共查询到20条相似文献,搜索用时 31 毫秒
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《Free radical research》2013,47(6):766-776
AbstractOxidative stress-induced cell damage is involved in many neurological diseases. Homer protein, as an important scaffold protein at postsynaptic density, regulates synaptic structure and function. Here, we reported that hydrogen peroxide (H2O2) induced the expression of Homer 1a. Down-regulation of Homer 1a with a specific small interfering RNA (siRNA) exacerbated H2O2-induced cell injury. Up-regulation of Homer 1a by lentivirus transfection did not affect the anti-oxidant activity, but significantly reduced the reactive oxygen species (ROS) production and lipid peroxidation after H2O2-induced oxidative stress. Overexpression of Homer 1a attenuated the loss of mitochondrial membrane potential (MMP) and ATP production induced by H2O2, and subsequently inhibited mitochondrial dysfunction-induced cytochrome c release, increase of Bax/Bcl-2 ratio and caspase-9/caspase-3 activity. Furthermore, in the presence of BAPTA-AM, an intracellular free-calcium (Ca2 +) chelator, overexpression of Homer 1a had no significant effects on H2O2-induced oxidative stress. These results suggest that Homer 1a has protective effects against H2O2-induced oxidative stress by reducing ROS accumulation and activation of mitochondrial apoptotic pathway, and these protective effects are dependent on the regulation of intracellular Ca2 + homeostasis. 相似文献
3.
Several neurodegenerative diseases and brain injury involve reactive oxygen species and implicate oxidative stress in disease mechanisms. Hydrogen peroxide (H2O2) formation due to mitochondrial superoxide leakage perpetuates oxidative stress in neuronal injury. Catalase, an H2O2-degrading enzyme, thus remains an important antioxidant therapy target. However, catalase therapy is restricted by its labile nature and inadequate delivery. Here, a nanotechnology approach was evaluated using catalase-loaded, poly(lactic co-glycolic acid) nanoparticles (NPs) in human neuronal protection against oxidative damage. This study showed highly efficient catalase encapsulation capable of retaining∼99% enzymatic activity. NPs released catalase rapidly, and antioxidant activity was sustained for over a month. NP uptake in human neurons was rapid and nontoxic. Although human neurons were highly sensitive to H2O2, NP-mediated catalase delivery successfully protected cultured neurons from H2O2-induced oxidative stress. Catalase-loaded NPs significantly reduced H2O2-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening and loss of cell membrane integrity and restored neuronal morphology, neurite network and microtubule-associated protein-2 levels. Further, catalase-loaded NPs improved neuronal recovery from H2O2 pre-exposure better than free catalase, suggesting possible applications in ameliorating stroke-relevant oxidative stress. Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders. 相似文献
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Agmatine, at concentrations of 10 μM or 100 μM, is able to induce oxidative stress in rat liver mitochondria (RLM), as evidenced by increased oxygen uptake, H2O2 generation, and oxidation of sulfhydryl groups and glutathione. One proposal for the production of H2O2 and, most probably, other reactive oxygen species (ROS), is that they are the reaction products of agmatine oxidation by an unknown mitochondrial amine oxidase. Alternatively, by interacting with an iron-sulfur center of the respiratory chain, agmatine can produce an imino radical and subsequently the superoxide anion and other ROS. The observed oxidative stress causes a drop in ATP synthesis and amplification of the mitochondrial permeability transition (MPT) induced by Ca2+. Instead, 1 mM agmatine generates larger amounts of H2O2 than the lower concentrations, but does not affect RLM respiration or redox levels of thiols and glutathione. Indeed, it maintains the normal level of ATP synthesis and prevents Ca2+-induced MPT in the presence of phosphate. The self-scavenging effect against ROS production by agmatine at higher concentrations is also proposed. 相似文献
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Weiliang He Lili Cui Cong Zhang Xiangjian Zhang Junna He Yanzhao Xie 《Neurochemical research》2016,41(4):687-695
Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression. 相似文献
6.
Effect of tumor suppressor PTEN gene on apoptosis and cell cycle of human airway smooth muscle cells
Luo Liang Gong Yuan Qi Qi XieFei Lai WenYan Lan Haibing Luo Yaling 《Molecular and cellular biochemistry》2013,373(1-2):1-9
Tumor necrosis factor-alpha (TNFα) plays a crucial role in inflammatory diseases such as rheumatoid arthritis and postmenopausal osteoporosis. Recently, it has been demonstrated that hydrogen gas, known as a novel antioxidant, can exert therapeutic anti-inflammatory effect in many diseases. In this study, we investigated the effect of treatment with hydrogen molecule (H2) on TNFα-induced cell injury in osteoblast. The osteoblasts isolated from neonatal rat calvariae were cultured. It was found that TNFα suppressed cell viability, induced cell apoptosis, suppressed Runx2 mRNA expression, and inhibited alkaline phosphatase activity, which was reversed by co-incubation with H2. Incubation with TNFα-enhanced intracellular reactive oxygen species (ROS) formation and malondialdehyde production increased NADPH oxidase activity, impaired mitochondrial function marked by increased mitochondrial ROS formation and decreased mitochondrial membrane potential and ATP synthesis, and suppressed activities of antioxidant enzymes including SOD and catalase, which were restored by co-incubation with H2. Treatment with H2 inhibited TNFα-induced activation of NFκB pathway. In addition, treatment with H2 inhibited TNFα-induced nitric oxide (NO) formation through inhibiting iNOS activity. Treatment with H2 inhibited TNFα-induced IL-6 and ICAM-1 mRNA expression. In conclusion, treatment with H2 alleviates TNFα-induced cell injury in osteoblast through abating oxidative stress, preserving mitochondrial function, suppressing inflammation, and enhancing NO bioavailability. 相似文献
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Ruo-Lian Dai Sheng-Yin Zhu Yuan-Peng Xia Ling Mao Yuan-Wu Mei Yu-Fang Yao Yu-Mei Xue Bo Hu 《Neurochemical research》2011,36(1):67-75
Oxidative stress is one of the most important pathological mechanisms in neurodegenerative diseases and ischemia. Recent studies
have indicated that the sonic hedgehog (SHH) signaling pathway is involved in these diseases, but the underlying mechanisms
remains elusive. Here we report that the SHH pathway was activated in primary cultured cortical neurons after exposure to
hydrogen peroxide (H2O2). H2O2 treatment decreased the cell viability of neurons, and inhibition of endogenous SHH signaling exacerbated its neurotoxicity.
Activation of SHH signaling protected neurons from H2O2-induced apoptosis and increased the cell viability while those effects were partially reversed by blocking SHH signals. Exogenous
SHH increased the activities of Superoxide dismutase (SOD) and Glutathione peroxidase (GSH-PX) in H2O2-treated neurons and decreased production of Malondialdehyde (MDA). It also promoted expression of the anti-apoptotic gene
Bcl-2 and inhibited expression of pro-apoptotic gene Bax. Activation of SHH signals upregulated both Neurotrophic factors
vascular endothelial growth factor (VEGF) and brain-derived neurotrophic factor (BDNF). Pretreatment with SHH inhibited the
activation of ERK (extracellular signal-regulated kinases) signals induced by H2O2. Our findings demonstrate that activation of SHH signaling protects cortical neurons against oxidative stress and suggest
a potential role of SHH for the clinic treatments of brain ischemia and neurodegenerative disorders. 相似文献
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Jia-Yong Tang Ai-Hua He Gang Jia Guang-Mang Liu Xiao-Ling Chen Jing-Yi Cai Hai-Ying Shang Jin-Qiu Liao Hua Zhao 《Biological trace element research》2018,181(1):44-53
Oxidative stress, as mediated by ROS (reactive oxygen species), is a significant factor in initiating the cells damaged by affecting cellular macromolecules and impairing their biological functions; SelX, a selenoprotein also known as MsrB1 belonging to the methionine sulfoxide reductase (Msr) family, is the redox repairing enzyme and involved in redox-related functions. In order to more precisely analyze the relationship between oxidative stress, cell oxidative damage, and SelX, we stably overexpressed porcine Selx full-length cDNA in human normal hepatocyte (LO2) cells. Cell viability, cell apoptosis rate, intracellular ROS, and the expression levels of mRNA or protein of apoptosis-related genes under H2O2-induced oxidative stress were detected. We found that overexpression of SelX can prevent the oxidative damage caused by H2O2 and propose that the main mechanism underlying the protective effects of SelX is the inhibition of LO2 cell apoptosis. The results revealed that overexpressed SelX reduced the H2O2-induced intracellular ROS generation, inhibited the H2O2-induced upregulation of Bax and downregulation of Bcl-2, and increased the mRNA and protein ratio of Bcl-2/Bax. Furthermore, it inhibited H2O2-induced p38 MAPK phosphorylation. Taken together, our findings suggested that SelX played important roles in protecting LO2 cells against oxidative damage and that its protective effect is partly via the p38 pathway by acting as a ROS scavenger. 相似文献
10.
Hydrogen peroxide (H2O2) overload may contribute to cardiac ischemia-reperfusion injury. We report utilization of a previously described cardiomyocyte model (J. Cell. Physiol., 149:347, 1991) to assess the effect of H2O2-induced oxidative stress on heart-muscle purine and pyrimidine nucleotides and high-energy phosphates (ATP, phosphocreatine). Oxidative stress induced by bolus H2O2 elicited the loss of cardiomyocyte purine and pyrimidine nucleotides, leading to eventual de-energization upon total ATP and phosphocreatine depletion. The rate and extent of ATP and phosphocreatine loss were dependent on the degree of oxidative stress within the range of 50 μM to 1.0 mM H2O2. At the highest H2O2 concentration, 5 min was sufficient to elicit appreciable cardiomyocyte highenergy phosphate loss, the extent of which could be limited by prompt elimination of H2O2 from the culture medium. Only H2O2 dismutation completely prevented ATP loss during H2O2-induced oxidative stress, whereas various freeradical scavengers and metal chelators afforded no significant ATP preservation. Exogenously-supplied catabolic substrates and glycolytic or tricarboxylic acidcycle intermediates did not ameliorate the observed ATP and phosphocreatine depletion, suggesting that cardiomyocyte de-energization during H2O2-induced oxidative stress reflected defects in substrate utilization/energy conservation. Compromise of cardiomyocyte nucleotide and phosphocreatine pools during H2O2-induced oxidative stress was completely dissociated from membrane peroxidative damage and maintenance of cell integrity. Cardiomyocyte de-energization in response to H2O2 overload may constitute a distinct nonperoxidative mode of injury by which cardiomyocyte energy balance could be chronically compromised in the post-ischemic heart. © 1993 Wiley-Liss, Inc. 相似文献
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We investigated the effects of H2O2-induced oxidative stress on the delayed-rectifier current (IKDR), neuronal physiological and morphological properties. Measurements were obtained from hippocampal CA1 neurons in control
solution and from the same neurons after exposure to oxidative stress (short- and long-term H2O2 external applications at 0.1, 1, and 10 mM). With short-term (6 min) H2O2 (1 mM) treatment, IKDR measured in the H2O2-containing solution (778 ± 23 pA, n = 20), was smaller than that measured in the control Ca2+-free Hepes solution (1,112 ± 38 pA, n = 20). Coenzyme Q10 (0.1 mM) pretreatment prevented the H2O2-induced inhibition of IKDR. With long-term (40, 80 min) H2O2 (0.1, 10 mM) treatment, the neuron lost its distinctive shape (rounded up) and the neurite almost disappeared. These results
suggest that oxidative stress, which inhibits IKDR, can alter neural activity. The morphological changes caused by H2O2 support the idea that oxidative stress causes intracellular damage and compromises neural function. 相似文献
12.
The present study was designed to investigate ex vivo the protective mechanisms of heat-shock response against H2O2-induced oxidative stress in peripheral blood mononuclear cells (PBMCs) of rats. Twenty-four hours later, heat-shock treatment
was executed in vivo; rat PBMCs were collected and treated with H2O2. The accumulation of reactive oxygen species and the mitochondrial membrane potential were evaluated by intracellular fluorescent
dHE and JC-1 dye staining, respectively, and expression of HSP72 and cytochrome c was detected by Western blot analysis. Cellular apoptosis was assayed by TUNEL staining and double staining of Annexin V
and PI. The results showed that H2O2-induced oxidative stress leads to intracellular superoxide accumulation and collapse of the mitochondrial membrane potential
in rat PBMCs. Moreover, cellular apoptosis was detected after H2O2 treatment, and the release of mitochondrial cytochrome c from mitochondria to cytosol was significantly enhanced. Heat-shock pretreatment decreases the accumulation of intracellular
superoxide in PBMCs during H2O2-induced oxidative stress. Moreover, heat-shock treatment prevents the collapse of the mitochondrial membrane potential and
cytochrome c release from mitochondria during H2O2-induced oxidative stress. In conclusion, mitochondria are critical organelles of the protective effects of heat-shock treatment.
Cellular apoptosis during H2O2-induced oxidative stress is decreased by heat-shock treatment through a decrease in superoxide induction and preservation
of the mitochondrial membrane potential. 相似文献
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《Journal of receptor and signal transduction research》2013,33(3):212-220
AbstractExogenous hydrogen peroxide (H2O2) can easily penetrate into biological membranes and enhance the formation of other reactive oxygen species (ROS). In the present study, we have investigated the neuroprotective effects of insulin on H2O2-induced toxicity of retinoic acid (RA)-differentiated SH-SY5Y cells. To measure the changes in the cell viability of SH-SY5Y cells at different concentrations of H2O2 for 24?h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT)-based assay was used and a 100?µM H2O2 was selected to establish a model of H2O2-induced oxidative stress. Further assays showed that 24?h of 100?µM H2O2-induced significant changes in the levels of lactate dehydrogenase (LDH), nitric oxide (NO), ROS, and calcium ion (Ca2+) in neuronal cells, but insulin can effectively diminish the H2O2-induced oxidative damages to these cells. Moreover, cells treated with insulin increased H2O2-induced suppression of glutathione levels and exerted an apparent suppressive effect on oxidative products. The results of insulin treatment with SH-SY5Y cells increased the Bcl-2 levels and decreased the Akt levels. The treatment of insulin had played a protective effect on H2O2-induced oxidative stress related to the Akt/Bcl-2 pathways. 相似文献
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Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H2O2-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H2O2-induced Leydig cells. Apoptosis was significant decreased in H2O2-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H2O2 in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H2O2. These data showed that DHEA could ameliorate H2O2-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA. 相似文献
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The purpose of our study was to investigate underlying basic mechanisms of hypothermia-induced cardioprotection during oxidative stress in a cardiomyocyte cell culture model. For hypothermic treatment we cooled H9c2 cardiomyocytes to 20 °C, maintained 20 min at 20 °C during which short-term oxidative damage was inflicted with 2 mM H2O2, followed by rewarming to 37 °C. Later on, we analyzed lactate dehydrogenase (LDH), caspase-3 cleavage, reactive oxygen species (ROS), mitochondrial activity, intracellular ATP production, cytoprotective signal molecules as well as DNA damage. Hypothermia decreased H2O2 damage in cardiomyocytes as demonstrated in a lower LDH release, less caspase-3 cleavage and less M30 CytoDeath staining. After rewarming H2O2 damaged cells demonstrated a significantly higher reduction rate of intracellular ROS compared to normothermic H2O2 damaged cardiomyocytes. This was in line with a significantly greater mitochondrial dehydrogenase activity and higher intracellular ATP content in cooled and rewarmed cells. Moreover, hypothermia preserved cell viability by up-regulation of the anti-apoptotic protein Bcl-2 and a reduction of p53 phosphorylation. DNA damage, proven by PARP-1 cleavage and H2AX phosphorylation, was significantly reduced by hypothermia. In conclusion, we could demonstrate that hypothermia protects cardiomyocytes during oxidative stress by preventing apoptosis via inhibiting mitochondrial dysfunction and DNA damage. 相似文献
16.
Guohua Gong Yuan Qin Wen Huang Shu Zhou Xiaohua Wu Xiaohua Yang Yinlan Zhao Dan Li 《Chemico-biological interactions》2010,184(3):366-375
Hyperlipidemia is a major cause of atherosclerosis and atherosclerosis-associated conditions in cardiovascular diseases. Oxidative stress, as a main risk factor causes vascular endothelial cell apoptosis, which is implicated in the pathogenesis of cardiovascular disorders. Diosgenin, an aglycone of steroidal saponins, has been reported to exert anti-proliferative and proapoptotic actions on cancer cells widely. In this study, we propose that diosgenin can protect the hyperlipidemic rats and prevent endothelial apoptosis under oxidative stress. We investigated the hypolipidemic and antioxidative effects of diosgenin on rats fed with high cholesterol and high fat diet for 6 weeks. Serum total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C), glutathione peroxidase (GSH-PX), nitric oxide synthase (NOS), hepatic malondialdehyde (MDA), lipoprotein lipase (LPL), hepaticlipase (HL) and superoxide dismutase (SOD) activities were evaluated. Then we explored the effects and mechanism of diosgenin against hydrogen peroxide-induced apoptosis of human vein endothelium cells (HUVECs). Intracellular reactive oxygen species (ROS), glutathione (GSH), nitric oxide (NO), DNA fragment formation and mitochondrial membrane potentials (ΔΨm) were determined. Diosgenin treatment increased LPL, HL, SOD, GSH-PX and NOS activities, thus attenuated oxygen free radicals, decreased MDA, TC, TG and LDL-C levels in hyperlipidemic rats. Diosgenin pretreatment significantly attenuated H2O2-induced apoptosis in HUVECs, intracellular ROS, GSH depletion, DNA fragment formation, and restored NO, ΔΨm. These results suggested that diosgenin is a very useful compound to control hyperlipidemia by both improving the lipid profile and modulating oxidative stress and prevent H2O2-induced apoptosis of HUVECs, in partly through regulating mitochondrial dysfunction pathway. 相似文献
17.
《Free radical research》2013,47(3):347-356
AbstractOxidative stress is induced by excess accumulation of reactive oxygen and nitrogen species (RONS). Astrocytes are metabolically active cells in the brain and understanding astrocytic responses to oxidative stress is essential to understand brain pathologies. In addition to direct oxidative stress, exogenous hydrogen peroxide (H2O2) can penetrate biological membranes and enhance formation of other RONS. The present study was carried out to examine the role of insulin in H2O2-induced oxidative stress in rat astrocytic cells. To measure changes in the viability of astrocytes at different concentrations of H2O2 for 3 h, a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-based assay was used and 500 μM H2O2 was selected to establish a model of H2O2-induced oxidative stress. Further assays showed that 3 h of 500 μM H2O2-induced significant changes in the levels of lactate dehydrogenase (LDH), reactive oxygen species (ROS) and calcium ion (Ca2+) in C6 cells, with insulin able to effectively diminish H2O2-induced oxidative damage to C6 cells. Western blotting studies showed that insulin treatment of astrocytes increased the levels of phosphorylated Akt and magnified the decrease in total Bcl-2 protein. The protective effect of insulin treatment on H2O2-induced oxidative stress in astrocytes by reducing apoptosis may relate to the PI3K/Akt pathway. 相似文献
18.
Xuemei Lin Songdi Wu Qing Wang Yaling Shi Guozheng Liu Jin Zhi Fang Wang 《Cellular and molecular neurobiology》2016,36(8):1365-1375
Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H2O2-induced apoptosis. The non-toxic level of SSD significantly mitigated the H2O2-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H2O2-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H2O2-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H2O2-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H2O2-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H2O2-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage. 相似文献
19.
Zhenling Ma Wenwen Zhang Yaru Wu Menghao Zhang Lei Wang Yihan Wang Yi Wang Wei Liu 《Bioscience reports》2021,41(1)
The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H2O2)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H2O2-induced A549 cells. Compared with H2O2-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H2O2 stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H2O2-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy. 相似文献
20.
Mitochondrial membrane potential (ΔΨm) is critical for maintaining the physiological function of the respiratory chain to generate ATP. A significant loss of ΔΨm renders cells depleted of energy with subsequent death. Reactive oxygen species (ROS) are important signaling molecules, but their accumulation in pathological conditions leads to oxidative stress. The two major sources of ROS in cells are environmental toxins and the process of oxidative phosphorylation. Mitochondrial dysfunction and oxidative stress have been implicated in the pathophysiology of many diseases; therefore, the ability to determine ΔΨm and ROS can provide important clues about the physiological status of the cell and the function of the mitochondria. Several fluorescent probes (Rhodamine 123, TMRM, TMRE, JC-1) can be used to determine Δψm in a variety of cell types, and many fluorescence indicators (Dihydroethidium, Dihydrorhodamine 123, H2DCF-DA) can be used to determine ROS. Nearly all of the available fluorescence probes used to assess ΔΨm or ROS are single-wavelength indicators, which increase or decrease their fluorescence intensity proportional to a stimulus that increases or decreases the levels of ΔΨm or ROS. Thus, it is imperative to measure the fluorescence intensity of these probes at the baseline level and after the application of a specific stimulus. This allows one to determine the percentage of change in fluorescence intensity between the baseline level and a stimulus. This change in fluorescence intensity reflects the change in relative levels of ΔΨm or ROS. In this video, we demonstrate how to apply the fluorescence indicator, TMRM, in rat cortical neurons to determine the percentage change in TMRM fluorescence intensity between the baseline level and after applying FCCP, a mitochondrial uncoupler. The lower levels of TMRM fluorescence resulting from FCCP treatment reflect the depolarization of mitochondrial membrane potential. We also show how to apply the fluorescence probe H2DCF-DA to assess the level of ROS in cortical neurons, first at baseline and then after application of H2O2. This protocol (with minor modifications) can be also used to determine changes in ∆Ψm and ROS in different cell types and in neurons isolated from other brain regions. 相似文献