一氧化碳对脂多糖诱导大鼠肺巨噬细胞损伤的影响

摘要： 目的 探讨一氧化碳（CO）对脂多糖（LPS）诱导大鼠肺巨噬细胞损伤的影响及可能机制。方法 用含有10%胎牛血清的细胞培养基, 在 37 ℃、 5%CO2细胞培养孵箱内培养大鼠肺巨噬细胞, 采用随机数字表法将其分为 4组（n=10）： 空白对照组（C 组）、 CO 组、 LPS 组、 LPS+CO 组。CO 组加入体外一氧化碳释放分子-2（CORM-2） 100μmol/L 孵育, LPS 组加入 LPS 10 mg/L, LPS+CO 组加入 CORM-2 100 μmol/L 预处理 1 h 后加入 LPS 10 mg/L 孵育, C组加入等量 PBS 液作为对照。每组细胞处理完毕后继续孵育 24 h, 采用 MTT 法测定细胞活力; 流式细胞仪测定细胞凋亡率和线粒体膜电位; ATP 酶含量试剂盒测定细胞内 ATP 含量; RT-PCR 法测定细胞中线粒体分裂相关蛋白Drp1 的 mRNA 含量; Western blot 法测定 Drp1 的蛋白表达。结果 与 C 组相比, LPS 组和 LPS+CO 组细胞活力、 ATP含量和线粒体膜电位下降, 细胞凋亡率、 线粒体分裂蛋白 Drp1 mRNA 及蛋白表达增加 （P < 0.05）, CO 组上述指标比较差异无统计学意义; 与 LPS 组比较, LPS+CO 组细胞活力、 ATP 含量和线粒体膜电位增加 （P < 0.05）, 细胞凋亡率、Drp1 mRNA 及蛋白表达减少（P < 0.05）。结论 CO 可减轻 LPS 诱导的大鼠肺巨噬细胞损伤, 其机制与下调线粒体分裂相关蛋白 Drp1 和改善线粒体功能有关。     

Effect of trehalose on manganese‐induced mitochondrial dysfunction and neuronal cell damage in mice

Chronic overexposure to manganese (Mn) has been verified to induce mitochondrial dysfunction, which is related to oxidative damage. The autophagic‐lysosomal degradation pathway plays a vital role in the removal of impaired mitochondria through a specific quality control mechanism termed mitophagy. However, trehalose functions as an inducer of autophagy by an mTOR‐independent mechanism, and little data report its effect on Mn‐induced mitochondrial dysfunction. To explore the possibility that trehalose could be effective in interfering with the Mn‐induced mitochondrial dysfunction, we used trehalose (2% and 4% (g/vol (mL))) in a mouse model of manganism. Our data showed that mice developed weary motor and behavioural deficits after exposure to Mn for 6 weeks. Overexposure to Mn resulted in mitochondrial dysfunction and neuronal cell damage in the basal nuclei of mice, which could be ameliorated by trehalose pre‐treatment. Moreover, our results indicated that trehalose pre‐treatment significantly reduced the oxidative damage and enhanced the activation of mitophagy. The findings clearly demonstrated that trehalose could relieve Mn‐induced mitochondrial and neuronal cell damage through its antioxidative and mitophagy‐inducing effects.     

Protection by apple peel polyphenols against indometacin‐induced oxidative stress,mitochondrial damage and cytotoxicity in Caco‐2 cells

Objectives Exposure of Caco‐2 cells to indometacin can be a useful model to assess some of the cytotoxic events that appear to underlie the gastrointestinal lesions associated with the use of this anti‐inflammatory agent. Using such a cellular model, we addressed here the cytoprotective potential of a recently standardized apple peel polyphenol extract, APPE. Methods We firstly characterized APPE in terms of its free radical scavenging and antioxidant properties, and subsequently investigated its potential to protect Caco‐2 cells against the deleterious effects of indometacin on cellular oxidative status (redox state, malondialdehyde, glutathione (GSH) and oxidized glutathione (GSSG) levels), mitochondrial function (ATP and mitochondrial membrane potential) and cell viability (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) leakage). For comparative purposes, the free radical scavenging properties and reducing capacity of quercetin, epicatechin and rutin were also estimated. Key findings In the absence of APPE, indometacin induced mitochondrial perturbations (reducing ATP and the mitochondrial membrane potential), enhanced the oxidative status (decreasing the GSH/GSSG ratio and increasing dichlorofluorescein oxidation and malondialdehyde) and lowered the cell viability (decreasing MTT reduction and increasing LDH leakage). APPE, whether pre‐added or co‐incubated with indometacin, concentration‐dependently prevented these mitochondrial, oxidative and cell viability alterations. Prompted by the recently recognized ability of indometacin to enhance the mitochondrial formation of reactive oxygen species, APPE was also characterized in terms of its free radical‐scavenging capacity. APPE was found to actively scavenge O₂·^‐, HO· and peroxyl radicals. Such free radical‐scavenging activity of APPE suggests that its ability to protect mitochondria and prevent the oxidative and lytic damage induced by indometacin arises from its potent antioxidant capacity. Conclusions In Caco‐2 cells APPE prevented mitochondrial oxidative and cell viability alterations induced by indometacin possibly through its ability to scavenge reactive oxygen species. These findings are of interest in view of the high prevalence of gastrointestinal side‐effects associated with the use of conventional anti‐inflammatory agents.     

Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes

Context: The serious side effect of Adriamycin (ADR) is cardiomyopathy. Cryptotanshinone (CRY) is widely and safely used as antioxidant with MTD more than 5?mg/g in rats (p.o).

Objective: The objective of this study is to study the protection effects of CRY against ADR-induced mitochondrial dysfunction in cardiomyocytes.

Materials and methods: The chemical administration lasted for 20 days with an effective dose of CRY (p.o.) at 50 mg/kg in rats. Mitochondrial respiratory chain complex activities, ATP generation, mitochondrial membrane potential (MMP), superoxide anion free radical, oxidative stress-relative enzymes, and mitochondrial biogenesis-relative factors in normal control, ADR (i.p., 1.25?mg/kg), and ADR (i.p., 1.25?mg/kg)?+?CYP (p.o., 50?mg/kg) groups were detected.

Results: 50?mg/kg CRY significantly promoted the energy production of ATP (16.99?±?2.38?nmol/g Pro) (Pro: Protein) by increasing the complexes activities except II (p?>?0.05). After the treatment of CRY, the suppressed MMP was increased while superoxide anion free radical (0.57?±?0.07/mg Pro) was inhibited markedly. Mitochondrial biogenesis-relative factors PGC-1α, NRF-1, and TFAM were also promoted. Remarkable augmentations of NO, inducible nitric oxide synthase (iNOS), and increased activity of GSH-PX (p?<?0.05) were also detected after the treatment of CRY, while no obvious changes on the activity of nitric oxide synthase (cNOS; p?>?0.05) were observed.

Discussion and conclusion: These results suggest that CRY protects against ADR-induced mitochondrial dysfunction in cardiomyocytes. It could be an ideal potential drug of cardioprotection.     

NF‐κB/p53‐activated inflammatory response involves in diquat‐induced mitochondrial dysfunction and apoptosis

Inflammation generated by environmental toxicants including pesticides could be one of the factors underlying neuronal cell damage in neurodegenerative diseases. In this study, we investigated the mechanisms by which inflammatory responses contribute to apoptosis in PC12 cells treated with diquat. We found that diquat induced apoptosis, as demonstrated by the activation of caspases and nuclear condensation, inhibition of mitochondrial complex I activity, and decreased ATP level in PC12 cells. Diquat also reduced the dopamine level, indicating that cell death induced by diquat is due to cytotoxicity of dopaminergic neuronal components in these cells. Exposure of PC12 cells to diquat led to the production of reactive oxygen species (ROS), and the antioxidant N‐acetyl‐cystein attenuated the cytotoxicity of caspase‐3 pathways. These results demonstrate that diquat‐induced apoptosis is involved in mitochondrial dysfunction through production of ROS. Furthermore, diquat increased expression of cyclooxygenase‐2 (COX‐2) and tumor necrosis factor‐α (TNF‐α) via inflammatory stimulation. Diquat induced nuclear accumulation of NF‐κB and p53 proteins. Importantly, an inhibitor of NF‐κB nuclear translocation blocked the increase of p53. Both NF‐κB and p53 inhibitors also blocked the diquat‐induced inflammatory response. Pretreatment of cells with meloxicam, a COX‐2 inhibitor, also blocked apoptosis and mitochondrial dysfunction. These results represent a unique molecular characterization of diquat‐induced cytotoxicity in PC12 cells. Our results demonstrate that diquat induces cell damage in part through inflammatory responses via NF‐κB‐mediated p53 signaling. This suggests the potential to generate mitochondrial damage via inflammatory responses and inflammatory stimulation‐related neurodegenerative disease.     

Curcumin induced HepG2 cell apoptosis-associated mitochondrial membrane potential and intracellular free Ca concentration

Curcumin is a phytochemicals which is able to inhibit carcinogenesis in a variety of cell lines. However little is known about its effect on the cell-surface and the interaction between cell-surface and the reacting drug. In this study, we found that curcumin could inhibit the growth of human hepatocellular carcinoma cell line (HepG2), change the cell-surface morphology and trigger the pro-apoptotic factor to promote cell apoptosis. Cell counting kit results indicated that the cell viability had a dose-dependent relationship with the curcumin concentration in 24 h. The 50% inhibiting concentration (IC50) was 17.5 ± 3.2 μM. It was clear that curcumin could lead to apoptosis, and the apoptosis increased as the reacting concentration goes up. Moreover, curcumin could also affect the disruption of mitochondrial membrane potential and the disturbance of intracellular free Ca²⁺ concentration. All these alterations changed the cell morphology and cell-surface ultrastructure with atomic force microscopy (AFM) detecting at nanoscale level. AFM results indicated that cells in control group clearly revealed a typical long spindle-shaped morphology. Cell tails was wide and unrolled. The ultrastructure showed that cell membrane was made up of many nanoparticles. After being treated with curcumin, cell tail was narrowed. The size of membrane nanoparticles became small. These results can improve our understanding of curcumin which can be potentially developed as a new agent for treatment of hepatocellular carcinoma since it has been reported to have a low cytotoxic effect on healthy cell. AFM can be used as a powerful tool for detecting ultrastructures.     

线粒体通透性转换在丁烯酸内酯细胞毒性中的作用

目的研究线粒体通透性转换在丁烯酸内酯(BUT)细胞毒性中的作用。方法HepG2细胞染毒BUT后,采用若丹明123荧光法测定线粒体膜电位(ΔΨm)的变化;线粒体膜通透性转换孔(MPTP)抑制剂环孢素A(CsA)预处理细胞后,采用MTT法测定线粒体膜通透性转换在BUT细胞毒性中的作用。结果BUT能够浓度依赖性地降低ΔΨm,而巯基化合物谷胱甘肽、N-乙酰半胱氨酸和二硫苏糖醇则能明显抑制该效应。CsA预处理能够明显减轻BUT的细胞毒性。结论BUT通过诱导HepG2细胞内氧化应激,导致MPTP的开放而最终引起细胞死亡。     

Hepatoprotective and antioxidant effects of gallic acid in paracetamol‐induced liver damage in mice

Objectives The aim of this research paper was to investigate the hepatoprotective and antioxidant effects of gallic acid in paracetamol‐induced liver damage in mice. Methods In the present study, the hepatoprotective and antioxidant effects of gallic acid were evaluated against paracetamol‐induced hepatotoxicity in mice and compared with the silymarin, a standard hepatoprotective drug. The mice received a single dose of paracetamol (900 mg/kg body weight i.p.). Gallic acid (100 mg/kg body weight i.p.) and silymarin (25 mg/kg body weight i.p.) were administered 30 min after the injection of paracetamol. After 4 h, liver marker enzymes (aspartate transaminase, alanine transaminase and alkaline phosphatase) and inflammatory mediator tumour necrosis factor‐alpha (TNF‐α) were estimated in serum, while the lipid peroxidation and antioxidant status (superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione‐S‐transferase and glutathione) were determined in liver homogenate of the control and experimental mice. Key findings Increased activities of liver marker enzymes and elevated TNF‐α and lipid peroxidation levels were observed in mice exposed to paracetamol (P < 0.05), whereas the antioxidant status was found to be depleted (P < 0.05) when compared with the control group. However gallic acid treatment (100 mg/kg body weight i.p.) significantly reverses (P < 0.05) the above changes by its antioxidant action compared to the control group as observed in the paracetamol‐challenged mice. Conclusions The results clearly demonstrate that gallic acid possesses promising hepatoprotective effects.     

Potential mechanisms of mitochondrial cytochrome‐C release during apoptosis

Cytochrome‐c release from the intermembrane space of mitochondria to the cytoplasm is a critical signaling event during many forms of apoptotic cell death. Due to the widespread involvement of apoptosis in pathobiology, this mitochondrial event is a logical target for the development of new therapeutic approaches to numerous diseases. Currently impeding such a development is our lack of understanding of the mechanism by which release of this component of the electron transport chain occurs in response to the wide variety of agents known to induce apoptosis. Release induced by the membrane permeability transition (a permeability change at the inner mitochondrial membrane) likely results from mitochondrial swelling, consequent rupture of the outer mitochondrial membrane, and nonspecific release of intermembrane space contents. An alternative mode of release may include the induction of a specific permeability change at the outer mitochondrial membrane. These two mechanisms present distinct molecular targets for drug development. Drug Dev. Res. 46:18–25, 1999. © 1999 Wiley‐Liss, Inc.     

Involvement of cytotoxicity and variation of the mitochondrial membrane potential induced by hybrid agent

The pyrrolo[2,1‐c][1,4]benzodiazepine (PBD) derivative DC‐81 is a potent antitumor antibiotic produced by Streptomyces species. The marked cytotoxic potential of this drug may be the result of its interaction with DNA. Because DC‐81 only recognizes three DNA base pairs this has precluded its clinical utility. Combination of DC‐81 with an indole carboxylate moiety was a hybrid designed to have much higher sequence selectivity in DNA Interactivity. In this paper, the association between cytotoxicity and the changes of mitochondria membrane potential ΔΨ_mt after exposing human melanoma cell line A375 to the hybrid agent was examined using MTS cell proliferation assay and flow cytometry using the fluorochrome rhodamine 123. Our results indicated that the hybrid induced cytotoxity and a significant reduction in ΔΨ_mt of A375 cells. We suggest that the hybrid agent is a potent inducer of cell apoptosis in A375 cells. Drug Dev. Res. 61: 1–5, 2004. © 2004 Wiley‐Liss, Inc.     

Hydroxyapatite nanoparticle‐induced mitochondrial energy metabolism impairment in liver cells: in vitro and in vivo studies

Hydroxyapatite nanoparticles (HAP‐NPs) have been extensively developed as drug carriers, bone implants, coating materials, etc. in the human body. However, research focusing on the potential side effects of HAP‐NPs on the mitochondria‐associated energy metabolism in liver cells is lacking. In this study, HAP‐NPs with a long diameter of 80 nm and a short diameter of 20 nm were evaluated for their ability to induce mitochondrial energy metabolism dysfunction in vitro and in vivo . In the in vitro system, the buffalo rat hepatocyte (BRL) cell line was directly exposed to the HAP‐NPs. The results of these experiments showed that the HAP‐NPs induced inhibition of mitochondrial dehydrogenase activity, which was accompanied by a decrease in the mitochondrial membrane potential (MMP). In addition, HAP‐NPs elevated the hepatic levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and decreased the levels of GSH and SOD. These data indicated that HAP‐NPs induced a lowered rate of electron transfer in the mitochondrial respiratory chain, accompanied by a decrease in the activity of the mitochondrial respiratory chain complexes I, II and III. Furthermore, HAP‐NPs induced a decline in the enzymatic expression in the Krebs cycle. We also investigated the role of Kupffer cells (KCs, rat‐derived) in the effects induced by the HAP‐NPs. The supernatant from the HAP‐NP‐treated KCs was used to stimulate the BRL cells. We observed that the HAP‐NPs had the ability to induce KC activation. The activation of KCs then led to the release of tumor necrosis factor‐α (TNF‐α), nitric oxide (NO) and reactive oxygen species (ROS), and induced the inhibition of mitochondrial respiratory chain complexes I, II and III in the BRL cells. In the in vivo study, the TEM examination revealed mitochondrial swelling and vacuolar degeneration in the HAP‐NP‐treated hepatocytes. In addition, the amount of succinate (Suc), an intermediate in the mitochondrial Krebs cycle, also declined in the ¹H NMR spectroscopic measurements. Copyright © 2017 John Wiley & Sons, Ltd.     

Mitochondrial and liver oxidative stress alterations induced by N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine: relevance for hepatotoxicity

The most significant toxicological effect of nitrosamines like N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) is their carcinogenic activity, which may result from exposure to a single large dose or from chronic exposure to relatively small doses. However, its effects on mitochondrial liver bioenergetics were never investigated. Liver is the principal organ responsible for BBN metabolic activation, and mitochondria have a central function in cellular energy production, participating in multiple metabolic pathways. Therefore any negative effect on mitochondrial function may affect cell viability. In the present work, ICR male mice were given 0.05% of BBN in drinking water for a period of 12 weeks and were sacrificed one week later. Mitochondrial physiology was characterized in BBN‐ and control‐treated mice. Transmembrane electric potential developed by mitochondria was significantly affected when pyruvate–malate was used, with an increase in state 4 respiration observed for pyruvate–malate (46%) and succinate (38%). A decrease in the contents of one subunit of mitochondrial complex I and in one subunit of mitochondrial complex IV was also observed. In addition, the activity of both complexes I and II was also decreased by BBN treatment. The treatment with BBN increases the susceptibility of liver mitochondria to the opening of the mitochondrial permeability transition pore. This susceptibility could be related with the increase in the production of H₂O₂ by mitochondria and increased oxidative stress confirmed by augmented susceptibility to lipid peroxidation. These results lead to the conclusion that hepatic mitochondria are one primary target for BBN toxic action during liver metabolism. Copyright © 2011 John Wiley & Sons, Ltd.     

Mitochondrial oxidative stress and dysfunction in arsenic neurotoxicity: A review

Arsenic is a toxic metalloid present ubiquitously on earth. Since the last decade, it has gained considerable attention due to its severe neurotoxic effects. Arsenic can cross the blood–brain barrier and accumulate in different regions of the brain suggesting its role in neurological diseases. Arsenic exposure has been associated with reactive oxygen species generation, which is supposed to be one of the mechanisms of arsenic‐induced oxidative stress. Mitochondria, being the major source of reactive oxygen species generation may present an important target of arsenic toxicity. It is speculated that the proper functioning of the brain depends largely on efficient mitochondrial functions. Multiple studies have reported evidence of brain mitochondrial impairment after arsenic exposure. In this review, we have evaluated the proposed mechanisms of arsenic‐induced mitochondrial oxidative stress and dysfunction. The understanding of molecular mechanism of mitochondrial dysfunction may be helpful to develop therapeutic strategies against arsenic‐induced neurotoxicity. The ameliorative measures undertaken in arsenic‐induced mitochondrial dysfunction have also been highlighted. Copyright © 2015 John Wiley & Sons, Ltd.     

N‐acetylcysteine alleviated paraquat‐induced mitochondrial fragmentation and autophagy in primary murine neural progenitor cells

The developing brain is uniquely vulnerable to toxic chemical exposures. Studies indicate that neural stem cell (NSC) self‐renewal is susceptible to oxidative stress caused by xenobiotics. However, the impact of antioxidants on NSC self‐renewal and the potential mechanisms remain elusive. In this study, primary murine neural progenitor cells (mNPCs) from the subventricular zone were used as a research model. In addition, paraquat (PQ) was used to elicit oxidative stress and N‐acetylcysteine (NAC) was used as a powerful antioxidant. mNPCs were treated with 80 μm PQ for 24 hours with or without 4 hours of NAC pretreatment. Our results showed that PQ treatment increased intracellular reactive oxygen species production, decreased cell viability and DNA synthesis, and promoted cell apoptosis. Meanwhile, pretreatment with NAC alleviated PQ‐induced cytotoxicity in mNPCs. To elucidate the mechanisms further, we found that NAC pretreatment prevented PQ‐induced reactive oxygen species production, mitochondrial fragmentation and autophagy in mNPCs. NAC‐pretreated cells showed increased anti‐apoptotic protein Bcl‐2 and decreased pro‐apoptotic protein Bax expression. Similarly, NAC pretreatment increased p‐mTOR and decreased LC3B‐II protein expression. Moreover, NAC decreased mitophagy related mRNA Pink1 and Parkin expression. Taken together, our results suggested that the antioxidant NAC treatment significantly attenuated PQ‐induced mNPC self‐renewal disruption through decreasing autophagy and salvaging mitochondrial morphology. These findings revealed a potential mechanism for neurological treatment relating to antioxidant and suggested potentially relevant implications for PQ‐related neurodegenerative disorders. Thus, our study also provided insight into therapeutic strategies for the neurotoxic effects of oxidative stress‐associated toxicants.