Reg2 protects mouse insulinoma cells from streptozotocin-induced mitochondrial disruption and apoptosis

We reported previously that pancreas-specific ablation of IGF-I in mice induced an increased expression of regenerating family proteins Reg2 and Reg3β in the pancreas and protected them from streptozotocin (Stz)-induced β-cell damage. We, therefore, assessed the effect of ectopically introduced Reg2 on Stz-induced apoptosis in MIN6 mouse insulinoma cells and report here that Reg2 protects MIN6 cells from Stz-induced apoptosis by attenuating its ability to disrupt mitochondrial membrane integrity, activate caspase-3 and promote poly-ADP ribose polymerase cleavage, and induce apoptosis. These changes correlated with suppression of c-jun N-terminal kinase (JNK) phosphorylation by Stz. Reg2 inhibited Stz-induced proapoptotic events as well as the inactivation of JNK. Inclusion of chemical inhibitor of JNK to Reg2 expressing cells rendered them sensitive to Stz. These data demonstrate that Reg2 protects insulin-producing cells against Stz-induced apoptosis by interfering with its cytotoxic signaling upstream of the intrinsic proapoptotic events by preventing its ability to inactivate JNK.     

Caspase activation during spontaneous and radiation‐induced apoptosis in the murine intestine

The aim of this study was to characterize the activation of caspase‐3 along the crypt/villus axis in the normal and irradiated intestine and to compare active caspase‐3 expression with existing apoptosis detection techniques. Small and large intestine were removed from mice at various time points after exposure to 8 Gy γ‐radiation. Positive apoptotic cells stained with an antibody against active caspase‐3, haematoxylin and eosin (H&E) or TUNEL were scored in histological sections of small and large intestinal crypts and villi. In the control intestine, active caspase‐3 expression was rarely observed; however, expression was markedly increased following exposure to radiation and was predominantly confined to apoptotic bodies. Measurement of apoptosis in intestinal crypts using active caspase‐3 expression gave similar results to apoptosis detected from H&E‐stained sections. In the normal villus, active caspase‐3 expression was observed infrequently and did not significantly increase following radiation, consistent with a lack of apoptotic body formation from H&E sections. This study indicates that caspase‐3 is activated in intestinal crypts but not in villi following γ‐radiation. Active caspase‐3 detection compared favourably with existing immunological techniques, suggesting that it is a suitable alternative method for apoptosis quantification. Copyright © 2001 John Wiley & Sons, Ltd.     

Notch1表达下调抑制病理性瘢痕成纤维细胞增殖并诱导其凋亡

目的探讨慢病毒介导的Notch1表达下调对病理瘢痕成纤维细胞(PSF)中caspase-9活化水平及线粒体膜电位的影响。方法分离培养增生性PSFs,感染Notch1 siRNA慢病毒和阴性对照慢病毒。分别采用RT-qPCR和Western blot检测细胞中Notch1mRNA和蛋白表达;MTT法检测细胞增殖;PI单染法检测细胞周期;Annexin V-FITC/PI法检测细胞凋亡;Western blot检测细胞周期蛋白D1(cyclin D1)、细胞周期依赖性蛋白激酶4(CDK4)、活化的caspase-3(c-caspase-3)和caspase-9(c-caspase-9)蛋白及胞质和线粒体中细胞色素C(cytochrome C)蛋白;JC-1法检测细胞线粒体膜电位。结果Notch1 siRNA慢病毒感染后的PSFs中Notch1表达水平明显低于阴性对照慢病毒和未感染的PSFs的表达水平(P<0.05)。下调Notch1后的PSFs增殖能力降低(P<0.05);细胞G0/G1期比例升高(P<0.05);细胞凋亡率升高(P<0.05);c-caspase-3和c-caspase-9蛋白表达升高(P<0.05);细胞线粒体膜电位明显下降(P<0.05);细胞中cyclin D1和CDK4蛋白水平降低(P<0.05);胞质内的cytochrome C蛋白含量升高(P<0.05)及线粒体中cytochrome C蛋白水平降低(P<0.05)。结论Notch1表达下调抑制PSFs增殖并诱导凋亡。     

线粒体凋亡途径参与表皮膜蛋白1诱导的细胞调亡

目的:探讨人表皮膜蛋白1(hEMP1)诱导细胞凋亡的信号途径.方法:构建包含hEMP1基因编码框的真核表达载体pcDNA3.1( )-EMP1,瞬转HEK293细胞后荧光倒置相差显微镜、流式细胞术检测Caspase-3、Caspase-8、Caspase-9的活力以及线粒体膜电位的变化.结果:荧光倒置相差显微镜、流式细胞术结果显示,过表达EMP1后细胞Caspase-3、Caspasc-9活力显著增强.线粒体膜电位改变增多,而Caspase-8的活力变化不明显.结论:线粒体凋亡途径参与hEMP1诱导的细胞凋亡.     

选择性线粒体分裂抑制剂抑制大鼠急性脊髓损伤后的细胞凋亡

目的 检测mdivi-1预处理对大鼠急性脊髓损伤(ASCI)后线粒体膜电位,凋亡诱导因子(AIF)释放及细胞凋亡的影响.方法 大鼠随机分为:假手术组(Sham组)、单纯脊髓损伤组(SCI组)、二甲基亚砜预处理组(DMSO组),mdivi-1预处理组(mdivi-1组).采用Allen's方法制备ASCI模型.JC-1标记法检测线粒体膜电位(MMP),Western blot方法检测线粒体及细胞核内AIF,TUNEL法检测细胞凋亡.结果 与Sham组相比,SCI组线粒体中AIF先降低后升高,最低点是8h(P＜0.01),而细胞核中AIF却呈相反趋势(P＜0.01),术后8 h MMP明显降低(P＜0.01),凋亡细胞数目明显增多(P＜0.01).与SCI 8 h组相比,mdivi-1组MMP明显升高(P＜0.01),线粒体中AIF明显增多(P＜0.01),细胞核中AIF明显降低(P＜0.01),凋亡细胞数目明显减少(P＜0.01).结论 Mdivi-1具有保护大鼠ASCI后MMP,抑制线粒体中AIF的释放和细胞凋亡的作用.     

黄芪总皂苷对心衰大鼠心肌细胞凋亡和线粒体膜电位的影响

目的研究黄芪总皂苷(ASTs)对心衰大鼠心肌细胞凋亡和线粒体膜电位的影响和机制。方法将大鼠分成对照组(normal)、心衰组(model)、AST低、中、高剂量干预组(10、20、40 mg/kg灌胃),每组12只,检测大鼠心功能指标。取大鼠心肌组织,TUNEL法检测细胞凋亡;黄嘌呤氧化酶法检测SOD活性;二硫代二硝基甲基苯法检测GSH-PX活性;硫代巴比妥酸法检测MDA含量;JC-1法检测线粒体膜电位;Western blot检测c-caspase-3、c-caspase-9、p-AKT、p-p38MAPK蛋白和线粒体、胞质中cyt-c蛋白表达水平。结果与对照组比较,model组大鼠LVSP、+dp/dt_max和-dp/dt_max降低(P<0.05);细胞凋亡率和c-caspase-3、c-caspase-9蛋白水平升高(P<0.05);SOD、GSH-PX活性降低(P<0.05);MDA含量升高(P<0.05);线粒体膜电位降低(P<0.05);线粒体中cyt-c蛋白水平降低(P<0.05);胞质中cy...     

芹菜素诱导人胃癌细胞凋亡作用及机制研究

目的：研究芹菜素（apigenin, API）致人胃癌细胞凋亡作用及其机制。方法：培养人胃癌BGC823细胞株，加入不同浓度的API，孵育48 h。PI染色流式细胞术（FCM）分析测定凋亡率；罗丹明染色FCM分析测定细胞线粒体跨膜电位(Δψm)；Caspase-9分光光度法检测试剂盒测定caspase-9活性；Western印迹检测线粒体凋亡信号转导通路相关蛋白的表达，包括bax，bcl-2，caspase-9和caspase-3。结果： API（20，40和80 μg/mL）作用48 h能呈浓度依赖性地诱导BGC823细胞凋亡。而且，API也能降低BGC823细胞的Δψm，增加caspase-9活性，促进细胞色素c（Cyt c）释放，上调bax，caspase-9和caspase-3蛋白的表达，同时下调bcl-2蛋白表达，且呈剂量依赖性。结论：API通过活化线粒体信号转导途径诱导人胃癌细胞凋亡。     

双歧杆菌的完整肽聚糖对小鼠腹腔巨噬细胞线粒体膜电位的影响

为探讨双歧杆菌的完整肽聚糖(WPG)对小鼠腹腔巨噬细胞线粒体膜电位的影响,首先分离培养昆明小鼠腹腔巨噬细胞,然后以WPG刺激巨噬细胞,用四甲基罗丹明乙酯(TMRE)标记线粒体膜电位,最后用激光共聚焦显微镜检测巨噬细胞内荧光强度的变化。和对照组相比,WPG刺激巨噬细胞后,巨噬细胞内反映线粒体膜电位的荧光强度明显增强。双歧杆菌的WPG在激活巨噬细胞的过程中可提高其线粒体膜电位。     

碘乙酸钠诱导原代大鼠软骨细胞凋亡

背景：近年碘乙酸钠多用于诱导骨关节炎的发生,病理结果观察到软骨区域凋亡的发生,但有关碘乙酸钠诱导软骨细胞凋亡机制的研究较少。 目的：探讨不同剂量碘乙酸钠诱导原代大鼠软骨细胞凋亡的发生机制。 方法：使用酶消化法建立大鼠关节软骨细胞培养体系;荧光显微镜和流式细胞仪检测软骨细胞凋亡;激光共聚焦显微镜和荧光分光光度计评估线粒体膜电位的改变;荧光分光光度法测定活性氧水平改变;免疫印迹法检测凋亡调控蛋白细胞色素C和caspase-3的表达。 结果与结论：碘乙酸钠剂量依赖性地诱导软骨细胞凋亡,使活性氧水平显著升高(P < 0.05）,线粒体膜电位水平明显降低(P < 0.05）,凋亡调控蛋白的表达明显增加。提示碘乙酸钠诱导的原代大鼠软骨细胞凋亡主要与活性氧的产生及线粒体介导的caspase-3活化有关。     

高三尖杉酯碱通过激活Caspase-3诱导鼻咽癌细胞凋亡

目的　了解高三尖杉酯碱 (HHT)是否可通过激活Caspase 3诱导鼻咽癌细胞CNE 2Z凋亡。方法　将细胞分为 4组 :对照组、1mg/LHHT处理组 (HHT组 ) ,以及分别用Caspase抑制剂 (z VAD fmk)和Caspase 3抑制剂 (DEVD fmk)预处理 2h后 ,再用 1mg/LHHT处理的z VAD fmk +HHT组和DEVD fmk +HHT组。采用流式细胞术及荧光染色法 ,检测 4组细胞的凋亡率 ,以比色法检测它们中Caspase 3的相对活性。结果　流式细胞术和荧光染色均发现 ,HHT组的凋亡率明显高于其它各组 (P <0 .0 1) ;Caspase 3的活性于 2h开始升高 ,8h达高峰 ,明显高于其它各组 (P <0 .0 1) ,2 4h时同其它各组的差异无显著性 (P >0 .0 5 )。结论　HHT可通过激活Cas pase 3诱导CNE 2Z细胞凋亡 ,Caspase 3的活性升高具有时间依赖性     

高三尖杉酯碱通过激活Caspase-3诱导T-淋巴细胞白血病细胞Molt-3凋亡

目的：研究高三尖杉酯碱（ＨＨＴ）诱导Ｔ－淋巴细胞白血病细胞凋亡的作用机制。方法：采用流式细胞仪等技术对ＨＨＴ诱导Ｔ－淋巴细胞白血病细胞（Ｍｏｌｔ－３）的凋亡作用进行探讨。结果：ＨＨＴ诱导Ｍｏｌｔ－３凋亡,凋亡率与作用浓度和时间成正比;细胞被阻滞在Ｇ１期。ＨＨＴ作用２４ｈ后细胞内Ｃａｓｐａｓｅ－３活性增高,Ｃａｓｐａｓｅ－３抑制剂Ｚ－ＤＥＶＤ－ＦＭＫ能特异地阻断ＨＨＴ对Ｍｏｌｔ－３凋亡的诱导;Ｆａｓ     

柯萨奇病毒B3通过caspase依赖途径诱导HeLa细胞凋亡

目的 评价柯萨奇病毒B3（CVB3）致HeLa细胞死亡的方式及分子机制。方法 用CVB3作用于HeLa细胞,在不同时间收集细胞,通过相差显微镜、电子显微镜、流式细胞仪以及分子生物学手段,HeLa细胞的病变及caspase-3基因mRNA和蛋白质的表达。结果 CVB3作用于HeLa细胞后,细胞很快发生变性和坏死,24h后较多细胞凋亡;caspase基因在病毒作用早期即被活化,表现在caspase-3 mRNA在病毒作用后6h内,迅速增高达峰值。在24h内,又降至接近病毒作用前的水平。caspase-3蛋白表达在42h内逐渐增高。结论CVB3可诱导HeLa细胞发生坏死和凋亡两种反应,坏死早于凋亡,细胞凋亡与caspase-3的表达密切相关。     

Death signals from the B cell antigen receptor target mitochondria, activating necrotic and apoptotic death cascades in a murine B cell line, WEHI-231.

B cell antigen receptor (BCR)-mediated cell death has been proposed as a mechanism for purging the immune repertoire of anti-self specificities during B cell differentiation in bone marrow. Mitochondrial alterations and activation of caspases are required for certain aspects of apoptotic cell death, but how the mitochondria and caspases contribute to BCR-mediated cell death is not well understood. In the present study, we used the mouse WEHI-231 B cell line to demonstrate that mitochondrial alterations and activation of caspases are indeed participants in BCR-mediated cell death. The peptide inhibitor of caspases, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk), blocked cleavage of poly(ADP-ribose) polymerase and various manifestation of nuclear apoptosis such as nuclear fragmentation, hypodiploidy and DNA fragmentation, indicating that signals from the BCR induced the activation of caspases. In addition, z-VAD-fmk delayed apoptosis-associated changes in cellular reduction-oxidation potentials as determined by hypergeneration of superoxide anion, as well as exposure of phosphatidylserine residues in the outer plasma membrane. By contrast, although z-VAD-fmk retarded cytolysis, it was incapable of preventing disruption of the plasma membrane even under the same condition in which it completely blocked nuclear apoptosis. Mitochondrial membrane potential loss was also not blocked by z-VAD-fmk. Bongkrekic acid, a specific inhibitor of mitochondrial permeability transition pores, suppressed not only the mitochondrial membrane potential but also the change of plasma membrane permeability. Overexpression of Bcl-xL prevented mitochondrial dysfunction, nuclear apoptosis and membrane permeability cell death triggered by BCR signal transduction. These observations indicate that death signals from BCR may first cause mitochondrial alterations followed by activation of both necrotic and apoptotic cascades.     

MOMP in the absence of BH3-only proteins

The minimum requirement for mitochondrial apoptosis has been controversial ever since the discovery of BCL-2 as a cell death regulator. In this issue of Genes & Development, O''Neill and colleagues (pp. 973–988) end a long-standing debate by creating a cellular system free of BCL-2 family proteins, thereby identifying the outer mitochondrial membrane rather than BH3-only proteins as the only requirement for BAX/BAK activation and mitochondrial outer membrane permeabilization (MOMP).     

超抗原SEA对Molt-4细胞线粒体膜电位的影响

目的:超抗原葡萄球菌肠毒素A(SEA)诱导Molt-4细胞活化与线粒体膜电位变化的关系。方法:应用CCK-8法体外检测不同浓度和时间SEA刺激传代T细胞株Molt-4细胞增殖活性,应用JC-1标记细胞线粒体,利用流式细胞术测定比较不同时间点SEA刺激对Molt-4细胞线粒体膜电位的影响。结果:经CCK-8检测,SEA的浓度在1 mg/L时Molt-4细胞的增殖最显著,以该浓度的SEA作用于Molt-4细胞180、60、30、10 min,10 min时间点膜电位升高明显高于其他时间点。SEA刺激10、30 min后线粒体膜电位升高低于有丝分裂原PHA刺激的结果。结论:SEA可诱导Molt-4细胞线粒体膜电位升高,且升高作用主要发生在细胞增殖的早期阶段。其升高作用略低于有丝分裂原PHA。     

Alterations in mitochondrial membrane potential during preimplantation stages of mouse and human embryo development

Mitochondria are cellular organelles regulating metabolism andcell death pathways. This study examined changes in mitochondrialmembrane potential (m) throughout the stages of preimplantationdevelopment in mouse embryos conceived either in vivo or invitro and human embryos donated to research from IVF. Embryosstained with the m-sensitive dye (JC-1) were quantified forthe ratio of high- to low-polarized mitochondria using a deconvolutionmicroscope. Overall, mouse zygotes and early embryos containa subset of high-polarized mitochondria with a progressive increasein the ratio of m observed with increasing cleavage. A transientincrease in the ratio of high to low m was observed in in vivofertilized 2-cell stage embryos, coincident with embryonic genomeactivation in the mouse, but not in 2-cell embryos obtainedthrough IVF. We further observed that arrested mouse 2-cellembryos possessed an increased ratio of m compared with non-arrestedembryos. In human 8-cell embryos we observed an increased ratioof high- to low-polarized mitochondria with increasing degreesof embryo fragmentation. We concluded that the pattern of mitochondrialmembrane potential progressively changes throughout preimplantationdevelopment, and that an aberrant shift in m could contributeto, or is associated with, decreased developmental potential.     

2型登革病毒通过线粒体途径诱导EA.hy926细胞凋亡

 目的：探讨登革病毒诱导EA.hy926细胞(人脐静脉内皮细胞融合细胞株)相对活力的变化与线粒体膜电位(mitochondrial membrane potential,Δψm)改变及线粒体凋亡途径的关系。方法：用2型登革病毒(dengue virus type 2, DENV-2)感染EA.hy926细胞,MTT法检测感染前后EA.hy926细胞的相对活力,荧光显微镜和流式细胞术分别观察感染前后JC-1在EA.hy926细胞线粒体内的聚集情况以检测Δψm的改变,通过比色法检测caspase-9的活性变化。结果：DENV-2感染EA.hy926细胞24 h、36 h及48 h后,细胞活性受到显著抑制,550 nm处的A值均低于未感染组,差异有统计学意义(P<0.05,P<0.01);JC-1染色显示,感染后各时点,代表正常线粒体的红色荧光均较未感染组减弱,而代表Δψm下降的绿色荧光较未感染组逐渐增强。流式细胞术检测Δψm平均荧光密度比未感染组减低,差异有统计学意义。DENV-2 感染后早期即可出现caspase-9活性的上升,与未感染组相比,各时点的活性差异均有统计学意义(P<0.01)。结论：DENV-2感染EA.hy926细胞后可诱发Δψm下降,增强caspase-9活性,进而启动线粒体的凋亡途径。     

Mechanisms Underlying Production And Externalization of Oxidized Phosphatidylserine in Apoptosis: Involvement of Mitochondria

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.