地塞米松诱导小鼠胸腺细胞线粒体去极化与凋亡进程研究

目的研究地塞米松(DEX)诱导小鼠胸腺细胞凋亡进程中线粒体去极化作用特点。方法无菌获取Balb/c小鼠胸腺细胞,设对照组和DEX组;在5 h时点,利用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡;利用DiOC6(3)/PI双染流式细胞术检测细胞线粒体去极化与死亡;利用DiOC6(3)/Annexin V-PE双染流式细胞术检测凋亡过程中的去极化现象。结果在1×10-6mol/L DEX诱导下,小鼠胸腺细胞在5 h凋亡百分率为(36.20±5.11)%,对照组为(4.10±0.98)%,差异显著(P<0.01);DEX组坏死百分率为(4.07±0.24)%,对照组为(1.25±0.25)%,差异显著(P<0.01)。DEX组线粒体去极化增强仍存活的细胞所占百分率为(46.77±6.21)%,显著高于对照组的(12.80±4.55)%(P<0.01)。DEX组线粒体去极化增强且已启动凋亡的细胞为(35.34±4.19)%,显著高于对照组的(7.21±0.61)%(P<0.01)。DEX组线粒体去极化作用增强未凋亡的细胞为(13.68±1.27)%,显著高于对照组的(6.85±0.92)%(P<0.01)。结论DEX诱导小鼠胸腺细胞发生典型细胞凋亡和线粒体去极化增强,在该进程中,线粒体去极化增强发生在细胞膜磷脂酰丝氨酸外翻之前。     

Molecular mechanism of satratoxin-induced apoptosis in HL-60 cells: activation of caspase-8 and caspase-9 is involved in activation of caspase-3

Satratoxins have been recognized as potential immunomodulatory agents in outbreaks of building-related illness. Here we report that satratoxin G-treated human leukemia HL-60 cells underwent apoptosis through the action of caspase-3 which was activated by both caspase-8 and caspase-9. Western blot analysis of caspase-3 in the satratoxin G-treated cells apparently indicated the appearance of a catalytically active fragment of 17 kDa. Increased caspase-3 activity was also detected by using a fluorogenic substrate, DEVD-AMC. Next, exposure to satratoxin G led to cleavage of PARP from its native 116 kDa form to a 85 kDa product. Moreover, DFF-45/ICAD were cleaved into a 12.5 kDa fragment via satratoxin G treatment. Enzymic assay on IETD-AMC revealed that caspase-8 is strongly activated by exposure to satratoxin G while T-2 toxin (T-2) could not activate caspase-8 at an early stage of apoptosis. Furthermore, satratoxin G caused a release of cytochrome c from mitochondria into the cytosol and increased the activity of caspase-9 against LEHD-AMC. These findings indicate that satratoxin G-induced apoptosis involves activation of caspase-3 and DFF-40/CAD through both activation of caspase-8 and cytosolic accumulation of cytochrome c along with activation of caspase-9.     

Cytochrome c release, mitochondrial membrane depolarization, caspase-3 activation, and Bax-alpha cleavage during IFN-alpha-induced apoptosis in Daudi B lymphoma cells.

Interferon-alpha (IFN-alpha) displays antitumor action by inducing direct cytotoxicity against tumor cells in addition to generation of cytotoxic cells. The IFN-alpha-induced direct cytotoxicity is at least partly due to induction of apoptosis. In the present study, we examined signaling pathways implicated in IFN-alpha-induced apoptosis in Daudi cells. Release of cytochrome c from mitochondria to cytosol was found after 12 h incubation with IFN-alpha, followed by a decline in mitochondrial membrane potential (Delta psi(m)) and procaspase-3 activation at 24 and 36 h, respectively. Cleavage of endogenous Bax-alpha (21 kDa), generating an 18-kDa fragment (p18 Bax-alpha), was found at 36 h. Although the endogenous p21 Bax-alpha was located in both cytosol and mitochondrial membranes, the p18 Bax-alpha resided only on mitochondrial membranes. IFN-alpha-induced apoptosis occurred 48 h after stimulation, with a further increase in proportion up to 72 h. Pretreatment with pancaspase inhibitor Z-VAD-fmk substantially inhibited the IFN-alpha-mediated Bax-alpha cleavage and apoptosis, but not the decline in Delta psi(m), suggesting the possibility that caspase-3 activation is implicated in the Bax-alpha cleavage, probably leading to amplification of the apoptotic processes. Our results suggest that modulation of endogenous p21 Bax-alpha is implicated in IFN-alpha-induced apoptosis.     

HIV gp41-induced apoptosis is mediated by caspase-3-dependent mitochondrial depolarization, which is inhibited by HIV protease inhibitor nelfinavir

Apoptotic loss of CD4+ T cells has been proposed as a mechanism of T cell depletion in human immunodeficiency virus (HIV) infections resulting in immunodeficiency. The Env glycoprotein has been implicated in apoptosis of uninfected bystander cells via gp120 binding to CD4/CXC chemokine receptor 4 as well as the fusion/hemifusion process mediated by gp41. Using an in vitro model of coculture of Env-expressing cells as effectors and CD4+ T cells as targets, we find that apoptosis mediated by Env glycoprotein in bystander cells in fact correlates with gp41-induced hemifusion. Further, the apoptotic pathway initiated by this interaction involves caspase-3-dependent mitochondrial depolarization and reactive oxygen species production. HIV gp41-induced mitochondrial depolarization is inhibited by protease inhibitor nelfinavir but not by other HIV protease inhibitors or inhibitors of calpain and cathepsin. This "kiss of death" (hemifusion) signaling pathway is independent of p38 mitogen-activated protein kinase and p53, making it distinct from the apoptosis seen in syncytia. We also show that virion-induced apoptosis is gp41-dependent. Our findings provide new insights into the mechanism via which HIV gp41 mediates apoptosis in bystander cells.     

Gleditsia sinensis fruit extract-induced apoptosis involves changes of reactive oxygen species level, mitochondrial membrane depolarization and caspase 3 activation

Recently, we have shown that the anomalous fruit extract of Gleditsia sinensis (GSE) processes apoptotic activity on numerous solid tumour and leukaemia cell lines as well as primary cultured leukaemia cells obtained from bone marrow aspirate of patients. GSE treated cancer cells exhibited apoptotic features as readily illustrated by morphological investigation, DNA fragmentation analysis and TUNEL labelling methods. Elevation of intracellular superoxide dismutase activity was observed. However, the detailed mechanism still remains undefined. Here we further demonstrated that cell cycle arrest, increment of hydrogen peroxide production, changes of intracellular acid-base equilibrium and mitochondrial membrane potential depolarization (DeltaPsi(m)) were induced from cancer cells after GSE incubation. Caspase 3 protease activity was significantly enhanced upon GSE treatment. Taken together, a defined signaling pathway for the mechanistic action of GSE on cancer cells was worked out.     

地塞米松介导胸腺细胞凋亡过程中线粒体和细胞结构蛋白的变化

目的：研究地塞米松(DEX)介导的小鼠胸腺细胞凋亡过程中线粒体质量和结构蛋白变化特点。 方法： 以地塞米松（DEX）诱导小鼠胸腺细胞凋亡为模型，利用Annexin V-FITC/PI双染流式细胞术研究细胞凋亡和坏死，JC-1染色流式细胞术检测线粒体膜电势（△Ψm）和线粒体质量，利用CFDA-SE染色流式细胞术检测细胞结构蛋白变化。 结果： 在1×10-6mol/L DEX诱导下，小鼠胸腺细胞在6 h凋亡比率为(51.25±5.51)%，对照组为(12.03±2.00)%，差异显著（P<0.01）； DEX组坏死比率为(30.25±3.67)%，对照组为(10.11±1.11)%，差异显著（P<0.01）。DEX组在6h时点的线粒体质量显著低于对照组（P<0.01），FL1平均荧光强度分别为（561.62±54.27）和（900.25±38.80）。DEX同时引起线粒体膜电势的显著下降（P<0.01），对照组FL2平均荧光强度为（267.51±26.48），DEX组为（133.17±12.29）。成熟T细胞培养48 h，CFDA-SE法仅检测到亲代单一细胞峰；而在Con A刺激条件下出现3个子代峰。对照组小鼠胸腺细胞在CFDA-SE染色培养6 h条件下，存在(5.25±1.15)％的低荧光强度细胞群，而在DEX刺激下，该群细胞占（47.39±9.76）％，并且在直方图结果上形成明显的细胞峰。 结论： DEX诱导小鼠胸腺细胞凋亡过程中，线粒体质量和细胞结构蛋白均有所下降；CFDA-SE染色流式细胞术可以作为基于细胞结构蛋白变化的凋亡定量检测方法。     

Fibroblast apoptosis and caspase-8 activation in aseptic loosening

The presence of apoptosis has been investigated in the interface membranes collected during revision surgery of loosened total hip joint arthroplasty (THAs). Terminal deoxyrobonucleotidyl transferase (TdT) assay for apoptotic DNA fragmentation quantification revealed a statistically significant presence of apoptosis in aseptic samples, obtained from both cementless (2.37+/-0.6%) and cemented (12.01+/-1%) prosthesis compared to septic samples where apoptosis was almost absent. Activated caspase-8 immunostaining was almost undetectable in septic samples, while in the aseptic samples active caspase-8 was present weakly in the cementless samples (1.35+/-0.22%) and strongly in the cemented ones (9.0+/-0.40%). The caspase-8 cytoplasmatic staining allowed the morphological recognition of positive cells both as fibroblast-like and immunocompetent cells. In aseptic cemented samples fibroblast-like cells were the most represented subpopulation in the caspase-8 positive population scored (76.6%) compared to the immunocompetent cells (23.4%). Caspase-8 activation is an upstream event in the apoptotic pathway triggered by the activation of cytokines receptors such as TNF-alpha receptor 1 (TNFR-1), and the presence of caspase-8 activation in fibroblast-like cells in the aseptic interface membranes of THAs suggests a possible TNF-alpha dependent apoptosis.     

Apoptin基因通过激活caspase-3诱导人黑色素瘤细胞A375凋亡

目的研究caspase-3在肿瘤特异性凋亡基因诱导人黑色素瘤细胞A375凋亡中的作用。方法用含有apoptin基因的真核表达载体瞬间转染体外培养的人黑色瘤细胞A375;采用RT-PCR、DNA凝胶电泳、流式细胞术检测A375细胞的凋亡;以比色法检测caspase-3的相对活性。结果Ap-optin基因瞬间转染的A375细胞可出现典型的细胞凋亡所具有的DNA梯状带;流式细胞术发现实验组细胞凋亡率明显高于其他各组(P<0.01);转染后24h实验组caspase-3的活性开始升高,72h达高峰,明显高于其他各组(P<0.01)。结论Apoptin基因可通过激活caspase-3诱导人黑色素瘤细胞A375凋亡。     

Activated monocytes induce human retinal pigment epithelial cell apoptosis through caspase-3 activation

Dysfunction and loss of human retinal pigment epithelial (HRPE) cells is a significant component of many ocular diseases, in which mononuclear phagocyte infiltration at the HRPE-related interface is also observed. In this study, we investigated whether HRPE cell apoptosis may be induced by overlay of IFN-gamma-activated monocytes. Human monocytes primed with IFN-gamma overlaid directly onto HRPE cells elicited significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive HRPE cells (p < 0.0001) and decreases of proliferating cell nuclear antigen-positive (p < 0.0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by the caspase-3 inhibitor, Z-DEVD-fmk. However, co-incubations in which activated monocytes were prevented from direct contact with HRPE cells or in which the monocytes were separated from the HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Function-blocking anti-CD18 and anti-intercellular adhesion molecule-1 (ICAM-1) antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells by 48% (p = 0.0051) and 38% (p = 0.046), respectively. Anti-CD18 and anti-ICAM-1 antibodies significantly inhibited caspase-3 activity by 56% (p < 0.0001) and 45% (p < 0.0001), respectively. However, antibodies to vascular cell adhesion molecule-1, TNF-alpha, IL-1beta, or TNF-related apoptosis-inducing ligand did not inhibit apoptosis or caspase-3 activation. Direct overlay of monocytes also induced reactive oxygen metabolites (ROM) within HRPE cells. The intracellular HRPE cell ROM production was inhibited by the anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase, presumably due to its failure to penetrate into HRPE cells. Accordingly, neither superoxide dismutase nor N(G)-monomethyl-L-arginine had significant effects on HRPE cell apoptosis or caspase-3 activation. Our results suggest that activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis. These mechanisms may compromise HRPE cell function and survival in a variety of retinal diseases.     

Apoptosis via the B cell antigen receptor requires Bax translocation and involves mitochondrial depolarization, cytochrome C release, and caspase-9 activation

Various routes to apoptosis can be active during B cell development. In a model system of mature B cells, differences in caspase-3 processing have suggested that antigen receptor (BCR)-mediated apoptosis may involve a zVAD-insensitive initiator protease(s). In search of the events leading to caspase-3 activation, we now establish that both CD95- and BCR-mediated apoptosis depend on Bax activation and cytochrome C (cytC) release. Nevertheless, the timing and caspase-dependence of mitochondrial membrane depolarization differed considerably after CD95- or BCR-triggering. To delineate events subsequent to cytC release, we compared apoptosis induced via BCR triggering and via direct mitochondrial depolarization by CCCP. In both cases, partial processing of caspase-3 was observed in the presence of zVAD. By expression in 293 cells we addressed the potential of candidate initiator caspases to function in the presence of zVAD, and found that caspase-9 efficiently processed caspase-3, while caspase-2 or -8 were inactive. Finally, retroviral expression of dominant-negative caspase-9 inhibited both CD95- and BCR-mediated apoptosis. In conclusion, we obtained no evidence for involvement of a BCR-specific protease. Instead, our data show for the first time that the BCR-signal causes Bax translocation, followed by mitochondrial depolarization, and cytC release. Subsequent caspase-9 activation can solely account for events further downstream.     

The equine arteritis virus induces apoptosis via caspase-8 and mitochondria-dependent caspase-9 activation

We have previously showed that equine arteritis virus (EAV), an arterivirus, induces apoptosis in vitro. To determine the caspase activation pathways involved in EAV-induced apoptosis, target cells were treated with peptide inhibitors of apoptosis Z-VAD-FMK (pan-caspase inhibitor), Z-IETD-FMK (caspase-8-specific inhibitor) or Z-LEHD-FMK (caspase-9-specific inhibitor) 4 h prior to infection with the EAV T1329 Canadian isolate. Significant inhibition of apoptosis was obtained with all peptide inhibitors used. Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given the activation of Bid and the translocation of cytochrome c within the cytoplasm, the overall results indicate that EAV induces apoptosis initiated by caspase-8 activation and subsequent mitochondria-dependent caspase-9 activation.     

Induction of apoptosis by Hypericin through activation of caspase-3 in human carcinoma cells

Potent photosensitizer Hypericin (HY), is a lipid soluble perylquinone derivative of the genus Hypericum and has a strong photodynamic effect on tumors and viruses. However, the mechanisms of tumor cell death induced by this compound is still unclear. Furthermore, there are no reports on mechanisms in cell apoptosis induced by perylquinones in human nasopharyngeal carcinoma (NPC) and other mucosal cells. We studied the photodynamic effects of HY compound in poorly differentiated (CNE2) and moderately differentiated (TW0-1) human NPC cells as well as human mucosal colon (CCL-220.1) and bladder (SD) cells. Using these cell lines we investigated few hall marks of apoptotic commitments in a drug and light dose dependent manner. Tumor cells photoactivated with HY showed cell size shrinkage and an increase in the sub-diploid DNA content. A loss of membrane phospholipid asymmetry associated with apoptosis was induced in all tumor cell lines as evidenced by the externalization of phosphatidylserine. Under apoptotic conditions, Western blot analysis of poly (ADP-ribose) polymerase, a caspase substrate, showed the classical cleavage pattern (116-85 kDa) associated with apoptosis in PDT-treated cell lysates. In addition, 85 kDa cleaved product was blocked by using tetrapeptide caspase inhibitors such as DEVD-CHO or z-VAD-fmk. These results demonstrate that tumor cell death induced by photoactivated HY is mediated by caspase proteases. This study also identifies that CNE2, CCL-220.1 (colon) and SD (bladder) cell lines are more sensitive than TW0-1 cell line to PDT using perylquinone HY.     

二甲基亚砜通过激活caspase-3以及抑制PARP-1引起A549细胞凋亡

目的 检测肺上皮癌细胞（A549）细胞经二甲基亚砜（DMSO）诱导发生凋亡后聚腺苷酸二磷酸核糖转移酶（PARP-1）的活性是否受到抑制。方法 通过MTT法检测caspase-3的活性升高以及TUNEL等方法验证二甲基亚砜可引起A549细胞凋亡后,采用western blotting 验证PARP-1是否被切割成俩个片段。结果 DMSO可诱导A549细胞发生凋亡,凋亡过程中caspase-3的活性上调,其下游底物PARP-1大量被切割成小片段。结论 DMSO诱导的A549细胞凋亡受到caspase-3-PARP-1信号分子的调节。     

Overexpression of caspase-9 triggers its activation and apoptosis in vitro

Aim

To investigate the consequences of increased expression of caspase-9: 1) whether the caspase-9 overexpression resulted in cell death through apoptosis, 2) whether apoptosis could be triggered in normal and tumor cells, and 3) what is the role of caspase-9 in the process.

Methods

The caspase-9 fused to green fluorescent protein was expressed in primary cultures of anterior pituitary cells and of HeLa tumor cells. The expressed caspase-9 and the number of apoptotic and necrotic cells were determined using fluorescence microscopy.

Results

Overexpression of caspase-9 resulted in cell death of primary pituitary cells and HeLa cells. More than 94% of the cells died of apoptosis, which was triggered by the activation of caspase-9, since the cell deaths were prevented in the presence of caspase-9 specific inhibitor. HeLa cells were about 50% more resistant to apoptosis than pituitary cells.

Conclusions

Caspase-9 overexpression and its activation leads to apoptosis. It occurs both in normal and tumor cells. Since the majority of cancer therapy treatments initiate apoptosis through the caspase-9 activation, the modulation of caspase-9 expression may be exploited in designing new ways to control apoptosis in neurodegenerative or malignant diseases.There are two types of cell death: necrosis and apoptosis. Apoptosis or programmed cell death is a process which controls the number and the quality of cells (1). Caspases are proteases, which have a central role in triggering and executing apoptosis (2,3). The two major pathways of triggering apoptosis are the intrinsic and extrinsic pathway (4). The main intrinsic pathway is characterized by mitochondrial dysfunction, with the release of cytochrome c, activation of caspase-9, and subsequently of caspase-3. The extrinsic pathway is activated at the cell surface through death receptor mediated activation of caspase-8 or caspase-10, followed by caspase-3 activation. This pathway may be amplified by caspase-9 activation (intrinsic pathway). Therefore, caspase-9 is one of the main initiator caspases (5). Like the other members of the caspase family, it is synthesized as an inactive zymogen and is activated through proteolytic processing. There are two main pathways for caspase-9 activation: within the apoptosome, a large protein complex, which consists of caspase-9, cytochrome c, and Apaf-1 (6-8), or by proteolytic cleavage by a previously activated caspase, which involves its dimerization (9-11).Among other processes, caspase-9 triggers apoptosis early in the development of nervous system (12,13). Failure of caspase-9 activation is also associated with the resistance for apoptosis in testicular tumors (14). In addition, the gene for caspase-9 maps to the locus disrupted in human tumors, a hotspot for loss of heterozygosity in several cancers, including neuroblastomas (15). A number of initial studies supported the claim that cancer therapy triggered apoptosis by activating the extrinsic pathway of apoptosis (16). Subsequently, there is compelling evidence that the majority of cytotoxic drugs initiate cell death by triggering the cytochrome c/Apaf-1/Caspase-9-dependent pathway through the mitochondria (intrinsic pathway) (16). Embryonic stem cells with disrupted caspase-9 and thymocytes with disrupted Apaf-1 are resistant to cytotoxic drugs, but sensitive to death receptor triggering (extrinsic pathway) (13). In contrast, embryonic fibroblasts disrupted with caspase-8 are sensitive to cytotoxic drugs (17). The relative contributions of intrinsic and extrinsic pathways to apoptosis induced by cytotoxic drugs may also depend on the drug, dose, and kinetics (16). While there are data on the consequences of reduced amounts of caspase-9 in the cells, its overexpression has not been systematically investigated so far.Here we report that caspase-9 overexpression triggers the activation of caspase-9 and apoptosis. It is likely that caspase-9 autoactivates itself under these conditions, since the caspase-9 specific inhibitor prevents the cell deaths. The apoptosis due to overexpression seems to be a ubiquitous process, since it occurs in normal and in tumor transformed cells. HeLa cells are only 50% less sensitive to cell death due to caspase-9 overexpression than the primary pituitary cells.     

TRAIL和顺铂对横纹肌肉瘤细胞凋亡时caspase-3活性和线粒体膜电位的影响

 目的: 探讨肿瘤坏死因子相关诱导凋亡配体（TRAIL）蛋白和顺铂协同抑制横纹肌肉瘤细胞生长和诱导凋亡作用及其机制。方法: 将不同浓度的TRAIL和顺铂作用于培养的人RD胚胎型横纹肌肉瘤细胞，通过MTT比色法、形态学改变、流式细胞仪检测细胞凋亡、caspase-3活性和线粒体膜电位的改变，分析其对横纹肌肉瘤细胞的作用及和顺铂协同作用的效果和机制。结果: TRAIL浓度为1.0、10.0、100.0 μg/L时，细胞毒性指数分别为18.9%、20.8%、43.5%；顺铂浓度为1.0、5.0、10.0 mg/L时，细胞毒性指数分别为9.8%、23.4%和43.8%。而浓度为100 μg/L的TRAIL与浓度为5 mg/L的顺铂联合作用时，细胞毒性指数明显高达66.4%，FCM分析显示联合应用后细胞线粒体跨膜电位降低，同时提高caspase-3的活性,与细胞凋亡率增加相一致。结论: TRAIL和顺铂联合应用对横纹肌肉瘤细胞具有明显的协同杀伤效果，这一作用与增加caspase-3活性及降低线粒体跨膜电位有关。     

超声对软骨细胞凋亡、caspase-8和caspase-3表达的影响

背景：超声治疗能够缓解疼痛,改善膝骨关节炎患者的运动功能,但目前关于超声治疗的文献缺乏一致性。 目的：进一步验证超声治疗的膝骨关节炎有效性。 方法：24 只兔子随机分为3组,正常组不干预;模型组兔行前交叉韧带离断诱导膝骨关节炎,不接受任何治疗;超声组兔建模后接受超声波治疗10 min/次,1次/d,0.3 W/cm2,1 MHz,共治疗10次。采用苏木精-伊红染色进行兔关节软骨组织学观察,运用免疫印迹和 RT-PCR技术检测兔软骨中的caspases3和caspases8 的表达,TUNEL技术检测兔膝骨关节软骨细胞凋亡率。 结果与结论：正常兔软骨组织软骨细胞呈柱状整齐的排列,模型中软骨层变薄,软骨细胞排列无序而少。超声治疗后软骨细胞重新排列,趋于整齐,细胞数增加。模型组为膝骨关节炎模型;与正常组比较,模型组和超声组改良Mankin 评分较高,模型组和超声组软骨细胞凋亡指数更高,模型组高于超声组。与正常组比较,模型组和超声组caspase-3 和caspase-8表达较高,超声治疗后两项指标表达下降。结果表明,超声可以改善软骨组织结构、降低caspase-3、caspase -8的表达,降低软骨细胞凋亡。提示超声治疗膝骨关节炎是有效的。     

阻断ERK途径对地塞米松诱导胸腺细胞凋亡中线粒体膜电势的影响

目的： 研究阻断ERK途径对地塞米松（DEX）诱导的胸腺细胞凋亡中线粒体膜电势的影响。 方法： 利用PD098059（PD）阻断小鼠胸腺细胞ERK途径，分别设对照组（control）、单纯PD组（PD only）、DEX组和PD＋DEX组；在3 h、5 h和7 h时点，利用Annexin V-FITC/PI双染流式细胞术检测细胞凋亡；在3 h、7 h和11 h，利用JC-1染色流式细胞术检测线粒体膜电势（△ψm）变化。 结果： 在1 μmol·L-1 DEX刺激下，小鼠胸腺细胞在3 h、5 h和7 h凋亡率分别为(19.63±0.35)%、(41.84±1.67)%和(67.00±2.43)%，对照组分别为(4.98±0.39)%、(6.08±0.33)%和(9.31±0.34)%，差异显著（P<0.01）；相同时点下，PD only组细胞凋亡率分别为(7.95±0.60)%、(10.69±0.48)%和(22.20±1.24)%，显著高于对照组（P<0.01）；PD＋DEX组在3 h和5 h时点细胞凋亡率显著高于DEX组（P<0.01），而在7 h时点，两组差异不显著（P>0.05）。在3 h、7 h和11 h，DEX组△ψm降低的细胞比率分别为(21.23±1.43)%、(55.34±1.78)%和(70.88±2.87)%，对照组分别为(5.25±1.22)%、(8.01±0.97)%和(12.88±1.10)%，差异显著（P<0.01）；相同时点下，PD only组△ψm降低的细胞比率分别为(11.09±2.00)%、(16.21±2.25)%和(21.15±3.70)%，显著高于对照组（P<0.01）；PD＋DEX组在3 h和5 h时点细胞凋亡率显著高于DEX组，分别为(30.55±2.99)%和(65.22±4.32)%（P<0.01），11 h时点两组差异不显著（P>0.05）。 结论： DEX诱导小鼠胸腺细胞凋亡至少部分通过ERK途径，阻断ERK途径在该凋亡过程中具有重要生物学意义。     

Coenzyme Q protects cells against serum withdrawal-induced apoptosis by inhibition of ceramide release and caspase-3 activation

Coenzyme Q10 (CoQ10) is a component of the antioxidant machinery that protects cell membranes from oxidative damage and decreases apoptosis in leukemic cells cultured in serum-depleted media. Serum deprivation induced apoptosis in CEM-C7H2 (CEM) and to a lesser extent in CEM-9F3, a subline overexpressing Bcl-2. Addition of CoQ10 to serum-free media decreased apoptosis in both cell lines. Serum withdrawal induced an early increase of neutral-sphingomyelinase activity, release of ceramide, and activation of caspase-3 in both cell lines, but this effect was more pronounced in CEM cells. CoQ10 prevented activation of this cascade of events. Lipids extracted from serum-depleted cultures activated caspase-3 independently of the presence of mitochondria in cell-free in vitro assays. Activation of caspase-3 by lipid extracts or ceramide was prevented by okadaic acid, indicating the implication of a phosphatase in this process. Our results support the hypothesis that plasma membrane CoQ10 regulate the initiation phase of serum withdrawal-induced apoptosis by preventing oxidative damage and thus avoiding activation of downstream effectors as neutral-sphingomyelinase and subsequent ceramide release and caspase activation pathways.