Smac/Diablo与X-连锁凋亡抑制蛋白在ATP耗竭再恢复诱导肾小管上皮细胞凋亡中的作用

目的 研究线粒体蛋白Smac/Diablo和细胞X-连锁凋亡抑制蛋白(XIAP)在ATP耗竭及再恢复导致肾小管上皮细胞凋亡中的作用和机制。 方法 应用代谢抑制剂暂时性阻断人肾小管上皮细胞(HK-2细胞)内ATP的生成,再换用含糖的培养液使细胞内ATP再恢复,诱导肾小管上皮细胞凋亡。应用Hoechst33342检测肾小管上皮细胞凋亡的发生。用间接免疫荧光检测Smac/Diablo在细胞内的分布。分别提取胞质蛋白和细胞总蛋白,以Western印迹检测胞质中Smac/Diablo、XIAP和活化半胱氨酸天冬氨酸蛋白酶3前体(pro-caspase-3)的蛋白水平。 结果 肾小管上皮细胞内ATP耗竭及再恢复时,Hoechst33342染色可见HK-2细胞核固缩和凋亡小体的形成;间接免疫荧光可见Smac/Diablo由线粒体释放至胞质;Western印迹可见胞质内Smac/Diablo的含量增多( P < 0.01);XIAP和pro-caspase 3的蛋白水平降低(P < 0.05)。 结论 肾小管上皮细胞内ATP耗竭及再恢复时,Smac/Diablo释放至胞质,XIAP蛋白水平降低,进而激活caspase 3,介导肾小管上皮细胞凋亡。     

热休克蛋白72抑制ATP耗竭时细胞色素C释放所诱导的肾小管上皮细胞凋亡

目的 研究热休克蛋白(HSP)72对ATP耗竭时细胞色素C释放所导致的肾小管上皮细胞凋亡的保护作用及其分子机制。方法 应用代谢抑制剂暂时性阻断细胞内ATP的生成,引起细胞凋亡。应用热处理细胞或编码HSP72 RNA的腺病毒感染细胞,诱导HSP72的表达。以Western印迹检测释放于胞浆内的细胞色素C。荧光肽法测定半胱氨酸天冬氨酸蛋白酶(caspase)3活性。Hoechst33342染色观察细胞凋亡的发生情况。结果 肾小管上皮细胞内ATP耗竭时,释放至胞浆内的细胞色素C的含量增多,caspase 3活性增强;细胞内ATP再恢复时,细胞色素C的释放和caspase 3活性进一步增加,细胞体积缩小,核浓染、固缩,形成凋亡小体。预先热处理后,各组细胞色素C的释放明显减少,caspase 3活性显著抑制(P<0.05,n=3)。高表达HSP72时,各时间点caspase 3活性的抑制程度与热处理组相似,细胞体积缩小,核浓染、固缩,凋亡小体的形成明显减少。结论 HSP72可抑制ATP耗竭时细胞色素C所导致的肾小管上皮细胞凋亡,其机制是抑制凋亡通路中细胞色素C的释放和caspase 3的激活。     

细胞色素C在ATP耗竭诱导的肾小管上皮细胞凋亡中的作用

目的 研究线粒体细胞色素C的释放在ATP耗竭导致的肾小管上皮细胞凋亡中的作用和机制。方法 应用代谢抑制剂暂时性阻断细胞内ATP的生成，诱导细胞凋亡。利用JC-1测定线粒体膜电位的变化。以间接免疫荧光和western印迹检测细胞色素C在细胞内的分布。荧光肽法测定Caspase3的活性。琼脂糖凝胶电泳分析DNA碎解。结果 肾小管上皮细胞内ATP耗竭时，线粒体膜电位显著降低，细胞浆内细胞色素C的含量增多，Caspase3活性增强。细胞内ATP再恢复时，细胞色素C的释放和Caspase3活性继续增加，并出现DNA的碎解。结论 肾小管上皮细胞ATP耗竭时，线粒体细胞色素C的释放在介导Caspase依赖的细胞凋亡通路中发挥重要作用。     

肾小管上皮细胞ATP耗竭再恢复时热休克蛋白72与Paxillin 的相互作用

目的 研究肾小管上皮细胞ATP耗竭再恢复时热休克蛋白(HSP)72与桩蛋白(Paxillin)的相互作用和意义。方法 应用代谢抑制剂暂时性阻断肾小管上皮细胞ATP的生成，引起细胞内的ATP耗竭； 换用含10 mmol/L葡萄糖的DMEM培养液，使细胞内ATP再恢复；以热处理细胞或编码HSP72 RNA的腺病毒感染细胞，诱导细胞高表达HSP72。Western印迹检测HSP72水平；间接免疫荧光和Western印迹检测Paxillin在细胞内的分布变化。免疫共沉淀观察HSP72与Paxillin的相互作用。结果 肾小管上皮细胞ATP耗竭再恢复时，对照组细胞内的Paxillin由局部黏附结构区域向细胞浆内弥散分布；HSP72由细胞浆转移至细胞膜。细胞高表达HSP72后，HSP72在细胞浆和细胞膜中的蛋白含量均增加(P < 0.05)；Paxillin由细胞浆向细胞膜的转移明显减少(P < 0.05)；Paxillin在细胞内的正常分布改善；HSP72和Paxillin之间的相互作用显著增加(P < 0.05)。 结论 肾小管上皮细胞ATP耗竭再恢复时，HSP72可保持Paxillin在细胞内的正常分布特点和区域，其机制可能是HSP72增加与Paxillin的相互作用，发挥分子伴侣的功能。     

bcl-2高表达提高肾小管上皮细胞抗凋亡能力的研究

目的 观察凋亡相关基因bcl-2高表达抑制过氧化氢诱导肾小管上皮细胞凋亡的作用.方法 构建含有人bcl-2 cDNA的逆转录病毒真核表达载体PLXSN,脂质体法将重组质粒转染PA317细胞,G418筛选阳性克隆,鉴定后浓缩收集病毒上清,浓缩病毒液,感染人肾小管上皮细胞株HK-2,Western blot检测bcl-2 mRNA表达.5 mmol/L过氧化氢(HO2)诱导细胞凋亡后,流式细胞仪检测细胞凋亡发生变化.结果 流式细胞仪分析显示5 mmol/L H2O2:成功诱导肾小管上皮细胞凋亡,bel-2逆转录病毒感染肾小管上皮细胞后,bcl-2蛋白水平呈高表达,HK bcl-2组细胞凋亡数量(12.41±3.46)较HK-2组(19.62±4.20)显著减少(P<0.05).而转染空载体的对照组无明显变化(19.62±4.20,P<0.05).结论 bel-2蛋白高表达显著抑制氧化剂诱导的肾小管上皮细胞凋亡.     

KB-R7943对造影剂诱导NRK52E细胞凋亡及p38MAPK信号通路的影响

目的 探讨经钠钙离子交换体介导的细胞内钙超载及p38MAPK信号通路是否参与了造影剂诱导的肾小管上皮细胞损伤及反向模式钠钙离子交换体抑制剂KB-R7943对造影剂诱导的肾小管上皮细胞凋亡及p38MAPK信号通路的影响.方法 鼠肾小管上皮细胞(NRK52E)被分成6组:A正常对照组;B造影剂组;C甘露醇组;D CCB(1×10-5mol/L);E KB-R7943(1×10-5mol/L);F KB-R7943(1×10-6mol/L).造影剂作用1 h后,细胞损伤采用LDH检测,细胞形态变化及细胞凋亡分别由倒置显微镜和流式细胞仪检测;细胞内钙通过Fluo-4染色共聚焦微镜测定;钠钙离子交换体mRNA表达采用RT-PCR测定;p38蛋白的表达采用Western blot测定.结果 造影剂作用1 h后,细胞损伤、细胞凋亡、细胞内钙明显增加,显著高于相同渗透压甘露醇组;p-p38表达30min时增加、60 min时下降.KB-R7943显著降低细胞损伤、细胞凋亡、细胞内钙水平、抑制p-p38的高表达述并显示出剂量效应;钠钙离子交换体mRNA表达没有变化.结论 经反向模式钠钙离子交换体导致的细胞内钙超载诱导了造影剂诱导的肾小管上皮细胞凋亡及p-p38蛋白的高表达;KB-R7943以呈剂量方式降低造影剂诱导的.肾小管上皮细胞凋亡及p-p38蛋白的表达.     

中性粒细胞明胶酶相关脂质运载蛋白对大鼠肾脏缺血再灌注损伤肾小管上皮细胞的保护作用

目的 探讨中性粒细胞明胶酶相关脂质运载蛋白(NGAL)对大鼠缺血再灌注损伤肾脏肾小管上皮细胞凋亡的保护作用及机制.方法 建立大鼠肾脏缺血再灌注模型,雄性SD大鼠随机分为对照组、缺血再灌注模型组、NGAL组 ;HE染色观察3组大鼠肾组织病理变化 ;TUNEL法检测肾小管上皮细胞凋亡 ;实时定量PCR、Western印迹法检测凋亡蛋白fas、bcl-2的表达变化.结果 与缺血再灌注模型组比较,NGAL组肾小管上皮细胞凋亡数量显著减少[(8.6±3.4)/HP比(20.8±3.7)/HP,P＜0.05] ;NGAL组肾组织fas mRNA(2.34±0.51比6.84±2.34,P＜0.05)、fas蛋白(0.65±0.05比0.95±0.08,P＜0.05)表达显著下调,bcl-2蛋白(0.33±0.05比0.24±0.03,P＜0.05)表达显著上调,但bcl-2 mRNA表达无明显改变.结论 NGAL对大鼠缺血再灌注损伤肾小管上皮细胞有保护作用,其作用可能与减少细胞凋亡、改变凋亡蛋白的表达有关.     

cys蛋白酶-3对离体培养的骨骼肌蛋白降解的影响

目的:通过体外培养骨骼肌细胞,测定凋亡效应相关蛋白酶cys蛋白酶-3(caspase-3)及其抑制剂对骨骼肌细胞蛋白降解率的影响,探讨骨骼肌细胞凋亡的可能的调控因子。方法:0.5μg、1μg、2μg、4μg、8μg的肌动球蛋白复合体的反应体系中加入1单位的重组Caspase-3,通过Western Blot电泳。体外培养骨骼肌细胞,并刺激其形成肌管,将培养的骨骼肌分为3组,分别为对照组、实验组A加入1单位Caspase-3,实验组B加入50 nM Caspase-3的抑制剂Ac-DEVD-CHO,体系中均加入2mM ATP(三磷酸腺苷)。用氨基酸全谱分析仪测定酪氨酸和3-甲基组氨酸的浓度。结果:0.5μg、1μg、2μg、4μg、8μg的肌动球蛋白复合体和1单位的重组Caspase-3,均可见到14kDa-actin条带,而肌动球蛋白复合体在0.5μg、1μg剂量时条带消失,在2μg、4μg、8μg时条带逐渐增大。与对照组相比,给予caspase-3后骨骼肌细胞总蛋白降解率增加36.9%,肌纤维蛋白降解率增加66.4%,给予DEVD后总蛋白降解率下降17.7%,肌纤维蛋白降解率下降27.7%。结论:caspase 3在体外可以降解肌动球蛋白复合体,而且还能改变骨骼肌蛋白降解率。caspase-3在骨骼肌蛋白降解中发挥了重要作用,参与了骨骼肌细胞的凋亡。     

高糖加重造影剂诱导的肾小管上皮细胞凋亡

目的探讨高糖对造影剂诱导的肾小管上皮细胞凋亡的影响及其分子机制。方法将NRK52E细胞分成6组：正常对照组（A组）;造影剂组（B组）;葡萄糖（5mmol／L）＋造影剂组（C组）;葡萄糖（15retool／L）＋造影剂组（D组）;葡萄糖（30mmol／L）＋造影剂组（E组）;8]3203580＋葡萄糖（30mmol／L）＋造影剂组（F组）。F组SB203580（20μmol／L）在造影剂加入30min前加入。结果C组细胞葡萄糖作用6、12、24h及D、E组细胞葡萄糖作用6h,未见造影剂诱导的肾损伤和肾小管上皮细胞凋亡加重。D、E组细胞葡萄糖作用12h,造影剂肾损伤和肾小管上皮细胞凋亡重于B组,葡萄糖作用24h,肾损伤和肾小管上皮细胞凋亡重于葡萄糖作用12h,且E组重于D组（P〈0．05）。高糖显著增加造影剂诱导的肾小管上皮细胞ROS水平及p-p38和caspase3蛋白表达,SB203580抑制1〉p38和caspase3蛋白表达。ROS与p-p38、caspase3及p-p38与caspase3呈显著正相关（r分别为0．70、0．68和0．65,P〈0．05）。结论高糖以时间和剂量依赖方式加重造影剂诱导的肾小管上皮细胞损伤,其分子机制与高糖显著增加细胞内ROS水平及上调p-p38、caspase3表达有关。     

红细胞生成素对高糖诱导肾小管细胞凋亡的影响

目的 探讨红细胞生成素（EPO）是否可以抑制高糖诱导的大鼠近端肾小管上皮细胞凋亡及其相关机制。 方法 传代培养大鼠近端肾小管上皮细胞（NRK-52E）,分为正常对照组（NC组）、渗透浓度对照组（OC组）、高糖组（HG组）、高糖+EPO 50 U/ml组（E1组）和高糖+EPO 100 U/ml组（E2组）。免疫荧光检测NRK-52E细胞有无EPO受体（EPOR）表达。Western印迹检测高糖对EPOR表达的影响。流式细胞仪Annexin V-FITC/PI双染法检测细胞凋亡指数。荧光探针CM-H2DCFDA检测细胞内活性氧（ROS）的水平。RT-PCR检测bcl-2、bax、capases-3 mRNA的表达。 结果 （1）NRK-52E细胞表达EPOR,且高糖可刺激EPOR表达增加。（2）高糖可诱导NRK-52E细胞凋亡,与葡萄糖相同渗透浓度的甘露醇不能明显诱导细胞凋亡。E1、E2组细胞早、晚期凋亡率显著低于HG组（P < 0.05）。（3）高糖刺激 NRK-52E细胞后,细胞内ROS产生增多,bcl-2 mRNA的表达下调,bax、caspase-3 mRNA的表达上调。EPO可以抑制细胞内ROS的产生,上调bcl-2 mRNA 表达,下调bax、caspase-3 mRNA 表达。 结论 EPO可能通过EPOR的介导,缓解高糖诱导的氧化应激,上调bcl-2 mRNA表达,下调bax、caspase-3 mRNA表达,抑制NRK-52E细胞凋亡。     

Prior heat stress inhibits apoptosis in adenosine triphosphate-depleted renal tubular cells.

BACKGROUND: This study tested the following hypotheses: (a) renal tubular epithelial cells subjected to transient adenosine triphosphate (ATP) depletion undergo apoptosis, and (b) induction of heat stress proteins (HSPs) inhibits cell death following ATP depletion, possibly by interacting with anti-apoptotic signal proteins. METHODS: To simulate ischemia in vivo, cells derived from opossum kidney proximal tubule (OK) were subjected to ATP depletion (5 mM cyanide, 5 mM 2-deoxy-D-glucose, and 0 mM glucose) for 1 to 1. 5 hours, followed by recovery (10 mM glucose without cyanide). The presence of apoptosis was assessed by morphological and biochemical criteria. The effect of prior heat stress or caspase inhibition on apoptosis and cell survival were assessed. RESULTS: In the ATP-depleted cell, both Hoechst dye and electron microscopy revealed morphological features that are typical of apoptosis. On an agarose gel, a "ladder pattern" typical of endonucleosomal DNA degradation was observed. Prior heat stress reduced the number of apoptotic-appearing cells, significantly decreased DNA fragmentation, and improved cell survival compared with controls (73.0 +/- 1% vs. 53.0 +/- 1.5%; P < 0.05). Two different caspase inhibitors also improved survival, suggesting that apoptosis is a cause of cell death in this model. Compared with ATP-depleted controls, prior heat stress inhibited the pro-apoptotic changes in the ratio of Bcl2 to BAX, proteins known to regulate the apoptotic set point in renal cells. HSP 72, a known cytoprotectant, co-immunoprecipitated with Bcl2, an anti-apoptotic protein. Prior heat stress markedly increased the interaction between HSP 72 and Bcl2. CONCLUSIONS: Transient ATP depletion causes apoptosis in tubular epithelial cells. Prior HS inhibits apoptosis and improves survival in these cells. Novel interactions between HSP 72 and Bcl2 may be responsible, at least in part, for the protection afforded by prior heat stress against ATP depletion injury.     

Nitric oxide mediates dendritic cell apoptosis by downregulating inhibitors of apoptosis proteins and upregulating effector caspase activity

BACKGROUND: Dendritic cells (DCs) play a crucial role in the amplification of the immune response by promoting antigen presentation, T-lymphocyte proliferation, and proinflammatory cytokine and nitric oxide (NO) production. We have previously shown that the exogenous NO donor, s-nitroso-N-acetyl-penicillamine, promotes DC apoptosis by disrupting the mitochondrial membrane potential, which induces cytochrome-C release and activates caspase 3. To further elucidate the signaling pathway, we examined the expression of cellular inhibitors of apoptosis proteins (cIAPs) and poly (ADP-ribose) polymerase cleavage (PARP), a terminal event in the apoptotic cascade. METHODS: DC2.4 were exposed to 250 micromol/L s-nitroso-N-acetyl-penicillamine for various intervals. Apoptosis and necrosis were measured by terminal deoxynucleotidyl transferase nick-end labeling assay or flow cytometry with Annexin V and propidium iodide. DC2.4 were cultured with the pan-caspase inhibitor, ZVAD (100 micromol/L). cIAP, pro-caspases, and PARP expression or activation was measured by Western blot. Caspase enzyme activity was confirmed with the use of specific substrates. RESULTS: NO-induced DC apoptosis correlated with the downregulation of cIAP expression. Caspase 3 and 6 were upregulated by SNAP and significantly inhibited by ZVAD. Maximal PARP cleavage occurred at 8 hours and coincided with the downregulation of cIAP and peak caspase 3 and near maximal caspase 6 activity. CONCLUSIONS: NO-induced DC apoptosis is associated with the downregulation of cIAP expression, which facilitates caspase cascade activation and subsequent PARP cleavage.     

Endoplasmic reticulum stress-associated caspase 12 mediates cisplatin-induced LLC-PK1 cell apoptosis

Reactive oxygen metabolites are important mediators in cisplatin-induced apoptosis in renal tubular epithelial cells (LLC-PK1). Mitochondria have been implicated to play a principal role in cisplatin-induced apoptosis. Caspase 12, an endoplasmic reticulum (ER)-specific caspase, participates in apoptosis under ER stress. Cytochrome P450 system is crucial to the generation of reactive oxygen metabolites and is present at high concentration in the ER. The direct role of caspase 12 in any model of renal injury has not previously been described. In this study, cleavage of procaspase 12 preceded that of caspases 3 and 9 after cisplatin treatment of LLC-PK1 cells. The active form of caspase 8 was not detected throughout the course of study. Preincubation of the LLC-PK1 cells with the caspase 9 inhibitor did not attenuate caspase 3 activation and provided no significant protection. Caspase 3 inhibitor provided only modest protection against cisplatin-induced apoptosis. LLC-PK1 cells that were transfected with anti-caspase 12 antibody significantly attenuated cisplatin-induced apoptosis. Taken together, these data indicate that caspase 12 plays a pivotal role in cisplatin-induced apoptosis. It is proposed that the oxidative stress that results from the interaction of cisplatin with the ER cytochrome P450 leads to activation of procaspase 12, resulting in apoptosis.     

Caspase 3 inhibition improves survival and reduces early graft injury after ischemia and reperfusion in rat liver transplantation

BACKGROUND: Apoptosis plays a crucial role after ischemia-reperfusion in organ transplantation. It is executed by caspases and influenced by the rheostat of pro- and anti-apoptotic proteins of the bcl-2 family. This study investigated the effect of specific inhibition of caspases 3 and 7 on graft function, survival, and hepatic bcl-2 levels after liver transplantation. METHODS: Lewis rats underwent syngeneic orthotopic liver transplantation after 16 hr of cold graft storage (in University of Wisconsin solution). Livers of donor animals treated with D(OMe)E(OMe)VD(OMe)-fluoromethylketone (specific inhibitor of apoptosis executor caspases 3 and 7), and appropriate control groups, were investigated. Early graft injury was quantified by measurement of bile flow and determination of microvascular graft injury by using in vivo fluorescence microscopy. Apoptosis and its regulation were examined by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining and Western blot analysis of cell death effectors, respectively. RESULTS: After specific in vivo caspase inhibition, Western blot analysis revealed inhibition of caspase-induced cleavage of poly-ADP-ribose-polymerase. Inhibition of caspases 3 and 7 resulted in a significantly decreased number of apoptotic endothelial cells and improved microvascular perfusion. A cell protective effect was also suggested by an increase of bcl-2 levels at 7 days. Most important, specific caspase blockade resulted in improved rat survival after liver transplantation. CONCLUSION: Specific inhibition of apoptosis executor caspases effectively reduces graft ischemia-reperfusion injury and improves survival in liver transplantation. Better tissue preservation after caspase inhibition correlates with reduced apoptosis execution, improved microvascular perfusion, and bcl-2 up-regulation. Therefore, specific caspase inhibition represents a promising regimen for clinical use in liver transplantation.     

ABL-N诱导前列腺癌细胞凋亡及其机制

目的 探讨旋覆花内酯(ABL)-N诱导前列腺癌细胞凋亡的作用及其机制.方法 0～40μmol/L ABL-N分别处理前列腺痛细胞后,利用噻唑蓝(MTF)检测对细胞增长的抑制作用;流式细胞学、TUNEL染色等方法检测其诱导凋亡的作用,并检测Capase活性,Western blot测定bax、bel-2水平变化.结果 ABL-N明显抑制前列腺癌细胞PC3、LNCaP及DU145的生长,并呈剂量依赖性.40 μmol/L ABL-N作用24 h后,(73.34±4.41)%的PrEC细胞存活,而3种前列腺癌细胞分别为(11.92±2.31)%、(12.55±1.94)%、(13.28±2.26)%.膜联蛋白/碘化丙锭(Annexin V/PI)及TUNEL染色表明ABL-N呈剂量依赖性诱导PC3凋亡.ABL-N可激活Caspase活性,尤其Caspase-3,20μmol/L ABL-N作用24 h后其活性是对照组的4.23倍;bax/bcl-2比率随浓度增加明显增高.结论 ABL-N可通过Caspase途径及bax/bcl-2蛋白途径,诱导PC3等前列腺癌细胞凋亡,抑制前列腺癌细胞增殖.

Abstract：

Objective To explore the ABL-N-induced apoptosis of human prostate cancer cells and the mechansim. Methods After administration of 0-40 μmol/L ABL-N for 24 h, the effects of ABL-N on the induction of apoptosis in human prostate cancer cells PC3 were measured by methyl thiazol tetrazolium ( MTT) colorimetry, Annexin V/propidium iodide staining and TUNEL staining. The levels of bax and bcl-2 were tested by Western blotting. Caspase activity was assayed. Results ABL-N treatment to PC3,LNCaP, and DU145 cells resulted in a dose-dependent inhibition of cell growth without any substantial effect on normal human prostate epithelial PrEc cells. About (73. 34 ±4. 41)% of PrEC cells were viable following a 24-h exposure to 40 μmol/L ABL-N, whereas only (11. 92 ± 2. 31) % of PC3, (12. 55 ±1. 94) % of LNcap, and (13. 28 ± 2. 26) % of DU145 cells survived under similar conditions of ABL-N treatment. ABL-N treatment resulted in a dose-dependent induction of apoptosis of PC3 cells. Furthermore,ABL-N induced the activation of Caspases, especially Caspase 3. The Caspase-3 activity of PC3 cells treated with ABL-N (20 μmol/L) (0. 95) was significantly increased by about 4. 2-fold of the untreated cells (0. 24) at 24 h. The ratio of bax/bcl-2 was also increased significantly. Conclusion ABL-N induces apoptosis though the activation of Caspase 3 and pro- and anti-apoptotic Bcl-2 family proteins in prostate cancer cells.