scholarly journals Measurement of Mitochondrial Mass by Flow Cytometry during Oxidative Stress

2017 ◽  
Author(s):  
Edward Doherty ◽  
Andras Perl
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Mitochondrial Mass

scholarly journals Overexpression of Mitochondrial Leishmania major Ascorbate Peroxidase Enhances Tolerance to Oxidative Stress-Induced Programmed Cell Death and Protein Damage

2009 ◽  
Vol 8 (11) ◽  
pp. 1721-1731 ◽  
Author(s):  
Subhankar Dolai ◽  
Rajesh K. Yadav ◽  
Swati Pal ◽  
Subrata Adak
Keyword(s):  
Oxidative Stress ◽  
Flow Cytometry ◽  
Confocal Microscopy ◽  
Ascorbate Peroxidase ◽  
Leishmania Major ◽  
Mitochondrial Respiratory Chain ◽  
Protein Carbonyl ◽  
Endonuclease G ◽  
Mitochondrial Membrane Depolarization ◽  
Induced Apoptosis

ABSTRACT Ascorbate peroxidase from Leishmania major (LmAPX) is one of the key enzymes for scavenging of reactive oxygen species generated from the mitochondrial respiratory chain. We have investigated whether mitochondrial LmAPX has any role in oxidative stress-induced apoptosis. The measurement of reduced glutathione (GSH) and protein carbonyl contents in cellular homogenates indicates that overexpression of LmAPX protects Leishmania cells against depletion of GSH and oxidative damage of proteins by H2O2 or camptothecin (CPT) treatment. Confocal microscopy and fluorescence spectroscopy data have revealed that the intracellular elevation of Ca2+ attained by the LmAPX-overexpressing cells was always below that attained in control cells. Flow cytometry assay data and confocal microscopy observation strongly suggest that LmAPX overexpression protects cells from H2O2-induced mitochondrial membrane depolarization as well as ATP decrease. Western blot data suggest that overexpression of LmAPX shields against H2O2- or CPT-induced cytochrome c and endonuclease G release from mitochondria and subsequently their accumulation in the cytoplasm. Caspase activity assay by flow cytometry shows a lower level of caspase-like protease activity in LmAPX-overexpressing cells under apoptotic stimuli. The data on phosphatidylserine exposed on the cell surface and DNA fragmentation results show that overexpression of LmAPX renders the Leishmania cells more resistant to apoptosis provoked by H2O2 or CPT treatment. Taken together, these results indicate that constitutive overexpression of LmAPX in the mitochondria of L. major prevents cells from the deleterious effects of oxidative stress, that is, mitochondrial dysfunction and cellular death.

Melatonin attenuates radiation-induced cortical bone-derived stem cells injury and enhances bone repair in postradiation femoral defect model

2021 ◽  
Author(s):  
Wei Hu ◽  
Jiawu Liang ◽  
Song Liao ◽  
Zhidong Zhao ◽  
Yuxing Wang ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stem Cells ◽  
Flow Cytometry ◽  
Genomic Stability ◽  
Bone Defects ◽  
Self Renewal ◽  
Tnf Α ◽  
Radiation Induced ◽  
Γ Ray

Abstract Background Ionizing radiation poses a challenge to the healing of bone defects. Radiation therapy and accidental exposure to gamma-ray (γ-ray) radiation inhibit bone formation and increase the risk of fractures. Cortical bone-derived stem cells (CBSCs) are essential for osteogenic lineages, bone maintenance, and repair. This study aimed to investigate the effects of melatonin on postradiation CBSCs and bone defects. Methods CBSCs were extracted from C57/BL6 mice and were identified by flow cytometry. The effects of exogenous melatonin on the self-renewal and osteogenic capacity of postradiation CBSCs were detected in vitro. The underlying mechanisms in terms of genomic stability, apoptosis and oxidative stress-related signaling were further analyzed by western blotting, flow cytometry and immunofluorescence. Finally, the effects of melatonin on healing in postradiation bone defects were evaluated in vivo by micro-CT and immunohistochemical analysis. Results The radiation-induced reduced self-renewal and osteogenic capacity were partially reversed in postradiation CBSCs treated with melatonin. Melatonin maintained the genomic stability and apoptosis of postradiation CBSCs, and intracellular oxidative stress was decreased significantly while antioxidant-related enzymes were enhanced. Western blotting verified the anti-inflammatory effect of melatonin by downregulating the levels of IL-6 and TNF-α via extracellular regulated kinase (ERK)/nuclear factor erythroid 2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, distinct from its antioxidant effect via NRF2 signaling. In vivo experiments demonstrated that the newly formed bone in the melatonin plus Matrigel group had higher trabecular bone volume per tissue volume (BV/TV) and bone mineral density (BMD) values, and lower levels of IL-6 and TNF-α than those in the irradiation and the Matrigel groups. Conclusions This study suggested the potential of melatonin to protect CBSCs against γ-ray radiation and to assist the healing of postradiation bone defects.

scholarly journals A Reproducible, Objective Method Using MitoTracker® Fluorescent Dyes to Assess Mitochondrial Mass in T Cells by Flow Cytometry

2018 ◽  
Vol 95 (4) ◽  
pp. 450-456 ◽  
Author(s):  
Genevieve Clutton ◽  
Katie Mollan ◽  
Michael Hudgens ◽  
Nilu Goonetilleke
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Fluorescent Dyes ◽  
Objective Method ◽  
Mitochondrial Mass

scholarly journals Inhibition of TLR4 Signaling Affects Mitochondrial Fitness Overcoming Bortezomib Resistance in Myeloma Plasma Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3073-3073
Author(s):  
Cesarina Giallongo ◽  
Daniele Tibullo ◽  
Giuseppina Camiolo ◽  
Fabrizio Puglisi ◽  
Daniela Cambria ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Er Stress ◽  
Mitochondrial Respiration ◽  
Research Funding ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Depolarization ◽  
Tlr4 Signaling ◽  
Mitochondrial Mass ◽  
Atp Production ◽  
Inflammatory Microenvironment

BACKGROUND Multiple myeloma (MM) is a B-cell malignancy critically dependent for survival and proliferation on signals coming from its inflammatory microenvironment in which toll-like receptors (TLR) may be potential linking elements between inflammation and cancer. It has been recently demonstrated that TLR4 pathway provides a protective effect against bortezomib (BTZ)-induced endoplasmic reticulum (ER) stress and pre-treatment of MM cells with LPS significantly reduces BTZ-induced apoptosis. AIM Since the acquisition of BTZ resistance is accompanied by an increased reliance on mitochondrial respiration, we investigated the role of TLR4 as stress-responsive mechanism that protect mitochondria during BTZ-induced ER stress as potential mechanism of drug resistance. RESULTS The activation of TLR4 signaling by LPS increased mitochondrial mass in human MM cell lines (HMCL: U266, MM1.S, OPM2, NCI-H929) and induced up-regulation of mitochondrial biogenesis markers (PGC1a, PRC and TFAM). After treatment with BTZ for 24h, all HMCL over-expressed TLR4 and its signaling was functional as suggested by up-regulation of MyD88 and MAPK activation. Compared to BTZ-sensitive cells (U266-S), U266-R showed higher levels of TLR4, p-p38 and p-ERK proteins and higher mitochondrial mass. Using a selective TLR4 inhibitor (TAK-242), we next treated U266-R cells with either 15nM BTZ, 20 μM TAK-242 or their combination. Combinatorial treatment significantly induced cell apoptosis (about 52%; p<0.001) that appeared to result from the deleterious effects of oxidative stress. Indeed, BTZ-induced intracellular ROS returned to normal levels after 3h and cells were able to up-regulate two anti-oxidant enzymes (GPX1 and GSTP1). On the contrary, TAK-242/BTZ activated a strong pro-oxidant status incresing ROS and RNS (reactive nitrogen species) levels, decreasing GSH ones and down-regulating GPX1 and GSTP1. Analyzing the effect of each treatment on mitochondrial polarization status, we observed about 6,7% of depolarized mitochondria after BTZ treatment, while TAK-242/BTZ combination induced a mitochondrial depolarization of about 69,3% (p<0.001). Moreover, cells treated with BTZ alone showed a compensatory up-regulation of the OXPHOS- (NDUFA-6 and MT-ND4) and mitochondrial fusion-related genes (mitofusin and OPA1) and TFAM. On the contrary, all these genes were down-regulated by TAK-242/BTZ combination. Also a dramatic drop in mitochondrial respiration was observed with a marked decrease in ATP production, consequent accumulation of AMP and a decreased NAD+/NADH and NADP+/NADPH ratio. Since high levels of oxidative stress and mitochondrial impairment activate mitophagy sensitizing cells to apoptosis, we evaluated co-localization of mitochondria (stained with MitoTracker) with the autophagosome marker LC3 using confocal microscopy. BTZ and TAK-242/BTZ increased Mitotracker/LC3 co-localization respectively of about 4,5 and 50 fold compared with control (BTZ vs combination: p<0.001). To evaluate whether TLR4 inhibition resensitizes resistant primary cells, CD138+ cells derived from 5 refractory/relapsed MM patients were treated with 5nM BTZ, 10mM TAK-242 or their combination. Compared to BTZ alone, combination treatment induced higher mitochondrial depolarization after 24h and significantly decreased viability of CD138+ cells after 48h. TLR4 inhibitor alone or in combination did never show cytotoxicity toward CD138- cells. CONCLUSION Taken together, these findings indicate thatTLR4 signaling is involved in the acquisition of bortezomib resistance protecting mitochondria during BTZ exposure and sustaining mitochondrial dynamics in BTZ-resitant cells. Inhibition of TLR4 may overcome bortezomib resistance in patients with relapsed/refractory MM. Disclosures Conticello: Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria, Research Funding. Palumbo:Celgene: Honoraria; Amgen: Honoraria; Hospira: Honoraria; Teva: Honoraria; Novartis: Honoraria; Janssen: Honoraria. Di Raimondo:Takeda: Consultancy; Amgen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding.

scholarly journals Active Vitamin D and Vitamin D Receptor Help Prevent High Glucose Induced Oxidative Stress of Renal Tubular Cells via AKT/UCP2 Signaling Pathway

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
XiaoJuan Zhu ◽  
ShengHua Wu ◽  
HanCheng Guo
Keyword(s):  
Oxidative Stress ◽  
Vitamin D ◽  
Flow Cytometry ◽  
High Glucose ◽  
Mitochondrial Membrane ◽  
Renal Tubular Cell ◽  
Active Vitamin ◽  
Akt Inhibitor ◽  
Renal Tubular ◽  
Hk2 Cells

Background. It has been documented that vitamin D supplementation showed an improvement of symptoms of diabetic nephropathy; however, the underlying mechanisms remain unknown. We here tested the hypothesis that active vitamin D is able to up-regulate AKT/UCP2 signaling to alleviate oxidative stress of renal tubular cell line HK2.Methods. There are eight groups in the present study: normal glucose, osmotic control (5.5 mmol/L D-glucose+24.5 mmol/L D-mannitol), NAC control (30 mmol/L D-glucose + 1.0 mmol/L N-Methylcysteine), high glucose, high glucose+VD, high glucose (HG)+VD+siVDR, HG+VD+AKT inhibitor (AI), and high glucose+VD+UCP2 inhibitor (Gelipin). Concentration of superoxide dismutase (SOD) and malondialdehyde (MDA) was analyzed by ELISA. Reactive oxygen species (ROS), mitochondrial membrane potential and apoptosis were measured by flow cytometry. JC-1 was evaluated by flow cytometry. The presence of VDR, AKT, and UCP2 in HK cells was assessed using RT-PCR and western blot analyses.Results. VD administration significantly upregulated the SOD activation and downregulated MDA levels compared to HG group. siVDR, AKT inhibitor, and UCP2 inhibitor significantly suppressed the activation of SOD and increased the expression of MDA compared to VD group. ROS generation and apoptosis of HK2 cells in HG+VD group were significantly lower than those in HG, HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group. ΔΨm in HG+VD group was obviously higher than those in HG, HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group. Decreased mRNA and protein levels of VDR, p-AKT, and UCP2 were observed in HG+VD+siVDR, HG+VD+AI, and HG+VD+Gelipin group compared to those in HG+VD group.Conclusions. siVDR, AKT inhibitor, and UCP2 inhibitor elevated the ROS and apoptosis of HK2 cells while attenuating the mitochondrial membrane potential, suggesting that vitamin D protects renal tubular cell from high glucose by AKT/UCP2 signaling pathway.

scholarly journals Effects of GH/IGF on the Aging Mitochondria

Cells ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 1384 ◽  
Author(s):  
Sher Bahadur Poudel ◽  
Manisha Dixit ◽  
Maria Neginskaya ◽  
Karthik Nagaraj ◽  
Evgeny Pavlov ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Growth Hormone ◽  
Cell Differentiation ◽  
Growth Factor ◽  
Mitochondrial Biogenesis ◽  
Mitochondrial Function ◽  
Cellular Aging ◽  
Mitochondrial Mass ◽  
Atp Production ◽  
And Function

The mitochondria are key organelles regulating vital processes in the eukaryote cell. A decline in mitochondrial function is one of the hallmarks of aging. Growth hormone (GH) and the insulin-like growth factor-1 (IGF-1) are somatotropic hormones that regulate cellular homeostasis and play significant roles in cell differentiation, function, and survival. In mammals, these hormones peak during puberty and decline gradually during adulthood and aging. Here, we review the evidence that GH and IGF-1 regulate mitochondrial mass and function and contribute to specific processes of cellular aging. Specifically, we discuss the contribution of GH and IGF-1 to mitochondrial biogenesis, respiration and ATP production, oxidative stress, senescence, and apoptosis. Particular emphasis was placed on how these pathways intersect during aging.

scholarly journals Administration of Enalapril Started Late in Life Attenuates Hypertrophy and Oxidative Stress Burden, Increases Mitochondrial Mass, and Modulates Mitochondrial Quality Control Signaling in the Rat Heart

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 177 ◽  
Author(s):  
Anna Picca ◽  
Giuseppe Sirago ◽  
Vito Pesce ◽  
Angela Maria Serena Lezza ◽  
Riccardo Calvani ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quality Control ◽  
Transcription Factor ◽  
Oxidative Damage ◽  
Mitochondrial Quality Control ◽  
Mitochondrial Transcription ◽  
Mitochondrial Mass ◽  
Mitochondrial Transcription Factor ◽  
Mitochondrial Transcription Factor A ◽  
And Oxidative Stress

Mitochondrial dysfunction is a relevant mechanism in cardiac aging. Here, we investigated the effects of late-life enalapril administration at a non-antihypertensive dose on mitochondrial genomic stability, oxidative damage, and mitochondrial quality control (MQC) signaling in the hearts of aged rats. The protein expression of selected mediators (i.e., mitochondrial antioxidant enzymes, energy metabolism, mitochondrial biogenesis, dynamics, and autophagy) was measured in old rats randomly assigned to receive enalapril (n = 8) or placebo (n = 8) from 24 to 27 months of age. We also assessed mitochondrial DNA (mtDNA) content, citrate synthase activity, oxidative lesions to protein and mtDNA (i.e., carbonyls and the abundance of mtDNA4834 deletion), and the mitochondrial transcription factor A (TFAM) binding to specific mtDNA regions. Enalapril attenuated cardiac hypertrophy and oxidative stress-derived damage (mtDNA oxidation, mtDNA4834 deletion, and protein carbonylation), while increasing mitochondrial antioxidant defenses. The binding of mitochondrial transcription factor A to mtDNA regions involved in replication and deletion generation was enhanced following enalapril administration. Increased mitochondrial mass as well as mitochondriogenesis and autophagy signaling were found in enalapril-treated rats. Late-life enalapril administration mitigates age-dependent cardiac hypertrophy and oxidative damage, while increasing mitochondrial mass and modulating MQC signaling. Further analyses are needed to conclusively establish whether enalapril may offer cardioprotection during aging.

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