Mitochondria-targeted antioxidants prevent TNFα-induced endothelial cell damage

Increased serum level of tumor necrosis factor α (TNFα) causes endothelial dysfunction and leads to serious vascular pathologies. TNFα signaling is known to involve reactive oxygen species (ROS). Using mitochondria-targeted antioxidant SkQR1, we studied the role of mitochondrial ROS in TNFα-induced apoptosis of human endothelial cell line EAhy926. We found that 0.2 nM SkQR1 prevents TNFα-induced apoptosis. SkQR1 has no influence on TNFα-dependent proteolytic activation of caspase-8 and Bid, but it inhibits cytochrome c release from mitochondria and cleavage of caspase-3 and its substrate PARP. SkQ analogs lacking the antioxidant moieties do not prevent TNFα-induced apoptosis. The antiapoptotic action of SkQR1 may be related to other observations made in these experiments, namely SkQR1-induced increase in Bcl-2 and corresponding decrease in Bax as well as p53. These results indicate that mitochondrial ROS production is involved in TNFα-initiated endothelial cell death, and they suggest the potential of mitochondria-targeted antioxidants as vasoprotectors.     

Activation of AMPK participates hydrogen sulfide-induced cyto-protective effect against dexamethasone in osteoblastic MC3T3-E1 cells

Long-time glucocorticoids (GCs) usage causes osteoporosis. In the present study, we explored the potential role of hydrogen sulfide (H₂S) against dexamethasone (Dex)-induced osteoblast cell damage, and focused on the underlying mechanisms. We showed that two H₂S-producing enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), were significantly downregulated in human osteonecrosis tissues as well as in Dex-treated osteoblastic MC3T3-E1 cells. H₂S donor NaHS as well as the CBS activator S-adenosyl-l-methionine (SAM) inhibited Dex-induced viability reduction, death and apoptosis in MC3T3-E1 cells. NaHS activated adenosine monophosphate (AMP)-activated protein kinase (AMPK) signaling, which participated its cyto-protective activity. AMPK inhibition by its inhibitor (compound C) or reduction by targeted-shRNA suppressed its pro-survival activity against Dex in MC3T3-E1 cells. Further, we found that NaHS inhibited Dex-mediated reactive oxygen species (ROS) production and ATP depletion. Such effects by NaHS were again inhibited by compound C and AMPKα1-shRNA. In summary, we show that H₂S inhibits Dex-induced osteoblast damage through activation of AMPK signaling. H₂S signaling might be further investigated as a novel target for anti-osteoporosis treatment.     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

CWJ-081, a novel 3-arylisoquinoline derivative,induces apoptosis in human leukemia HL-60 cells partially involves reactive oxygen species through c-Jun NH2-terminal kinase pathway

In the present study, we investigated the effect of a novel 3-arylisoquinoline derivative 3-(6-ethyl-benzo[1,3]dioxol-5-yl)-7,8-dimethoxy-2-methyl-2H-isoquinolin-1-one (CWJ-081) on the induction of apoptosis and the putative molecular mechanism of its action in human leukemia cells. Treatment with CWJ-081 exhibited a characteristic feature of apoptosis including externalization of phosphatidylserine and formation of DNA fragmentation in human leukemia cell lines (HL-60, U-937, K-562). In addition, stimulation of HL-60 cells with CWJ-081 induced a series of intracellular events: (1) the activations of caspase-8, -9, and -3; (2) the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1); (3) the loss of mitochondrial membrane potential (ΔΨ_m); (4) the release of cytochrome c; and (5) the modulation of Bcl-2 family proteins. We further demonstrated that CWJ-081 induces reactive oxygen species (ROS) production and c-Jun NH₂-terminal kinase (JNK) activation. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) markedly inhibited the CWJ-081-induced JNK activation and apoptosis. Moreover, CWJ-081-induced apoptosis was suppressed in the presence of SP600125, a specific JNK inhibitor. Taken together, these data suggest that CWJ-081 induces apoptosis via the mitochondrial apoptotic pathway in HL-60 cells, and ROS-mediated JNK activation plays a key role in the CWJ-081-induced apoptosis.     

Protective effect of augmenter of liver regeneration on hydrogen peroxide-induced apoptosis in SH-SY5Y human neuroblastoma cells

Background: Hydrogen peroxide, as other reactive oxygen species (ROS) produced during redox processes, induces lipid membrane peroxidation and protein degeneration causing cell apoptosis. ROS are recently considered as messengers in cell signalling processes, which, through reversible protein disulphide bridges formation, activate regulatory factors of cell proliferation and apoptosis. Disulphide bridges formation is catalysed by sulphydryl oxidase enzymes.

Aim: The neuroprotective effect of ALR protein (Alrp), a sulphydryl oxidase enzyme, on H₂O₂-induced apoptosis in SH-SY5Y cells has been evaluated.

Methods: Cell viability, flow cytometric evaluation of apoptotic cells, fluorescent changes of nuclear morphology, immunocytochemistry Alrp detection, Western blot evaluation of mitochondrial cyt c release and mitochondrial swelling were determined.

Results: Alrp prevents the H₂O₂-induced cell viability loss, apoptotic cell death and mitochondrial swelling in SH-SY5Y cells in culture.

Conclusions: The data demonstrate that Alrp improves SH-SY5Y cells survival in H₂O₂-induced apoptosis. It is speculated that this effect could be related to the Alrp enzymatic activity.     

Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway

ABSTRACT

Honokiol is one of the main active components of Magnolia officinalis, and has been demonstrated to have multiple pharmacological activities against a variety of diseases. Recently, this phenolic compound is known to have antioxidant activity, but its mechanism of action remains unclear. The purpose of the current study was to evaluate the preventive effects of honokiol against oxidative stress-induced DNA damage and apoptosis in C2C12 myoblasts. The present study found that honokiol inhibited hydrogen peroxide (H₂O₂)-induced DNA damage and mitochondrial dysfunction, while reducing reactive oxygen species (ROS) formation. The inhibitory effect of honokiol on H₂O₂-induced apoptosis was associated with the up-regulation of Bcl-2 and down-regulation of Bax, thus reducing the Bax/Bcl-2 ratio that in turn protected the activation of caspase-9 and -3, and inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blocking of cytochrome c release to the cytoplasm. Collectively, these results demonstrate that honokiol defends C2C12 myoblasts against H₂O₂-induced DNA damage and apoptosis, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS.     

Mitochondrial K<Subscript>ATP</Subscript> channels-derived reactive oxygen species activate pro-survival pathway in pravastatin-induced cardioprotection

Reactive oxygen species (ROS) are important intracellular signaling molecules and are implicated in cardioprotective pathways including ischemic preconditioning. Statins have been shown to have cardioprotective effects against ischemia/reperfusion injury, however, the precise mechanisms remain to be elucidated. We hypothesized that ROS-mediated signaling cascade may be involved in pravastatin-induced cardioprotection. Cultured rat cardiomyocytes were exposed to H₂O₂ for 30 min to induce cell injury. Pravastatin significantly suppressed H₂O₂-induced cell death evaluated by propidium iodide staining and the MTT assay. Incubation with pravastatin activated catalase, and prevented a ROS burst induced by H₂O₂, which preserved mitochondrial membrane potential. Protective effects were induced very rapidly within 10 min, which was concordant with the up-regulation of phosphorylated ERK1/2. L-NAME, 5HD, N-acetylcysteine (NAC) and staurosporine inhibited ERK1/2 phosphorylation and also reduced pravastatin-induced cardioprotection, suggesting NO, mitochondrial K_ATP (mitoK_ATP) channels, ROS and PKC should be involved in the cardioprotective signaling. We also demonstrated that pravastatin moderately up-regulated ROS generation in a 5HD-inhibitable manner. In isolated perfused rat heart experiments, pravastatin administered 10 min prior to no-flow global ischemia significantly improved left ventricular functional recovery, and also reduced infarct size, which were attenuated by the treatment with NAC, 5HD, L-NAME or staurosporine. Administration of pravastatin from the beginning of reperfusion also conferred cardioprotection. Pravastatin protected the cardiomyocytes against oxidative stress by preventing the ROS burst and preserving mitochondrial function. Moderately up-regulated ROS production by mitoK_ATP channels opening is involved in the pro-survival signaling cascade activated by pravastatin.     

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Protective effect of Homer 1a against hydrogen peroxide-induced oxidative stress in PC12 cells

Abstract

Oxidative 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 (Ca^{2 +}) 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 Ca^{2 +} homeostasis.     

Prooxidative effects of green tea polyphenol (−)-epigallocatethin-3-gallate on the HIT-T15 pancreatic beta cell line

Epigallocatechin-3-gallate (EGCG) is the main polyphenolic constituent in green tea and is believed to function as an antioxidant. However, increasing evidence indicates that EGCG produces reactive oxygen species (ROS) and subsequent cell death. In this study, we investigated the prooxidative effects of EGCG on the HIT-T15 pancreatic beta cell line. Dose-dependent cell viability was monitored with the cell counting kit-8 assay, while the induction of apoptosis was analyzed by a cell death ELISA kit and comet assay. Extracellular H₂O₂ was determined using the Amplex Red Hydrogen Peroxide Assay Kit. Intracellular oxidative stress was measured by fluorometric analysis of 2′,7′-dichlorofluorescin (DCFH) oxidation using DCFH diacetate (DA) as the probe. Treatment with EGCG (5–100 μM) decreased the viability of pancreatic beta cells, caused concomitant increases in apoptotic cell death, and increased the production of H₂O₂ and ROS. Catalase, the iron-chelating agent diethylenetriaminepentaacetic acid, and the Fe(II)-specific chelator o-phenanthroline all suppressed the effects of EGCG, indicating the involvement of both H₂O₂ and Fe(II) in the mechanism of action of EGCG. The antioxidant N-acetyl-cysteine and alpha-lipoic acid also suppressed the effects of EGCG. Furthermore, EGCG did not scavenge exogenous H₂O₂, but rather, it synergistically increased H₂O₂-induced oxidative cell damage in pancreatic beta cells. Together, these findings suggest that in the HIT-T15 pancreatic beta cell line, EGCG mediated the generation of H₂O₂, triggering Fe(II)-dependent formation of a highly toxic radical that in turn induced oxidative cell damage.     

Intermolecular disulfide bond formation in the NEMO dimer requires Cys54 and Cys347

NEMO is an essential regulatory component of the IκB kinase (IKK) complex, which controls activation of the NF-κB signaling pathway. Herein, we show that NEMO exists as a disulfide-bonded dimer when isolated from several cell types and analyzed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. Treatment of cells with hydrogen peroxide (H₂O₂) induces further formation of NEMO dimers. Disulfide bond-mediated formation of NEMO dimers requires Cys54 and Cys347. The ability of these residues to form disulfide bonds is consistent with their location in a NEMO dimer structure that we generated by molecular modeling. We also show that pretreatment with H₂O₂ decreases TNFα-induced IKK activity in NEMO-reconstituted cells, and that TNFα has a diminished ability to activate NF-κB DNA binding in cells reconstituted with NEMO mutant C54/347A. This study implicates NEMO as a target of redox regulation and presents the first structural model for the NEMO protein.     

Cytoprotective effects of 12-oxo phytodienoic acid,a plant-derived oxylipin jasmonate,on oxidative stress-induced toxicity in human neuroblastoma SH-SY5Y cells

BackgroundJasmonates are plant lipid-derived oxylipins that act as key signaling compounds when plants are under oxidative stress, but little is known about their functions in mammalian cells. Here we investigated whether jasmonates could protect human neuroblastoma SH-SY5Y cells against oxidative stress-induced toxicity.MethodsThe cells were pretreated with individual jasmonates for 24 h and exposed to hydrogen peroxide (H₂O₂) for 24 h. Before the resulting cytotoxicity, intracellular reactive oxygen species (ROS) levels, and mitochondrial membrane potential were measured. We also measured intracellular glutathione (GSH) levels and investigated changes in the signaling cascade mediated by nuclear factor erythroid 2-related factor 2 (Nrf2) in cells treated with 12-oxo phytodienoic acid (OPDA).ResultsAmong the jasmonates, only OPDA suppressed H₂O₂-induced cytotoxicity. OPDA pretreatment also inhibited the H₂O₂-induced ROS increase and mitochondrial membrane potential decrease. In addition, OPDA induced the nuclear translocation of Nrf2 and increased intracellular GSH level and the expression of the Nrf2-regulated phase II antioxidant enzymes heme oxygenase-1, NADPH quinone oxidoreductase 1, and glutathione reductase. Finally, the cytoprotective effects of OPDA were reduced by siRNA-induced knockdown of Nrf2.ConclusionsThese results demonstrated that among jasmonates, only OPDA suppressed oxidative stress-induced death of human neuroblastoma cells, which occurred via activation of the Nrf2 pathway.General significancePlant-derived oxylipin OPDA may have the potential to provide protection against oxidative stress-related diseases.     

1-Butanol triggers programmed cell death in Populus euphratica cell cultures

1-Butanol, which is a specific inhibitor of phospholipase D, usually inhibits phosphatidic acid (PA) production and blocks the PA-dependent signaling pathway under stress conditions. However, the effects of 1-butanol on plant cells under non-stress condition are still unclear. In this study, we report that 1-butanol induced a dose dependent cell death in poplar (Populus euphratica) cell cultures. In contrast, the control 2-butanol and ethanol had no effects on cell viability. 1-Butanol-treated cells displayed hallmark features of programmed cell death (PCD), such as shrinkage of the cytoplasm, DNA fragmentation, condensed or stretched chromatin and the activation of caspase-3-like protease. Exogenous application of PA markedly inhibited the 1-butanol-induced PCD. 1-Butanol also caused a burst of mitochondrial H₂O₂ ([H₂O₂]_mit) that was usually accompanied by a loss of mitochondrial membrane potential (?Ψm). Supplement of PA, antioxidant enzyme (catalase) and antioxidant (ascorbic acid) reversed this effect. Moreover, a significant increase of nitric oxide (NO) was observed in 1-butanol-treated poplar cells. This NO burst was suppressed by PA or inhibitors of NO synthesis. Further pharmacological experiments indicate that the burst of NO contributed to the 1-butanol-induced inhibition of antioxidant enzymes and subsequent H₂O₂-dependent PCD. In conclusion, we propose that 1-butanol is a potent inducer of PCD in plants and this process is regulated by the PA, NO and H₂O₂.     

1Alpha,25-dihydroxyvitamin D3 modulation of adipocyte reactive oxygen species production

Objective: We have previously shown 1α,25‐dihydroxyvitamin D₃ [1α,25‐(OH)₂D₃] to inhibit mitochondrial uncoupling protein 2 (UCP2) expression in adipocytes and that in vivo suppression of calcitriol levels with calcium‐rich diets increases UCP2 expression. Because UCP2 plays a significant role in the clearance of reactive oxygen species (ROS), we studied the effect of calcitriol on ROS production and ROS‐induced adipocyte proliferation. Research Methods and Procedures: ROS production in human and murine adipocytes was stimulated by high glucose (30 mM) or H₂O₂ (100 nM). Results: Both approaches resulted in increased ROS production by 27% to 100% (p < 0.05) and increased cell proliferation by 15% to 39% (p < 0.03). These effects were augmented by the addition of mitochondrial uncoupling inhibitor guanosine 5′‐diphosphate (GDP; 100 μM) or 1α,25‐(OH)₂D₃ (10 nM) and attenuated by UCP2 overexpression, suggesting that inhibition of mitochondrial uncoupling suppresses clearance of ROS and increases adipocyte proliferation. The addition of α ± tocopherol (1 μM) inhibited cell proliferation in adipocytes treated with either H₂O₂ or high glucose, indicating that ROS plays a major role in the regulation of cell proliferation in adipocytes. Moreover, stimulation of ROS with high glucose and H₂O₂ resulted in a 2‐ to 5‐fold increase in adipocyte intracellular calcium ([Ca²⁺]i; p < 0.001), and calcium channel antagonism (nifedipine, 10 μM) suppressed ROS induced calcium influx and cell proliferation, indicating that [Ca²⁺]i may also regulate ROS production and exert a mitogenic effect in adipocytes. Discussion: These data support a role of 1α,25‐(OH)₂D₃, UCP2, and [Ca²⁺]i in the regulation of adipocyte ROS production.     

Over-expression of mitochondrial heat shock protein 70 suppresses programmed cell death in rice

In this study, we identified and functionally characterized the mitochondrial heat shock protein 70 (mtHsp70). Over-expression of mtHsp70 suppressed heat- and H₂O₂-induced programmed cell death (PCD) in rice protoplasts, as reflected by higher cell viability, decreased DNA laddering and chromatin condensation. Mitochondrial membrane potential (Δψ_m) after heat shock was destroyed gradually in protoplasts, but mtHsp70 over-expression showed higher Δψ_m relative to the vector control cells, and partially inhibited cytochrome c release from mitochondria to cytosol. Heat treatment also significantly increased reactive oxygen species (ROS) generation, a phenomenon not observed in protoplasts over-expressing mtHsp70. Together, these results suggest that mtHsp70 may suppress PCD in rice protoplasts by maintaining mitochondrial Δψ_m and inhibiting the amplification of ROS.     

Neuroprotective effects of aucubin on hydrogen peroxide-induced toxicity in human neuroblastoma SH-SY5Y cells via the Nrf2/HO-1 pathway

BackgroundWhen redox balance is lost in the brain, oxidative stress can cause serious damage that leads to neuronal loss, in congruence with neurodegenerative diseases. Aucubin (AU) is an iridoid glycoside and that is one of the active constituents of Eucommia ulmoides, has many pharmacological effects such as anti-inflammation, anti-liver fibrosis, and anti-atherosclerosis.PurposeThe present study aimed to evaluate the inhibitory effects of AU on cell oxidative stress against hydrogen peroxide (H₂O₂)-induced injury in SH-SY5Y cells in vitro.MethodsSH-SY5Y cells were simultaneously treated with AU and H₂O₂ for 24 h. Cell viability was measured by CCK-8. Additionally, mitochondrial membrane depolarization, reactive oxygen species (ROS) generation, and cell apoptosis were measured by flow cytometry.ResultsThe results showed that AU can significantly increase the H₂O₂-induced cell viability and the mitochondrial membrane potential, decrease the ROS generation, malondialdehyde (MDA), and increase glutathione (GSH) contents and the superoxide dismutase (SOD) activity. We also found that H₂O₂ stimulated the production of nitric oxide (NO), which could be reduced by treatment with AU through inhibiting the inducible nitric oxide synthase (iNOS) protein expression. In H₂O₂-induced SH-SY5Y cells, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) content and cell apoptosis were significantly reduced by AU treatment through nuclear factor E2-related factor 2/hemo oxygenase-1 (Nrf2/HO-1) activation, inhibiting the expression of p-NF-κB/NF-κB and down-regulating MAPK and Bcl-2/Bax pathways.ConclusionThese results indicate that AU can reduce inflammation and oxidative stress through the NF-κB, Nrf2/HO-1, and MAPK pathways.     

A critical role of the transient receptor potential melastatin 2 channel in a positive feedback mechanism for reactive oxygen species-induced delayed cell death

Transient receptor potential melastatin 2 (TRPM2) channel activation by reactive oxygen species (ROS) plays a critical role in delayed neuronal cell death, responsible for postischemia brain damage via altering intracellular Zn²⁺ homeostasis, but a mechanistic understanding is still lacking. Here, we showed that H₂O₂ induced neuroblastoma SH-SY5Y cell death with a significant delay, dependently of the TRPM2 channel and increased [Zn²⁺]_i, and therefore used this cell model to investigate the mechanisms underlying ROS-induced TRPM2-mediated delayed cell death. H₂O₂ increased concentration-dependently the [Zn²⁺]_i and caused lysosomal dysfunction and Zn²⁺ loss and, furthermore, mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation. Such effects were suppressed by preventing poly(adenosine diphosphate ribose, ADPR) polymerase-1-dependent TRPM2 channel activation with PJ34 and 3,3′,5,5′-tetra-tert-butyldiphenoquinone, inhibiting the TRPM2 channel with 2-aminoethoxydiphenyl borate (2-APB) and N-(p-amylcinnamoyl)anthranilic acid, or chelating Zn²⁺ with N,N,N,N-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Bafilomycin-induced lysosomal dysfunction also resulted in mitochondrial Zn²⁺ accumulation, fragmentation, and ROS generation that were inhibited by PJ34 or 2-APB, suggesting that these mitochondrial events are TRPM2 dependent and sequela of lysosomal dysfunction. Mitochondrial TRPM2 expression was detected and exposure to ADPR-induced Zn²⁺ uptake in isolated mitochondria, which was prevented by TPEN. H₂O₂-induced delayed cell death was inhibited by apocynin and diphenyleneiodonium, nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) inhibitors, GKT137831, an NOX1/4-specific inhibitor, or Gö6983, a protein kinase C (PKC) inhibitor. Moreover, inhibition of PKC/NOX prevented H₂O₂-induced ROS generation, lysosomal dysfunction and Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, fragmentation and ROS generation. Collectively, these results support a critical role for the TRPM2 channel in coupling PKC/NOX-mediated ROS generation, lysosomal Zn²⁺ release, and mitochondrial Zn²⁺ accumulation, and ROS generation to form a vicious positive feedback signaling mechanism for ROS-induced delayed cell death.     

Tiliroside,a dietary glycosidic flavonoid,inhibits TRAF-6/NF-κB/p38-mediated neuroinflammation in activated BV2 microglia

Background

Tiliroside is a dietary glycosidic flavonoid which has shown in vivo anti-inflammatory activity. This study is aimed at evaluating the effect of tiliroside on neuroinflammation in BV2 microglia, and to identify its molecular targets of anti-neuroinflammatory action.

Methods

BV2 cells were stimulated with LPS + IFNγ in the presence or absence of tiliroside. TNFα, IL-6, nitrite and PGE₂ production was determined with ELISA, Griess assay and enzyme immunoassay, respectively. iNOS, COX-2, phospho-p65, phospho-IκBα, phospho-IKKα, phospho-p38, phospho-MK2, phosopho-MKK3/6 and TRAF-6 were determined by western blot analysis. NF-κB activity was also investigated using a reporter gene assay in HEK293 cells. LPS-induced microglia ROS production was tested using the DCFDA method, while HO-1 and Nrf2 activation was determined with western blot.

Results

Tiliroside significantly suppressed TNFα, IL-6, nitrite and PGE₂ production, as well as iNOS and COX-2 protein expression from LPS + IFNγ-activated BV2 microglia. Further mechanistic studies showed that tiliroside inhibited neuroinflammation by targeting important steps in the NF-κB and p38 signalling in LPS + IFNγ-activated BV2 cells. This compound also inhibited LPS-induced TRAF-6 protein expression in BV2 cells. Antioxidant activity of tiliroside in BV2 cells was demonstrated through attenuation of LPS + IFNγ-induced ROS production and activation of HO-1/Nrf2 antioxidant system.

Conclusions

Tiliroside inhibits neuroinflammation in BV2 microglia through a mechanism involving TRAF-6-mediated activation of NF-κB and p38 MAPK signalling pathways. These activities are possibly due, in part, to the antioxidant property of this compound.

General Significance

Tiliroside is a potential novel natural compound for inhibiting neuroinflammation in neurodegenerative disorders.