Involvement of caspase-8, -9, and -3 in high glucose-induced apoptosis in PC12 cells

Hyperglycemia, which occurs under the diabetic condition, is widely recognized as the causal link between diabetes and its serious complications. Diabetic neuropathies, which are among the most frequent complications of diabetes, affect sensory, motor, and autonomic nerves. The exact molecular mechanisms of high glucose-induced toxicity on neuronal cells, is still unclear. We previously reported that high glucose can induce apoptosis in PC12 cells, as evidenced by DNA fragmentation and high Bax/Bcl-2 ratio. The present study examined the involvement of caspase-3, the executioner, and two initiators of apoptosis, caspase-8 and caspase-9, during high glucose-induced apoptosis in PC12 cells, a neuronal cell line. Cells were exposed to high glucose with or without z-VAD-fmk, a pan-caspase inhibitor. Cell viability was measured by MTT assay. Caspase activity was determined spectrophotometrically using enzyme specific substrates. To correlate and confirm the caspase activity with changes in protein expression, procaspase-8, -9, and -3 were evaluated by Western blot analysis. The DNA-fragmentation was determined by DNA ladder using gel electrophoresis. The PC12 cell viability on high glucose exposure was decreased compared to controls, which was reversed by z-VAD-fmk. The activities of caspase-8, -9, and -3 were significantly increased in treated cells compared to controls. Moreover, high glucose exposure induced a significant decrease in protein levels of procaspases, indicating conversion of pro-form into the mature caspases. Finally, DNA fragmentation (Ladder) was shown in treated cells by high glucose. Based on the current data, it could be concluded that high glucose-induced apoptosis in PC12 cells is mediated, in part, by activation of caspase-8, -9, and -3 dependent pathways.     

Reactive nitrogen intermediates and monokines induce caspase-3 mediated macrophage apoptosis by anaerobically stressed Salmonella typhi

A successful pathogen manipulates its host for its own benefit. After ingestion, on reaching the intestine Salmonella encounters the resident tissue macrophages. Rather than being destroyed by these professional phagocytes after internalization, Salmonella survives intracellularly. Invasive Salmonella has been reported to induce apoptosis of macrophages as a part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under different host environments, including the anaerobic stress encountered by the pathogen in the gut, remains to be examined. The present study is aimed at investigating the apoptotic potential of S. enterica serovar Typhi (S. typhi) grown under anaerobic conditions simulating the in vivo situation encountered by the pathogen. Apoptotic cell death was determined by assessment of nucleosomal DNA and flow cytometric analysis. Evaluation of the data revealed that anaerobically grown S. typhi could induce apoptosis in significantly more number of macrophages compared to the bacterial cells grown under aerobic conditions. A significantly enhanced generation of reactive nitrogen intermediates and caspase-3 activity during macrophage apoptosis induced by anaerobic S. typhi correlated with the increased generation of tumour necrosis factor-alpha, interleukin (IL)-1alpha and IL-6. The results indicate that reactive nitrogen intermediates and monokines induce caspase-3 mediated apoptosis of macrophages by S. typhi under anaerobic conditions. These findings may be relevant for clearer understanding of the Salmonella-macrophage interactions and may be of clinical importance in the development of preventive intervention against the infection.     

Involvement of bcl-2 and caspase-3 in apoptosis induced by cigarette smoke extract in the gastric epithelial cell

Previous studies have shown that short-term passive cigarette smoking can increase apoptosis in rat gastric mucosa. However, the mechanism is not yet defined. Chloroform and ethanol extracts were used to investigate whether cigarette smoke could induce apoptosis in a human gastric epithelial cell line (AGS) as well as the roles of bcl-2, caspase-3, and cytochrome c in this process. AGS cell lines were treated with either chloroform extract (CE) or ethanol extract (EE) for 5 hours, and the level of bcl-2, the activity of caspase-3, and the level of cytosolic cytochrome c in these cells were determined. Time course studies on the effects of cigarette smoke extracts (CSEs) on DNA fragmentation and cytochrome c relocalization were also performed. Data showed that only CE induced apoptosis in a dose- and time-dependent manner in AGS cells, along with a decrease of bcl-2 and an increase of caspase-3 activity. Pretreatment with Z-DEVD-FMK (specific inhibitor of caspase-3) dose-dependently blocked the DNA fragmentation induced by the CE. Moreover, CE could time- and dose-dependently increase the level of cytochrome c in the cytoplasm, which might activate caspase-3. In conclusion, CSE triggers apoptosis in AGS cells through the inhibition of bcl-2 and the activation of a mitochondria-related pathway.     

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。     

Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration

Increasing evidences suggest that activated microglia may contribute to neurodegeneration in Parkinson's disease (PD). In the present study, primary ventral mesencephalic (VM) cultures from E14 rats and PC12 cells were utilized as in vitro models to examine the mechanism underlying microglia activation mediated dopaminergic neurodegeneration. Using lipopolysaccharide (LPS) (1-100 ng/ml) as a tool, we observed that microglia activation-mediated a selective dopaminergic neurodegeneration in VM neuron-glia cultures, which was supported by the further study showing that conditioned medium (CM) from microglia-enriched cultures treated with LPS (10-100 ng/ml) decreased PC12 cell viability. The results from antibody neutralization, NO inhibition and superoxide neutralization suggested that the dopaminergic cell death was due to the production of microglia-derived proinflammatory factors (TNF-alpha, NO and superoxide), among which reactive oxygen species (ROS) might outweigh proinflammatory cytokines. Apoptosis assay on PC12 cells and primary dopaminergic neurons showed that apoptosis was a mechanism for both microglia activation-mediated dopaminergic cell death. Through Western blot and immunocytochemistry, we found that caspase-3 activation was involved in both dopaminergic cell injuries. Finally, the results from laser scanning confocal microscope demonstrated that PC12 cell intracellular free Ca(2+) ([Ca(2+)](i)) increased early after CM treatment. [Ca(2+)](i) increase involved influx of calcium from the extracellular milieu and release from intracellular stores and participated in the CM-induced PC12 cell apoptosis. Further investigations indicated that TNF-alpha, IL-1beta, NO and superoxide contributed at different degrees to CM-induced [Ca(2+)](i) increase and apoptosis in PC12 cells. Using primary VM cultures and PC12 cells, our study shows the roles of proinflammatory factors, apoptosis, caspase-3 activation and Ca(2+) disturbance in microglia activation-mediated dopaminergic cell degeneration. Understanding the mechanism for microglia activation-mediated dopaminergic neurodegeneration may contribute to the development of new neuroprotective strategies against PD.     

Phospholipase A2 activity and lipid peroxidation during endotoxemia under conditions of experimental peritonitis

The severity of endotoxemia in peritonitis depends on morphofunctional state of the intestine determined by the intensity of free-radical lipid peroxidation and phospholipase A₂ activity, which are the highest on day 1 postoperation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 31–33, January, 2000     

Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium

The enteric pathogen Salmonella enterica serotype Typhimurium induces apoptosis in infected macrophages. This process is rapid, specific, and depends on the type III protein secretion system encoded within Salmonella pathogenicity island 1 (SPI1). Here, we demonstrate that serotype Typhimurium can activate programmed macrophage cell death independently of SPI1. SPI1 independent induction of apoptosis in infected macrophages is observed as early as 12 to 13 h postinfection, even in the absence of intracellular bacterial replication. Delayed activation of programmed macrophage cell death is not observed with serotype Typhimurium strains mutated in ompR or SPI2. Even though SPI2 mutants have a defect in intracellular proliferation, our results indicate that long-term intracellular survival and growth are not required for delayed macrophage killing per se, since Salmonella mutants that are severely defective in intracellular growth still induce delayed apoptosis. Inactivation of genes required for either rapid or delayed induction of apoptosis results in a conditional noncytotoxic phenotype, whereas simultaneous inactivation of genes required for both rapid and delayed induction of apoptosis renders serotype Typhimurium noncytotoxic under all conditions tested. Our hypothesis is that differential activation of programmed macrophage cell death by serotype Typhimurium occurs under discrete physiological conditions at distinct locations within an infected host.     

槲皮素对ox-LDL所致的小鼠巨噬细胞脂质蓄积和过氧化的影响

 目的：研究槲皮素(quercetin,QUE)预处理对氧化低密度脂蛋白 (oxidized low-density lipoprotein,ox-LDL)所致小鼠巨噬细胞脂质蓄积和过氧化的影响,并探讨可能的分子机制。方法：体外培养小鼠RAW264.7巨噬细胞,给予20、40和80 μmol/L QUE预处理30 min,再加入ox-LDL (100 mg/L)继续培养24 h。采用油红O染色检测细胞内脂质蓄积,测定培养液乳酸脱氢酶(lactic dehydrogenase,LDH)、丙二醛(malondialdehyde,MDA)和细胞内活性氧(reactive oxygen species,ROS)水平,以评价细胞膜完整性和脂质过氧化程度。分别采用real-time PCR和免疫印迹技术检测清道夫受体CD36 mRNA和蛋白表达变化。结果：QUE (20,40和80 μmol/L)预处理显著抑制ox-LDL所诱导的巨噬细胞内脂质蓄积和泡沫细胞形成,且呈浓度依赖性。ox-LDL组细胞LDH释放增加,而QUE预处理则显著抑制ox-LDL的上述细胞毒性。与ox-LDL组比较,QUE预处理组细胞内ROS含量和培养液中MDA水平明显降低。另外QUE预处理在mRNA和蛋白水平均明显抑制ox-LDL所诱导的CD36表达上调。结论: QUE可减轻ox-LDL所诱导的小鼠巨噬细胞脂质蓄积和过氧化,其机制可能部分是通过下调CD36表达实现的。     

虎杖苷抑制ox-LDL诱导的脂质过氧化并下调巨噬细胞清道夫受体CD36表达

目的:研究虎杖苷抑制ox-LDL诱导的巨噬细胞氧化反应和调节巨噬细胞清道夫受体CD36表达的功效。方法:将C57/BL6小鼠随机分为生理盐水组、虎杖苷组,分别经腹腔给予1.5%生理盐水、1.5%虎杖苷溶液,连续3d,第4d停止注射,第5d处死老鼠,取腹腔巨噬细胞培养。将培养的巨噬细胞给予氧化修饰的低密度脂蛋白(ox-LDL)刺激,观察腹腔巨噬细胞超氧化物歧化酶(SOD)的活性,ox-LDL诱导的巨噬细胞呼吸爆发及脂质过氧化物(LPO)含量,以及清道夫受体CD36的表达调控。结果:与生理盐水组相比,虎杖苷组巨噬细胞的SOD活性明显提高,巨噬细胞的LPO蓄积也明显降低;并且虎杖苷组ox-LDL诱导的巨噬细胞呼吸爆发水平也显著低于生理盐水对照组;虎杖苷组巨噬细胞CD36的表达同样明显低于生理盐水组。结论:虎杖苷可有效抑制巨噬细胞脂质过氧化应激情况下的呼吸爆发,下调其清道夫受体CD36的表达,提示虎杖苷可能具有预防动脉粥样硬化早期病变的功能。     

Contribution of TNF-alpha converting enzyme and proteinase-3 to TNF-alpha processing in human alveolar macrophages

Membrane-associated TNF-alpha cleavage is required to yield the 17.5-kD soluble product. This process is poorly understood in human cells, and no studies have related this process to the alveolar macrophage (AM). TNF-alpha-converting enzyme (TACE) is known to cleave TNF at the Ala-76-Val-77 site. We have evaluated the expression, regulation, and catalytic function of TACE in healthy human AMs. TACE was detected on the surface of AMs using flow cytometry. TACE protein can be upregulated by LPS (P = 0.036) and IFN-gamma. LPS-induced expression is downregulated by IL-10 (P = 0.04) and TNF-alpha. TACE regulation was observed at the mRNA level. TACE catalytic activity as assessed by cleavage of glutathione S-transferase-proTNF fusion protein correlates significantly with TACE protein expression (P = 0.04). However, cleavage and soluble TNF-alpha release by AMs was inhibited by matrix metalloproteinase and serine protease inhibitors, suggesting a role for a serine protease in this process. We confirmed the presence of proteinase-3 (PR-3) on the AM surface that was functionally capable of TNF cleavage. PR-3 mRNA expression was not found in AMs. However, we determined that PR-3 from neutrophil supernatants could bind to the AM membrane, suggesting that AM-derived PR-3 is from an exogenous source, which is important in the context of inflammation.     

Serum protein peroxidation in rats selected by the rate of active avoidance conditioning under normal conditions and during stress

Serum protein peroxidation was studied in rats with different strategy of adaptive behavior at rest and during short-term immobilization stress. Oxidative destruction of serum proteins was less intensive in KLA rats compared to that in KHA rats. Stress stimulated peroxidation of serum proteins in KHA rats.     

The activation of caspase-3 and DNA fragmentation in B cells phagocytosed by macrophages

Apoptotic signaling of mammalian cells involves two pathways: the death receptor and mitochondrial pathways. In this in vivo study, we investigated apoptotic signaling of B cells in mouse germinal centers (GCs) of gut-associated lymphoid tissues (GALTs) using transmission electron microscopy (TEM), terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL), immunofluorescence of members of caspase family and cFLIP(L), and caspase activity assay. It was very difficult to ultrastructurally differentiate B cells undergoing apoptosis from B cells differentiating into memory cells or plasma cells among B cells constituting GCs. Isolated B cells in GCs showed no active form of caspase-3 or TUNEL immunoreactivity, but expressed cFLIP(L). Contrary to isolated B cells, apoptotic B cells phagocytosed by macrophages exhibited immunoreactivity of the active form of caspase-3 and TUNEL, but lacked the cFLIP(L) expression. The caspase activity assay in GALTs clearly showed intense activity of caspase-3, caspase-9, and caspace-8 that was high in order. Therefore, the death receptor pathway accompanying the increased activity of caspase-3 and caspase-8 may be blocked by the expression of cFLIP(L) in B cells of GALTs. Moreover, both the activation of caspase-3 and DNA fragmentation first occur only when B cells are phagocytosed by macrophages.     

MAPK和caspase-3在异基因CD8+T淋巴细胞诱导血管内皮细胞凋亡中的作用

目的： 探讨MAPK和caspase-3在异基因CD8+T细胞诱导血管内皮细胞凋亡中的作用。方法：免疫磁珠阳性分选异基因CD8+T细胞,AnnexinⅤ/FITC试剂盒检测异基因CD8+T细胞诱导的HUVECs和HDMECs凋亡率,Western blotting检测血管内皮细胞内caspase-3、MAPK表达。观察SB203580 (p38MAPK抑制剂)、SP600125 (JNK抑制剂)、PD98059（ERK抑制剂）、Z-DEVD-FMK（caspase-3抑制剂）对内皮细胞凋亡的影响。结果：异基因CD8+T细胞作用24 h和48 h后,HUVECs凋亡率分别为41.7％±10.1％和29.4％±8.3％,HDMECs凋亡率分别为28.9％±7.2％和15.2％±4.8％,与对照组相比均具有显著差异（P<0.01）。异基因CD8+T细胞作用后,HUVECs和HDMECs内磷酸化p38MAPK表达、caspase-3裂解增强,而磷酸化JNK、ERK无明显变化。Z-DEVD-FMK和SB203580可显著抑制异基因CD8+T细胞诱导的HUVECs和HEMEC凋亡,并降低内皮细胞caspase-3表达。结论：p38MAPK和caspase-3介导了异基因CD8+T细胞诱导的血管内皮细胞凋亡。     

Effect of cobalt and chromium ions on bcl-2, bax, caspase-3, and caspase-8 expression in human U937 macrophages

The bcl-2 and caspase families of proteins play a central role in the modulation of apoptosis. The purpose of this study was to analyze the effect of Co(2+) and Cr(3+) ions on the expression of bcl-2, bax, caspase-3 and caspase-8 to better understand the mechanisms leading to ion-induced apoptosis in macrophages. U937 human macrophages were exposed to Co(2+) and Cr(3+) ions. The expression of proteins was measured by Western blot while caspase activities were measured by colorimetric assay. Results show that Co(2+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 52% inhibitory effect after 24 h. Co(2+) stimulated bax expression with a significant stimulation (p<0.05) after 8 h and a maximal 1.75-fold increase after 16 h. Co(2+) also stimulated the expression of the active fragment of caspase-3 as well as caspase-3 activity maximal increase after 24 h. Co(2+) ions had no effect on caspase-8 expression or activity.Cr(3+) ions inhibited bcl-2 expression with significant effect (p<0.05) after 16 h and a maximal 43% inhibitory effect after 24 h. Cr(3+) stimulated bax expression with significant stimulation (p<0.01) after 8h and a maximal 2.25-fold increase after 24 h. Cr(3+) ions also stimulated the expression of the active fragments of caspase-3 and -8, as well as the activities of both proteases. The effect of Cr(3+) ions on the expression of both caspase active fragments was maximal after 16 h incubation. In conclusion, our results suggest that the modulation of the expression of proteins from the bcl-2 and the caspase families of proteins are implicated in the induction of macrophage apoptosis by Co(2+) and Cr(3+) ions.     

Characterization of coxsackievirus B3-caused apoptosis under in vitro conditions

Among several mechanisms of pathogenesis of the frequent and sometimes serious infections with coxsackievirus B3 (CVB3), one detail is apoptosis. Recently, a new apoptotic mechanism involving the specific interaction between the capsid protein VP2 of the highly virulent variant CVB3H3 and the proapoptotic host protein Siva was identified. The relevance of this observation for virus pathogenicity was shown in a BALB/c mouse model using CVB3H3 and the interaction-deficient mutant virus CVB3H310A1. In this study these results were verified and extended under in vitro conditions. The different apoptotic capability of CVB3H3 versus CVB3H310A1 was demonstrated by apoptotic nuclear condensation, DNA fragmentation, expression of Siva mRNA, and caspase-3 activation. The virus-specific differences were caused by the VP2 capsid proteins, which was shown by overexpression of the single VP2H3 and VP2H310A1 protein. Furthermore, the involvement of apoptosis in virus progeny production and the associated appearance of the cytopathic effect was demonstrated by application of the pan-caspase inhibitor Z-VAD-FMK. These in vitro results indicate that the induction of apoptosis during CVB3H3 infection is based on the interaction between the capsid protein VP2 and the proapoptotic protein Siva, independently from the complex situation in vivo.     

Dependence of lipid peroxidation changes and antioxidant activity in myocardium under acute and chronic stress from organism thyroid status

In experiments on 122 unbreed white male rats with body weight 250-300 g investigated influence of thyroid status on evoked by chronic stress (CS--immobilization on 3 h during 5 days) and acute stress (AS--once immobilization during 3 h) changes of lipid peroxidation (LP) intensivity and antioxidant activity (AOA) in myocardium. It was shown that chronic stress, which decreases triiodothyronine and thyroxine levels in blood, leads to more market LP activation and lowering of superoxiddismutase activity in myocardium than acute stress, which reducts only T3 concentration. Close correlation between T3 and T4 concentration in blood and LP speed, level of its products, general AOA in myocardium was founded in both stresses. Hypothyrosis (1.2 mg/kg body weight during 14 days intragastrally) leads to more market accumulation of initial lipid peroxidation products and general AOA depression in myocardium under acute and, especially, chronic stress. Like to physiological doses of thyroxine (from 5.0 to 8.0 mkg/kg body weight during 28 days same way) prevent LP activation and limit AOA decreasing in myocardium under both stresses.     

Impact of ellagic acid on adriamycin-induced testicular histopathological lesions,apoptosis, lipid peroxidation and sperm damages

The aim of the present study was to investigate whether ellagic acid (EA) has protective effect on adriamycin (ADR)-induced testicular and spermatozoal toxicity associated with the oxidative stress in male rats. Thirthy-two healthy 8-week-old male Sprague–Dawley rats were equally divided into four groups. The first (EA) group was treated with EA (2 mg/kg/every other day) by gavage. The second (ADR) group received ADR (2 mg/kg/once a week) intraperitoneally, while the combination of ADR and EA was given to the third (ADR + EA) group. The forth (control) group was treated with placebo. At the end of the 8-week treatment period, reproductive organ weights, epididymal sperm parameters, histopathological changes and apoptosis via Bax and Bcl-2 proteins, testicular tissue lipid peroxidation, and antioxidant enzyme activities, were investigated. ADR administration was determined to cause significant decreases in reproductive organ weights, epididymal sperm concentration and motility, plasma testosterone concentration, diameter of seminiferous tubules, germinal cell layer thickness, Johnsen's testicular score and Bcl-2 positive antiapoptotic cell rate, wherease it caused significant increases in level of lipid peroxidation and glutathione, catalase activity, abnormal sperm rates and Bax positive apoptotic cell rates along with degeneration, necrosis, immature germ cells, congestion and atrophy in testicular tissue when compared with the control group. EA administration to ADR-treated rats provided significant improvements in ADR-induced disturbed oxidant/antioxidant balance, decreased testosterone concentration, testicular apoptosis and mild improvements in the histopathological view of the testicular tissue. However, EA failed to improve decreased reproductive organ weights and deteriorated sperm parameters due to ADR administration. It is concluded that while ADR has direct or indirect (lipid peroxidation) negative effects on sperm structure and testicular apoptosis in rats, EA has protective effects on ADR-induced testicular lipid peroxidation and apoptosis.