NOD8对H2O2诱导的人肝细胞凋亡的影响

 目的:探讨NOD8对H2O2诱导的人肝细胞L02凋亡的影响。 方法: pEGFP-C2 及pEGFP-NOD8重组质粒经JetPRIME介导转染L02细胞;用H2O2诱导细胞凋亡。实验分为pEGFP-C2组、pEGFP-C2+H2O2组和pEGFP-NOD8+H2O2组。采用MTT法检测细胞活性,Western blotting检测细胞NOD8的蛋白表达,Hoechst 33342染色检测细胞凋亡情况,流式细胞术检测细胞凋亡率,比色法检测细胞caspase-3活性。 结果: 通过MTT检测不同浓度（0.2～2 mmol/L）H2O2刺激6 h后的细胞活性,确定1 mmol/L H2O2为诱导细胞凋亡的剂量。Western blotting检测结果显示,转染pEGFP-NOD8质粒的细胞NOD8蛋白表达明显增加。Hoechst 33342染色法观察发现,pEGFP-C2+H2O2组有较多细胞出现强蓝色荧光细胞核,细胞凋亡较多,而pEGFP-NOD8+H2O2组细胞凋亡明显减少。流式细胞术分析显示,pEGFP-C2+H2O2组的细胞凋亡率明显升高,pEGFP-NOD8+H2O2组的细胞凋亡率则显著下降。pEGFP-C2+H2O2组细胞的caspase-3活性明显升高,而pEGFP-NOD8+H2O2组细胞的caspase-3活性显著下降。 结论: NOD8可抑制H2O2诱导的L02细胞凋亡,其作用机制可能与NOD8抑制细胞的caspase-3活性有关。     

Alginate oligosaccharide protects against endoplasmic reticulum- and mitochondrial-mediated apoptotic cell death and oxidative stress

Oxidative stress is a major component of harmful cascades activated in neurodegenerative disorders. We sought to elucidate possible effects of alginate oligosaccharide (AOS) on H(2)O(2)-induced cell death and to determine the underlying molecular mechanisms in neuron-like PC12 cells. We found that AOS treatment protected PC12 cells against H(2)O(2)-induced endoplasmic reticulum (ER) and mitochondrial-dependent apoptotic cell death. AOS promoted Bcl-2 expression, while blocked Bax expression and inhibited H(2)O(2)-induced caspase-3 activation. It also blocked PARP cleavage. AOS acted on key molecules in apoptotic cell death pathway and reduced p53, p38, c-June NH2-terminal kinase phosphorylations, inhibited NFkB, and enhanced Nrf2 activation. These results suggest that treatment of PC12 cells with AOS can block H(2)O(2)-induced oxidative stress and caspase-dependent apoptotic cascades originating from both ER and mitochondria. Our in?vivo experiments further confirm the neuroprotective potential of AOS against Aβ-induced neural damage. According to our data, the involvement of caspase-independent pathway in AOS-induced protection appears to be unlikely.     

Caffeine induces apoptosis in human neuroblastoma cell line SK-N-MC

Caffeine is one of the most widely consumed neuroactive drugs, coming mostly from everyday beverages such as coffee and tea. To investigate whether caffeine induces apoptosis in the central nervous system, 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, flow cytometric analysis, DNA fragmentation assay, and caspase-3 enzyme assay were performed on SK-N-MC human neuroblastoma cells. Cells treated with caffeine at concentrations as high as 10 mM exhibited several characteristics of apoptosis. In addition, caffeine was shown to increase the caspase-3 activity. These results suggest that high-dose of caffeine induces apoptosis in human neuroblastoma cells, probably by increasing the caspase-3 enzyme activity.     

St john's wort (Hypericum perforatum) counteracts deleterious effects of the chronic restraint stress on recall in rats

This study aimed at verifying a hypothesis that St. John's wort (Hypericum perforatum) alleviates stress-induced memory impairments. Administration of Hypericum perforatum (350 mg kg(-1) daily for 21 days) significantly enhanced recall of passive avoidance behavior (PAB), but had no effect on the acquisition of conditioned avoidance responses (CARs). Rats stressed chronically (2 h daily for 21 days) displayed diminished recall of the PAB and this effect was abolished by St John's wort. Chronic administration of the "equivalent" to the stress dose of exogenous corticosterone (5 mg kg(-1) daily for 21 days) also impaired recall of PAB, and this effect was also reversed by Hypericum perforatum. None of our treatments produced significant motor coordination impairments as tested in a 'chimney' test. It appears that H. perforatum prevents stress-induced deterioration of memory in rats.     

Fibroblast growth factor-10 attenuates H2O2-induced alveolar epithelial cell DNA damage: role of MAPK activation and DNA repair

Fibroblast growth factor-10 (FGF-10), an alveolar epithelial cell (AEC) mitogen that is critical for lung development, may promote AEC repair. We determined whether FGF-10 attenuates H2O2-induced, A549 and rat alveolar type II cell DNA damage. We show that FGF-10 prevents H2O2-induced DNA damage assessed by an alkaline elution, ethidium bromide fluorescence as well as by a comet assay. Mitogen-activated protein kinase inhibitors abolished the protective effect of FGF-10 against H2O2-induced DNA damage yet had no effect on H2O2-induced DNA damage. A Grb2-SOS inhibitor (SH3 binding peptide), an Ras inhibitor (farnesyl transferase inhibitor 277), and an Raf-1 inhibitor (forskolin) each prevented FGF-10- and H2O2-induced A549 cell ERK1/2 phosphorylation. Also, FGF-10 and H2O2 each induced negligible ERK1/2 phosphorylation in Ras dominant-negative (N17) cells. Inhibitors of Ras and Raf-1 blocked the protective effect of FGF-10 against H2O2-induced DNA damage but had no effect on H2O2-induced DNA damage. Furthermore, cold conditions and aphidicolin, an inhibitor of DNA polymerase-alpha, -delta, and -epsilon, each blocked the protective effects of FGF-10, suggesting a role for DNA repair. We conclude that FGF-10 attenuates H2O2-induced AEC DNA damage by mechanisms that involve activation of Grb2-SOS/Ras/RAF-1/ERK1/2 pathway and DNA repair.     

3,4-dihydroxybenzoic acid from Smilacis chinae rhizome protects amyloid beta protein (25-35)-induced neurotoxicity in cultured rat cortical neurons

The neuroprotective effect of 3,4-dihydroxybenzoic acid (3,4-DHBA) isolated from Smilacis chinae rhizome against Abeta (25-35)-induced neurotoxicity on cultured rat cortical neurons was found in this study. The protective effect of 3,4-DHBA against Abeta (25-35)-induced neuronal cell death was investigated by measuring cell viability via a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and Hoechst 33342 staining. 3,4-DHBA (1 and 10 microM) concentration-dependently inhibited 10 microM Abeta (25-35)-induced neuronal apoptotic death. 3,4-DHBA (1 and 10 microM) inhibited 10 microM Abeta (25-35)-induced elevation of cytosolic Ca(2+) concentration ([Ca(2+)](c)), which was measured by a fluorescent dye, Fluo-4 AM. 3,4-DHBA also inhibited glutamate release into medium, reactive oxygen species (ROS) generation, and caspase-3 activation, which were induced by 10 microM Abeta (25-35). These results suggest that 3,4-DHBA prevents Abeta (25-35)-induced neuronal cell damage by interfering with the increase of [Ca(2+)](c), and then by inhibiting glutamate release, generation of ROS and caspase-3 activity.     

GP7 can induce apoptotic DNA fragmentation of human leukemia cells through caspase-3-dependent and -independent pathways

GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy)amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin, is a promising anticancer drug of podophyllotoxin class. The primary effect of GP7 is the anticancer activity on transplanted mouse tumors and cultured tumor cells. However, its molecular mechanism of action is still obscure. In this study, we investigated the activity of GP7 to induce apoptosis in human leukemia HL-60 and Jurkat cells. Apoptosis was determined by detection of DNA fragmentation in agarose gel electrophoresis. GP7 induced apoptotic DNA fragmentation of HL-60 and Jurkat cells in time- and dose-dependent manner. We further investigated the activity of caspase-3 in GP7-induced apoptotic DNA fragmentation of HL-60 and Jurkat cells. GP7 also induced time- and dose-dependent caspase-3 activation in both cell lines, and the kinetics of caspase-3 activation induced by GP7 was well correlated with that of apoptotic DNA fragmentation. To determine the role of caspase-3 in GP7-induced apoptotic DNA fragmentation, we examined the effect of specific caspase-3 inhibitor, Ac-DEVD-CHO, on GP7-induced apoptotic DNA fragmentation. Ac-DEVD-CHO prevented GP7-induced caspase-3 activation in both HL-60 and Jurkat cells, however, it only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. We then employed L-carnitine to investigate the role of caspase-3 in GP7-induced apoptotic DNA fragmentation. L-carnitine treatment prevented GP7-induced caspase-3 activation in both cell lines in a dose-dependent manner. Similar to Ac-DEVD-CHO, L-carnitine only inhibited GP7-induced apoptotic internucleosomal DNA fragmentation in HL-60 cells. These findings suggest that GP7 exerts an anti-leukemic effect by both caspase-3-dependent and -independent apoptotic signaling pathways.     

Protection against CD95-induced apoptosis by chlamydial infection at a mitochondrial step

Chlamydiae are obligate intracellular bacteria that infect human epithelial and myeloid cells. Previous work has established that chlamydiae are able to protect a cell against apoptosis induced by certain experimentally applied stimuli. Here we provide an analysis of this protective activity against the signal transduction during CD95-induced apoptosis. In HeLa cells overexpressing CD95, infection with Chlamydia trachomatis inhibited the appearance of apoptotic morphology, effector caspase activity, the activation of caspase-9 and -3, and the release of cytochrome c from mitochondria. However, caspase-8-processing and activity (measured as cleavage of Bid) were unaffected by the chlamydial infection. Similarly, infection with the species C. pneumoniae did not prevent the activation of caspase-8 but inhibited the appearance of effector caspase activity upon signaling through CD95. Furthermore, infection with C. trachomatis was able to inhibit CD95-induced apoptosis in Jurkat lymphoid cells, where a mitochondrial contribution is required, but not in SKW6.4 lymphoid cells, where caspase-8 directly activates caspase-3. Taken together, these data show that chlamydial infection can protect cells against CD95-induced apoptosis but only where a mitochondrial signaling step is necessary for apoptotic signal transduction.     

内质网应激介导蜕皮甾酮对H2 O2 诱导的心肌细胞损伤的保护作用

目的:探讨蜕皮甾酮(EDS)如何通过调节内质网应激对过氧化氢(H2 O2 )诱导的心肌细胞损伤起保护作用。方法:大鼠H9c2 心肌细胞随机分为对照(Control)组,正常心肌细胞;H2 O2 组,不同浓度H2 O2(1*10-3 、1*10-4 、1*10-5 mol/ L)诱导损伤细胞;EDS 组,在H2 O2 组基础上,加入不同浓度的EDS(25、50、100 μmol/ L)处理。MTT 法和流式细胞术分别检测实验各组细胞活力及细胞凋亡率。Western blot 检测各组细胞中Bcl-2、Bax、cleaved-caspase-3、caspase-4、caspase-7、caspase-12、 GRP78 和CHOP 的蛋白水平,同时检测各组细胞中丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果:与Control 组相比,H2 O2 组的细胞活力减弱,凋亡率升高,MOD 含量升高,SOD 活性降低,GRP78 和CHOP 的表达升高(均P<0.05)。H2 O2 组加入EDS 处理后,细胞活力提升,凋亡率下降,MOD 含量降低,SOD 活性升高,GRP78 和CHOP 的表达降低(均P<0.05)。结论:通过抑制内质网的应激过程,蜕皮甾酮能减轻H2 O2 诱导的心肌细胞损伤。     

Protection by puerarin against MPP+-induced neurotoxicity in PC12 cells mediated by inhibiting mitochondrial dysfunction and caspase-3-like activation

Puerarin, a main isoflavone glycoside distributed in Pueraria lobata (Willd.) Ohwi, showed inhibitory activity on H2O2-induced PC12 cells damage in our previous work. However, there is insufficient evidence in protective mechanism of puerarin, especially that relating to the mitochondrial function. In this study, when cells were pretreated with puerarin prior to 0.4 mM MPP+, protective roles were accompanied by a reduction of cell viability loss, morphological changes of apoptosis and apoptotic rate. To explore the protective mechanism of puerarin in MPP+-induced PC12 cells, mitochondrial function and caspase-3-like activity were measured. The results indicated that puerarin inhibited the release of mitochondrial cytochrome c to cytosol and the loss of mitochondrial membrane potentials. In addition, puerarin also reduced MPP+-induced caspase-3-like activation. Taken together, the above results suggest that pretreatment of PC12 cells with puerarin could block MPP+-mediated apoptosis by mitochondria-dependent caspase cascade.     

Spinal cord histopathology of MOG peptide 35-55-induced experimental autoimmune encephalomyelitis is time- and score-dependent

The neuroprotective effects of Jatrorrhizine from Coptidis Rhizoma against hydrogen peroxide (H(2)O(2))-induced rat pheochromocytoma line PC12 injury and its potential mechanisms were evaluated in the present study. When cells were exposed to H(2)O(2) (200 μM) for 12h, there was a significant reduction in cell survival and activity of antioxidant enzyme (SOD and HO-1) and LDH release. In addition, increased ROS production, declined MMP and increased production of malondialdehyde (MDA) were observed. Preincubation of cells with Jatrorrhizine (0.01-10.0 μM) 24h prior to H(2)O(2) exposure markedly elevated cell viability and activities of antioxidant enzyme (SOD and HO-1), prevented LDH release and lipid peroxidation (MDA) production, attenuated the decrease of MMP and scavenged ROS formation. Jatrorrhizine also attenuated caspase-3 activation of the downstream cascade following ROS. Our results suggest that Jatrorrhizine holds potential for neuroprotective effects against H(2)O(2)-induced injury.     

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.     

Infection with Langat Flavivirus or expression of the envelope protein induces apoptotic cell death

Langat (LGT) flavivirus, derived from infectious full-length cDNA clone 636, was investigated for its apoptotic activities in mouse neuroblastoma (Neuro-2a) and simian kidney (Vero and LLC-MK(2)) cells. The hallmark of apoptosis, cleavage of cellular DNA, was observed 48 h after infection of Vero, LLC-MK(2), and Neuro-2a cells by electrophoresis analysis. Apoptosis in infected cells was also confirmed by TUNEL assay. LGT-infected Neuro-2a cells showed an increase in caspase-3-like protease (DEVDase) activity. Expression of the major envelope glycoprotein (E) alone reduced cell viability in both Vero and Neuro-2a cells, and the baculovirus P35 protein, which inhibits multiple caspases, completely blocked this effect. Cleavage of cellular DNA was observed in E gene-transfected Vero cells by TUNEL assay. Expression of E protein or caspase-9 resulted in activation of caspase-3-like proteases in Neuro-2a cells. The caspase-3-like protease specific inhibitor, Ac-DEVD-CHO peptide, partially inhibited E protein- or caspase-9-induced apoptosis in Neuro-2a cells. These observations indicate that infection of cells with LGT virus or expression of LGT virus E protein induces apoptosis through a caspase-3-like protease pathway.     

Vitamin E but not St. John's wort mitigates leukopenia caused by cancer chemotherapy in rats

Dietary supplements are used by most patients with cancer. As nutraceuticals can interact with many drugs, this study investigated the effect of herbal remedies and vitamins on the toxicity of representative cancer chemotherapeutic agents. Fisher 344 rats were fed a standard cereal-based diet or the same diet with additional vitamin E in low (50 mg/kg) or high (750 mg/kg) concentrations, or with added St. John's wort (400 mg/kg). The LD50 was determined after the administration of chemotherapy drugs. Neither low or high vitamin E supplements nor St. John's wort significantly changed the LD50 for doxorubicin, docetaxel, or cyclophosphamide. The nadir white blood cell (WBC) count was significantly higher (P = 0.004) after docetaxel in rats supplemented with low-dose vitamin E, but the drop in WBC count from initial to nadir levels (Nfall) was greater in rats fed a diet containing high vitamin E supplementation (P = 0.04). Similarly, the Nfall was greater in the standard and high vitamin E dietary groups than in the low vitamin E group after cyclophosphamide (P = 0.03). No effect of vitamin E or St. John's wort supplementation occurred on doxorubicin pharmacokinetics. Neither vitamin E nor St. John's wort had an important effect on the mitochondrial deoxyribonucleic acid (DNA) damage caused by either doxorubicin or docetaxel. These data suggest that the leucopenia caused by some chemotherapeutic agents can be modified by dietary supplementation with vitamin E, but the effect seems to be dose-dependent. St. John's wort had neither a beneficial nor a detrimental effect on chemotherapy-induced toxicity.     

Induction of apoptotic cell death by mycelium extracts of Phellinus linteus in human neuroblastoma cells

Phellinus linteus is a well-known Oriental medicinal fungus that has various biological activities, including immunomodulatory and anti-tumor activities, the mechanisms of which are poorly understood. In the present study, we investigated the effects of mycelium extracts of P. linteus (MEPL) on the growth of human neuroblastoma SK-N-MC cells. Upon treatment with MEPL, a concentration-dependent inhibition of cell proliferation was observed and cells developed many of the hallmark features of apoptosis, including condensation of chromatin and an increase in the sub-G1 population. The anti-proliferative and apoptotic effects of MEPL were associated with a marked induction of the Bax and cyclin-dependent kinase inhibitor p21. Western blotting and in vitro caspase-3 activity assay demonstrated that the processing/activation of caspases accompanies the generation of MEPL-mediating apoptotic cell death. In addition, the proteolytic cleavage of specific target proteins such as poly(ADP-ribose) polymerase and beta-catenin were observed. Taken together, the present results suggest that apoptotic signals evoked by MEPL in human neuroblastoma SK-N-MC cells may converge caspase-3 activation through an up-regulation of Bax rather than a down-regulation of Bcl-2.     

Melissa Officinalis L. Extracts Protect Human Retinal Pigment Epithelial Cells against Oxidative Stress-Induced Apoptosis

Background: We evaluated the protective effect of ALS-L1023, an extract of Melissa officinalis L. (Labiatae; lemon balm) against oxidative stress-induced apoptosis in human retinal pigment epithelial cells (ARPE-19 cells).Methods: ARPE-19 cells were incubated with ALS-L1023 for 24 h and then treated with hydrogen peroxide (H₂O₂). Oxidative stress-induced apoptosis and intracellular generation of reactive oxygen species (ROS) were assessed by flow cytometry. Caspase-3/7 activation and cleaved poly ADP-ribose polymerase (PARP) were measured to investigate the protective role of ALS-L1023 against apoptosis. The protective effect of ALS-L1023 against oxidative stress through activation of the phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) was evaluated by Western blot analysis.Results: ALS-L1023 clearly reduced H₂O₂-induced cell apoptosis and intracellular production of ROS. H₂O₂-induced oxidative stress increased caspase-3/7 activity and apoptotic PARP cleavage, which were significantly inhibited by ALS-L1023. Activation of the PI3K/Akt pathway was associated with the protective effect of ALS-L1023 on ARPE-19 cells.Conclusions: ALS-L1023 protected human RPE cells against oxidative damage. This suggests that ALS-L1023 has therapeutic potential for the prevention of dry age-related macular degeneration.     

雌激素减轻H2O2诱导PC12细胞凋亡

目的观察雌激素对H2O2诱导细胞凋亡的作用,探讨雌激素保护作用机制。方法在PC12细胞建立H2O2诱导细胞凋亡的实验模型。用MTT法检测细胞存活率,比色法测定乳酸脱氢酶(LDH)活性,Hoechst染色检测细胞凋亡,碘化丙啶(PI)染色流式细胞术检测细胞凋亡率,比色法测定caspase-3活性。结果H2O2明显降低PC12细胞的存活率,使LDH释放增加,促进细胞凋亡,并能明显地升高caspase-3的活性。雌激素能显著地减轻上述变化。结论雌激素对抗H2O2诱导的细胞凋亡,抑制caspase-3的激活是其细胞保护机制之一。