Upregulation of Bax and Bcl-2 following prenatal cocaine exposure induces apoptosis in fetal rat brain

Cocaine abuse during pregnancy has been associated with numerous adverse perinatal outcomes. Aims: The present study was to determine whether prenatal cocaine exposure induced apoptosis and the possible role of Bcl-2 family genes in the programming cell death in fetal rat brain. Main methods: Pregnant rats were treated with cocaine subcutaneously (30 & 60 mg/kg/day) from day 15 to 21 of gestation. Then the fetal and maternal brains were isolated. Key findings: Cocaine produced a dose-dependent decrease in fetal brain weight and brain/body weight ratio (P<0.05). Apoptotic nuclei in fetal brain were increased from 2.6 ± 0.1 (control) to 8.1± 0.6 (low dose) and 10.4 ± 0.2% (high dose) (P<0.05). In accordance, cocaine dose dependently increased activities of caspase-3, caspase-8, and caspase-9 (% of control) in the fetal brain by 177%, 155%, 174%, respectively, at 30 mg/kg/day, and by 191%, 176%, 274%, respectively, at 60 mg/kg/day. In contrast, cocaine showed no effect on caspase activities in the maternal brain. Cocaine produced a dose-dependent increase in both Bcl-2 and Bax protein expression in the fetal brain, and increased the ratio of Bax/Bcl-2 at dose of 30 mg/kg/day (P<0.05). Significance: Our study has demonstrated that prenatal cocaine exposure induces apoptosis in the fetal brain, and suggested that up-regulating Bax/Bcl-2 gene expression may be involved in cocaine-induced apoptosis. The increased apoptosis of neuronal cells in the fetal brain is likely to play a key role in cocaine-induced neuronal defects during fetal development.     

第三丁基过氧化氢损伤皮层神经元线粒体并诱导凋亡

目的：探讨第三丁基过氧化氢（t-BHP）诱导大鼠皮层神经元凋亡的可能机制。 方法： 体外培养大鼠皮层神经元，MTT法测定细胞存活率，DNA断裂评价细胞凋亡，流式细胞术测定线粒体跨膜电位（ΔΨm），分光光度计法测定细胞内谷胱甘肽（GSH）浓度，Western blot法测定Bcl-2和Bax蛋白和胞浆细胞色素c以及活化型半胱氨酸天冬氨酸蛋白酶3（caspase-3）和多聚（ADP-核糖）聚合酶（PARP）水平。 结果： tBHP（25－400 μmol/L）可明显抑制皮层神经元的生长，引起ΔΨm下降和线粒体内细胞色素c向胞浆释放，同时细胞内GSH浓度以及Bcl-2蛋白水平下降，Bax蛋白水平增加，caspase-3和PARP得以激活并最终导致神经细胞凋亡。 结论： tBHP引起的氧化应激可通过损伤线粒体诱导皮层神经元凋亡。     

Sequential expression patterns of apoptosis- and cell cycle-related proteins in neuronal response to severe or mild transient hypoxia

Severe hypoxia was shown to induce apoptotic death in developing brain neurons, whereas mild hypoxia was demonstrated to stimulate neurogenesis. Since the apoptotic process may share common pathways with mitosis, expression profiles of proteins involved in apoptosis or the cell cycle were analyzed by immunohistochemistry and/or western blotting, in relation with cell outcome of cultured neurons from fetal rat forebrain subjected to either lethal (6 h) or non-lethal (3 h) hypoxia (95% N(2)/5% CO(2)). Hypoxia for 6 h led to apoptosis that was inhibited by the cell cycle blocker olomoucine. Transient overexpression of proliferating cell nuclear antigen was followed by increasing expression of p53, p21, Bax and caspases, whereas Bcl-2 and heat shock proteins were progressively repressed. Conversely, a 3-h hypoxic insult initiated neuronal mitosis, with increased thymidine incorporation. In these conditions, levels of proliferating cell nuclear antigen, Rb, Bcl-2 and heat shock proteins were persistently elevated, while expression of p53, p21, Bax and caspases gradually decreased.These data confirm that hypoxia promotes cell cycle activation, whatever the stress intensity. This process is then aborted following apoptosis-inducing hypoxia, whereas sublethal insult would trigger neurogenesis, at least in developing brain neurons in vitro, by stimulating timed expression of neurogenic and survival-associated proteins.     

CART肽抑制NMDA诱导海马神经元凋亡的机制

目的：探讨可卡因－苯丙胺调节转录肽（CART）对兴奋性氨基酸N-甲基-D-天冬氨酸（NMDA）诱导海马神经元凋亡的作用和机制。方法：18 d SD大鼠胚胎（E18）海马神经元原代培养8 d,用MTT吸光度值、DNA ladder、Hoechst染色、Western blotting方法观察NMDA引起的神经元凋亡,以及CART肽对NMDA诱导的细胞凋亡的影响,分析CART肽的作用机制。结果：CART肽预处理组海马神经元存活率明显高于NMDA组（P<0.05）;DNA ladder和核形态Hoechst染色证明,CART肽明显抑制NMDA诱导的海马神经元凋亡（P<0.01）。CART肽预处理明显促进NMDA组海马神经元中Bcl-2表达（P<0.01）,增加Bcl-2/Bax比值,抑制caspase-9及caspase-3活化（P<0.01）;但CART肽对Bax的表达及caspase-8活化无显著影响。结论： CART肽抑制NMDA诱导的海马神经元凋亡,呈明显的神经保护作用,其神经保护机制主要是提高抗凋亡分子Bcl-2表达,抑制线粒体凋亡途径启动分子caspase-9和执行分子caspase-3的活化。     

培补肝肾方药对帕金森模型小鼠黑质内Bax和Bcl-2表达的影响

目的：观察培补肝肾方药对帕金森模型小鼠黑质内Bax和Bcl-2表达的影响，探讨此方药预防帕金森病的机制。方法：成年C57-BL雄性小鼠分为对照组、模型组和中药治疗组。应用免疫组织化学染色技术，观察各组小鼠黑质内Bax和Bcl-2表达的变化情况。结果：Bax和Bcl-2阳性神经元主要位于黑质致密部，其中Bax和Bcl-2阳性神经元的数量在对照组和中药治疗组均较少且组间无显著差异；MPTP造模后，Bax阳性神经元的数量随时间明显递增，Bcl-2阳性神经元的数量亦呈逐渐递增的趋势。结论：培补肝肾方药可能通过抑制黑质神经元内Bax基因表达的途径抑制黑质神经元的凋亡，从而对帕金森病发挥预防和治疗作用。     

AZD8055对胆管癌细胞自噬和凋亡的影响

目的:探讨哺乳动物雷帕霉素靶蛋白(mTOR)双重抑制剂AZD8055对人胆管癌HuCCT1细胞自噬和凋亡的影响。方法:采用MTT法检测不同浓度的AZD8055对HuCCT1细胞活力的抑制作用;吖啶橙染色检测AZD8055处理胆管癌细胞后,细胞自噬小体形成情况;Western blot法观察细胞凋亡相关蛋白Bcl-2、Bax和cleaved caspase-3以及自噬相关标志蛋白beclin 1、LC3和p62的表达;AnnexinⅤ-FITC/PI双染法检测细胞凋亡。结果:AZD8055显著抑制胆管癌细胞的活力(P0.05)。吖啶橙染色后,AZD8055用药组橙红色颗粒增多。Western blot实验显示,AZD8055处理后,与对照组相比自噬标志蛋白beclin 1表达上调,LC3-II/LC3-I比例上调,p62表达下调;cleaved caspase-3表达降低,促凋亡蛋白Bax表达降低,抑凋亡蛋白Bcl-2表达升高(P0.05)。流式细胞术显示,AZD8055可抑制细胞凋亡。结论:AZD8055可抑制胆管癌细胞的生长,其机制可能与诱导细胞自噬相关。     

Increased osteoblast apoptosis in apert craniosynostosis: role of protein kinase C and interleukin-1

Apert syndrome is an autosomal dominant disorder characterized by premature cranial ossification resulting from fibroblast growth factor receptor-2 (FGFR-2)-activating mutations. We have studied the effects of the prominent S252W FGFR-2 Apert mutation on apoptosis and the underlying mechanisms in human mutant osteoblasts. In vivo analysis of terminal deoxynucleotidyl transferase-mediated nick-end labeling revealed premature apoptosis of mature osteoblasts and osteocytes in the Apert suture compared to normal coronal suture. In vitro, mutant osteoblasts showed increased apoptosis, as demonstrated by terminal deoxynucleotidyl transferase-mediated nick-end labeling analysis, trypan blue staining, and DNA fragmentation. Mutant osteoblasts also showed increased activity of caspase-8 and effector caspases (-3, -6, -7) constitutively. This was related to protein kinase C activation because the selective protein kinase C inhibitor calphostin C inhibited caspase-8, effector caspases, and apoptosis in mutant osteoblasts. Apert osteoblasts also showed increased expression of interleukin (IL)-1alpha, IL-1beta, Fas, and Bax, and decreased Bcl-2 levels. Specific neutralizing anti-IL-1 antibody reduced Fas levels, Bax expression, effector caspases activity, and apoptosis in mutant cells. Thus, the Apert S252W FGFR-2 mutation promotes apoptosis in human osteoblasts through activation of protein kinase C, overexpression of IL-1 and Fas, activation of caspase-8, and increased Bax/Bcl-2 levels, leading to increased effector caspases and DNA fragmentation. This identifies a complex FGFR-2 signaling pathway involved in the premature apoptosis induced by the Apert S252W FGFR-2 mutation in human calvaria osteoblasts.     

阿司匹林对同源不同辐射抗拒能力鼻咽癌细胞的凋亡诱导作用

目的:探讨阿司匹林诱导同源不同辐射抗拒能力鼻咽癌细胞株CNE2R/CNE2凋亡的作用及其机制。方法:采用MTT法、流式细胞术和Western blot法检测并比较阿司匹林对CNE2R和CNE2细胞活力、细胞凋亡及凋亡相关蛋白procaspase-3、cleaved caspase-3、procaspase-9、procaspase-12、PARP、cleaved PARP、Bcl-2和Bax,以及PI3K p110α、Akt和p27蛋白水平的影响。结果:阿司匹林抑制同源不同辐射抗拒能力细胞CNE2R/CNE2的活力(阿司匹林对CNE2细胞作用24、48和72 h的IC50分别为6.18、3.92和3.06 mmol/L,对CNE2R细胞作用24、48和72 h的IC50分别为7.05、3.90和2.20 mmol/L,两者差异无统计学显著性)。阿司匹林作用48 h后,CNE2R细胞凋亡率升高,明显高于CNE2细胞(P0.05)。阿司匹林作用48 h后,CNE2和CNE2R细胞中procaspase-3、procaspase-9、procaspase-12和PARP的蛋白水平降低,cleaved caspase-3和cleaved PARP的蛋白水平升高(P0.05);PI3K p110α和Akt蛋白水平下降,Bcl-2水平降低,Bax水平升高,Bcl-2/Bax比值降低,p27水平升高(P0.05)。结论:阿司匹林对同源不同辐射抗拒能力鼻咽癌细胞株CNE2R和CNE2具有同等细胞活力抑制作用;并且可以诱导细胞凋亡,且对抗辐射细胞株CNE2R的凋亡诱导作用更明显。阿司匹林的抗癌作用可能与其影响PI3K/Akt信号通路及其下游蛋白的水平有关。     

Activation of multiple molecular mechanisms for apoptosis in human malignant glioblastoma T98G and U87MG cells treated with sulforaphane

Glioblastoma is the most malignant and prevalent brain tumor that still remains incurable. Recent studies reported anti-cancer effect of the broccoli-derived compound sulforaphane. We explored the mechanisms of sulforaphane-mediated apoptosis in human glioblastoma T98G and U87MG cells. Wright staining and ApopTag assay confirmed apoptosis in glioblastoma cells treated with sulforaphane. Increase in intracellular free Ca2+ was detected by fura-2 assay, suggesting activation of Ca2+-dependent pathways for apoptosis. Western blotting was used to detect changes in expression of Bax and Bcl-2 proteins resulting in increased Bax:Bcl-2 ratio that indicated a commitment of glioblastoma cells to apoptosis. Upregulation of calpain, a Ca2+-dependent cysteine protease, activated caspase-12 that in turn caused activation of caspase-9. With the increased Bax:Bcl-2 ratio, cytochrome c was released from mitochondria to cytosol for sequential activation of caspase-9 and caspase-3. Increased calpain and caspase-3 activities generated 145 kD spectrin breakdown product and 120 kD spectrin breakdown product, respectively. Activation of caspase-3 also cleaved the inhibitor-of-caspase-activated-DNase. Accumulation of apoptosis-inducing-factor in cytosol suggested caspase-independent pathway of apoptosis as well. Two of the inhibitor-of-apoptosis proteins were downregulated because of an increase in 'second mitochondrial activator of caspases/Direct inhibitor-of-apoptosis protein binding protein with low pI.' Decrease in nuclear factor kappa B and increase in inhibitor of nuclear factor kappa B alpha expression favored the process of apoptosis. Collectively, our results indicated activation of multiple molecular mechanisms for apoptosis in glioblastoma cells following treatment with sulforaphane.     

自噬水平变化对心脏骤停心肺复苏后大鼠海马神经元凋亡的影响

目的探讨自噬水平变化对心脏骤停心肺复苏(CA/CPR)后大鼠海马神经元凋亡的影响。方法将40只大鼠随机分为假手术组(sham)、CA/CPR模型组(model)、雷帕霉素(Rapa)组(CA/CPR+Rapa)及3-甲基腺嘌呤(3-MA)组(CA/CPR+3-MA)。采用呼气末夹闭气管窒息法复制大鼠CA/CPR动物模型,分别给予自噬激动剂Rapa 0.2 mg/kg及自噬抑制剂3-MA 10 mg/kg进行干预。采用神经功能缺陷评分(NDS)评价CA/CPR大鼠神经功能;用TUNEL染色法检测大鼠海马神经元的凋亡变化;用RT-PCR和Western blotting法检测大鼠海马内微管蛋白轻链3(LC3)、Beclin-1、Bax、Bcl-2及Caspase-3 mRNA和蛋白的表达水平。结果与假手术组比较,CA/CPR模型组大鼠NDS评分明显降低;海马神经元TUNEL染色阳性细胞数明显增多,凋亡率显著升高;海马内LC3、Beclin-1、Caspase-3、Bax表达上调,Bcl-2表达下调(P<0.05,P<0.01)。与模型组比较,CA/CPR+Rapa大鼠NDS评分明显降低,而海马神经元凋亡率明显有所增加,海马内LC3、Beclin-1、Caspase-3、Bax表达明显上调,而Bcl-2表达则明显有所下降;CA/CPR+3-MA大鼠NDS评分明显升高,而海马神经元凋亡率下降,海马内LC3、Beclin-1、Caspase-3、Bax表达明显下调,而Bcl-2表达则有所升高(P<0.05,P<0.01)。结论 CA/CPR后自噬水平升高促进海马神经元凋亡,自噬水平降低抑制海马神经凋亡,两者相互作用共同参与CA/CPR的病理过程。     

抑制miR-421表达增强宫颈癌细胞放疗敏感性

目的:研究抑制miR-421表达增强宫颈癌细胞放疗敏感性的分子机制。方法:运用脂质体2000将miR-421 inhibitor转染4株宫颈癌细胞系HeLa、SiHa、C33A和CaSki,以转染阴性核苷酸为对照,real-time PCR检测子宫内膜上皮细胞ESC细胞和4株宫颈癌细胞中miR-421的表达水平;转染的细胞经不同剂量电离辐射(0、2、4、6和8 Gy)处理48 h后运用MTT法、ELISA和Annexin V-FITC/PI染色法联合流式细胞术分别测定细胞活力、乳酸脱氢酶(LDH)漏出率和细胞凋亡率;运用Western blot检测细胞中cleaved caspase-9、caspase-9、cleaved PARP、PARP、Bcl-2和Bax蛋白的水平。结果:miR421在ESC细胞中低表达,而在4株宫颈癌细胞中高表达,转染miR-421 inhibitor后miR-421显著低于阴性对照组(P0.05)。相同条件下,辐照后低表达miR-421的宫颈癌细胞活力和LDH漏出率显著低于阴性对照组,72 h的细胞凋亡率明显增加(P0.05)。Western blot结果显示,电离辐射后与阴性对照组比较,低表达miR-421的宫颈癌细胞中cleaved caspase-9、cleaved PARP和Bcl-2的蛋白水平明显增高,而Bax蛋白水平显著降低(P0.05)。结论:miR-421在正常子宫内膜上皮ESC细胞中低表达,而在宫颈癌细胞系中高表达;下调miR-421表达能抑制宫颈癌细胞的活力,显著增强宫颈癌细胞的辐射敏感性,其机制至少部分通过激活caspase-9细胞凋亡通路进而促进抗凋亡蛋白Bcl-2表达和抑制促凋亡蛋白Bax表达而实现。     

Bax cleavage implicates caspase-dependent H2O2-induced apoptosis of hepatocytes

Oxidative stress plays an important role in the development of ischemia/reperfusion (I/R)-induced apoptosis of hepatocytes. We aimed to examine the involvement of caspases and calpains in H2O2-induced hepatic cell apoptosis. TUNEL-positive apoptotic cells appeared in parallel with poly(ADP-ribose) polymerase (PARP) cleavage and procaspase-3 proteolysis by H2O2 treatment in a dose-dependent manner (250-1,000 micro M). Bcl-xL and intact Bax expression levels decreased when H2O2 was >250 micro M. The cleaved form of Bax appeared prior to caspase-3 activation, increasing in a dose-dependent manner. A pan-caspase inhibitor, Z-VAD-fmk, completely blocked H2O2-induced procaspase-3 proteolysis and PARP cleavage without changing Bax cleavage, but partially attenuated H2O2-induced apoptosis. Calpeptin, a calpain inhibitor, did not inhibit caspase-3 activation, Bax cleavage or apoptosis. Our results indicate that Bax cleavage is upstream signal of caspase-dependent apoptosis in hepatocytes exposed to H2O2, but not independent upon calpain. Molecular targeting of Bax cleavage may allow the development of strategies to prevent hepatic I/R injury.     

Nitric oxide is involved in ischemia-induced apoptosis in brain: a study in neuronal nitric oxide synthase null mice

Nitric oxide can promote or inhibit apoptosis depending on the cell type and coexisting metabolic or experimental conditions. We examined the impact of nitric oxide on development of apoptosis 6, 24, and 72 h after permanent middle cerebral artery occlusion in mutant mice that lack the ability to generate nitric oxide from neuronal nitric oxide synthase. Adjacent coronal sections passing through the anterior commissure were stained with hematoxylin and eosin or terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL). Immunoblotting was used to identify changes in the anti- and proapoptotic proteins Bcl-2 and Bax, respectively. Activation of caspases was assessed by appearance of actin cleavage products using a novel antiserum directed against 32-kDa actin fragment (fractin). In the neuronal nitric oxide synthase mutant mouse, infarct size and TUNEL positive apoptotic neurons were reduced compared to the wild-type controls. At 6 h, Bcl-2 levels in the ischemic hemisphere were increased in mutants but decreased in the wild-type strain. Bax levels did not change significantly. Caspase-mediated actin cleavage appeared in the ischemic hemisphere at this time point, and was significantly less in mutant brains at 72 h compared to the wild-type. The reduction in the number of TUNEL and fractin positive apoptotic cells appears far greater than anticipated based on the smaller lesion size in mutant mice.Hence, from these data we suggest that a deficiency in neuronal nitric oxide production slows the development of apoptotic cell death after ischemic injury and is associated with preserved Bcl-2 levels and delayed activation of effector caspases.     

miR-29b通过调控Bax基因抑制LPS诱导的肺泡上皮细胞系A549凋亡

目的探讨miR-29b对脂多糖(LPS)诱导的肺泡上皮细胞系A549凋亡的影响及其机制。方法将体外培养的A549细胞分为对照组、LPS组(给予10 mg/L的LPS处理)、LPS+miR-NC组(转染miR-29b mimics阴性对照后给予LPS处理)、LPS+miR-29b组(转染miR-29b mimics后给予LPS处理);用RT-qPCR检测细胞中miR-29b的表达水平;MTT法检测细胞存活率;流式细胞仪检测细胞凋亡率;Western blot检测Bcl-2、Bax和cleaved caspase-3蛋白的表达;双荧光素酶报告基因实验检测Bax和miR-29b的靶向关系。结果与对照组相比,LPS组、LPS+miR-NC组和LPS+miR-29b组细胞中miR-29b、Bcl-2蛋白的表达水平和细胞存活率均明显降低,而细胞凋亡率和Bax、cleaved caspase-3蛋白的表达水平均明显升高(P<0.05);与LPS组相比,LPS+miR-29b组细胞中miR-29b、Bcl-2蛋白的表达水平和细胞存活率均明显升高,而细胞凋亡率和Bax、cleaved caspase-3蛋白的表达水平均明显降低(P<0.05)。双荧光素酶报告基因实验证实Bax是miR-29b的潜在靶基因。结论miR-29b可抑制LPS诱导的A549细胞凋亡,其作用机制可能与靶向调控Bax表达有关。     

Neural Cell Apoptosis Induced by Microwave Exposure Through Mitochondria-dependent Caspase-3 Pathway

To determine whether microwave (MW) radiation induces neural cell apoptosis, differentiated PC12 cells and Wistar rats were exposed to 2.856GHz for 5min and 15min, respectively, at an average power density of 30 mW/cm². JC-1 and TUNEL staining detected significant apoptotic events, such as the loss of mitochondria membrane potential and DNA fragmentation, respectively. Transmission electron microscopy and Hoechst staining were used to observe chromatin ultrastructure and apoptotic body formation. Annexin V-FITC/PI double staining was used to quantify the level of apoptosis. The expressions of Bax, Bcl-2, cytochrome c, cleaved caspase-3 and PARP were examined by immunoblotting or immunocytochemistry. Caspase-3 activity was measured using an enzyme-linked immunosorbent assay. The results showed chromatin condensation and apoptotic body formation in neural cells 6h after microwave exposure. Moreover, the mitochondria membrane potential decreased, DNA fragmentation increased, leading to an increase in the apoptotic cell percentage. Furthermore, the ratio of Bax/Bcl-2, expression of cytochrome c, cleaved caspase-3 and PARP all increased. In conclusion, microwave radiation induced neural cell apoptosis via the classical mitochondria-dependent caspase-3 pathway. This study may provide the experimental basis for further investigation of the mechanism of the neurological effects induced by microwave radiation.     

Apoptotic proteins in the course of aging of central nervous system in the rat

Studies were performed on the level of cells with damaged DNA (TUNEL), the level of protein engaged in DNA repair (PARP) and the level of proteins indicating the extent of apoptosis (Bax:Bcl-2) (Western blot). The studies were performed on cerebral cortex (GM), white matter (WM), medulla oblongata (MO), cerebellum (C) of rats, 3.0-3.5-, 12-, 24-months of age. The highest levels of DNA injury in GM of 1-year-old rats and in MO of 2-year-old rats were accompanied by peak levels of PARP. In the remaining structures (WM, C) levels of DNA injury showed no correspondence with levels of PARP. Levels of Bax proteins exceeded levels of Blc-2 protein in all cerebral structures of young rats. In old animals, Bax protein continued to exceed Blc-2 levels both in GM and in MO, in which most pronounced fragmentation of DNA was observed. The data indicated that in spite of high level of TUNEL positive cells in aged brain PARP and Bcl-2 are probably engaged in protection of the cells against death.     

沉默Apaf-1 基因对氧糖剥夺/ 复氧复糖PC12 细胞线粒体凋亡通路的影响

目的:探讨RNA干扰沉默Apaf-1基因对氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路的影响。方法:PC12细胞随机分为3组:正常组(Control)、模型组(Model)、Apaf-1基因沉默组(Apaf-1-siRNA)。正常组于CO2培养箱内正常培养,其余2组给予氧糖剥夺2 h、复氧复糖24 h处理,Apaf-1-siRNA组于造模前将化学合成的siRNA通过脂质体转染于PC12细胞靶向沉默Apaf-1基因。用荧光标记的siRNA检测Apaf-1转染效率,Western blot检测转染后PC12细胞Apaf-1蛋白表达,CCK-8检测细胞存活率,TUNEL染色检测细胞凋亡指数,流式细胞术检测细胞凋亡率,免疫荧光染色检测Bax/Bcl-2比值,Western blot检测线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达。结果:Apaf-1-siRNA可有效沉默PC12细胞Apaf-1蛋白表达(P<0.05)。与Control组相比,Model组细胞存活率明显降低(P<0.05),细胞凋亡指数和凋亡率显著升高(P<0.05),Bax/Bcl-2比值升高(P<0.05),线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达显著升高(P<0.05);与Model组相比,Apaf-1-siRNA组细胞存活率显著升高(P<0.05),细胞凋亡指数和凋亡率显著降低(P<0.05),Bax/Bcl-2比值降低(P<0.05),Apaf-1、caspase-9、caspase-3蛋白表达均明显降低(P<0.05)。结论:靶向沉默Apaf-1基因可有效降低氧糖剥夺/复氧复糖PC12细胞线粒体凋亡通路关键蛋白Apaf-1、caspase-9、caspase-3表达,抑制细胞凋亡,提高细胞存活率。     

Ectopic expression of Bcl-2 and Bcl-xL inhibits apoptosis induced by TNF-related apoptosis-inducing ligand (TRAIL) through suppression of caspases-8, 7, and 3 and BID cleavage in human acute myelogenous leukemia cell line HL-60.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is one of the latest members of the TNF superfamily known to induce apoptosis in a wide variety of tumor cells. Some cell types, however, are quite resistant to TRAIL. We investigated the effect of ectopic expression of Bcl-2 and Bcl-xL on TRAIL-induced apoptosis in human acute myelogenous leukemia HL-60 cells. We found that HL-60 cells, which express TRAIL receptors (also called death receptor, DR) DR4, DR5, and Dc (decoy) R2, are highly sensitive to TRAIL-induced cytotoxicity. Greater than 90% killing occurred within 24 h of TRAIL treatment. The expression of Bcl-2 and Bcl-xL, however, completely abolished the TRAIL-induced cytotoxic effects. Treatment of HL-60 cells with TRAIL induced caspase-8 activation within 2-4 h, but no activation could be seen in Bcl-2-expressing or Bcl-xL-expressing cells. TRAIL also induced cleavage of BID, which was also abolished by Bcl-2 and Bcl-xL. Similarly, TRAIL activated caspase-3 and caspase-7 in control cells but not in cells expressing Bcl-2 or Bcl-xL. Cleavage of the caspase-3 substrate poly(ADP-ribose) polymerase (PARP), was abrogated by ectopic expression of Bcl-2 and Bcl-xL. Inhibition of caspases by the pan-caspase inhibitor, benzyloxycarbonyl-valine-alanine-aspartate-fluoromethylketone (zVAD-fmk) abolished the TRAIL-induced apoptosis. Overall, these results indicate that TRAIL-induced apoptosis involves activation of caspase-8, caspase-7, caspase-3, and BID cleavage, and Bcl-2 and Bcl-xL prevents TRAIL-induced apoptosis by abrogating caspase activation and BID cleavage.     

叶黄素抑制叔丁基过氧化氢诱导的视网膜神经节细胞凋亡

目的:观察叶黄素(lutein)对叔丁基过氧化氢(t-BHP)处理的视网膜神经节细胞(RGC-5细胞系)的保护效应并探讨其作用机制。方法:用免疫荧光染色检测视网膜神经节特异性蛋白Brn-3和神经微管结合蛋白MAP-2的表达来鉴定RGC-5细胞;将RGC-5细胞随机分为对照组、t-BHP处理组、t-BHP和lutein共同处理组、lutein处理组,培养24 h,MTT实验检测细胞活力;Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;免疫细胞化学技术检测caspase-3蛋白的活化情况;Western blot检测Bcl-2/Bax、cleaved caspase-3、JNK和c-Jun蛋白的变化。结果:MTT实验和流式细胞检测结果显示,lutein能提高t-BHP处理的RGC-5细胞的活力,并降低t-BHP诱导的RGC-5细胞凋亡;免疫荧光结果显示lutein能抑制t-BHP诱导的caspase-3的活化;与对照组比较,t-BHP处理后RGC-5细胞抗凋亡蛋白Bcl-2表达下调(P0.05),Bax/Bcl-2比率升高,cleaved caspase-3表达上调(P0.05),JNK和c-Jun蛋白的磷酸化水平增加(P0.05),t-BHP的上述作用可被lutein部分逆转。结论:Lutein能够降低t-BHP诱导的RGC-5细胞凋亡,其机制与其上调Bcl-2的表达、抑制caspase-3的活化并降低JNK和c-Jun蛋白的磷酸化有关。     

ATG5敲低后抑制人肺癌细胞H1299自噬并增强南蛇藤素诱导的细胞凋亡

目的构建自噬相关基因5(autophagy-related gene 5,ATG5)低表达肺癌细胞株,观察肺癌细胞自噬活性,检测南蛇藤素对肺癌细胞凋亡的影响。方法用ATG5 shRNA技术构建ATG5敲低的人肺癌H1299细胞株作为ATG5敲低组,未敲低ATG5的H1299细胞作为对照组;荧光定量PCR和Western blot检测肺癌细胞中ATG5的表达和自噬标志物微管相关轻链蛋白3(microtubule-associated protein 1A/1B-light chain 3,LC3)及死骨片蛋白1的(sequestosome 1,P62)表达,并转染红色荧光标记的LC3质粒观察LC3斑点聚集情况;经南蛇藤素刺激后,以流式细胞术检测细胞凋亡,最后使用Western blot检测cleaved caspase-3、Bcl-2和Bax等蛋白的表达。结果慢病毒感染组ATG5较对照组mRNA和蛋白表达水平明显降低(P0.05);ATG5敲低后LC3-Ⅱ水平下降,P62水平上升,并且转染后RFP-LC3斑点聚集减少(P0.05)。相比对照组,南蛇藤素明显促进ATG5敲低细胞凋亡(P0.01);ATG5敲低组中促凋亡分子Bax、cleaved caspase-3表达比对照组明显增加(P0.05),抗凋亡蛋白Bcl-2表达减少(P0.05)。结论 ATG5敲低抑制肺癌细胞自噬后,南蛇藤素能够明显增强人肺癌细胞的凋亡,提示抑制肺癌细胞自噬可能为针对性处理肺癌细胞耐药提供新的思路。