蒿甲醚引起线粒体跨膜电位下降在神经毒性中的作用

目的　用嗜铬细胞瘤细胞(PC12细胞)、原代培养的大鼠神经元细胞和大鼠脑线粒体作为体外模型观察线粒体跨膜电位(mitochondrialmembranepotential,MMP)和细胞膜通透性改变在蒿甲醚引起的神经毒性中的作用。　方法　用流式细胞仪测定蒿甲醚对线粒体跨膜电位和细胞膜通透性的影响,用分光光度法分析蒿甲醚对线粒体肿胀度的影响。　结果　蒿甲醚能够降低两种细胞的MMP,使其峰值左移,摄入的Rh123荧光强度降低,量效关系明显;能增加两种细胞的细胞膜通透性,使细胞内的PI摄入增加;能引起大鼠脑线粒体肿胀,有一定的时间效应和剂量效应关系。结论　MMP下降是蒿甲醚引起神经毒性的重要环节,可通过影响线粒体膜通透性转运孔,改变线粒体跨膜电位和线粒体肿胀度,并能增加细胞膜通透性,引起细胞能量代谢障碍,最终导致神经毒性     

Brain cellular and mitochondrial respiration in media of altered pH

This study was designed to investigate the effects of altered pH on cellular aerobic energy metabolism in the immature and adult rat cerebral cortex. Cerebral cortical slice respiration was measured polarographically in acid and alkaline media. In separate experiments, the extracellular pH was changed by altering the HCO ₃ ^– concentration or the intracellular pH and extracellular pH were changed by altering the CO₂. Respiratory rates and oxidative phosphorylation in adult rat cerebral mitochindria also were measured in media with an altered pH. Increased intracellular pH inhibited respiratory rates in cortical slices from immature rats more than in tissue from adults. Decreasing the pH to 6.7 produced no changes in respiration in mature cortical slices and moderate inhibition of immature tissue respiration. In cerebral mitochondria, altered pH caused inhibition of State 3 respiration, respiratory control ratios, and ADP/O ratios. These changes were greater and occurred with smaller pH changes in the alkaline compared to the acid direction. From the results of these studies, we conclude that brain cellular respiration is not affected by moderate decreases in intracellular pH. With increased pH, there is inhibition of cellular and mitochondrial respiration, which may be the mechanism for the rise in lactic acid previously observed to result from hypocarbiain vivo.Abbreviations used BSA bovine serum albumin - DNP dinitrophenol - RCR respiratory control ratio     

Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane

The pivotal step on the mitochondrial pathway to apoptosis is permeabilization of the mitochondrial outer membrane (MOM) by oligomers of the B-cell lymphoma-2 (Bcl-2) family members Bak or Bax. However, how they disrupt MOM integrity is unknown. A longstanding model is that activated Bak and Bax insert two α-helices, α5 and α6, as a hairpin across the MOM, but recent insights on the oligomer structures question this model. We have clarified how these helices contribute to MOM perforation by determining that, in the oligomers, Bak α5 (like Bax α5) remains part of the protein core and that a membrane-impermeable cysteine reagent can label cysteines placed at many positions in α5 and α6 of both Bak and Bax. The results are inconsistent with the hairpin insertion model but support an in-plane model in which α5 and α6 collapse onto the membrane and insert shallowly to drive formation of proteolipidic pores.Commitment of cells to apoptosis is determined primarily by interactions within the B-cell lymphoma-2 (Bcl-2) protein family on the mitochondrial outer membrane (MOM) (1–4). The proapoptotic members Bcl-2 antagonist/killer (Bak) and Bcl-2–associated X protein (Bax) mediate the pivotal step of MOM permeabilization, which releases proteins, such as cytochrome c, that promote the proteolytic demolition by caspases. Two other Bcl-2 subfamilies tightly control Bak and Bax activation. Their activation is promoted by the Bcl-2 homology domain 3 (BH3)-only proteins, such as BH3-interacting domain death agonist (Bid), the truncated form of which (tBid) can directly bind both. Conversely, prosurvival family members can bind and inhibit activated Bak and Bax, as well as the BH3-only proteins.Like their prosurvival relatives, Bak and Bax in healthy cells are globular monomers, comprising similar helical bundles with a hydrophobic α-helix (α5) surrounded by amphipathic helices (5, 6). Their C-terminal helix (α9) is a hydrophobic transmembrane (TM) domain that anchors them in the MOM. In healthy cells Bak is already anchored there, presumably solely by α9, whereas Bax is primarily cytosolic (5), accumulating at the MOM after an apoptotic signal and inserting its α9. Other major conformational changes in both Bak and Bax, reviewed in ref 4, include exposure of their BH3 (α2) and its reburial within the surface groove of another activated Bak or Bax molecule (7–10). These novel “symmetric” homo-dimers can multimerize via association of α6 helices (8, 11, 12).Although oligomeric Bak and Bax are highly implicated in MOM permeabilization, how they interact with the membrane to form pores remains a mystery. The first structure of a Bcl-2 family member, the prosurvival protein Bcl-x_L (13), and later those of Bax (5) and Bak (6), provided a tantalizing clue: similarities with the pore-forming domains of bacterial toxins, such as diphtheria toxin or colicin A. To form pores, these toxins are thought to insert their two hydrophobic core helices as a hairpin across the membrane (14), suggesting that the central helices of Bak and Bax (α5 and α6) might penetrate the MOM similarly (reviewed in refs 3, 15, and 16). Consistent with this hairpin insertion model, α5 and α6 peptides can permeabilize membranes (17–19). More pertinently, Bax α5 and α6 were reported to insert into and span the MOM as a hairpin before oligomerization (20).This longstanding model, however, does not fit well with recent evidence on the structure of Bak and Bax oligomers, as recently reviewed (2, 4). Analysis of Bak oligomers in liposomes by electron paramagnetic resonance (EPR) suggests that α6 inserts only shallowly in the lipid bilayer (21). Additionaly, the first 3D structures of activated forms of Bax (10) suggest that, early in its activation, α5 and α6 separate. Moreover, a Bax core domain containing only helices α2 to α5 generated a BH3:groove symmetric dimer in which two α4 and two α5 helices form an aromatic face that might sit on the bilayer (10). These findings fit better with an “in-plane model” in which only α9 is a TM domain and other helices (including α5 and α6) insert only shallowly into the bilayer.The nature of the apoptotic pores remains uncertain. Some findings favor a proteinaceous pore (22), but studies with model membranes suggest that Bax oligomers can perturb the bilayer and produce lipidic pores (i.e., pores not bounded entirely by protein) (23–26).These important unresolved questions about the pivotal event in apoptosis prompted us to explore the membrane topology of Bak, before, during, and after an apoptotic signal, and to reinvestigate that of Bax. In accord with recent Bax structures (10) and recent EPR studies on Bak (21, 27), the results show that neither oligomeric Bak nor Bax inserts an α5–α6 hairpin across the MOM. We propose instead that the α5 and α6 helices lie in the bilayer plane and disrupt membrane integrity by imposing tension and curvature to the membrane that provoke its permeabilization.     

番茄红素对过氧化氢诱导的小鼠来源神经瘤母细胞损伤的保护作用及机制研究

目的观察番茄红素(LY)对过氧化氢(H2O2)诱导的神经瘤母(N2a)细胞损伤的保护作用及可能机制。方法用H2O2处理小鼠来源N2a细胞6h,建立细胞氧化损伤模型。用LY预孵N2a细胞24h后,加入100μmol/LH2O2共同作用6h,以探讨LY的保护作用。实验分对照组、LY组、损伤组及保护组。采用MTT比色法测定N2a细胞活力;Hochest33258染色观察凋亡细胞形态;流式细胞仪测定N2a细胞凋亡率;Western blot法测定细胞质Bcl-2、Bax及Caspase-3蛋白表达。结果与对照组比较,损伤组明显造成细胞损伤(P<0.01),导致细胞凋亡,同时降低Bcl-2表达,增加Bax表达,Bcl-2/Bax比值显著降低,增加Caspase-3蛋白水平(P<0.01)。与损伤组比较,保护组予LY预处理后,N2a细胞活力明显增加(P<0.01),凋亡细胞减少,Bcl-2/Bax比值增高,Caspase-3蛋白表达降低(P<0.05)。结论 LY能有效保护H2O2对N2a细胞的损伤,并通过提高Bcl-2/Bax比值,抑制Caspase-3蛋白活化,发挥保护作用。     

Platelets of type 2 diabetic patients are characterized by high ATP content and low mitochondrial membrane potential

Platelet dysfunction plays a critical role in vascular complications of type 2 diabetes mellitus (T2DM). But the relationship between platelet hyperactivity and its energy metabolic process remains unclear. This study was designed to explore alterations of platelet mitochondrial ATP production and the possible mechanism. A total of 39 T2DM patients without macrovascular and microvascular complications and 32 normal controls were fasting sampled. Platelet ATP content was measured by a high performance liquid chromatograph (HPLC). The flow cytometry technique was adopted to evaluate mitochondrial membrane potential (ΔΨm), the stored force for platelet ATP production. Consequently, T2DM patients exhibited obvious hyperglycemia, hyperlipidemia and hypertension, but normal platelet morphology. Platelet ATP content was significantly higher in T2DM (0.032 ± 0.010 µmol/10⁹ platelets versus 0.017 ± 0.006 µmol/10⁹ platelets, p < 0.001) than in the control group. Interestingly, ΔΨm was markedly decreased in T2DM patients (0.79 ± 0.18 versus 2.70 ± 1.03, p < 0.001) compared with normal controls. For whole subjects, a stepwise regression showed that plasma glycated hemoglobin A1c (HbA1c) level positively correlated to platelet ATP content (β = 0.552, 95% CI = 0.072–1.451), and fasting plasma glucose (FPG) level was negatively correlated to ΔΨm (β = ?0.372, 95% CI = ?0.471 to ?0.089). These data support that hyperglycemia of T2DM promotes platelet mitochondria to generate more ATP, but decreases platelet mitochondrial potential. The discordance between them requires further researches to elucidate.     

Melatonin protects against MPTP/MPP+ -induced mitochondrial DNA oxidative damage in vivo and in vitro

The effects of melatonin on the mitochondrial DNA (mtDNA) damage induced by 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP) and 1-methyl-4-phenylpyridine ion (MPP(+)) were investigated both in vivo and in vitro. MPTP (24 mg/kg, s.c.) induced a rapid increase in the immunoreactivity of 8-hydroxyguanine (8-oxoG), a common biomarker of DNA oxidative damage, in the cytoplasm of neurons in the Substantia Nigra Compact of mouse brain. Melatonin preinjection (7.5, 15 or 30 mg/kg, i.p.) dose-dependently prevented MPTP-induced DNA oxidative damage. In SH-SY5Y cells, MPP(+) (1 mm) increased the immunoreactivity of 8-oxoG in the mitochondria at 1 hr and in the nucleus at 3 hr after treatment. Melatonin (200 microm) preincubation significantly attenuated MPP(+)-induced mtDNA oxidative damage. Furthermore, MPP(+) time-dependently increased the accumulation of mitochondrial oxygen free radicals (mtOFR) from 1 to 24 hr and gradually decreased the mitochondrial membrane potential (Psim) from 18 to 36 hr after incubation. At 72 hr after incubation, MPP(+) caused cell death in 49% of the control. However, melatonin prevented MPP(+)-induced mtOFR generation and Psim collapse, and later cell death. The present results suggest that cytoprotection of melatonin against MPTP/MPP(+)-induced cell death may be associated with the attenuation of mtDNA oxidative damage via inhibition of mtOFR generation and the prevention of Psim collapse.     

肺泡巨噬细胞凋亡在老年多器官功能衰竭肺启动中作用的研究

目的探讨肺泡巨噬细胞(AM)凋亡在老年多器官功能衰竭(MOF)肺启动中的可能机制。方法选择SD大鼠40只,随机分为油酸+脂多糖组20只和对照组20只。应用油酸-内毒素序贯打击,复制以肺功能障碍为首发的MOF模型,分别在造模6、12、24 h后处死2组大鼠各5只,通过支气管肺泡灌洗和细胞贴壁的方法获取AM,用碘化丙啶染色流式细胞仪测定AM凋亡率。结果造模后6、12、24 h,油酸+脂多糖组大鼠AM的多器官功能障碍综合征发生率、凋亡率和游离钙含量逐渐升高,线粒体膜电位逐渐变小(P<0.01);而对照组各时间点AM的凋亡率、游离钙含量及线粒体膜电位无明显变化(P>0.05)。与对照组比较,油酸+脂多糖组造模后各时间点多器官功能障碍综合征发生率、AM的凋亡率和游离钙含量均明显升高(P<0.01),线粒体膜电位明显降低(P<0.01)。结论 AM凋亡率逐渐增加严重影响了其功能的发挥,可能是MOF发生的重要原因。AM细胞内游离钙含量逐渐升高、线粒体膜电位逐渐变小,是诱导老年大鼠AM凋亡增加的重要机制。     

庚醇预处理对兔缺血再灌注心肌凋亡和线粒体结构与功能的影响

目的探讨庚醇预处理对兔心肌缺血再灌注心肌凋亡和线粒体结构与功能的影响及可能机制。方法大白兔80只,随机分为假手术组、心肌缺血再灌注组(IR组)、缺血预处理组(IP组)、庚醇预处理组(HT组)以及庚醇预处理加5-HD干预组(5-HD组)。采用TUNEL法检测各组缺血心肌细胞凋亡,用透射电镜观察心肌细胞的超微结构改变,同时检测线粒体膜电位、Ca~(2+)、MDA、超氧化物歧化酶(SOD)浓度。结果假手术组心肌线粒体结构正常。与IR组和5-HD组比较,HT组和IP组心肌凋亡指数明显减少,心肌线粒体形态结构改变明显减轻(P0.05);线粒体膜电位明显升高、Ca~(2+)浓度明显下降(P0.05,P0.01)。与IR组比较,IP组SOD浓度明显升高、MDA浓度明显下降(P0.05)。结论庚醇可改善心肌凋亡以及保护心肌线粒体结构,其机制可能与线粒体ATP敏感性钾通道有关。     

Dinitrophenol pretreatment of rat ventricular myocytes protects against damage by metabolic inhibition and reperfusion

We have investigated the protective effects of pretreatment with the mitochondrial uncoupler 2,4-dinitrophenol on the cellular damage induced by metabolic inhibition (with cyanide and iodoacetic acid) and reperfusion in freshly isolated adult rat ventricular myocytes. Damage was assessed from changes in cell length and morphology measured using video microscopy. Intracellular Ca(2+), mitochondrial membrane potential, and NADH were measured using fura-2, tetramethylrhodamine ethyl ester and autofluorescence, respectively. During metabolic inhibition myocytes developed rigor, and on reperfusion 73.6+/-8.1% hypercontracted and 10.8+/-6.7% recovered contractile function in response to electrical stimulation. Intracellular Ca(2+) increased substantially, indicated by a rise in the fura-2 ratio (340/380 nm) on reperfusion from 0.86+/-0.04 to 1.93+/-0.18. Myocytes pretreated with substrate-free Tyrode containing 50 microm dinitrophenol showed reduced reperfusion injury: 29.0+/-7.4% of cells hypercontracted and 65.3+/-7.3% recovered contractile function (P<0.001 vs control). The fura-2 ratio on reperfusion was also lower at 1.01+/-0.08. Fluorescence measurements showed that dinitrophenol caused mitochondrial depolarisation, and decreased NADH. The presence of the substrates glucose and pyruvate reduced these effects, and abolished the protection against damage by metabolic inhibition and reperfusion. However protection was unaffected by block of ATP-sensitive potassium channels. Thus the protective effects of pretreatment with dinitrophenol may result from a reduction in NADH in response to mitochondrial depolarisation.     

Bacterial endotoxin lipopolysaccharide and reactive oxygen species inhibit Leydig cell steroidogenesis via perturbation of mitochondria

Chronic inflammatory disease and acute infection are well known to inhibit gonadal steroidogenesis. Previous studies have demonstrated that immune activation in response to lipopolysaccharide (LPS) results in reductions in serum testosterone, and this is a direct effect on the Leydig cell. We hypothesize that during the early onset of LPS endotoxemia in vivo, testicular macrophages produce reactive oxygen species (ROS) leading to perturbation of Leydig cell mitochondria and an inhibition in steroidogenesis. To investigate the mechanism of LPS inhibition of Leydig cell steroidogenesis, alterations in mitochondria and markers of oxidative stress were assessed in vivo and in Leydig cell primary culture. After a single injection of mice with LPS, serum testosterone was significantly decreased within 2 h. LPS injection of mice resulted in significant reductions in steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydogenase-Δ⁴-Δ⁵ isomerase (3β-HSD) proteins. LPS significantly increased lipid peroxidation of Leydig cell membranes, indicating that LPS results in oxidative damage in vivo. Mitochondria in Leydig cells isolated from LPS-injected mice were disrupted and showed a marked reduction in the mitochondrial membrane potential (Δψ_m). Similar to the effects of LPS, treatment of Leydig cells with hydrogen peroxide acutely inhibited steroidogenesis, reduced StAR and 3β-HSD protein levels, and disrupted Δψ_m. These results suggest that LPS acutely inhibits Leydig cell function by ROS-mediated disruption of Leydig cell mitochondria. Taken together, these results demonstrate the necessity of having respiring mitochondria with an intact Δψ_m to facilitate StAR function and Leydig cell steroidogenesis. The acute effects of LPS demonstrate how sensitive Leydig cell mitochondrial steroidogenesis is to inflammation-induced oxidative stress.     

Different sensitivity of lamina propria T-cell subsets to nitric oxide-induced apoptosis explains immunomodulatory activity of a nitric oxide-releasing derivative of mesalamine in rodent colitis

BACKGROUND & AIMS: Uncontrolled T-cell activation plays a critical role in the pathogenesis of inflammatory bowel diseases. Therefore, pharmacological strategies directed toward restoring the normal responsiveness of the immune system could be effective in the treatment of these pathologic conditions. The addition of a nitric oxide-releasing moiety to conventional drugs, such as aspirin and other anti-inflammatory analgesic drugs, results in new chemical entities with potent immunomodulatory activities. The aim of this study was to investigate the immunomodulatory activity of a nitric oxide-releasing derivative of mesalamine (NCX-456), as compared with standard mesalamine, in 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice. METHODS: Cells and tissues from mice with 2,4,6-trinitrobenzene sulfonic acid-induced colitis and from interleukin 10-deficient mice with spontaneous chronic colitis receiving treatment with several doses of NCX-456 or mesalamine were analyzed for morphology, cytokine production, and apoptosis. RESULTS: NCX-456, but not mesalamine, administration resulted in a marked reduction in clinical, histological, and immunologic signs of colitis in both models. NCX-456 inhibited the release of T-helper type 1-derived cytokines and increased the release of the regulatory T cell-derived cytokines interleukin 10 and transforming growth factor beta. In vitro analyses showed that NCX-456 inhibited proliferation and caused selective apoptosis of the subset of activated lamina propria T-helper type 1 cells, whereas it was ineffective for regulatory T-cell function and survival. CONCLUSIONS: Collectively, these data show that NCX-456 inhibits lamina propria T-helper type 1 function and stimulates the activity of interleukin 10- and transforming growth factor beta-secreting cells, thus restoring mucosal immune homeostasis and suppressing intestinal inflammation.     

Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell–Derived Cardiomyocytes Restore Energetics of Ischemic Myocardium

BackgroundMitochondrial dysfunction results in an imbalance between energy supply and demand in a failing heart. An innovative therapy that targets the intracellular bioenergetics directly through mitochondria transfer may be necessary.ObjectivesThe purpose of this study was to establish a preclinical proof-of-concept that extracellular vesicle (EV)-mediated transfer of autologous mitochondria and their related energy source enhance cardiac function through restoration of myocardial bioenergetics.MethodsHuman-induced pluripotent stem cell–derived cardiomyocytes (iCMs) were employed. iCM-conditioned medium was ultracentrifuged to collect mitochondria-rich EVs (M-EVs). Therapeutic effects of M-EVs were investigated using in vivo murine myocardial infarction (MI) model.ResultsElectron microscopy revealed healthy-shaped mitochondria inside M-EVs. Confocal microscopy showed that M-EV–derived mitochondria were transferred into the recipient iCMs and fused with their endogenous mitochondrial networks. Treatment with 1.0 × 10⁸/ml M-EVs significantly restored the intracellular adenosine triphosphate production and improved contractile profiles of hypoxia-injured iCMs as early as 3 h after treatment. In contrast, isolated mitochondria that contained 300× more mitochondrial proteins than 1.0 × 10⁸/ml M-EVs showed no effect after 24 h. M-EVs contained mitochondrial biogenesis-related messenger ribonucleic acids, including proliferator-activated receptor γ coactivator-1α, which on transfer activated mitochondrial biogenesis in the recipient iCMs at 24 h after treatment. Finally, intramyocardial injection of 1.0 × 10⁸ M-EVs demonstrated significantly improved post-MI cardiac function through restoration of bioenergetics and mitochondrial biogenesis.ConclusionsM-EVs facilitated immediate transfer of their mitochondrial and nonmitochondrial cargos, contributing to improved intracellular energetics in vitro. Intramyocardial injection of M-EVs enhanced post-MI cardiac function in vivo. This therapy can be developed as a novel, precision therapeutic for mitochondria-related diseases including heart failure.