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.

基于Caspase-3/Bcl-2/Bax信号通路探究加味旋覆代赭汤治疗食管癌前病变的作用机制

目的:探究加味旋覆代赭汤对食管癌前病变大鼠Caspase-3/Bcl-2/Bax信号通路的调控作用机制。方法:将48只雄性SD大鼠随机分为4组,空白组、模型组、中药组、西药组,每组各12只。除空白组外均采用复合造模法制作食管癌前病变大鼠模型,造模成功后,分别进行药物灌胃干预,空白组、模型组用生理盐水灌胃,中药组和西药组分别给予加味旋覆代赭汤、西药(雷贝拉唑+莫沙必利)灌胃,给药8周取材。利用光学显微镜观察食管上皮组织的形态学变化;分别应用蛋白质印迹法(Western-blot)及聚合酶链式反应(PCR)检测食管上皮组织Caspase-3、Bcl-2及Bax的表达水平。结果:与空白组比较,模型组大鼠食管上皮病理积分升高(P<0.01);与模型组比较,中药组、西药组大鼠食管上皮病理积分均降低(P<0.01)。PCR及Western-blot检测结果显示,与空白组比较,模型组大鼠Caspase-3、Bax mRNA、蛋白表达均降低(P<0.01);与模型组比较,中药、西药组大鼠Caspase-3、Bax mRNA、蛋白表达均增高(P<0.05)。与空白组比较,模型组大鼠食管组织中Bcl-2 m RNA及蛋白表达水平均升高(P<0.05);与模型组比较,中药组和西药组Bcl-2 mRNA及蛋白表达水平均降低(P<0.05)。结论:加味旋覆代赭汤可能通过下调Bcl-2/Bax比值,增加线粒体外膜通透性,释放凋亡因子,激活Caspase-3,使病变组织发生凋亡,扭转食管上皮异型增生,从而起到治疗食管癌前病变的作用。

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.

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.

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.

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.

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.

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.

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.

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.

Effect of bax,bcl-2 and bcl-xL on regulating apoptosis in tissues of normal liver and hepatocellular carcinoma

AIM: To investigate the expression of bax, bcl-2 and bcl-xL mRNA in the tissues of normal liver and hepatocellular carcinoma (HCC), and analyze the relationship between the expression of bax, bcl-2 and bcl-xL mRNA and clinical parameters of HCC patients.METHODS: The expression of bax, bcl-2 and bcl-xL mRNA of normal liver and HCC was measured by Northern blot. Statistical analyses were made by t test and correlation analysis.RESULTS: A very low mRNA level was indicated at bax,bcl-2 and bcl-xL in the HCC tissues in contrast to the tissues of normal liver by Northern blot analysis. The analyses of mRNA level revealed that HCC tissues exhibited a mean 7.6-fold decrease in bax, 4.2-fold in bcl-2 and 3.5-fold in bcl-xL in comparison with normal control tissues, respectively. Positive correlation was found between bax and bcl-xL (r=0.7061,P＜0.01). There was no significance between the mRNA expression of these three genes and age, gender, tumor differentiation and tumor stage of HCC patients. CONCLUSION: The results are consistent with the fact that apoptosis rarely occurs in normal livers but increases in HCC, indicating that bcl-2 and bcl-xL may play a very important role in regulating the apoptosis of normal liver and HCC.

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.

黄芪多糖调控Bcl-2/Bax信号通路抑制卵巢腺癌Caov-3细胞生长的实验研究

目的研究黄芪多糖调控Bcl-2/Bax信号通路对卵巢腺癌Caov-3细胞生长的影响。方法将黄芪多糖分为低、中、高剂量组,分别加入10、50、100 mg/mL黄芪多糖,流式细胞术检测10、50、100 mg/mL黄芪多糖作用Caov-3细胞后对细胞周期的影响,显微镜下观察Caov-3细胞凋亡情况和形态变化,Western blot法检测3组药物作用Caov-3细胞后对Bcl-2、Bax蛋白表达水平及对Bcl/Bax的影响;同时,比较阿司匹林联合黄芪多糖与单用黄芪多糖对Caov-3细胞的抑制率。结果黄芪多糖低、中、高剂量组对Caov-3细胞的抑制率分别为40%、50%、65%,黄芪多糖的IC_(50)为50 mg/mL,与对照组比较,黄芪多糖高剂量组对Caov-3细胞的抑制效果最为明显,差异具有统计学意义(P<0.05);黄芪多糖联合阿司匹林的抑制效果优于单纯使用黄芪多糖(P<0.05)。与对照组比较,黄芪多糖高剂量组对Caov-3细胞周期的影响更为显著,差异具有统计学意义(P<0.05)。与对照组比较,黄芪多糖高剂量组Caov-3细胞凋亡的数目显著增加,Bcl-2蛋白表达水平显著降低,Bax蛋白表达水平显著升高,Bcl-2/Bax降低,差异均有统计学意义(P<0.05)。结论黄芪多糖能有效抑制Caov-3细胞的生长,联合阿司匹林效果更好。其机制可能与黄芪多糖调控Bcl-2/Bax信号通路,促进细胞凋亡有关。

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.

Expression of apoptosis related gen e Bcl-2 and Bax in osteosarcoma and their relations hip with the prognosis

Objective Apoptosis related gene Bcl-2and Bax in osteosarcoma patients with diffe rent clinical appearance were being studied to analyze the prognosis of the patient s.Method The cases were divided into two diffe rent groups according to the results of the follow-up.33cases in high-risk group and 18cases in low risk group.Expression of Bcl-2and B ax were immunohistochemically stai ned by ABC method.Result Positive expression rate of Bcl-2wa s 61%in high risk group(20/23)and 33%in low risk group(1/8).Positive expression of Bax was22%in high risk group(6/27)and 67%in low risk group(12/18).Conclusion Expression of Bcl-2and Bax was relat ed to the prognosis of osteosarcoma.Positively expressed Bcl-2in osteosarcoma cells may i ndicate bad prognosis.If Bax is high ly expressed in osteosarcoma cells,this may indicated a good prognosis.

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.

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.

Total saponins in Rubus parvifolius L.induce lymphoma cells apoptosis through upregulated Bax/Fas and downregulated Bcl-2 in vivo and in vitro

Purpose:To investigate the effect of total saponins of Rubus parvifolius L.(TSRP)on lymphoma Raji cells and further discuss its mechanism.Methods:The model of nude mice bearing Raji cells was established,the volume,weight and inhibition rate of the transplanted tumor were analyzed and compared after different concentrations of TSRP treatment.Cell apoptosis and expression of Bcl-2,Bax,Fas proteins were detected by TUNEL and immunohistochemiscal method respectively.Effects of TSRP on cell proliferation were tested with MTT assay in vitro.Cell apoptosis and expression of Caspase-9,Caspase-3,Bcl-2,Bax and Fas proteins were tested with DAPI staining and Western blot.Results:TSRP significantly reduced the volume and tumor weight of Raji subcutaneous transplanted tumor and induced the apoptosis of Raji cells in vivo.The tumor inhibition rate of high-dose(100 mg/kg)TSRP is 90.84%.The TUNEL test results show that the fluorescence intensity of the tumor issue treated with TSRP is significantly improved.Compared with the control group,the fluorescence intensity of high-concentration TSRP is 82.43 ± 7.81,which is significantly different(P<0.001).The results of immunohistochemistry test showed that the Bcl-2 expression of Raji cell treated with TSRP is obviously reduced,and Bax expression is obviously increased.Meanwhile,compared with that of control group,Fas expression is obviously reduced.MTT assay showed that TSRP can significantly inhibit proliferation of Raji cells with dose dependence.The inhibition rate of 400 μg/mL TSRP is 53.46 ± 4.90%(P<0.001).DAPI staining results showed that TSRP can significantly induce cell apoptosis.According to Western blot results,it is found that TSRP can significantly inhibit activity of Bcl-2 and increase Bax expression,and TSRP can also inhibit Fas expression.Meanwhile,expression of Caspase-9 and Caspase-3 is also increased.Conclusion:TSRP could inhibit the proliferation of lymphoma via induction of apoptosis in a time and dose-dependent manner.Apoptotic signaling induced by TSRP was characterized by up-regulating Bax,Fas and Caspase-8 protein expression,and down-regulating of Bcl-2 protein expression.