Role of Bmbuffy in hydroxycamptothecine-induced apoptosis in BmN-SWU1 cells of the silkworm, Bombyx mori

Bcl-2 family proteins have been reported previously to play important roles in the mitochondrial apoptotic pathway. Particularly, Bmbuffy has been identified as a key homologue of Bcl-2 in silkworm; however, its exact function is unknown. In this study, we investigated the role of Bmbuffy in hydroxycamptothecine (HCPT)-induced apoptosis of BmN-SWU1 cells. By conducting confocal microscopy studies, we found that Bmbuffy is located on the outer membrane of mitochondria and endoplasmic reticulum (ER). Furthermore, we discovered that the hydrophobic transmembrane domain at the COOH terminus is a putative anchor for the subcellular localization of Bmbuffy. Overexpression of Bmbuffy inhibited cytochrome c release, activation of caspase-3 and cell apoptosis, while RNAi-mediated silencing of Bmbuffy promoted apoptosis. In the absence of a hydrophobic membrane anchor, we revealed that Bmbuffy is unable to block apoptosis. These results indicate that Bmbuffy acts as an anti-apoptotic protein, located on the mitochondrial outer membrane and is involved in the mitochondrial apoptotic pathway. Moreover, in HCPT-induced apoptosis, we showed that the translocation of endogenous Bmp53 from the nucleus to the mitochondria is a slow and progressive process, followed by cytochrome c release. This suggests that mitochondrial Bmp53 accumulation may contribute to membrane permeability. The co-localization of Bmp53 and Bmbuffy suggests the interaction of the two proteins, which was further confirmed by Co-IP assay. In addition, overexpression of Bmp53 increased cytochrome c release and the cell apoptotic rate, whereas Bmbuffy overexpression blocked these. All the data suggest that Bmbuffy functions as an anti-apoptotic protein and interacts with Bmp53 in HCPT-induced apoptosis of silkworm cells.     

The Effect of Selenium on the Cd-Induced Apoptosis via NO-Mediated Mitochondrial Apoptosis Pathway in Chicken Liver

Cd-induced apoptosis and the protective effects of Se against Cd-induced injury have been reported in previous studies. However, little is known regarding the effects of Cd-induced apoptosis in hepatic cells and the antagonistic effects of Se on Cd in poultry. In the present study, 128 healthy 31-week-old laying hens were randomly divided into four groups, which were fed basic diets, with the addition of Se (Na₂SeO₃, 2 mg/kg), Cd (CdCl₂, 150 mg/kg), or Se + Cd (150 mg/kg of CdCl₂ and 2 mg/kg of Na₂SeO₃) for 90 days. Ultrastructural changes, nitric oxide (NO) concentrations, inducible nitric oxide synthase (iNOS) activities, results of the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay of apoptosis, and the expression of iNOS and apoptosis-related genes in livers were determined. It was observed that Cd treatment significantly increased the concentrations of NO and iNOS activity in chicken livers. The production of excessive NO initiated the mitochondrial apoptotic pathway. Exposure to Cd increased the mRNA and the protein expression levels of iNOS, caspase-3, Bax, p53, and Cyt-c. Furthermore, the ratio of Bax/Bcl-2 increased, while the expression of Bcl-2 decreased. Treatment with Se significantly alleviated Cd-induced apoptosis in chicken livers, as evidenced by a reduction in the production of NO, iNOS activity, the number of apoptotic cells, and mRNA and protein expression levels of iNOS, caspase-3, Bax, and Cyt-c. It indicated that Cd induced NO-mediated apoptosis through the mitochondrial apoptotic pathway and Se exerted antagonizing effects. The present study provides new insights as to how Se affects Cd-induced toxicity in the chicken liver.     

The green algae Ulva fasciata Delile extract induces apoptotic cell death in human colon cancer cells

This study investigated the mechanisms underlying the cytotoxicity of the green algae Ulva fasciata Delile. U. fasciata extract (UFE) inhibited the growth of HCT 116 human colon cancer cells by 50% at a concentration of 200 μg/ml. In addition, UFE stimulated the production of intracellular reactive oxygen species, an effect that was abolished by pretreatment with N-acetyl cysteine, which also inhibited the cytotoxic effects of UFE. UFE also induced morphological changes indicative of apoptosis, such as the formation of apoptotic bodies, DNA fragmentation, an increase in the population of apoptotic sub-G₁ phase cells, and mitochondrial membrane depolarization. Concomitant activation of the mitochondria-dependent apoptotic pathway occurred via modulation of Bax and Bcl-2 expression, resulting in disruption of the mitochondrial membrane potential and activation of caspase-9 and caspase-3. This is the first report to demonstrate the cytotoxic effect of U. fasciata on human colon cancer cells and to provide a possible mechanism for this activity.     

Oxidative Stress and Ca2+ Signals Involved on Cadmium-Induced Apoptosis in Rat Hepatocyte

Cadmium (Cd) is an important industrial and environmental pollutant. In animals, the liver is the major target organ of Cd toxicity. In this study, rat hepatocytes were treated with 2.5～10 μM Cd for various durations. Studies on nuclear morphology, chromatin condensation, and apoptotic cells demonstrate that Cd concentrations ranging within 2.5～10 μM induced apoptosis. The early-stage marker of apoptosis, i.e., decreased mitochondrial membrane potential, was observed as early as 1.5 h at 5 μM Cd. Significant (P?P?2+ concentration ([Ca²⁺] _i ) of Cd-exposed cells significantly increased (P?2+] _i may play an important role in apoptosis. Overall, these results showed that oxidative stress and Ca²⁺ signaling were critical mediators of the Cd-induced apoptosis of rat hepatocytes.     

Induction of mitochondrial biogenesis protects against caspase-dependent and caspase-independent apoptosis in L6 myoblasts

Apoptotic signaling plays an important role in skeletal muscle degradation, atrophy, and dysfunction. Mitochondria are central executers of apoptosis by directly participating in caspase-dependent and caspase-independent cell death signaling. Given the important apoptotic role of mitochondria, altering mitochondrial content could influence apoptosis. Therefore, we examined the direct effect of modest, but physiological increases in mitochondrial biogenesis and content on skeletal muscle apoptosis using a cell culture approach. Treatment of L6 myoblasts with SNAP or AICAR (5 h/day for 5 days) significantly increased PGC-1, AIF, cytochrome c, and MnSOD protein content as well as MitoTracker staining. Following induction of mitochondrial biogenesis, L6 myoblasts displayed decreased sensitivity to apoptotic cell death as well as reduced caspase-3 and caspase-9 activation following exposure to staurosporine (STS) and C2-ceramide. L6 myoblasts with higher mitochondrial content also exhibited reduced apoptosis and AIF release following exposure to hydrogen peroxide (H₂O₂). Analysis of several key apoptosis regulatory proteins (ARC, Bax, Bcl-2, XIAP), antioxidant proteins (catalase, MnSOD, CuZnSOD), and reactive oxygen species (ROS) measures (DCF and MitoSOX fluorescence) revealed that these mechanisms were not responsible for the observed cellular protection. However, myoblasts with higher mitochondrial content were less sensitive to Ca^2 +-induced mitochondrial permeability transition pore formation (mPTP) and mitochondrial membrane depolarization. Collectively, these data demonstrate that increased mitochondrial content at physiological levels provides protection against apoptotic cell death by decreasing caspase-dependent and caspase-independent signaling through influencing mitochondrial Ca^2 +-mediated apoptotic events. Therefore, increasing mitochondrial biogenesis/content may represent a potential therapeutic approach in skeletal muscle disorders displaying increased apoptosis.     

Role of microRNA-195 in cardiomyocyte apoptosis induced by myocardial ischaemia–reperfusion injury

This study aims to investigate microRNA-195 (miR-195) expression in myocardial ischaemia–reperfusion (I/R) injury and the roles of miR-195 in cardiomyocyte apoptosis though targeting Bcl-2. A mouse model of I/R injury was established. MiR-195 expression levels were detected by real-time quantitative PCR (qPCR), and the cardiomyocyte apoptosis was detected by TUNEL assay. After cardiomyocytes isolated from neonatal rats and transfected with miR-195 mimic or inhibitor, the hypoxia/reoxygenation (H/R) injury model was established. Cardiomyocyte apoptosis and mitochondrial membrane potential were evaluated using flow cytometry. Bcl-2 and Bax mRNA expressions were detected by RT-PCR. Bcl-2, Bax and cytochrome c (Cyt-c) protein levels were determined by Western blot. Caspase-3 and caspase-9 activities were assessed by luciferase assay. Compared with the sham group, miR-195 expression levels and rate of cardiomyocyte apoptosis increased significantly in I/R group (both P<0.05). Compared to H/R + negative control (NC) group, rate of cardiomyocyte apoptosis increased in H/R + miR-195 mimic group while decreased in H/R + miR-195 inhibitor group (both P<0.05). MiR-195 knockdown alleviated the loss of mitochondrial membrane potential (P<0.05). MiR-195 overexpression decreased Bcl-2 mRNA and protein expression, increased BaxmRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). While, downregulated MiR-195 increased Bcl-2 mRNA and protein expression, decreased Bax mRNA and protein expression, Cyt-c protein expression and caspase-3 and caspase-9 activities (all P<0.05). Our study identified that miR-195 expression was upregulated in myocardial I/R injury, and miR-195 overexpression may promote cardiomyocyte apoptosis by targeting Bcl-2 and inducing mitochondrial apoptotic pathway.     

The bacterial exotoxin AIP56 induces fish macrophage and neutrophil apoptosis using mechanisms of the extrinsic and intrinsic pathways

It has been previously shown that the exotoxin of the important fish pathogen Photobacterium damselae ssp. piscicida is a key pathogenicity factor and is responsible for the extensive systemic apoptosis of macrophages and neutrophils seen in acute fish photobacteriosis. The focus of the present study was to further characterize the AIP56-induced apoptosis of sea bass professional phagocytes by assessing the involvement of caspases, mitochondria and oxidative stress. The resulting data indicate that the apoptotic response in peritoneal macrophages and neutrophils treated ex vivo with AIP56 involves activation of caspase-8, -9 and -3, and mitochondria as shown by loss of mitochondrial membrane potential, release of cytochrome c and over-production of ROS. These results together with previous data from this laboratory suggest that both the extrinsic and intrinsic apoptotic pathways are involved in the AIP56-induced phagocyte apoptosis.     

Mechanism of immunotoxicological effects of tributyltin chloride on murine thymocytes

Tributyltin-chloride, a well-known organotin compound, is a widespread environmental toxicant. The immunotoxic effects of tributyltin-chloride on mammalian system and its mechanism is still unclear. This study is designed to explore the mode of action of tributyltin-induced apoptosis and other parallel apoptotic pathways in murine thymocytes. The earliest response in oxidative stress followed by mitochondrial membrane depolarization and caspase-3 activation has been observed. Pre-treatment with N-acetyl cysteine and buthionine sulfoximine effectively inhibited the tributyltin-induced apoptotic DNA and elevated the sub G₁ population, respectively. Caspase inhibitors pretreatment prevent tributyltin-induced apoptosis. Western blot and flow cytometry indicate no translocation of apoptosis-inducing factor and endonuclease G in the nuclear fraction from mitochondria. Intracellular Ca²⁺ levels are significantly raised by tributyltin chloride. These results clearly demonstrate caspase-dependent apoptotic pathway and support the role of oxidative stress, mitochondrial membrane depolarization, caspase-3 activation, and calcium during tributyltin-chloride (TBTC)-induced thymic apoptosis.     

Chlorpromazine protects against apoptosis induced by exogenous stimuli in the developing rat brain

Background

Chlorpromazine (CPZ), a commonly used antipsychotic drug, was found to play a neuroprotective role in various models of toxicity. However, whether CPZ has the potential to affect brain apoptosis in vivo is still unknown. The purpose of this study was to investigate the potential effect of CPZ on the apoptosis induced by exogenous stimuli.

Methodology

The ethanol treated infant rat was utilized as a valid apoptotic model, which is commonly used and could trigger robust apoptosis in brain tissue. Prior to the induction of apoptosis by subcutaneous injection of ethanol, 7-day-old rats were treated with CPZ at several doses (5 mg/kg, 10 mg/kg and 20 mg/kg) by intraperitoneal injection. Apoptotic cells in the brain were measured using TUNEL analysis, and the levels of cleaved caspase-3, cytochrome c, the pro-apoptotic factor Bax and the anti-apoptotic factor Bcl-2 were assessed by immunostaining or western blot.

Findings

Compared to the group injected with ethanol only, the brains of the CPZ-pretreated rats had fewer apoptotic cells, lower expression of cleaved caspase-3, cytochrome c and Bax, and higher expression of Bcl-2. These results demonstrate that CPZ could prevent apoptosis in the brain by regulating the mitochondrial pathway.

Conclusions

CPZ exerts an inhibitory effect on apoptosis induced by ethanol in the rat brain, intimating that it may offer a means of protecting nerve cells from apoptosis induced by exogenous stimuli.     

β-Carotene-induced apoptosis is mediated with loss of Ku proteins in gastric cancer AGS cells

High dietary intakes and high blood levels of β-carotene are associated with a decreased incidence of various cancers. The anticancer effect of β-carotene is related to its pro-oxidant activity. DNA repair Ku proteins, as a heterodimer of Ku70 and Ku80, play a crucial role in DNA double-strand break repair. Reductions in Ku70/80 contribute to apoptosis. Previously, we showed that reactive oxygen species (ROS) activate caspase-3 which induces degradation of Ku proteins. In the present study, we investigated the mechanism of β-carotene-induced apoptosis of gastric cancer AGS cells by determining cell viability, DNA fragmentation, apoptotic indices (increases in cytochrome c and Bax, decrease in Bcl-2), ROS levels, mitochondrial membrane potential, caspase-3 activity, Ku70/80 levels, and Ku-DNA-binding activity of the cells treated with or without antioxidant N-acetyl cysteine and caspase-3 inhibitor z-DEVED-fmk. As a result, β-carotene induced apoptosis (decrease in cell viability, increases in DNA fragmentation and apoptotic indices) and caspase-3 activation, but decreased Ku70/80 levels and Ku-DNA-binding activity. β-Carotene-induced alterations (increase in caspase-3 activity, decrease in Ku proteins) and apoptosis were inhibited by N-acetyl cysteine and z-DEVED-fmk. Increment of intracellular and mitochondrial ROS levels and loss of mitochondrial membrane potential were suppressed by N-acetyl cysteine, but not by z-DEVED-fmk in β-carotene-treated cells. Therefore, β-carotene-induced increases in ROS and caspase-3 activity may lead to reduction of Ku70/80 levels, which results in apoptosis in gastric cancer cells. Loss of Ku proteins might be the underlying mechanism for β-carotene-induced apoptosis in gastric cancer cells.     

Induction of Apoptosis and Antiproliferative Activity of Naringenin in Human Epidermoid Carcinoma Cell through ROS Generation and Cell Cycle Arrest

A natural predominant flavanone naringenin, especially abundant in citrus fruits, has a wide range of pharmacological activities. The search for antiproliferative agents that reduce skin carcinoma is a task of great importance. The objective of this study was to analyze the anti-proliferative and apoptotic mechanism of naringenin using MTT assay, DNA fragmentation, nuclear condensation, change in mitochondrial membrane potential, cell cycle kinetics and caspase-3 as biomarkers and to investigate the ability to induce reactive oxygen species (ROS) initiating apoptotic cascade in human epidermoid carcinoma A431 cells. Results showed that naringenin exposure significantly reduced the cell viability of A431 cells (p<0.01) with a concomitant increase in nuclear condensation and DNA fragmentation in a dose dependent manner. The intracellular ROS generation assay showed statistically significant (p<0.001) dose-related increment in ROS production for naringenin. It also caused naringenin-mediated epidermoid carcinoma apoptosis by inducing mitochondrial depolarization. Cell cycle study showed that naringenin induced cell cycle arrest in G₀/G₁ phase of cell cycle and caspase-3 analysis revealed a dose dependent increment in caspase-3 activity which led to cell apoptosis. This study confirms the efficacy of naringenin that lead to cell death in epidermoid carcinoma cells via inducing ROS generation, mitochondrial depolarization, nuclear condensation, DNA fragmentation, cell cycle arrest in G₀/G₁ phase and caspase-3 activation.     

Ginsenoside Rd Attenuates Myocardial Ischemia/Reperfusion Injury via Akt/GSK-3β Signaling and Inhibition of the Mitochondria-Dependent Apoptotic Pathway

Evidence suggests Ginsenoside Rd (GSRd), a biologically active extract from the medical plant Panax Ginseng, exerts antioxidant effect, decreasing reactive oxygen species (ROS) formation. Current study determined the effect of GSRd on myocardial ischemia/reperfusion (MI/R) injury (a pathological condition where ROS production is significantly increased) and investigated the underlying mechanisms. The current study utilized an in vivo rat model of MI/R injury and an in vitro neonatal rat cardiomyocyte (NRC) model of simulated ischemia/reperfusion (SI/R) injury. Infarct size was measured by Evans blue/TTC double staining. NRC injury was determined by MTT and lactate dehydrogenase (LDH) leakage assay. ROS accumulation and apoptosis were assessed by flow cytometry. Mitochondrial membrane potential (MMP) was determined by 5, 5′, 6, 6′-tetrachloro-1, 1′, 3, 3′-tetrathylbenzimidazol carbocyanine iodide (JC-1). Cytosolic translocation of mitochondrial cytochrome c and expression of caspase-9, caspase-3, Bcl-2 family proteins, and phosphorylated Akt and GSK-3β were determined by western blot. Pretreatment with GSRd (50 mg/kg) significantly augmented rat cardiac function, as evidenced by increased left ventricular ejection fraction (LVEF) and ±dP/dt. GSRd reduced myocardial infarct size, apoptotic cell death, and blood creatine kinase/lactate dehydrogenase levels after MI/R. In NRCs, GSRd (10 µM) inhibited SI/R-induced ROS generation (P<0.01), decreased cellular apoptosis, stabilized the mitochondrial membrane potential (MMP), and attenuated cytosolic translocation of mitochondrial cytochrome c. GSRd inhibited activation of caspase-9 and caspase-3, increased the phosphorylated Akt and GSK-3β, and increased the Bcl-2/Bax ratio. Together, these data demonstrate GSRd mediated cardioprotective effect against MI/R–induced apoptosis via a mitochondrial-dependent apoptotic pathway.     

Cadmium-Induced Injury and the Ameliorative Effects of Selenium on Chicken Splenic Lymphocytes: Mechanisms of Oxidative Stress and Apoptosis

Cadmium (Cd) is an important environmental pollutant present in soil, water, air, and food. Selenium (Se) can antagonize some metal element toxicity including Cd. To investigate the cytotoxicity of Cd and the protective effects of Se on bird immunocytes in vitro, chicken splenic lymphocytes with CdCl₂ (10^?6 mol/L), Na₂SeO₃ (10^?7 mol/L), and the mixture (10^?7 mol/L Na₂SeO₃ and 10^?6 mol/L CdCI₂) were incubated for 12, 24, 36, and 48 h, respectively. A high level of malondialdehyde (MDA) and reactive oxygen species (ROS) productions were observed in Cd treatment group; the activities of catalase (CAT), glutathione peroxidise (GSH-Px), superoxide dismutase (SOD), and the mitochondrial inner transmembrane potential (ΔΨm) were significantly lower in Cd treatment group than those in controls (P?P?mRNA level of Bak, p53, caspase-3, caspase-9, and cytochrome c (Cyt c) and decreased Bcl-2, Bcl-xl, and CaM were observed in Cd treatment group. Se ameliorated ΔΨm and [Ca²⁺]i for mitochondria function restoring, and Se was able to modulate the expression of relative genes. In conclusion, concurrent treatment with Se reduced the Cd-induced morphological changes and oxidative stress, ion disorder, and apoptosis, suggesting that the toxic effects of Cd on the chicken splenic lymphocytes were partly meliorated by Se.     

The Effect of Sodium Fluoride on Cell Apoptosis and the Mechanism of Human Lung BEAS-2B Cells In Vitro

Sodium fluoride (NaF) is a source of fluoride ions used in many applications. Previous studies found that NaF suppressed the proliferation of osteoblast MC3T3 E1 cells and induced the apoptosis of chondrocytes. However, little is known about the effects of NaF on human lung BEAS-2B cells. Therefore, we investigated the mode of cell death induced by NaF and its underlying molecular mechanisms. BEAS-2B cells were treated with NaF at concentrations of 0, 0.25, 0.5, 1.0, 2.0, and 4.0 mmol/L. Cell viability decreased and apoptotic cells significantly increased as concentrations of NaF increased over specific periods of time. The IC₅₀ of NaF was 1.9 and 0.9 mM after 24 and 48 h, respectively. The rates of apoptosis increased from 4.8 to 37.7% after NaF exposure. HE staining, electron microscopy, and single cell gel electrophoresis revealed that morphological changes of apoptosis increased with exposure concentrations. RT-PCR and Western blotting were used to detect the apoptotic pathways. The expressions of bax, caspase-3, caspase-9, p53, and the cytoplasmic CytC of the NaF groups increased, while bcl-2 and mitochondrial CytC decreased compared with that of the control group (P < 0.05). Further, the fluorescence intensities of ROS in the NaF groups were higher than those in the control group, and the membrane potential of mitochondria in the NaF group was significantly lower than that of the control group (P < 0.05). These findings suggested that NaF induced apoptosis in the BEAS-2B cells through mitochondria-mediated signal pathways. Our study provides the theoretical foundation and experimental basis for exploring the mechanisms of human lung epithelial cell damage and cytotoxicity induced by fluorine.     

Functional Consequences for Apoptosis by Transcription Elongation Regulator 1 (TCERG1)-Mediated Bcl-x and Fas/CD95 Alternative Splicing

Here, we present evidence for a specific role of the splicing-related factor TCERG1 in regulating apoptosis in live cells by modulating the alternative splicing of the apoptotic genes Bcl-x and Fas. We show that TCERG1 modulates Bcl-x alternative splicing during apoptosis and its activity in Bcl-x alternative splicing correlates with the induction of apoptosis, as determined by assessing dead cells, sub-G1-phase cells, annexin-V binding, cell viability, and cleavage of caspase-3 and PARP-1. Furthermore, the effect of TCERG1 on apoptosis involved changes in mitochondrial membrane permeabilization. We also found that depletion of TCERG1 reduces the expression of the activated form of the pro-apoptotic mitochondrial membrane protein Bak, which remains inactive by heterodimerizing with Bcl-x_L, preventing the initial step of cytochrome c release in Bak-mediated mitochondrial apoptosis. In addition, we provide evidence that TCERG1 also participates in the death receptor-mediated apoptosis pathway. Interestingly, TCERG1 also modulates Fas/CD95 alternative splicing. We propose that TCERG1 sensitizes a cell to apoptotic agents, thus promoting apoptosis by regulating the alternative splicing of both the Bcl-x and Fas/CD95 genes. Our findings may provide a new link between the control of alternative splicing and the molecular events leading to apoptosis.     

Viability inhibition effect of gambogic acid combined with cisplatin on osteosarcoma cells via mitochondria-independent apoptotic pathway

We previously demonstrated that gambogic acid (GA) is a promising chemotherapeutic compound for human osteosarcoma treatment. The aim of this study was to detect whether the combination of lower-dose GA (0.3 mg/L) and cisplatin (CDDP) (1 mg/L) could perform a synergistic effect on inhibiting tumor in four osteosarcoma cell lines. Our results showed that the combination between GA at lower dose and CDDP significantly exerts a synergistic effect on inhibiting the cellular viability in MG63, HOS, and U2OS cells. In contrast, an antagonistic character was detected in SAOS2 cells exposed to the combined use of lower-dose GA (0.3 mg/L) and CDDP (1 mg/L). Then, analysis of cell cycle showed the combination of both drugs significantly induced the G₂/M phase arrest, without any difference relative to GA treatment alone, in MG63 cells. Flow-cytometric analysis of cell apoptosis displayed that the apoptotic rate in the combination group is higher than that in GA treatment alone in MG63, HOS, and U2OS cells. The combined use of both drugs had no effect on mitochondrial membrane potential, but promoted the apoptosis-inducing function through triggering of CDDP in the three cell lines. By measurement of mitochondrial membrane potential, the activity of caspase-3 and the expressions of caspase-8 and caspase-9, it was showed that the apoptosis-promoting effect of the combined use of both drugs could be dependent on the death receptor apoptosis pathway, not dependent on the mitochondria apoptosis mechanism. This research, for the first time, demonstrates that GA could increase the chemotherapeutic effect of CDDP in human osteosarcoma treatment through inducing the cell cycle arrest and promoting cell apoptosis.     

MitoQ对高糖诱导的心肌细胞线粒体功能影响

目的:探讨MitoQ对高糖诱导的心肌细胞线粒体功能影响。方法:常规获取与纯化SD大鼠新生仔鼠心肌细胞,分为对照组、高糖组、实验组。对照组用含10%血清的DMEM培养基(5.5 mmol/L葡萄糖)培养;高糖组用含血清的高糖DMEM培养基(33mmol/L葡萄糖)培养;实验组用含血清的高糖DMEM培养基(33 mmol/L葡萄糖)和MitoQ。MTT法检测心肌细胞存活率,氯离子荧光探针检测细胞内氯离子浓度,流式细胞术检测各组心肌细胞凋亡率,超氧化物阴离子荧光染色检测心肌细胞活性氧(reactive oxygen,ROS)含量,利用ATP检测试剂盒检测心肌细胞中的ATP水平,Western blot法检测心肌细胞胱天蛋白酶3(caspase-3)蛋白水平。结果:与对照组相比,高糖组的心肌细胞增凋亡率、ROS产生、氯离子相对浓度均明显增加,ATP显著降低(P0.05),细胞内caspase-3蛋白表达显著上升(P0.05);与高糖组相比,实验组凋亡率降低,ROS产生、细胞内caspase-3蛋白表达均显著降低(P0.05)。结论:高糖会引起心肌细胞线粒体障碍,造成心肌细胞凋亡,MitoQ可降低细胞内ROS和caspase-3水平,抑制心肌细胞凋亡,改善心肌细胞线粒体功能。     

Pro-apoptotic complexes of BAX and BAK on the outer mitochondrial membrane

In multicellular organisms the regulated cell death apoptosis is critically important for both ontogeny and homeostasis. Mitochondria are indispensable for stress-induced apoptosis. The BCL-2 protein family controls mitochondrial apoptosis and initiates cell death through the pro-apoptotic activities of BAX and BAK at the outer mitochondrial membrane (OMM). Cellular survival is ensured by the retrotranslocation of mitochondrial BAX and BAK into the cytosol by anti-apoptotic BCL-2 proteins. BAX/BAK-dependent OMM permeabilization releases the mitochondrial cytochrome c (cyt c), which initiates activation of caspase-9. The caspase cascade leads to cell shrinkage, plasma membrane blebbing, chromatin condensation, and apoptotic body formation. Although it is clear that ultimately complexes of active BAX and BAK commit the cell to apoptosis, the nature of these complexes is still enigmatic. Excessive research has described a range of complexes, varying from a few molecules to several 10,000, in different systems. BAX/BAK complexes potentially form ring-like structures that could expose the inner mitochondrial membrane. It has been suggested that these pores allow the efflux of small proteins and even mitochondrial DNA. Here we summarize the current state of knowledge for mitochondrial BAX/BAK complexes and the interactions between these proteins and the membrane.     

Caspase-2 permeabilizes the outer mitochondrial membrane and disrupts the binding of cytochrome c to anionic phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.