： （1） sham-operated rats in which sutures were inserted and immediately pulled out; （2） Tongqiao Jiannao capsule-treated rats that were intragastrically administered 6.5 g/kg/d Tongqiao Jiannao capsule preparation for seven successive days prior to middle cerebral artery occlusion （MCAO）; and （3） MCAO rats without any other treatments. MAIN OUTCOME MEASURES： The levels of neural cell apoptosis and Bcl-2 and Bax proteins at 24 hours post-surgery. RESULTS： In the MCAO group, the numbers of apoptotic cells and Bax-positive cells were significantly increased, while the numbers of Bcl-2-positive cells were slightly decreased compared with the sham-operated group. Bcl-2- and Bax-positive cells and apoptotic cells were primarily distributed in the ischemic penumbra. In the Tongqiao Jiannao capsule-treated group, neuronal apoptosis was inhibited, and the number of Bcl-2-positive cells was significantly increased （P 〈 0.01）, while the number of Bax-positive cells was significantly decreased （P 〈 0.01）, compared with the MCAO group. CONCLUSION： Tongqiao Jiannao capsules elevated Bcl-2 expression, lowered Bax expression, and inhibited cellular apoptosis during the process of cerebral ischemia/reperfusion injury.

Intermittent hypoxia attenuates ischemia/reperfusion induced apoptosis in cardiac myocytes via regulating Bcl-2/Bax expression

Intermittent hypoxia has been shown to provide myocardial protection against ishemia/reperfusion-induced injury.Cardiac myocyte loss through apoptosis has been reported in ischemia/reperfusion injury. Our aim was to investigate whether intermittent hypoxia could attenuate ischemia/reperfusion-induced apoptosis in cardiac myocytes and its potential mechanisms. Adult male Sprague-Dawley rats were exposed to hypoxia simulated 5000 m in a hypobaric chamber for 6 h/day, lasting 42 days. Normoxia group rats were kept under normoxic conditions. Isolated perfused hearts from both groups were subjected to 30 min of global ischemia followed by 60 min reperfusion.Incidence of apoptosis in cardiac myocytes was determined by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) and DNA agarose gel electrophoresis. Expressions of apoptosis related proteins,Bax and Bcl-2, in cytosolic and membrane fraction were detected by Western Blotting. After ischemia/reperfusion,enhanced recovery of cardiac function was observed in intermittent hypoxia hearts compared with normoxia group.Ischemia/reperfusion-induced apoptosis, as evidenced by TUNEL-positive nuclei and DNA fragmentation, was significantly reduced in intermittent hypoxia group compared with normoxia group. After ischemia/reperfusion,expression of Bax in both cytosolic and membrane fractions was decreased in intermittent hypoxia hearts compared with normoxia group. Although ischemia/reperfusion did not induce changes in the level of Bcl-2 expression in cytosolic fraction between intermittent hypoxia and normoxia groups, the expression of Bcl-2 in membrane fraction was upregulated in intermittent hypoxia group compared with normoxia group. These results indicated that the cardioprotection of intermittent hypoxia against ischemia/reperfusion injury appears to be in part due to reduce myocardial apoptosis. Intermittent hypoxia attenuated ischemia/reperfusion-induced apoptosis via increasing the ratio of Bcl-2/Bax, especially in membrane fraction.

Signal transduction mediated by Bid,a pro-death Bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways

Two major apoptosis pathways have been defined in mammalian cells, the Fas/TNF-R1 death receptor pathway and the mitochondria pathway. The Bcl-2 family proteins consist of both anti-apoptosis and pro- apoptosis members that regulate apoptosis, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by caspase 8 in response to Fas/TNF-R1 death receptor signals. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates downstream caspases. Such a connection between the two apoptosis pathways could be important for induction of apoptosis in certain types of cells and responsible for the pathogenesis of a number of human diseases.

Correlation between expression of gastrin, somatostatin and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma

AIM: To explore the correlation between expression of somatostatin (SS), gastrin (GAS) and cell apoptosis regulation gene bcl-2/bax in large intestine carcinoma.METHODS: Sixty-two large intestine cancer tissue samples were randomly and retrospectively selected from patients with large intestine carcinoma. Immunohistochemical staining for bcl-2, bax, GAS, SS was performed according to the standard streptavidin-biotin-peroxidase (S-P) method.According to the semi-quantitative integral evaluation, SS and GAS were divided into three groups as follows. Scores1-3 were defined as the low expression group, 4-8 as the intermediate expression group, 9-16 as the high expression group. Bax and bcl-2 protein expressions in different GAS and SS expression groups of large intestine carcinoma were assessed.RESULTS: The positive expression rate of bax had a prominent difference between SS and GAS high, intermediate and low expression groups (P＜0.05, x2ss = 9.246; P＜0.05,x2GAS = 6.981). The positive expression rate of bax in SS high (80.0%, 8/10) and intermediate (76.5%, 13/17)expression groups was higher than that in low expression group (40.0%, 14/35) (P＜0.05, x2high vs low = 5.242; P＜0.05,x2middle vs low = 6.097). The positive expression rate of bax in GAS high expression group (27.3%, 3/8) was lower than that in low expression group (69.4%, 25/36) (P＜0.05,x2 = 4.594). However, bax expression in GAS intermediate expression group (46.7%, 7/15) was lower than that in low expression group, but not statistically significant. The positive expression rate of bcl-2 had a prominent difference between SS and GAS high, intermediate and low expression groups (P＜0.05, x2ss = 7.178; P＜0.05, x2GAS = 13.831). The positive expression rate of bcl-2 in GAS high (90.9%, 10/11)and intermediate (86.7%, 13/15) expression groups was higher than that in low expression group (44.4%, 16/36)(P＜0.05,x2high vs low = 5.600; P＜0.05, x2 middle vs low = 7.695).However, the positive expression rate of bcl-2 in SS high (40.0%, 4/10) and intermediate (47.1%, 8/9) expression groups was lower than that in low expression group (77.1%, 27/35)(P＜0.05, x2 high vs low = 4.710; P＜0.05, x2 middle vs low = 4.706).There was a significant positive correlation between the integral ratio of GAS to SS and the integral of bcl-2 (P＜0.01,r=0.340). However, there was a negative correlation between the integral ratio of GAS to the SS and bax the integral of (P＜0.05, r = -0.299).CONCLUSION: The regulation and control of gastrin,somatostatin in cell apoptosis of large intestine carcinoma may be directly related to the abnormal expression of bcl-2, bax.

Dioscin-induced Apoptosis of Human LNCaP Prostate Carcinoma Cells through Activation of Caspase-3 and Modulation of Bcl-2 Protein Family

Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin（1, 2 and 4 μmol/L） could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.

Exogenous phosphatidylethanolamine induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway

AIM: To investigate the signaling pathways implicated in phosphatidylethanolamine (PE)-induced apoptosis of human hepatoma HepG2 cells. METHODS: Inhibitory effects of PE on human hepatoma HepG2 cells were detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell cycle, apoptosis and mitochondrial transmembrane potential (ΔΨm) were analyzed by flow cytometry. Immunocytochemical assay and Western blotting were used to examine Bcl-2, Bax and caspase-3 protein levels in HepG2 cells treated with PE. RESULTS: PE inhibited the growth of HepG2 cells in a doseand timedependent manner. It did notaffect the cell cycle, but induced apoptosis. PE significantly decreased ΔΨm at 0.25, 0.5 and 1 mmol/L, respectively, suggesting that PE induces cell apoptosis by decreasing the mitochondrial transmembrane potential. The Bcl-2 expression level induced by different concentrations of PE was lower than that in control groups. However, the Bax expression level induced by PE was higher than that in the control group. Meanwhile, PE increased the caspase-3 expression in a doseand time-dependent manner. CONCLUSION: Exogenous PE induces apoptosis of human hepatoma HepG2 cells via the bcl-2/bax pathway.

Effect of Hypoxic Preconditioning on Neural Cell Apoptosis and Expression of Bcl-2 and Bax in Cerebral Ischemia-Reperfusion in Rats

In order to investigate the protective effect of hypoxic preconditioning on the cerebral ischemia-reperfusion injury, the expression of Bcl-2 and Bax was detected by using immunohistochemical staining after 3 h cerebral ischemia followed by 1, 6, 12, 24 and 48 h reperfusion respectively in rats treated with or without hypoxic preconditioning before cerebral ischemia. In addition, the apoptosis of neural cells and the behavioral scores for neurological functions recovery were evaluated by TUNEL staining and ＂crawling method＂, respectively. Compared with control group （cerebral ischemia-reperfusion without hypoxic preconditioning）, the expression of Bcl-2 was significantly increased, but that of Bax decreased in the hypoxic preconditioning group （cerebral ischemiareperfusion with hypoxie preconditioning）, both P〈0.05. The pre-treatment with hypoxic preconditioning could reduce the apoptosis of neural cells and promote the neurological function recovery as compared to control group. It was suggested that hypoxic preconditioning may have protective effects on the cerebral ischemia-reperfusion injury by inhibiting the apoptosis of neural cells, increase the expression of Bcl-2 and decrease the expression of Bax.

Gambogic acid induces mitochondria-dependent apoptosis by modulation of Bcl-2 and Bax in mantle cell lymphoma JeKo-1 cells

Objective： To study the mechanisms in gambogic acid （GA） -induced JeKo-1 human Mantle Cell Lymphoma cell apoptosis in vitro. Methods： The proliferation of GA-treated JeKo-1 cells was measured by CCK-8 assay and Ki-67 immunocytochemical detection. Apopt0sis, cell cycle and mitochondrial membrane potential were measured by flow cytometric analysis. Caspase-3, -8 and -9 were detected by colorimetric assay. Bcl-2 and Bax were analyzed by Western blotting. Results： GA inhibited cell growth in a time- and dose- dependent manner. GA induces apoptosis in JeKo- 1 cells but not in normal bone marrow cells, which was involved in reducing the membrane potential of mitochondria, activating caspases-3, -8 and -9 and decreasing the ratio of Bd-2 and Bax without cell cycle arresting. Conclusions： GA induced apoptosis in human MCL JeKo-1 cells by regulating Bcl-2/Bax and activating caspase-3, -8 and -9 via mitochondrial pathway without affecting cell cycle.

Effectof Ginsenoside Re on Cardiomyocyte Apoptosis and Expression of Bcl-2/Bax Gene after Ischemia and Reperfusion in Rats

To observe the effectof ginsenoside Re on cardiomyocyte apoptosis and Bcl- 2 / Bax gene expression after ischemia (30 m in) and reperfusion (6 h) in rats and to elucidate the possible m echanism s of ginsenoside Re on inhibition of cardiom yocyte apoptosis,the ischem ia/ reperfusion heart m odel was established by ligating the left anterior descending branch of coronary artery in Wistar rats.The apoptotic cardiom yocytes were confirmed by transm ission electron m icroscopy and counted by in situ nick end labeling(TU NEL) method and lightm icroscopy.The m RNA and protein expression of Bcl- 2 and Bax genes were studied by in situ hybridization and im munohis- tochemical staining.Mean optical density (OD) value of the positive fields of m RNA and protein expression was quantitatively exam ined by im age analysis system.The results were as follows: (1) The apoptotic cardiomyocytes were found in ischemic fields in the ischem ia/ reperfusion group and weren't observed in the sham- operation group by transmission electron microscopy;(2 ) The num bers of the apoptotic cells were134.4 5± 4 5 .6 1/ field in the ischemia/ reperfusion group,and 90 .6 6± 19.2 2 / field in the ginsenoside Re- treated group.The differences was significant between two groups(P<0 .0 1) ;(3) Gene expression of Bcl- 2 and Bax were increased significantly in the is- chemia/ reperfusion group and ginsenoside Re- treated group when compared with the sham - opera- tion group.There was no significant difference in the gene expression of Bcl- 2 between the gin- senoside Re- treated group and ischemia/ reperfusion group(P>0 .0 5 ) ,but gene expression of Bax was decreased significantly in the ginsenoside Re- treated group as compared with the ischem ia/ reperfusion group(P<0 .0 1) .The ratio of Bcl- 2 / Bax was increased significantly in the ginseno- side Re- treated group when com pared with the ischem ia/ reperfusion group and sham- operation group.These findings suggest that m yocardial ischem ia- reperfusion can induce cardiom yocyte apoptosis,and ginsenoside Re can significantly inhibit cardiom yocyte apoptosis induced by ischemi- a- reperfusion in rats.It is concluded that ginsenoside Re inhibits cardiomyocyte apoptosis by in- hibiting expression of pro- apoptotic Bax gene and raising the ratio of Bcl- 2 / Bax.

Simvastatin inhibits apoptosis of endothelial cells induced by sepsis through upregulating the expression of Bcl-2 and downregulating Bax

BACKGROUND: Many studies have showed that apoptosis of endothelial cells plays a curial role in the progress of sepsis. But the role of simvastatin in apoptosis of endothelial cells induced by sepsis is not clear. The present study aimed to investigate the role of simvastatin in apoptosis of endothelial cells induced by sepsis and its mechanism.METHODS: Human umbilical vein endothelial cells(HUVECs) were randomly divided into three groups: control group, sepsis serum intervention group(sepsis group) and simvastatin+sepsis serum intervention group(simvastatin group). After 24-hour incubation with corresponding culture medium, the relative growth rate of HUVECS in different groups was detected by MTT assay; the apoptosis of HUVECs was detected by Hoechst33258 assay and fl ow cytometry; and the expression of the Bcl-2 and Bax genes of HUVECs was detected by PCR.RESULTS: Compared with the sepsis group, HUVECs in the simvastatin group had a higher relative growth rate. Apoptotic HUVECs decreased significantly in the simvastatin group in comparison with the sepsis group. Expression of the Bcl-2 gene in HUVECs decreased obviously, but the expression of the Bax gene increased obviously after 24-hour incubation with sepsis serum; however, the expression of the Bcl-2 and Bax genes was just the opposite in the simvastatin group.CONCLUSIONS: Our study suggests that simvastatin can inhibit apoptosis of endothelial cells induced by sepsis through upregulating the expression of Bcl-2 and downregulating Bax. It may be one of the mechanisms for simvastatin to treat sepsis.

Propofol blocks apoptosis and Bcl-2 and Bax expression induced by hypoxia-reoxygenation in primary cultures of rat hippocampal astrocytes

BACKGROUND： Cerebral hippocampal astrocytes are more sensitive.to ischemic injury than neurons. Hypoxic-ischemic brain injury induces profound astrocyte apoptosis, and propofol may protect against astrocyte apoptosis. OBJECTIVE： To verify the protective effects of propofol against astrocyte apoptosis and to investigate anti-apoptotic Bcl-2 and pro-apoptotic Bax expression in primary cultures of rat hippocampal astrocytes exposed to hypoxia-reoxygenation for different periods of time following propofol treatment. DESIGN, TIME, AND SETTING： In vitro neural immunocytochemistry was performed at the Central Laboratory of Yunyang Medical College between September 2007 and March 2008.MATERIALS： A total of 30 Wistar rats, aged 1-3 days, wJth equal numbers of males and females, were included for isolation and culture of .hippocampal astrocytes. METHODS： Hippocampal astrocytes were purified and cultured for 3 weeks and treated with four culture conditions： 50 μL Hank＇s solution （normal control）; 0.2 mL/L Intralipid; 50 μL Hank＇s solution for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 or 72 hours; propofol （250 μmol/L final） for 10 minutes followed by hypoxic incubation for 4 hours and normoxic incubation for 12, 24, 36, 48, 60 and 72 hours. MAIN OUTCOME MEASURES： （1） Morphologic changes in hippocampal astrocytes. （2） Levels of astrocyte apoptosis and Bcl-2 and Bax expression. RESULTS： Hypoxia and reoxygenation increased apoptosis over time, with Bcl-2 expression peaking at 24 hours and decreasing gradually （P 〈 0.01 ）; Bax expression peaked at 72 hours （P 〈 0.01）; the ratio of Bcl-2/Bax was 1.4, 0.8, and 0.6, respectively, at 24, 48 and 72 hours. Non-apoptotic astrocytes showed significant proliferation and swelling. Propofol treatment decreased apoptosis after hypoxia-reoxygenation （P 〈 0.01）, as well as Bct-2 and Bax expression （P 〈 0.05, P 〈 0.01）, with Bcl-2/Bax ratios of 1.6-1.8. Propofol treatmentalso blocked astrocyte proliferation and swelling. No apoptotic cells or Bcl-2/Bax expression was detected in astrocytes cultured in Hank＇s or Intralipid solution. CONCLUSION： Propofol protects astrocytes against injury caused by hypoxia and reoxygenation via a mechanism that involves maintaining high ratios of Bcl-2/Bax.

THE EXPERIMENTAL STUDY ON THE CELL APOPTOSIS AND EXPRESSION OF BCL-2 PROTEIN IN INTRACEREBRAL HEMORRHAGE IN MODEL OF RATS

Objective To study whether there is the apoptosis of neural cells and the expression of Bcl-2 protein in intracerebral hemorrhage (ICH) in model of rats, for the further understanding the mechanism of the delayed damage of the neural cells around the hematoma after ICH. Methods Fifty SD rats were randomly divided into 5 groups, ten in each. With the Group A as the control, the rest 40 were used to set up intracerebral hemorrhage model. The brains were taken out at 12 th , 24 th , 48 th and 72 th hours, respectively. Apoptosis cells were detected with terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL), and the expression of Bcl-2 protein was detected with immunochemical stainging methed (SP). Results In the control group, no apoptosis cells and Bc1-2 protein were detected. In rest groups, the apoptosis cells and Bc1-2 protein were expressed in different degree. Apoptosis rates verified and corresponded with the time after ICH, with the peak at 48 th -72 th hour after hemorrhage. The peak rate of apoptosis cells was (24.50±2.69)% and Bcl-2 protein expression was (20.76±1.97)% . There was significant difference between the experimental groups and control (P<0.05), and no linear relationship between the apoptosis rate and the expression of Bcl-2 protein. Conclusion Apoptosis may be an important factor in the secondary trauma of ICH. There is a time leg after hemorrhage. All this is instructive to clinical treatment in time. Bcl-2 protein keeps increasing in a certain time after hemorrhage, but not synchronize with the cell apoptosis. This indicates that bcl-2 has the effect to reduce the apoptosis of neural cells.

Effect of exogenous phosphocreatine on cardiomycytic apoptosis and expression of Bcl-2 and Bax after cardiopulmonary resuscitation in rats

BACKGROUND： Ischemia-reperfusion injury in the myocardium after cardiac arrest （CA）and cardiopulmonary resuscitation （CPR） is an important pathologic basis of post-cardiac arrestof syndrome （PCAS）, and apoptosis is one of the major mechanisms in myocardial ischemiareperfusioninjury. To lessen myocardial ischemia-reperfusion injury after cardiac arrest and CPR,it is important to reduce energy consumption and to increase energy supply in the myocardium.This study aimed to observe changes of cell apoptosis and expression of Bcl-2 and Bax proteinon the myocardium after CPR in rats, and the protective effects of different doses of exogenousphosphocreatine （creatine phosphate, CP） on them.METHODS： A total of 32 male adult Sprague-Dawley rats were randomly divided into 4 groups：control group （group A）, CPR group （group B）, low-dose CP group （group C, CP 0.5 g/kg at beginning ofCPR and 1.0 g/kg at 2 hours after CPR） and high-dose CP group （group D, CP 1.0 g/kg at beginning ofCPR and 2.0 g/kg at 2 hours after CPR）. Cardiac arrest was induced by asphyxiation and CPR startedat 7 minutes after asphyxiation in groups B, C and D. Myocardium samples were taken at 24 hoursafter CPR. Cardiomycytic apoptosis was detected by the TdT-mediated dUTP-biotin nick end labeling（TUNEL） method. The expression of Bcl-2 and Bax protein was measured by immunohistochemistry.RESULTS： Cardiomyocytic apoptosis index （AI） and expression of Bcl-2 and Bax proteinincreased more significantly in groups B, C and D than in group A （P〈0.01）, but Bcl-2/Bax ratiosignificantly decreased （P〈0.01）. Cardiomyocytic AI and expression of Bcl-2 and Bax proteindecreased more significantly in groups C and D than in group B （P〈0.01）, but Bcl-2/Bax ratioincreased more significantly （P〈0.01）. Cardiomyocytic AI and expression of Bcl-2 and Bax proteindecreased more signifi cantly in group D than in group C （P〈0.05）, but Bcl-2/Bax ratio increased moresignifi cantly （P〈0.05）.CONCLUSION： Exogenous phosphocreatine, especially at a large dose, could inhibitcardiomyocytic apoptosis and alleviate myocardial injury after CPR in rats.

Regulatory Effect of Bcl-2 Family Proteins in CPB-induced Cardiomyocyte Apoptosis in Dog Hearts

Summary: Whether conventional hypothermic CPB induces myocyte apoptosis in dog hearts and modulation of bcl-2, bcl-xl, bax, bad, and caspase-3 pathways in this setting was investigated. Ten healthy adult dogs were randomized into sham-operated and CPB groups. Samples of left ventricle were obtained before, during and 3 h after CPB. In situ TUNEL was used to detect apoptotic myocytes. Immunohistochemistry and flow cytometry were employed for detection of expressions of bcl-2, bcl-xl, bax and bad proteins. Z-DEVD-AMC substrate cleavage and TBARS methods were used to measure the activity of caspase-3 and the content of lipid peroxide in LV myocardium, respectively. After CPB, the number of apoptotic myocytes in CPB group was significantly increased. The results of immunohistichemistry demonstrated that bcl-2, bcl-xl, bax and bad proteins were constitutionally present on the sarcolemma of the LV myocytes. FACS results showed that, after CPB, expressions of bax and bad in CPB group were significantly upregulated, while the expressions of bcl-2 and bcl-xl were not significantly changed in both groups. The activity of caspase-3 and the content of lipid peroxide in LV myocardium in CPB group were also significantly increased after CPB. The present study shows that there exists myocardiocyte apoptosis in dog hearts undergoing conventional hypothermic CPB and the myocyte apoptosis is initiated by ischemia and performed during reperfusion. Moreover, the CPB-induced myocyte apoptosis was associated with upregulation of expressions of bax and bad proteins, activation of caspase-3 and increase of oxidative stress.

THE EXPRESSION AND CLINICAL VALUE OF APOPTOSIS CONTROL GENE Bcl-2 AND Bax IN BREAST CANCER

Objective: To study the expression and clinical value of apoptosis control gene bcl-2 and bax in breast cancer. Methods: Protein bax and bcl-2 in 41 breast cancers obtained from operations in our hospital in 1996 were detected using ABC immunohistochemical stain assay and compared with 10 cases with normal breast tissues. Results: The positive rate of bax in normal breast tissue was 90% and in breast cancer was 59%, with a significant statistical difference between them (P<0.05), but there was no statistical difference in bcl-2 protein expression. Among the 41 breast cancer, the group with lymph node metastasis (21 cases) had obviously low bax expression (43%) and high bcl-2 expression (76%), showing significant difference to the group without lymph node metastasis (P<0.05). Conclusion: The antiapoptosis function of bcl-2 was stronger than bax in breast cancer. Protein bax and bcl-2 assay may be useful in understanding the biological behaviors of breast cancer.