多聚（二磷酸腺苷-核糖）聚合酶介导过氧亚硝基阴离子致豚鼠气道反应性增高的研究

目的:探讨多聚(二磷酸腺苷-核糖)聚合酶(PARP)在ONOO^-致豚鼠气管高反应性中的作用。方法:建立离体豚鼠气管条对组胺反应的浓度反应曲线,观察PARP抑制剂3-氨基苯甲酰胺(3-AB)对过氧亚硝基阴离子(ONOO^-)诱导豚鼠气管反应性变化的影响。结果:ONOO^-(0.5 mmol/L)引起豚鼠气管上皮细胞明显受损,气管条对组胺的反应性及敏感性明显增高,3-AB(1 mmol/L或5 mmol/L)逆转了ONOO^-的上述作用;3-AB(5 mmol/L)本身对正常气管条收缩反应无明显影响。结论:PARP介导了ONOO^-所引起的气道上皮细胞的损伤和气道高反应性的形成,提示通过抑制PARP的过度活化可为防治哮喘气道高反应性提供新策略。     

过氧亚硝基阴离子对气道上皮细胞的损伤作用

目的:探讨过氧亚硝基阴离子(ONOO^-)对气道上皮细胞的损伤作用。方法:在培养的大鼠气道上皮(RTE)细胞观察外源性给予ONOO^-对RTE细胞线粒体呼吸、8-羟基脱氧鸟苷(8-OHdG)水平、乳酸脱氢酶(LDH)释放及凋亡细胞百分率的影响。结果:ONOO^-(0.25-1mmol/L)呈剂量依赖方式抑制RTE细胞线粒体呼吸功能、增高LDH释放率。并呈剂量依赖性引起8-OHdG水平升高。不同浓度(0.25mmol/L、0.5mmol/L及1mmol/L)ONOO^-均以时间依赖方式引起RTE细胞凋亡。结论:ONOO^-可引起培养的RTE细胞发生凋亡和坏死。低浓度的ONOO^-损伤RTE细胞以凋亡为主;高浓度的ONOO^-可能主要引起RTE细胞发生坏死。ONOO^-对RTE细胞线粒体呼吸抑制和DNA的氧化损伤可能是ONOO^-导致RTE细胞坏死和凋亡的主要原因。     

过氧亚硝酸阴离子供体通过蛋白激酶C途径抑制CA1区神经元延迟整流钾电流

目的: 过氧亚硝酸阴离子(ONOO^-)是一种性质活泼的自由基,可引起强的氧化性损伤,介导了一氧化氮(NO)的大部分毒性作用。本研究探讨ONOO^-对脑片海马神经元延迟整流钾电流(I_K)和动作电位时程(APD)的影响及其作用机制。方法: 应用全细胞脑片膜片钳技术记录I_K和动作电位。结果: ONOO^-供体SIN-1可抑制I_K电流峰值,使其激活曲线向超极化方向移动,并可延长APD。脑片预处理PKC抑制剂chelerythrine (2.5 μmol/L)可抑制SIN-1对I_K的作用。PKC激动剂PDBu (6 μmol/L)不仅加强而且模拟SIN-1对I_K的影响。然而,鸟苷酸环化酶(GC)抑制剂ODQ对SIN-1的作用无影响。结论: ONOO^-可能通过PKC-I_K-动作电位信号级联反应作用于海马神经元,并不依赖环磷鸟苷(cGMP)通路,这可能是ONOO^-神经毒性的机制之一。     

一氧化氮和过氧亚硝基阴离子在肢体缺血再灌注致肺损伤中的作用

目的:观察肢体缺血再灌注致肺损伤时肺组织中一氧化氮(NO)及过氧亚硝基阴离子(ONOO^-)的变化,以探讨二者在此种损伤中的作用。方法:采用夹闭大鼠腹主动脉下段造成双下肢缺血和再灌注后肺损伤模型,分别测定假手术组、缺血4h组、缺血4h再灌注1h组及再灌注4h组肺组织匀浆中超氧化物歧化酶(SOD)活性和丙二醛(MDA)、NO₂^-/NO₃^-含量变化;应用免疫组化方法测定上述各组肺组织中诱导型一氧化氮合酶(iNOS)及ONOO^-体内生成标志物硝基酪氨酸(NT)的变化。结果:肢体缺血再灌注后1h和4h肺组织中MDA和NO₂^-/NO₃^-的含量显著高于对照组和单纯缺血组(P＜0.05),而SOD活性则显著低于此两组(P＜0.05),并出现大量iNOS及NT阳性信号。结论:肢体缺血再灌注致肺损伤时肺组织中有大量NO和ONOO^-产生,脂质过氧化增强,提示ONOO^-参与介导此种肺损伤。     

一氧化氮和caspase-3在多巴胺诱导PC12细胞

目的：探讨一氧化氮（NO）和半胱氨酸蛋白酶3（caspase-3）在多巴胺诱导PC12细胞凋亡中的可能作用。方法：流式细胞仪定量检测PC12细胞的凋亡率,原位末端标记法（TUNEL）观察凋亡细胞的形态,Griess法测定NO₂^-的浓度,荧光分光光度计法检测caspase-3的活力,半定量RT-PCR法检测诱导型一氧化氮合酶（iNOS）mRNA的表达水平。结果：多巴胺（0.15-0.60 mmol/L）可剂量依赖性地诱导PC12细胞凋亡,表现为凋亡细胞的TUNEL染色阳性;iNOS mRNA的表达、NO的合成及caspase-3的活力均有明显的增加（P<0.01）;特异性的iNOS抑制剂aminoguanidine和caspase-3抑制剂Ac-DEVD-CHO通过减少NO的生成和抑制caspase-3的激活阻断PC12细胞凋亡。结论：NO的生成可能是多巴胺诱导PC12细胞凋亡的触发因子,caspase-3的激活是其中的效应因子。     

过氧亚硝基阴离子致大鼠肺微血管壁通透性增高的研究

目的:探讨过氧亚硝基阴离子(ONOO^-)对肺微血管内皮屏障功能的影响和在急性肺损伤发生中的意义。方法:向SD大鼠气管内推注不同浓度的ONOO^-、分解 ONOO^-或溶剂2 h后,检测肺微血管壁通透性变化和肺组织病理学变化,并检测ONOO^-引起正常肺匀浆MDA含量变化。结果: ONOO^-推入气管后,肺系数、肺湿干重比以及肺含水量和伊文思兰含量明显增高呈现剂量效应关系;并可导致肺泡明显萎陷、肺泡壁毛细血管明显扩张充血及局灶性出血,内皮细胞肿胀、核深染及脱落。ONOO^-造成正常肺匀浆MDA含量增加。 结论: 外源性ONOO^-气管推注后可导致明显的肺微血管内皮屏障功能障碍,提示肺内源性ONOO^-生成增多时可能参与介导急性肺损伤的发生。     

Caspase 3抑制剂抑制羟基喜树碱对人乳腺癌Bcap37细胞NF-κB的激活

目的： 探讨caspase 3抑制剂Ac-DEVD-CHO对caspase 3和NF-κB信号转导途径的影响。方法:采用MTT法测定细胞的增殖活力，琼脂糖凝胶电泳观察细胞凋亡，流式细胞仪进行细胞周期分析，Western blotting和DIG-EMSA研究细胞凋亡途径。结果:Caspase 3抑制剂Ac-DEVD-CHO可抑制羟基喜树碱(HCPT)对Bcap-37诱导的凋亡，抑制Pro-caspase 3的裂解，并能抑制IκBα 蛋白降解，阻止NF-κB活化。结论:Caspase 3抑制剂Ac-DEVD-CHO除特异性抑制Pro-caspase 3的裂解外，还可以抑制HCPT对Bcap37细胞NF-κB的活化。     

大鼠肺纤维化形成中内源性一氧化氮对肺泡上皮细胞凋亡的影响

目的:研究大鼠肺纤维化形成过程中异常增多的肺内源性一氧化氮对肺泡上皮细胞凋亡的影响。方法:气管内滴注博莱霉素,用硝酸还原法检测出肺血NO₂^-/NO₃^-含量;TdT介导的dUTP缺口末端标记法(TUNEL)和透射电镜观察肺泡上皮细胞凋亡。分别观察注BLMA₅后14 d和30 d以及用氨基胍(AG)阻断iNOS合成NO后上述指标的变化。结果:(1)注BLMA₅后14 d,出肺血NO₂^-/NO₃^-含量分别高于正常对照组和注BLMA₅后30 d组(均P<0.01);凋亡的肺泡上皮细胞数亦分别多于正常对照和注BLMA₅后30 d组(均P<0.01)。(2)AG阻止出肺血NO₂^-/NO₃^-含量增高的同时,亦抑制了肺泡上皮细胞的凋亡。结论:在肺纤维化形成过程中,内源性NO的增多,有诱导肺泡上皮细胞凋亡的作用。     

吸烟大鼠一氧化氮合酶和一氧化氮的变化

目的:观察吸烟对大鼠肺组织iNOS、eNOSmRNA和蛋白表达以及支气管肺泡灌洗液(BALF)中NO的影响, 探讨不同类型的NOS在吸烟所致慢性气道炎症中的作用。方法:选用Wistar大鼠80只随机分为对照组, 被动吸烟组, iNOS抑制剂L-NIL干预组及NOS抑制剂L-NAME干预组。用免疫组化法检测iNOS及eNOS的蛋白表达, 用RT-PCR检测iNOS及eNOSmRNA的表达, 用Griess法测定BALF中的NO^-₂/NO^-₃含量。结果:吸烟大鼠肺组织中iNOSmRNA及其蛋白表达增加, eNOSmRNA及蛋白表达下降, BALF中细胞总数及NO^-₂/NO^-₃显著增加(P＜0.05)。在体实验发现, L-NIL使BALF中细胞总数及NO^-₂/NO^-₃下降(P＜0.05);L-NAME对BALF中细胞总数及NO^-₂/NO^-₃无显著影响(P＞0.05)。结论:吸烟大鼠肺组织iNOSmRNA和蛋白表达增加, eNOSmRNA和蛋白表达减少。活化的iNOS产生大量NO促进炎症发展。     

人白细胞介素1β通过caspase途径诱导人黑色素瘤A375-S2细胞凋亡

目的:对interleukin-1β(IL-1β)诱导人黑色素瘤A375-S2细胞凋亡的信号转导途径进行研究。方法:使用倒置显微镜观察细胞形态学变化。通过MTT法测定IL-1β对A375-S2细胞的抑制作用以及细胞内半胱氨酸蛋白酶(caspases)与这种作用的关系。利用乳酸脱氢酶(LDH)测定法对IL-1β作用后细胞的损伤情况进行分析。琼脂糖凝胶电泳法检测IL-1β对细胞DNA降解的影响。结果: IL-1β对A375-S2细胞的抑制作用呈剂量和时间依赖性,在10^-9mol/L作用72 h时达到90%以上。caspase-1、-3、-8、-9和caspase-10的抑制剂能够部分抑制IL-1β早期诱导的细胞凋亡。 LDH活力测定显示,在IL-1β诱导的细胞死亡过程中,凋亡占主导地位,并呈现剂量和时间依赖性 。细胞经过10^-11mol/L IL-1β处理72 h后,出现凋亡典型的DNA梯状条带,与上述结果一致。 结论: IL-1β能够诱导人黑色素瘤A375-S2细胞凋亡,这种作用可能依赖于激活一类介导凋亡的caspase家族蛋白酶。     

Interfering with interferon-γ signalling in intestinal epithelial cells: selective inhibition of apoptosis-maintained secretion of anti-inflammatory interleukin-18 binding protein

The intestinal epithelial barrier represents an important component in the pathogenesis of inflammatory bowel diseases. Interferon (IFN)-γ, a T helper type 1 (Th1) cytokine, regulated by the interleukin (IL)-18/IL-18 binding protein (bp) system, modulates the integrity of this barrier. The aim of this work was to study functionally the consequences of IFN-γ on intestinal epithelial cells (IEC) and to interfere selectively with identified adverse IFN-γ effects. IEC lines were stimulated with IFN-γ. IL-18 and IL-18bp were assessed by enzyme-linked immunosorbent assay. Staining of phosphatidylserine, DNA laddering, lactate dehydrogenase (LDH) release, cleavage of poly-adenosine diphosphate-ribose-polymerase (PARP) and activation of caspase-3 were analysed to determine cell death. Inhibitors of tyrosine kinase, caspase-3 or p38 mitogen-activated kinase ((MAP) activity were used. Cytokines were measured in supernatants of colonic biopsies of healthy controls and inflammatory bowel disease (IBD) patients. In IEC lines, IFN-γ up-regulated IL-18bp selectively. Ex vivo, IFN-γ was present in supernatants from cultured biopsies and up-regulated with inflammation. Contrary to previous reports, IFN-γ alone induced apoptosis in IEC lines, as demonstrated by phosphatidylserin staining, DNA cleavage and LDH release. Further, activation of caspase-3, PARP cleavage and expression of pro-apoptotic Bad were induced. Partial inhibition of caspase-3 and of p38 but not JAK tyrosine kinase, preserved up-regulation of IL-18bp expression. Selective inhibition of IFN-γ mediated apoptosis, while preserving its beneficial consequences on the ratio of IL-18/IL-18bp, could contribute to the integrity of the mucosal barrier in intestinal inflammation.     

Photodynamic therapy induced Fas-mediated apoptosis in human carcinoma cells

Photodynamic therapy (PDT) is a clinical approach that utilizes light-activated drugs for the treatment of a variety of pathologic conditions. Human poorly (CNE2) and moderately differentiated (TW0-1) human nasopharyngeal carcinoma (NPC) cells undergo rapid apoptosis when treated with PDT sensitized with Hypocrellin A (HA) and Hypocrellin B (HB). It has been shown that these compounds have a strong photodynamic effect on tumors and viruses. The initiating events of PDT sensitized HA and HB-induced apoptosis are poorly defined. In the current study, we sought to determine whether Fas/FasL upregulation and involvement of mitochondrial events are an early event in HA and HB-treated PDT induced apoptosis. Loss of mitochondrial transmembrane potential, release of cytochrome c, involvement of caspases-8 and -3 and the status caspase-3 specific substrate PARP, were evaluated in PDT treated tumor cells. Photoactivation of HA and HB enhanced both CD95/CD95L expression and induced CD95-signaling dependent cell death in all tumor cell lines studied. CD95/ CD95L expression appeared within 2 h following light activation and appeared to be a primary event in PDT induced apoptosis. Furthermore, these results indicate that release of mitochondrial cytochrome c into the cytoplasm is a secondary event following the activation of initiator caspase-8 preceding caspase-3 activation, cleavage of PARP and DNA fragmentation. Cytochrome c appeared in the cytosol within 2-3 h post PDT. Cleavage of PARP was observed at 3-4 h following PDT and caspase-3 specific inhibitor DEVD-CHO and broad-spectrum caspases inhibitor z-VAD-fmk blocked caspase-3 activation and PARP cleavage suggesting that caspase-3 plays an important role in HA and HB-induced apoptosis.     

Activation of a caspase-dependent oxidative damage response mediates TGFbeta1 apoptosis in rat hepatocytes

Activation of transforming growth factor-beta type 1- (TGFbeta1) mediated signaling occurs in response to cell injury affecting stem-type cells and hepatocytes in liver. In this work we used WB stemlike liver epithelial cells and p53-defective CWSV-1 nontumorigenic rat hepatocytes to investigate the possible roles of caspases and oxidative stress in TGFbeta1 signaling. TGFbeta1 significantly increased the level of 4-hydroxy-2-nonenal (4-HNE), a stable product of lipid peroxidation. In addition, TGFbeta1-treated cells exhibited activation of caspases that accompanied by enhanced cleavage of the caspase substrate poly(ADP)-ribose polymerase (PARP) and induction of apoptosis. WB cells were twice as sensitive as sensitive as CWSV-1 cells to induction of TGFbeta1 apoptosis. TGFbeta1-apoptosis was significantly reduced when cells were treated with TGFbeta1 in the presence of inhibitors of caspase-1, -3, -8, and -9. Importantly, in addition to suppression of apoptosis, treatment of cells with the caspase-3 inhibitor Z-DEVD-FMK in the presence of TGFbeta1 suppressed the formation 4-HNE and restored mitotic activity. Together, these data suggest TGFbeta1 induces activation of a caspase signaling cascade that includes an oxidative damage response, PARP cleavage, and apoptosis that do not require intact p53 in rat hepatocytes.     

Poly (ADP-ribose) synthetase inhibitor has a heart protective effect in a rat model of experimental sepsis

The aim of this study is to investigate whether PARP inhibitor could reduce cell apoptosis and injury in the heart during sepsis. Materials and methods: 60 healthy male Sprague-Dawley (SD) rats were randomly divided into 4 groups---sham group, modal group, 3-AB pretreatment group and 3-AB treatment group, 15 rats per group. The cecal ligation and puncture (CLP) model of sepsis was used. The following were determined--levels of malondialdehyde (MDA), ATP and nicotinamide adenine dinucleotide (NAD+), expression of PARP, Bcl-2, Bax, cytochrome C and caspase 3 activity in the myocardium tissue, levels of serum creatine kinase muscle brain (CK-MB) fraction and troponin I. Results: Histological and molecular analyses showed that myocardial cells apoptosis were associated with mitochondria injury, with an increase in the amount of PARP and a decrease in ATP and NAD+ levels in model group. In addition, the levels of Bax, cytochrome C and caspase 3 activity, serum levels of CK-MB and troponin I increased, but levels of Bcl-2 significantly decreased. Inhibition of PARP upregulated the levels of ATP, NAD + and Bcl-2, and significantly reduced the activation of PARP and caspase 3, decreased the levels of MDA, cytochrome C, CK-MB and troponin I. As a result, apoptosis in the heart was attenuated. Conclusion: These results indicate that PARP activation may be involved in apoptosis in the heart induced by sepsis and 3-AB could improve it.     

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.     

Escherichia coli shiga-like toxins induce apoptosis and cleavage of poly(ADP-ribose) polymerase via in vitro activation of caspases

Shiga-like toxin-producing Escherichia coli causes hemorrhagic colitis and hemolytic-uremic syndrome in association with the production of Shiga-like toxins, which induce cell death via either necrosis or apoptosis. However, the abilities of different Shiga-like toxins to trigger apoptosis and the sequence of intracellular signaling events mediating the death of epithelial cells have not been completely defined. Fluorescent dye staining with acridine orange and ethidium bromide showed that Shiga-like toxin 1 (Stx1) induced apoptosis of HEp-2 cells in a dose- and time-dependent manner. Stx2 also induced apoptosis in a dose-dependent manner. Apoptosis induced by Stx1 (200 ng/ml) and apoptosis induced by Stx2 (200 ng/ml) were maximal following incubation with cells for 24 h (94.3% +/- 1.8% and 81.7% +/- 5.2% of the cells, respectively). Toxin-treated cells showed characteristic features of apoptosis, including membrane blebbing, DNA fragmentation, chromatin condensation, cell shrinkage, and the formation of apoptotic bodies, as assessed by transmission electron microscopy. Stx2c induced apoptosis weakly even at a high dose (1,000 ng/ml for 24 h; 26.7% +/- 1.3% of the cells), whereas Stx2e did not induce apoptosis of HEp-2 cells. Thin-layer chromatography confirmed that HEp-2 cells express the Stx1-Stx2-Stx2c receptor, globotriaosylceramide (Gb3), but not the Stx2e receptor, globotetraosylceramide (Gb4). Western blot analysis of poly(ADP-ribose) polymerase (PARP), a DNA repair enzyme, demonstrated that incubation with Stx1 and Stx2 induced cleavage, whereas incubation with Stx2e did not result in cleavage of PARP. A pan-caspase inhibitor (Z-VAD-FMK) and a caspase-8-specific inhibitor (Z-IETD-FMK) eliminated, in a dose-dependent fashion, the cleavage of PARP induced by Shiga-like toxins. Caspase-8 activation was confirmed by detection of cleavage of this enzyme by immunoblotting. Cleavage of caspase-9 and the proapoptotic member of the Bcl-2 family BID was also induced by Stx1, as determined by immunoblot analyses. We conclude that different Shiga-like toxins induce different degrees of apoptosis that correlates with toxin binding to the glycolipid receptor Gb3 and that caspases play an integral role in the signal transduction cascade leading to toxin-mediated programmed cell death.     

依托泊苷通过线粒体信号通路释放细胞色素C诱导Jurkat白血病细胞凋亡

目的：研究在人类Jurkat白血病细胞株中依托泊苷诱导凋亡的分子机制，揭示由依托泊苷启动的凋亡信号通路。 方法：分别用annexin V-FITC和碘化丙啶（PI）染色，通过流式细胞仪测定annexin V阳性和出现亚二倍体DNA的凋亡细胞。以3,3＇-dihexyloxyacarbocyanine iodide ［DiOC6(3)］为染色剂，采用流式细胞术检测细胞线粒体膜电位的变化。采用离心技术分离细胞的胞浆与线粒体。细胞色素c从线粒体转入胞浆，caspase-3的激活，多聚二磷酸腺苷核糖聚合酶（PARP）的切割等蛋白质的表达由免疫印迹技术（Western blotting）检测。 结果：依托泊苷诱导Jurkat白血病细胞凋亡，细胞凋亡与依托泊苷的作用时间呈线性关系。广谱的caspase抑制剂zVAD.fmk可抑制依托泊苷诱导的DNA片段化和磷脂酰丝氨酸外翻。依托泊苷引起的线粒体膜电位下降早于DNA片段化和磷脂酰丝氨酸外翻，形成明显对照的是zVAD.fmk不能阻断依托泊苷诱导的线粒体膜电位的下降。依托泊苷介导细胞色素c从线粒体释放到胞浆，激活caspase-3，caspase-3的底物PARP被切割。 结论：依托泊苷诱导Jurkat白血病细胞株凋亡的机制是降低线粒体膜电位和释放细胞色素c到细胞浆启动线粒体信号转导通路, 最终激活caspase而导致细胞凋亡。