Mitochondrial Reactive Oxygen Species Generation Research Articles

Diallyl Trisulfide Suppresses Angiotensin II-Induced Vascular Remodeling Via Inhibition of Mitochondrial Fission.

We have shown previously that diallyl trisulfide (DATS) ameliorates mitochondrial fission and oxidative stress in a hyperglycemia-induced endothelial apoptosis and diabetic mouse model. The aim of this study was to investigate whether DATS mitigates Ang II-induced vascular smooth muscle cell (VSMC) phenotypic switching and vascular remodeling, and if so, to determine the underlying molecular events. Male C57BL/6 mice were used to establish a vascular remodeling model by continuous 2-week Ang II infusion using a subcutaneous osmotic pump. Animals were intraperitoneally injected with DATS or vehicle. Physiological parameters, vascular morphology, and molecular markers were assessed. For in vitro studies, VSMCs were pretreated with or without DATS for 1h, then were stimulated with Ang II, and mitochondrial morphology and phenotypic switching of VSMCs were also measured. In primary mouse VSMCs, we found that Drp1-dependent mitochondrial fission regulated mitochondrial reactive oxygen species (mtROS) generation, which eventually promoted Ang II-induced VSMC proliferation, migration, and phenotypic switching. Moreover, Ang II was found to up-regulate the Rho-associated coiled coil-containing protein kinase 1 (ROCK1), which regulated mitochondrial fission and VSMC phenotypic switching by phosphorylating Drp1. However, the biological effect of Ang II was abrogated by DATS. Consistent with the effects in VSMCs, we found that DATS markedly alleviated mitochondrial fission, VSMC differentiation, and vessel wall thickening in an animal model of Ang II-induced vascular remodeling, which was regulated by the ROCK1/Drp1 signal. Our findings showed that DATS mitigated Ang II-induced vascular remodeling by suppressing Drp1-mediated mitochondrial fission in an ROCK1-dependent manner.

Equine herpesvirus type 1 affects mitochondrial network morphology and reactive oxygen species generation in equine dermal cell line.

In the present study, the influence of the infection with equine herpesvirus type 1 (non-neuro-pathogenic and neuropathogenic strains of EHV-1) on the morphology and distribution of mitochondrial network in equine dermal cell line was investigated. Our results indicate that EHV-1-infection caused changes in the mitochondrial morphology manifested mostly by fission and reactive oxygen species generation.

Unlike a high-fat diet model, mitochondrial ROS production does not appear to contribute to bed rest-induced insulin resistance.

Unlike a high-fat diet model, mitochondrial ROS production does not appear to contribute to bed rest-induced insulin resistance.

The iron–sulfur protein subunit of succinate dehydrogenase is critical in driving mitochondrial reactive oxygen species generation in Apostichopus japonicus

Succinate dehydrogenase (SDH) is a mitochondrial enzyme with the unique ability to participate in both the tricarboxylic acid cycle and the electron transport chain to produce reactive oxygen species (ROS). The B subunit of SDH is required for succinate oxidation, which is critical for pro-inflammatory response. In this study, we cloned the iron–sulfur protein subunit of SDH from Apostichopus japonicus (denoted as AjSDHB) via RACE technology and explored its role in the immune system as a response to pathogen infection. The full-length cDNA of AjSDHB was 1442 bp with a complete open reading frame of 858 bp encoding 286 amino acids. Simple modular architecture research tool analysis revealed that AjSDHB contained two conserved domains, including a 2Fe–2S iron–sulfur cluster binding domain and a 4Fe–4S dicluster domain, without a signal peptide. Multiple sequence alignment demonstrated that AjSDHB shared a high degree of structural conservation and sequence identities with other counterparts from invertebrates and vertebrates. Phylogenetic analysis supported the finding that AjSDHB is a new member of the SDHB protein subfamily. Tissue distribution analysis revealed that AjSDHB was expressed in all examined tissues and particularly highly expressed in the muscles. AjSDHB transcripts were markedly induced in coelomocytes both by Vibrio splendidus challenge in vivo and lipopolysaccharide exposure in vitro. Function analysis showed that siRNA-mediated AjSDHB knockdown could substantially reduce the mitochondrial membrane potential (ΔΨm) and further decrease mitochondrial ROS production in A. japonicus coelomocytes. By contrast, AjSDHB overexpression considerably increased ΔΨm and mitochondrial ROS production of A. japonicus coelomocytes. These results supported the idea that AjSDHB is involved in the innate immunity of A. japonicus through its participation in mitochondrial ROS generation.

Metal‐Based Nanoparticles: Mechanistic Differences in Cell Death Responses to Metal‐Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes (Small 21/2020)

In article number 2000528, André E. Nel, Tian Xia, and co-workers show how metal-based nanoparticles are taken up by liver cells including Kupffer cells and hepatocytes, where they induce tiered oxidative stress responses including mitochondrial reactive oxygen species generation, glutathione depletion, expression of phase II enzymes such as HO-1, production of pro-inflammatory cytokines including IL-1β and TNF-α, and cell death via different mechanisms including apoptosis (Ag, CuO, ZnO, V2O5) and pyroptosis (fumed SiO2).

Strongylocentrotus nudus lipids induce apoptosis in HepG2 cells through the induction of oxidative stress

Abstract: Strongylocentrotus nudus lipid composition is known but its biological effects are largely unknown. The anti-cancer effect of sea urchin lipids (SUL) and the underlying mechanism in a human hepatocellular carcinoma HepG2 cells were studied. The cell viability, apoptosis, mitochondrial transmembrane potential and reactive oxygen species (ROS) generation were studied. The protein levels of caspase-3, Bcl-2 and the mRNA levels of antioxidants including superoxide dismutase (SOD) and catalase (CAT) were also measured. The results showed that SUL treatment significantly inhibited the viability of HepG2 cells with reduced proliferation and elevated apoptosis as evidenced by the decreased Bcl-2 level and increased caspase-3 level. SUL treatment reduced mitochondrial membrane potential and induced ROS production in HepG2 cells. Finally, SUL up-regulated the gene expression levels of CuSOD and MnSOD in a dose-dependent manner but down-regulated the expression level of CAT, which may cause the accumulation of H2O2 in the HepG2 cells. These results suggested that the anti-cancer effect of SUL is mediated through the differential regulation of antioxidants, which contributes to the elevated ROS production, thereby, inducing apoptosis in the HepG2 cells.

Resveratrol attenuates hypoxia-induced neuronal cell death, inflammation and mitochondrial oxidative stress by modulation of TRPM2 channel

Hypoxia (HYPX) induced-overload Ca2+ entry results in increase of mitochondrial oxidative stress, inflammation and apoptosis in several neurons. Ca2+ permeable TRPM2 channel was gated by ADP-ribose (ADPR) and reactive oxygen species (ROS), although its activity was modulated in HYPX-exposed neurons by resveratrol (RSV). The aim of this study was to evaluate if a therapy of RSV can modulate the effect of HYPX in the TRPM2 expressing SH-SY5Y neuronal and HEK293 (no expression of TRPM2) cell lines. The SH-SY5Y and HEK293 cells were divided into four groups as control, RSV (50 μM and 24 hours), and HYPX and RSV + HYPX. For induction of HYPX in the cells, CoCl2 (200 μM and 24 hours) incubation was used. HYPX-induced intracellular Ca2+ responses to TRPM2 activation were increased in the SH-SY5Y cells but not in the HEK293 cells from coming H2O2 and ADPR. RSV treatment improved intracellular Ca2+ responses, mitochondrial function, suppressed the generation of cytokine (IL-1β and TNF-α), cytosolic and mitochondrial ROS in the SH-SY5Y cells. Intracellular free Zn2+, apoptosis, cell death, PARP-1, TRPM2 expression, caspase −3 and −9 levels are increased through activating TRPM2 in the SH-SY5Y cells exposed to the HYPX. However, the values were decreased in the cells by RSV and TRPM2 blockers (ACA and 2-APB). In SH-SY5Y neuronal cells exposed to HYPX conditions, the neuroprotective effects of RSV were shown to be exerted via modulation of oxidative stress, inflammation, apoptosis and death through modulation of TRPM2 channel. RSV could be used as an effective agent in the treatment of neurodegeneration exposure to HYPX.

Icariside II overcomes BRAF inhibitor resistance in melanoma by inducing ROS production and inhibiting MITF.

Metastatic melanoma is the most aggressive skin cancer. Although BRAF inhibitor treatment has achieved great success in melanoma, resistance develops within 12months. IcarisideII (IS), a natural compound extracted from HerbaEpimedii, exerts anticancer properties. In the present study, we determined by MTT, flow cytometry and western blotting, respectively that IS potentiated the PLX4032‑induced downregulation of cell viability and increase in apoptosis and autophagy in BRAF inhibitor‑resistant melanoma. In addition, we also revealed by flow cytometry and western blotting, respectively, that IS combined with PLX4032 increased mitochondrial and intracellular reactive oxygen species (ROS) generation and subsequently promoted depolarization of mitochondria and release of apoptotic proteins. N‑acetyl cysteine (NAC) and glutathione (GSH), ROS scavengers, reversed the IS‑induced enhancement of the response to PLX4032. Microphthalmia‑associated transcription factor (MITF) and tyrosine‑protein kinase Met (c‑Met) are well‑known factors that contribute to BRAF inhibitor resistance. Furthermore, c‑Met is a direct transcriptional target of MITF in melanocytes and melanoma cells. It was also revealed that IS markedly inhibited MITF and c‑Met expression partially by increasing ROS production in BRAF inhibitor‑resistant melanoma cells.

TRAF3 promotes ROS production and pyroptosis by targeting ULK1 ubiquitination in macrophages.

Disrupted mitochondrial function and reactive oxygen species (ROS) generation cause cellular damage and oxidative stress-induced macrophage inflammatory cell death. It remains unclear how mitochondrial dysfunction relates to inflammasome activation and pyroptotic cell death. In this study, we demonstrated that tumor necrosis factor receptor-associated factor 3 (TRAF3) regulates mitochondrial ROS production and promotes TLR agonist LPS plus nigericin (LPS/Ng)-induced inflammasome and pyroptosis in mouse primary macrophages and human monocyte THP-1 cells. Co-IP assays confirmed that TRAF3 forms a complex with TRAF2 and cIAP1 and mediates ubiquitin and degradation of Unc-51 like autophagy activating kinase 1 (ULK1). Moreover, knockdown of ULK1 in THP-1 cells significantly promoted LPS/Ng-induced inflammasome by activating caspase 1 and mature IL-1β. Apoptosis inducing factor (AIF) translocation from mitochondrial to nuclear was observed in ULK1-deficient THP-1 cells under LPS/Ng stimulation, which mediates LPS/Ng-induced cell death in ULK1 deficient macrophages. In conclusion, this study identified a novel role of TRAF3 in regulation of ULK1 ubiquitination and inflammasome signaling and provided molecular mechanisms by which ubiquitination of ULK1 controls mitochondrial ROS production, inflammasome activity, and AIF-dependent pyroptosis.

Astragaloside IV inhibits astrocyte senescence: implication in Parkinson\u2019s disease

BackgroundSenescent astrocytes have been implicated in the aging brain and neurodegenerative disorders, including Parkinson’s disease (PD). Astragaloside IV (AS-IV) is an antioxidant derivative from a traditional Chinese herbal medicine Astragalus membraneaceus Bunge and exerts anti-inflammatory and longevity effects and neuroprotective activities. However, its effect on astrocyte senescence in PD remains to be defined.MethodsLong culture-induced replicative senescence model and lipopolysaccharide/1-methyl-4-phenylpyridinium (LPS/MPP+)-induced premature senescence model and a mouse model of PD were used to investigate the effect of AS-IV on astrocyte senescence in vivo and in vitro. Immunocytochemistry, qPCR, subcellular fractionation, flow cytometric analyses, and immunohistochemistry were subsequently conducted to determine the effects of AS-IV on senescence markers.ResultsWe found that AS-IV inhibited the astrocyte replicative senescence and LPS/MPP+-induced premature senescence, evidenced by decreased senescence-associated β-galactosidase activity and expression of senescence marker p16, and increased nuclear level of lamin B1, and reduced pro-inflammatory senescence-associated secretory phenotype. More importantly, we showed that AS-IV protected against the loss of dopamine neurons and behavioral deficits in the mouse model of PD, which companied by reduced accumulation of senescent astrocytes in substantia nigra compacta. Mechanistically, AS-IV promoted mitophagy, which reduced damaged mitochondria accumulation and mitochondrial reactive oxygen species generation and then contributed to the suppression of astrocyte senescence. The inhibition of autophagy abolished the suppressive effects of AS-IV on astrocyte senescence.ConclusionsOur findings reveal that AS-IV prevents dopaminergic neurodegeneration in PD via inhibition of astrocyte senescence through promoting mitophagy and suggest that AS-IV is a promising therapeutic strategy for the treatment of age-associated neurodegenerative diseases such as PD.

Molecular Mechanism and Functional Role of Hypoxia‐induced Reactive Oxygen Species in Pulmonary Arterial Endothelial Cells

Hypoxia‐induced (Group 3) pulmonary hypertension (PH) is a common devastating lung disease. Pulmonary artery endothelial cells (PAECs) are a major contributor in hypoxic PH due to possible roles of reactive oxygen species (ROS). However, the production up‐and‐down, molecular mechanisms and functional roles of hypoxia‐induced ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H2O2) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed‐derived fluorescence (i.e., H2O2 production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O2−) production. Our assays showed that hypoxia largely increased O2− production. Hypoxia also enhanced H2O2 production in isolated mitochondria from PAECs. In support, using the genetically encoded H2O2 sensor HyPer, we revealed that hypoxia caused a large increase in HyPer‐derived fluorescence (ROS production) in PAECs, which was blocked by the mitochondrial inhibitor rotenone or myxothiazol. Hypoxia largely increased proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. The hypoxic cell proliferation was blocked by rotenone or myxothiazol. Like rotenone and myxothiazol, lentiviral shRNA‐mediated knockdown of Rieske iron‐sulfur protein nicotine (RISP), known to be a primary molecule in mitochondrial ROS generation, inhibited the hypoxic proliferation as well. Hypoxic exposure evoked a significant increase in migration of PAECs, evaluated by wound healing assay. Pharmacological and genetic inhibition of mitochondrial ROS generation with myxothiazol and RISP knockdown both diminished the hypoxic cell migration. Taken together, we conclude that hypoxia significantly increases intracellular ROS generation in human PAECs, the hypoxia‐induced intracellular ROS generation primarily occur in mitochondria, and RISP serves as an essential molecule in controlling the hypoxic mitochondrial ROS generation. Equally Importantly, and RISP‐dependent mitochondrial proliferation that is likely to be ROS‐dependent. Conceivably, hypoxia remarkably causes proliferation and migration of human PAECs, which is predominantly mediated by RISP‐dependent mitochondrial ROS, thereby making an essential contribution to hypoxic pulmonary hypertension.

Impact of oxidative stress on male and female germ cells: implications for fertility.

Male and female germ lines are vulnerable to oxidative stress. In spermatozoa, such stress triggers a lipid peroxidation cascade that culminates in the generation of electrophilic lipid aldehydes that bind to DNA and a raft of proteins involved in the delivery of functionally competent cells. One set of targets for these aldehydes are the proteins of the mitochondrial electron transport chain. When this interaction occurs, mitochondrial ROS generation is enhanced leading to the sustained generation of oxidative damage in a self-perpetuating cycle. Such damage affects all aspects of sperm function including motility, sperm-egg recognition, acrosomal exocytosis and sperm-oocyte fusion. Oxidative stress in the male germ line also attacks the integrity of sperm DNA with potential impacts on the developmental capacity of embryos and the health and wellbeing of the offspring. Potential pathways of reactive oxygen species (ROS) generation in male germ cells could involve enhanced lipoxygenase activity, activation of NADPH oxidase and/or electron leakage from mitochondria. Similarly, in the female germ line, both the induction of oocyte senescence following ovulation and the deterioration of oocyte quality with maternal age appear to involve the generation of oxidative damage. In this case, the mitochondria appear to be a particularly important source of ROS compromising the viability and fertilizability of the oocyte and interfering with the normal segregation of chromosomes during meiosis. In light of these considerations, antioxidants should have some role to play in the preservation of reproductive function in both men and women; however, we still await appropriate trials to test this hypothesis.

Treatment with Momordica charantia Fractions Disrupts Energy Metabolism and Elevated Reactive Oxygen Species’ Generation in Human Lung and Breast Cancer Cell Lines

The mechanism of action of mitochondrial targeted anti‐cancer drugs relies on their ability to disrupt the energy producing systems of cancer cell mitochondria. Momordica charantia (MC), an acclaimed anticancer plant in folk medicine, is here investigated for the probable mechanism by which it kills cancer cells. The ethyl acetate (D3), n‐hexane (D4) and dichloromethane (D5) fractions were obtained from the Crude Water‐Soluble extract (CWSE) of MC via solvent partitioning and then administered on MDA‐MB 436 and A549 cells at 100 and 125 μg/mL for 24 h. Cytofluorometric techniques, using TMRM and H2DCF‐DA as fluorescent probes were employed to assess the effect of MC on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) generation respectively, while ATP levels were measured using the luciferin‐luciferase reaction. Mean respiring cell numbers obtained from Dot graphs and Histograms were further analyzed, yielding significant (P<0.05) MMP depressions only at 125μg/mL in both MDA‐MB 436 (43.08±2.33) and A549 (24.84±2.51) cells treated with D3 in comparison with untreated cells (175.8±6.13 and 131.7±11.21 respectively). In the same vein, only MDA‐MB 436 cells treated with D3 and D5 at 100 and 125 μg/mL significantly elevated ROS levels in comparison with untreated (100.0±2.16) cells; the highest generation being 332.1±3.20, seen in D3‐treated cells at 125 μg/mL. Furthermore, all fractions (D3‐D5) tested on A549 cells at 100μg/mL and 125μg/mL generated significantly higher levels of ROS compared with the untreated (42±4.50) group. The highest observed levels of generated ROS were seen at 125μg/mL in the D5‐treated cells (536±4.00). For intracellular ATP generation, significant (P<0.05) depletion was observed in both MDA‐MB 436 cells [only at 125μg/mL of D3 (80.63±4.70) compared with the control group (106.7±5.69)] and A549 cells [D3 at 125 μg/mL (43.68±0.13) < D5 at 125 μg/mL (52.56±4.16) < D3 at 100 μg/mL (59.84±5.39) compared with the control group (98.57±0.66)]. On the whole, this study suggests that the tested fractions of MC induced a mitochondrial‐dependent cell death in selected cancer cell lines.Support or Funding InformationOrganization for Women in Science for the Developing World (OWSD) and Swedish International Development Cooperation Agency (SIDA)

Role of Mitochondrial Ca2+‐Activated Cl− Channel anoctamin‐1 and mitochondrial fusion protein OPA1 in pulmonary arterial hypertension

Activation of a Ca2+‐activated chloride channel, anoctamin‐1 (Ano1), is associated with enhanced cell proliferation in multiple cell types, such as endothelial cells (ECs). Recent proteomics studies indicate that Ano1 may interact with mitochondrial proteins in addition to plasma membrane (PM)‐localized proteins. However, the expression levels of Ano1 and its functions in other cellular compartments including mitochondria remain unclear. We previously showed that Ano1 is overexpressed in pulmonary artery ECs isolated from patients with idiopathic pulmonary arterial hypertension (PAH) and that Ano1 activation promotes apoptosis of pulmonary artery ECs from patients with idiopathic PAH, likely via depolarization of mitochondrial membrane potential and increased mitochondrial reactive oxygen species (ROS) generation. Therefore, we hypothesized that Ano1 exists not only at PM, but also in the mitochondria, which may contribute to a hyperproliferative phenotype of ECs under PAH. Here, we determined the mitochondrial expression of Ano1 and further investigated its role in the mitochondria. Subcellular localization of Ano1 was determined by immunostaining of Ano1 in Rat lung microvascular ECs (RLMVECs) and by live cell imaging of HEK293T cells expressing GFP‐tagged Ano1. The mitochondrial and PM localization of Ano1 was also confirmed by biochemical assays using protein fractionation from RLMVECs. Moreover, co‐immunoprecipitation assay showed that Ano1 interacts with a mitochondrial fusion protein OPA1 which is the critical regulator of mitochondrial bioenergetics and cristae formation and possesses a trans‐membrane domain at the inner mitochondrial membrane (IMM). These results indicate that 1) Ano1 expresses at the IMM in addition to the PM and 2) Ano1 and OPA1 interactions may be partly involved in the molecular mechanism underlying the hyperproliferation and apoptosis resistance of pulmonary artery ECs during PAH

Selective Toxicity of Ag/TiO2 Nanoparticles of Waste Water of industrial factories on muscle Mitochondria Isolated from Solendactylus scallop

Industrial wastewater is of global concern due to its severe effects on the environment. Compared with municipal wastewater, industrial wastewater generally contains the high concentration of toxic or no biodegradable pollutants. In recently year, scientific showed that scallop could filtration wastewater. Therefore, it was decided to determine the mechanistic toxicity of wastewater contained NPs (Ag and TiO2) towards isolated mitochondria via reliable methods. Isolated muscle scallop mitochondria were obtained by differential ultracentrifugation on before and after exposure to wastewater. Our results showed that two NPs (Ag and TiO2) induced mitochondrial dysfunction via an increase in mitochondrial reactive oxygen species (ROS) generation, lipid peroxidation (LPO) and mitochondrial membrane potential (MMP) collapse. Finally, Ag-NPs and TiO2-NPs have reduced the level of glutathione (GSH) and also induced apoptosis. Our results suggest that wastewater contained NPs -induced toxicity is the result of a disruptive effect on the mitochondrial respiratory chain, increasing the chance of cell death signaling.

Class III PI3‐Kinase in Angiotensin II‐Induced Cardiac Hypertrophy

Pathological cardiac hypertrophy could lead to heart failure and sudden cardiac death. Our previous study demonstrated that Class I PI3‐kinase (PI3KC1) contributes to the development of cardiac hypertrophy by elevating intracellular reactive oxygen species (ROS) levels. In the current study, we evaluated the role of Class III PI3‐kinase (PI3KC3) in Ang II‐induced cardiac hypertrophy. Treatment of cultured cardiomyocytes with Ang II (10−6 M) significantly increased the cellular surface area, which was measured under a fluorescence microscope after staining with anti‐alpha‐sarcomeric actin antibodies. In cardiomyocytes, Ang II (10−9‐10−5 M) induced a dose‐dependent activation of PI3KC3, detected using an ELISA kit. More interestingly, Ang II‐induced cardiac hypertrophy was dramatically exacerbated by co‐treatment with 3‐Methyladenine (3‐MA, 10−6 M), a PI3KC3 inhibitor, suggesting that activation of PI3KC3 may play a protective role in Ang II‐induced cardiac hypertrophy. To identify the intracellular signaling pathway involved in the PI3KC3‐mediated protective effect, mitochondrial ROS was measured using MitoSOX in cardiomyocytes treated with control, Ang II (10−6 M), Ang II plus 3‐MA (10−6 M), and 3‐MA alone. The results demonstrated that Ang II treatment significantly increased mitochondrial ROS production and that Ang II‐induced increases in ROS generation were potentiated by co‐treatment with 3‐MA. It is well known that the major intracellular source of ROS is the damaged mitochondria, which is scavenged by autophagy (also called mitophagy) in cardiomyocytes. Thus, we measured autophagy activity by detection of autophagosomes using staining method with MAP‐LC3 antibodies in cardiomyocytes treated with Ang II. Treatment of cardiomyocytes with Ang II (10−9 to 10−5 M) significantly increased autophagy activity with two phases (increased activity at low doses, decreased activity at high doses, peaked at 10−7 M). More interestingly, Ang II (10−6 M)‐induced increases in autophagy were dramatically attenuated by 3‐MA (10−6 M). Taken together, these data suggest that activation of PI3KCIII has a protective effect on Ang II‐induced hypertrophy by enhancing autophagy activity and decreasing mitochondrial ROS generation in cardiomyocytes.Support or Funding InformationNIH HL143519

Autophagy attenuates high glucose-induced oxidative injury to lens epithelial cells.

Purpose: Autophagic dysfunction and abnormal oxidative stress are associated with cataract. The purpose of the present study was to investigate the changes of cellular autophagy and oxidative stress and their association in lens epithelial cells (LECs) upon exposure to high glucose.Methods: Autophagy and oxidative stress-related changes were detected in streptozotocin-induced Type 1 diabetic mice and normal mouse LECs incubated in high glucose conditions. Rapamycin at a concentration of 100 nm/l or 50 μM chloroquine was combined for analysis of the relationship between autophagy and oxidative stress. The morphology of LECs during autophagy was observed by transmission electron microscopy. The expressions of autophagy markers (LC3B and p62) were identified, as well as the key factors of oxidative stress (SOD2 and CAT) and mitochondrial reactive oxygen species (ROS) generation.Results: Transmission electron microscopy indicated an altered autophagy activity in diabetic mouse lens tissues with larger autophagosomes and multiple mitochondria. Regarding the expressions, LC3B was elevated, p62 was decreased first and then increased, and SOD2 and CAT were increased before a decrease during 4 months of follow-up in diabetic mice and 72 h of culture under high glucose for mouse LECs. Furthermore, rapamycin promoted the expressions of autophagy markers but alleviated those of oxidative stress markers, whereas chloroquine antagonized autophagy but enhanced oxidative stress by elevating ROS generation in LECs exposed to high glucose.Conclusions: The changes in autophagy and oxidative stress were fluctuating in the mouse LECs under constant high glucose conditions. Autophagy might attenuate high glucose-induced oxidative injury to LECs.

Thrombospondin-1 mediates Drp-1 signaling following ischemia reperfusion in the aging heart.

BackgroundIschemia reperfusion (IR) injury leads to activation of dynamin‐related protein (Drp‐1), causing mitochondrial fission and generation of reactive oxygen species (ROS), but the molecular mechanisms that activate Drp‐1 are not known. The purpose of this study was to establish a link between Thbs‐1 and fission protein (Drp‐1) through Pgc‐1α following IR in advancing age.MethodsFemale Fischer‐344 rats were divided into four groups: Young Control, Young + IR, Old Control, and Old + IR. Heart function and coronary flow were evaluated at baseline and 72 hours after IR, hearts were explanted and mitochondrial ROS generation was measured using MitoPY1, as well as protein levels of Thbs‐1, Pgc‐1α, and Drp‐1. In vitro, rat aortic endothelial cells (RAEC) were treated with siRNA or plasmid for Pgc‐1α to evaluate Pgc‐1α effect on Drp‐1.ResultsMitochondrial ROS generation in heart tissue increased in both age groups following IR. Old animals exhibited diastolic dysfunction at baseline; after IR they displayed reduced systolic function and exacerbated diastolic dysfunction compared to young controls. IR increased Thbs‐1 and Drp‐1 expression in young and old hearts compared to control. siRNA to Pgc‐1α enhanced levels of Drp‐1 in RAECs and increased ROS generation after hypoxia, while Pgc‐1α plasmid ameliorates Drp‐1 expression in the presence of exogenous Thbs‐1.ConclusionThese results highlight a novel signaling pathway by which Thbs‐1 regulates mitochondrial fission protein (Drp‐1) and ROS generation during hypoxia, and presumably, following IR. Inhibiting Thbs‐1 immediately after IR may prevent Drp‐1‐mediated mitochondrial fission and is likely to improve the diastolic function of the heart by reducing ROS‐mediated cardiomyocyte damage in the aged population.

Evaluation of cytotoxicity, apoptosis, and genotoxicity induced by indium chloride in macrophages through mitochondrial dysfunction and reactive oxygen species generation.

Due to rapid advances in the era of electronic technologies, indium has played the important material for the production of liquid crystal display screens in the semiconductor and optoelectronic industries. The present study focuses on evaluating the toxic effects and related mechanisms of indium chloride (InCl3) on RAW264.7 macrophages. Cytotoxicity was induced by InCl3 in a concentration- and time-dependent manner. InCl3 had the ability to induce macrophage death through apoptosis rather than through necrosis. According to the cytokinesis-block micronucleus assay and alkaline single-cell gel electrophoresis assay, InCl3 induced DNA damage, also called genotoxicity, in a concentration-dependent manner. Cysteine-dependent aspartate-directed protease (caspase)-3, -8, and -9 were activated by InCl3 in a concentration-dependent manner. Mitochondria dysfunction and cytochrome c release from the mitochondria were induced by InCl3 in a concentration-dependent manner. Downregulation of BCL2 and upregulation of BAD were induced by InCl3 in a concentration-dependent manner. More, we proposed that InCl3 treatment generated reactive oxygen species (ROS) in a concentration-dependent manner. In conclusion, the current study revealed that InCl3 induced macrophage cytotoxicity, apoptosis, and genotoxicity via a mitochondria-dependent apoptotic pathway and ROS generation.

Trans-Fatty acids facilitate DNA damage-induced apoptosis through the mitochondrial JNK-Sab-ROS positive feedback loop

trans-Fatty acids (TFAs) are unsaturated fatty acids that contain one or more carbon-carbon double bonds in trans configuration. Epidemiological evidence has linked TFA consumption with various disorders, including cardiovascular diseases. However, the underlying pathological mechanisms are largely unknown. Here, we show a novel toxic mechanism of TFAs triggered by DNA damage. We found that elaidic acid (EA) and linoelaidic acid, major TFAs produced during industrial food manufacturing (so-called as industrial TFAs), but not their corresponding cis isomers, facilitated apoptosis induced by doxorubicin. Consistently, EA enhanced UV-induced embryonic lethality in C. elegans worms. The pro-apoptotic action of EA was blocked by knocking down Sab, a c-Jun N-terminal kinase (JNK)-interacting protein localizing at mitochondrial outer membrane, which mediates mutual amplification of mitochondrial reactive oxygen species (ROS) generation and JNK activation. EA enhanced doxorubicin-induced mitochondrial ROS generation and JNK activation, both of which were suppressed by Sab knockdown and pharmacological inhibition of either mitochondrial ROS generation, JNK, or Src-homology 2 domain-containing protein tyrosine phosphatase 1 (SHP1) as a Sab-associated protein. These results demonstrate that in response to DNA damage, TFAs drive the mitochondrial JNK-Sab-ROS positive feedback loop and ultimately apoptosis, which may provide insight into the common pathogenetic mechanisms of diverse TFA-related disorders.