Well-known Reactive Oxygen Species Research Articles

Imaging peroxynitrite in endoplasmic reticulum stress and acute lung injury with a near-infrared fluorescent probe

BackgroundThe cellular endoplasmic reticulum (ER) is responsible for various functions, including protein synthesis, folding, distribution, and calcium ion storage. Studies have linked ER stress with acute lung injury (ALI), which can result in oxidative stress and even cell death. Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) that contributes to various physiological and pathological processes in oxidative stress diseases. To understand the role of ER ONOO− in ALI, it is crucial to accurately measure its level in the ER. Unfortunately, there is currently no probe available to detect ER ONOO− in an ALI model. ResultsTo address this, we developed three near-infrared (NIR) fluorescent probes (DCM-F-ONOO, DCM-Cl-ONOO, and DCM-Br-ONOO) for the detection of ONOO− using pentafluorobenzenesulfonate (PFBS) moieties as fluorescence quenchers. Through comprehensive testing, we selected DCM-Br-ONOO as the best NIR fluorescent probe due to its rapid response (within 3 min), high selectivity, good sensitivity (LOD = 2.3 nM), and approximately 66-fold enhanced response to ONOO− in fluorescence intensity. The probe was successfully applied to detect changes in ONOO− levels induced by different drugs in the ER of living cells. Importantly, a significant increase in the level of ONOO− was observed in the ER of an ALI cell model (4.5-fold) and an ALI mouse model (2.5-fold) using the probe, which is essential for understanding the role of ONOO− in ER-associated diseases. SignificanceUsing DCM-Br-ONOO as a probe, present work further validated that the elevated levels of ONOO− secretion were accompanied by the ALI progressed. These findings may provide valuable results for figuring out the biological roles that ONOO− played in ALI.

Neuroprotective effect and possible mechanism of edaravone in rat models of spinal cord injury: a protocol for a systematic review and meta-analysis

BackgroundSpinal cord injury (SCI) is one of the most disabling neurological conditions, afflicting thousands of human beings. Edaravone, a well-known reactive oxygen species scavenger, is expanding its new scope in field of SCI. The objective of this systematic review is to determine the neuroprotective effects and discuss the underlying mechanism of edaravone in management of SCI.MethodsThe systematic review will include the controlled studies evaluating the neurological roles of edaravone on experiment rat models following SCI. The primary outcome will be the 21-point Basso, Beattie, and Bresnahan locomotor rating scale. The secondary outcomes will include the preservation of white matter areas and malondialdehyde levels. Two researchers will independently search PubMed, Embase, Web of Science, Scopus and Cochrane Library from their inception date. Following study selection, data extraction, and assessment of methodological quality in included studies using the SYRCLE’s RoB tool, data from eligible studies will be pooled and analyzed using random-effects models with RevMan 5.3 software. In case of sufficient data, subgroup analyses with respect to species, age, gender, injury characteristics, or administration details will be carried out to explore the factors modifying efficacy of edaravone. For exploring the appropriate dose of edaravone, a network meta-analysis approach will be conducted based on the Bayesian method. Importantly, the proposed mechanisms and changes of related molecules will be also extracted from included studies for comprehensively investigating the mechanisms underlying the neuroprotective effects of edaravone.DiscussionIn this study, we aim to quantitatively analyze the role of edaravone in locomotor recovery and tissue damage in SCI rat model. The efficacy of edaravone in distinct scenarios will be investigated by subgroup analyses, and we expect to predict the candidate dose that offers a superior treatment effect using network meta-analyses. Moreover, a comprehensive framework regarding the neuroprotective mechanisms behind edaravone will be constructed via a combination of systematic and traditional review. This study will bring implications for future preclinical studies and clinical applications of SCI. Nonetheless, in light of the anticipated limitations in animal experimental design and methodological quality, the results in this review should be interpreted with caution.

Evaluation of the Growth-Inhibitory Spectrum of Three Types of Cyanoacrylate Nanoparticles on Gram-Positive and Gram-Negative Bacteria.

The development of novel effective antibacterial agents is crucial due to increasing antibiotic resistance in various bacteria. Poly (alkyl cyanoacrylate) nanoparticles (PACA-NPs) are promising novel antibacterial agents as they have shown antibacterial activity against several Gram-positive and Gram-negative bacteria. However, the antibacterial mechanism remains unclear. Here, we compared the antibacterial efficacy of ethyl cyanoacrylate nanoparticles (ECA-NPs), isobutyl cyanoacrylate NPs (iBCA-NPs), and ethoxyethyl cyanoacrylate NPs (EECA-NPs) using five Gram-positive and five Gram-negative bacteria. Among these resin nanoparticles, ECA-NPs showed the highest growth inhibitory effect against all the examined bacterial species, and this effect was higher against Gram-positive bacteria than Gram-negative. While iBCA-NP could inhibit the cell growth only in two Gram-positive bacteria, i.e., Bacillus subtilis and Staphylococcus aureus, it had negligible inhibitory effect against all five Gram-negative bacteria examined. Irrespective of the differences in growth inhibition induced by these three NPs, N-acetyl-L-cysteine (NAC), a well-known reactive oxygen species (ROS) scavenger, efficiently restored growth in all the bacterial strains to that similar to untreated cells. This strongly suggests that the exposure to NPs generates ROS, which mainly induces cell growth inhibition irrespective of the difference in bacterial species and cyanoacrylate NPs used.

Apelinergic System: an in‐vitro Investigation of Inflammatory & Oxidative Stress in Chronic Kidney Disease

Over 37 million US adults have Chronic Kidney Disease (CKD). Of that population–35% are Black. With 4x higher predisposition to developing renal failure due to comorbidities (i.e. hypertension and diabetes), there is a dire need to address both preventative and therapeutic options in this population. Besides dialysis and transplant as reactionary treatments of CKD, vitamin E—an antioxidant—has been given to those in late stage CKD. Our study aims to address mechanisms behind CKD pathology. The 5/6 nephrectomy (5/6Nx) rat CKD model provides the opportunity to isolate the effects of impaired renal function without significant confounding comorbidities. A hallmark feature of both rat and human CKD is adverse vascular remodeling which is often preceded by endothelial cell (EC) dysfunction. With respect to EC dysfunction, inflammatory and oxidative imbalances are suspected to be involved. Our previous transcriptomic analysis of kidney tissues from the 5/6Nx rat identified a significant increase in apelin receptor (APLNR) expression compared to sham controls. In addition, the data revealed several pro‐inflammatory cytokines (IL‐6 and IL‐1β) preceding APLNR transcript increases. Treatment of Normal Rat Kidney epithelial (NRK) cells with IL‐6 caused a significant reduction in apelin (APLN) ligand, while IL‐1β augmented APLNR transcript levels. After further study of the 3 pathways, a common downstream target, protein kinase B (AKT), arose. Interestingly enough, AKT is responsible for various vascular cascades including interaction with well‐known reactive oxygen species (ROS) producer–NADPH Oxidase 4 (NOX4). Hydrogen Peroxide (H2O2), a ROS product of NOX4, coordinates vasoactivity through AKT signaling and has overlapping function with apelinergic (APJ) system in the vasculature. Due to the inflammatory and oxidative nature of CKD, we want to address the relationship between H2O2 and the APJ system. We hypothesize that pretreating NRK cells with APLN prior to administration of H2O2 will have 2 effects: oxidant attenuation and antioxidant augmentation. NRK cells were pretreated with 2.5ng/mL apelin or 30mg/mL vitamin E 48‐hours prior to addition of 20mM H2O2. At study termination, cells were collected/quantified using the Bradford protein assay. Protein expression was measured via Western blots against catalase, an antioxidant marker, and GPx, an oxidant protein. One‐Way ANOVA and Tukey Post‐Hoc test were used to establish statistical significance (p<0.05). Catalase: no significant expression changes after APLN or vitamin E treat (p = 0.10), but significant increases after H2O2 treat (p = 0.05) compared to control. GPx: significant increases in expression after APLN treat compared to [control (p=0.0002), vitamin E (p=0.04), and H2O2 + APLN (p=0.0001)] and vitamin E compared to H2O2 + Vitamin E treat (p=0.01). Data presented as mean ± SEM (n=3). These studies assessed functionality of the APJ system in an in‐vitro model of CKD. Both of the inflammatory cytokines are regulators of APJ system transcription, which is relevant for patients in a chronic inflammatory state. These studies also highlight the interaction between APJ system and oxidative stressors. Results here begin to lay the foundation for future studies involving the APJ system as a viable target for assessing CKD pathology.

Reactive Oxygen Species Detection Using Fluorescence in Enchytraeus crypticus-Method Implementation through Ag NM300K Case Study.

An imbalance between reactive oxygen species (ROS) and antioxidants in a living organism results in oxidative stress. Measures of such imbalance can be used as a biomarker of stress in ecotoxicology. In this study, we implemented the ROS detection method based on the oxidant-sensing probe dichloro-dihydro-fluorescein diacetate (DCFH-DA), detected by fluorescence microscopy, in Enchytraeus crypticus adults and cocoons, i.e., also covering the embryo stage. Hydrogen peroxide (H2O2), a well-known ROS inducer, was used both to optimize the method and as positive control. Implementation was successful, and the method was used to assess ROS formation in E. crypticus cocoons and adults when exposed to the reference silver nanomaterial Ag NM300K, at two effect concentrations (EC20 and EC50) for both hatching and reproduction over 3 and 7 days. The measured ROS levels varied with time, concentration, and developmental stage, with higher levels detected in adults compared with cocoons. In cocoons, ROS levels were higher at the EC20 than the EC50, which could be explained by non-monotonic concentration-response curve for hatching and reproduction, as previously observed. The increase in ROS levels at day 3 preceded the oxidative damage, as reported to occur later (day 7) in adults. The DCFH-DA method was successfully implemented here and can be further used as a new tool to detect ROS formation in E. crypticus, especially after short-term exposure to chemicals, including nanomaterials. We recommend the use of 3 and 7 days in the exposure design for this assessment.

ASSESSMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND PEROXIDASE ACTIVITIES IN ASPERGILLUS SP. AND CLADOSPORIUM SP.

Antioxidative enzymes are a family of powerful free radical scavenging proteins, naturally synthesized by all aerobic organisms. Family of antioxidant enzymes include superoxide dismutase (SOD), catalase (CAT), peroxidase (PRX), thioredoxins /thioredoxin reductase (TrxR), and glutaredoxins/glutathione reductase (GPx). These oxidative enzymatic machineries, sequentially sequester singlet oxygen (O2 −) and Hydrogen Peroxide, which are the well-known reactive oxygen species. These enzymes can be intracellular as well as extracellular. In the present study, two most common soil fungi namely, Aspergillus Sp. and Cladosporium Sp. were cultivated by submerged cultivation on Potato dextrose broth and pellets were separated by filtration. Pellets were disrupted by detergent lysis method (0.5% Triton X 100, 1% CTAB, 1.5% Tween 80, 5% SDS). Culture media and cellular extracts were analyzed for intracellular and extracellular total proteins and their panel of antioxidative enzymes. Enzyme activity of SOD was carried out by Beauchamp and Fridovich method. Catalase and Peroxidase activities were determined by simple spectrophotometric method. In Aspergillus Sp.1.33 U of superoxide dismutase, 0.115 U catalase, 0.00097 U peroxidase activities per mg of protein was observed. Similarly, in Cladosporium Sp. 1.38 U Superoxide dismutase, 0.1460 U catalase, and 0.00150 U peroxidase activities per mg protein was recorded. Significant amount of antioxidative enzyme activity observed in both the test cultures. And comparatively, Cladosporium Sp has given promising results. Probably, with altered culture conditions and improved extraction procedures, still higher amounts of antioxidative enzymes would be expected.

Remodeling the periodontitis microenvironment for osteogenesis by using a reactive oxygen species-cleavable nanoplatform

Modestly removing the excessive reactive oxygen species (ROS) plays a crucial role in regulating the microenvironment of periodontitis and provides favorable conditions for osteogenesis. However, the current strategy for scavenging ROS is not controllable, substantially limiting the outcomes in periodontitis. Herein, we introduced a controllable ROS-scavenging nanoplatform by encasing N-acetylcysteine (NAC, (a well-known ROS scavenger) into tailor-made ROS-cleavable amphiphilic polymer nanoparticles (PEG-ss-PCL NPs) as an intracellular delivery carrier. The existing ROS in the inflammatory microenvironment facilitated polymer degradation via breakage of thioketal bonds, and then led to encapsulated NAC release. NAC eliminated all ROS induced by lipopolysaccharide (LPS), while PssL-NAC adjusted the ROS level slightly higher than that of the control group. The percentage of apoptotic cells cultured with NAC and PssL-NAC decreased observably compared with that of cells cultured with 10 µg/ml LPS. The microenvironment regulated by PssL-NAC was highly suitable for osteogenic differentiation based on PCR and Western blot results, which showed higher expression levels of BMP2, Runx2, and PKA. Analysis of ALP activity and Alizarin red S staining showed consistent results. Additionally, the injection of PssL-NAC into the periodontitis area could alleviate the tissue destruction induced by ligation of the maxillary second molar. PssL-NAC showed a better ability to decrease osteoclast activity and inflammation, consequently improving the restoration of destroyed tissue. Our study suggests that ROS-responsive polymer nanoparticles loaded with NAC (PssL-NAC) can be new promising materials for the treatment of periodontitis. Statement of significanceMore and more studies indicate that periodontal tissue damage is closely related to the high reactive oxygen species (ROS) environment. Excessive ROS will aggravate periodontal tissue damage and is not conducive to tissue repair. However, as an essential signal molecule in human physiological activities, ROS absence is also useless for tissue repair. In this study, we proposed to improve ROS imbalance in the environment of periodontitis as a strategy to promote periodontal regeneration and successfully synthesized a smart drug-releasing nanoplatform that can respond to ROS. Besides, we validated its ability to regulate the ROS environment and promote osteogenesis through experimental data in vivo and in vitro.

Triggered Release from Cellulose Microparticles Inspired by Wood Degradation by Fungi

Triggered compound release has attracted considerable attention in recent years. However, the concepts and carriers reported for triggered release are often complex while nonapproved ingredients are mostly used to make the carriers. In this study, a bioinspired reactive oxygen species (ROS)-mediated delivery system, triggered by light, is introduced. We took inspiration from the well-known ROS mediated degradation of wood (cellulose) by brown-rot fungi and designed microparticles which consist of a core of cellulose nanocrystals, loaded with a drug, surrounded by a light sensitive shell. Incorporating indocyanine green, an FDA-approved photosensitizer, in the shell of the microparticles generates ROS upon NIR laser irradiation, which degrades the cellulose nanocrystals in the microparticles. It was revealed that doxorubicin, encapsulated in the cellulose core and used as a model compound in our study, becomes specifically released upon NIR irradiation of the microparticles. Attractive as well is that the microparticles are prepared by a simple one-step gas-shearing process and fully consist of compounds generally regarded as safe (GRAS).

An ICT-based fluorescent probe for ratiometric monitoring the fluctuations of peroxynitrite in mitochondria

Peroxynitrite (ONOO−) is a well-known reactive oxygen species (ROS) which closely involves in various physiological and pathological processes. It is reported that mitochondria are the major site of ONOO− production. Normal level of ONOO− benefits mitochondria biological functions, while its overproduction will induce oxidative stress, resulting in an imbalance of mitochondria homeostasis. Therefore, accurate monitoring of ONOO− in mitochondria will be beneficial for elucidating the physiological metabolism and fluctuations of ONOO−. Herein, a novel mitochondria-targeted ratiometric fluorescent probe Mito-NA for ONOO− was designed rationally by regulating its intramolecular charge transfer (ICT) effect. Once reaction with ONOO−, Mito-NA manifested remarkable ratiometric fluorescence changes (18-fold) with a large red-shift (∼104 nm) emission, which was ascribed to the enhanced ICT process caused by the elimination of the electron-withdrawing recognition group 3-(trifluoromethyl) cinnamic acid and the exposure of electron-donating hydroxyl on Mito-NA structure. Furthermore, Mito-NA exhibited satisfactory performances for ONOO− such as good sensitivity (LOD =0.12 μM), high selectivity to ONOO− over other ROS, fast response and good mitochondrial targeting. More importantly, Mito-NA was successfully applied for ratiometric imaging and monitoring the fluctuations of mitochondrial ONOO− in living cells.

Micheliolide Attenuates Lipopolysaccharide-Induced Inflammation by Modulating the mROS/NF-κB/NLRP3 Axis in Renal Tubular Epithelial Cells.

Chronic kidney disease is a common disease closely related to renal tubular inflammation and oxidative stress, and no effective treatment is available. Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is an important factor in renal inflammation, but the mechanism remains unclear. Micheliolide (MCL), which is derived from parthenolide, is a new compound with antioxidative and anti-inflammatory effects and has multiple roles in tumors and inflammatory diseases. In this study, we investigated the effect of MCL on lipopolysaccharide- (LPS-) induced inflammation in renal tubular cells and the related mechanism. We found that MCL significantly suppressed the LPS-induced NF-κB signaling and inflammatory expression of cytokines, such as tumor necrosis factor-α and monocyte chemoattractant protein-1 in a rat renal proximal tubular cell line (NRK-52E). MCL also prevented LPS- and adenosine triphosphate-induced NLRP3 inflammasome activation in vitro, as evidenced by the inhibition of NLRP3 expression, caspase-1 cleavage, and interleukin-1β and interleukin-18 maturation and secretion. Additionally, MCL inhibited the reduction of mitochondrial membrane potential and decreases the release of reactive oxygen species (ROS). Moreover, MCL can prevent NLRP3 inflammasome activation induced by rotenone, a well-known mitochondrial ROS (mROS) agonist, indicating that the mechanism of MCL's anti-inflammatory effect may be closely related to the mROS. In conclusion, our study indicates that MCL can inhibit LPS-induced renal inflammation through suppressing the mROS/NF-κB/NLRP3 axis in tubular epithelial cells.

Klebsiella pneumoniae ST258 Negatively Regulates the Oxidative Burst in Human Neutrophils.

The epidemic clone of Klebsiella pneumoniae (Kpn), sequence type 258 (ST258), carbapenamase producer (KPC), commonly infects hospitalized patients that are left with scarce therapeutic option since carbapenems are last resort antibiotics for life-threatening bacterial infections. To improve prevention and treatment, we should better understand the biology of Kpn KPC ST258 infections. Our hypothesis was that Kpn KPC ST258 evade the first line of defense of innate immunity, the polymorphonuclear neutrophil (PMN), by decreasing its functional response. Therefore, our aim was to evaluate how the ST258 Kpn clone affects PMN responses, focusing on the respiratory burst, compared to another opportunistic pathogen, Escherichia coli (Eco). We found that Kpn KPC ST258 was unable to trigger bactericidal responses as reactive oxygen species (ROS) generation and NETosis, compared to the high induction observed with Eco, but both bacterial strains were similarly phagocytized and cause increases in cell size and CD11b expression. The absence of ROS induction was also observed with other Kpn ST258 strains negative for KPC. These results reflect certain selectivity in terms of the functions that are triggered in PMN by Kpn, which seems to evade specifically those responses critical for bacterial survival. In this sense, bactericidal mechanisms evasion was associated with a higher survival of Kpn KPC ST258 compared to Eco. To investigate the mechanisms and molecules involved in ROS inhibition, we used bacterial extracts (BE) and found that BE were able to inhibit ROS generation triggered by the well-known ROS inducer, fMLP. A sequence of experiments led us to elucidate that the polysaccharide part of LPS was responsible for this inhibition, whereas lipid A mediated the other responses that were not affected by bacteria, such as cell size increase and CD11b up-regulation. In conclusion, we unraveled a mechanism of immune evasion of Kpn KPC ST258, which may contribute to design more effective strategies for the treatment of these multi-resistant bacterial infections.

Cell-penetrating peptide conjugates of gambogic acid enhance the antitumor effect on human bladder cancer EJ cells through ROS-mediated apoptosis.

BackgroundGambogic acid (GA) is the main active ingredient of resin gamboges and possesses anti-cancer activity toward various human cancer cells. However, clinical application of GA has been limited by its poor aqueous solubility and dose-limiting toxicities. Cell-penetrating peptides (CPPs) are widely used to deliver anti-cancer drugs into cancer cells and to enhance the water solubility of drugs.PurposeThe object of this study was to synthesize peptide-drug conjugates in which the cell-penetrating peptide TAT (trans-activator of transcription) was conjugated to GA and evaluated the anti-cancer activity of this GA-CPP conjugate (GA-TAT) in EJ bladder cancer cells.MethodsGA is built onto the TAT, and the GA-TAT conjugates are cleaved from the solid support and purified via HPLC. The equilibrium solubility of GA-TAT was measured using the shake-flask method. The effects of GA-TAT on EJ cell viability and proliferation were determined by MTT assay, Edu assay and colony formation assay, respectively. After treated with 1.0 μM GA-TAT for 24 h, the apoptosis rate of EJ cells were detected by Acridine orange/ethidium bromide (AO/EB) assay and flow cytometry assay. The proteins of caspase-3 (processing), caspase-9 (processing), Bcl-2 and Bax were analyzed by Western blotting, and the intracellular reactive oxygen species (ROS) production was evaluated by a reactive oxygen species assay.ResultsIn contrast to free GA, the solubility of GA-TAT in water was significantly improved. Meanwhile, GA-TAT significantly increased EJ cellular uptake, toxicity and apoptosis. Mechanistic analysis revealed that GA-TAT enhanced the anti-cancer effect of GA against EJ cells through ROS-mediated apoptosis. The results were demonstrated that GA-TAT increased the ROS level in EJ cells, and N-acetyl-L-cysteine (NAC; a well-known ROS scavenger) inhibited GA-TAT-induced ROS generation and apoptosis. Additionally, GA-TAT activated caspase-3 and caspase-9 and down-regulated the Bcl-2/Bax ratio, but these effects were largely rescued by NAC.ConclusionGA-TAT has outstanding potential for promoting tumor apoptosis and exhibits promise for use in bladder cancer therapy.

α-Syntrophin stabilizes catalase to reduce endogenous reactive oxygen species levels during myoblast differentiation.

α-Syntrophin is a component of the dystrophin-glycoprotein complex that interacts with various intracellular signaling proteins in muscle cells. The α-syntrophin knock-down C2 cell line (SNKD), established by infecting lentivirus particles with α-syntrophin shRNA, is characterized by a defect in terminal differentiation and increase in cell death. Since myoblast differentiation is accompanied by intensive mitochondrial biogenesis, the generation of intracellular reactive oxygen species (ROS) is also increased during myogenesis. Two-photon microscopy imaging showed that excessive intracellular ROS accumulated during the differentiation of SNKD cells as compared with control cells. The formation of 4-hydroxynonenal adduct, a byproduct of lipid peroxidation during oxidative stress, significantly increased in differentiated SNKD myotubes and was dramatically reduced by epigallocatechin-3-gallate, a well-known ROS scavenger. Among antioxidant enzymes, catalase was significantly decreased during differentiation of SNKD cells without changes at the mRNA level. Of interest was the finding that the degradation of catalase was rescued by MG132, a proteasome inhibitor, in the SNKD cells. This study demonstrates a novel function of α-syntrophin. This protein plays an important role in the regulation of oxidative stress from endogenously generated ROS during myoblast differentiation by modulating the protein stability of catalase.

Etoposide induced cytotoxicity mediated by ROS and ERK in human kidney proximal tubule cells.

Etoposide (ETO) is a commonly used chemotherapeutic drug that inhibits topoisomerase II activity, thereby leading to genotoxicity and cytotoxicity. However, ETO has limited application due to its side effects on normal organs, especially the kidney. Here, we report the mechanism of ETO-induced cytotoxicity progression in human kidney proximal tubule (HK-2) cells. Our results show that ETO perpetuates DNA damage, activates mitogen-activated protein kinase (MAPK), and triggers morphological changes, such as cell and nuclear swelling. When NAC, a well-known reactive oxygen species (ROS) scavenger, is co-treated with ETO, it inhibits an ETO-induced increase in mitochondrial mass, mitochondrial DNA (ND1 and ND4) copy number, intracellular ATP level, and mitochondrial biogenesis activators (TFAM, PGC-1α and PGC-1β). Moreover, co-treatment with ETO and NAC inhibits ETO-induced necrosis and cell swelling, but not apoptosis. Studies using MAPK inhibitors reveal that inhibition of extracellular signal regulated kinase (ERK) protects ETO-induced cytotoxicity by inhibiting DNA damage and caspase 3/7 activity. Eventually, ERK inhibitor treated cells are protected from ETO-induced nuclear envelope (NE) rupture and DNA leakage through inhibition of caspase activity. Taken together, these data suggest that ETO mediates cytotoxicity in HK-2 cells through ROS and ERK pathways, which highlight the preventive avenues in ETO-induced cytotoxicity in kidney.

Helicobacter pylori induces Snail expression through ROS-mediated activation of Erk and inactivation of GSK-3β in human gastric cancer cells.

Helicobacter pylori (H. pylori) infection has been known to be implicated in human gastric carcinogenesis. Snail, the zinc-finger transcription factor known as a key inducer of changes in the cell shape and morphogenetic movement, is aberrantly overexpressed and correlates with lymph node metastasis in gastric cancer. In the present study, we investigated whether H. pylori could induce Snail activation to provoke these changes. Using a cell scatter assay, we noticed that human gastric cancer AGS cells infected with H. pylori underwent morphological changes as well as disruption of cell-cell interaction, which was then reversed by silencing of Snail by use of small interfering RNA (siRNA). In addition, infection with H. pylori resulted in an increased intracellular level of Snail in gastric cancer cells, which was abrogated in the presence of U0126 and LY294002, inhibitors of MEK/Erk and PI3K/Akt pathways, respectively. Cycloheximide pulse-chase experiments coupled with immunocytochemical analysis revealed that the induction of Snail by H. pylori was regulated at multiple levels, including increased transcription of Snail mRNA, inhibition of protein degradation, and enhancement of nuclear translocation of Snail. Pre-treatment of AGS cells with N-acetylcysteine, a well-known reactive oxygen species (ROS) scavenger, attenuated the H. pylori-induced activation of Erk, its binding to Snail promoter, inactivation of GSK-3β, and accumulation of Snail. Collectively, these findings suggest that the upregulation of Snail expression induced by H. pylori and transformation to a spindle-like shape as a consequence in gastric cancer cells are attributable to ROS-mediated activation of Erk and the inhibition of GSK-3β signaling. © 2016 Wiley Periodicals, Inc.

Induction of Activating Transcription Factor 6 via Activation of ERK and ROS-P38 MAPK is Related to Methylglyoxal-Induced Cytotoxicity in Human Retinal Pigment Epithelial Cells

Methylglyoxal (MGO), a reactive α-oxoaldehyde produced by glucose metabolism, is elevated in several diabetic complications, including diabetic retinopathy. The breakdown of retinal pigment epithelial cells is implicated in the progression of diabetic retinopathy. Increased concentrations of MGO lead to retinal pigment epithelial cell death. In this study, we investigated the involvement of activating transcription factor 6 (ATF6) in MGO-mediated cytotoxicity in ARPE-19 cells. In response to high concentrations of MGO, unfolded protein response-related ATF6 was induced. Interestingly, the MGO also induced the generation of reactive oxygen species (ROS) and the phosphorylation of ERK and p38 MAPK. The induction of ATF6 was inhibited by ERK-specific and p38 MAPK-specific inhibitors (U0126 and SB202190) and by NAC, a well-known ROS scavenger. NAC also attenuated the phosphorylation of p38 MAPK. MGO induced cytotoxicity in the ARPE-19 cells, which was ameliorated by the inhibition of the ERK, p38 MAPK, and ROS pathways. Furthermore, the MGO-mediated cytotoxicity was inhibited by ATF6 siRNA. Taken together, these results clearly show that the induction of ATF6 via the ERK and ROS-p38 MAPK pathways is implicated in MGO-induced cytotoxicity in ARPE-19 cells.

Pro-Oxidant Activity of Amine-Pyridine-Based Iron Complexes Efficiently Kills Cancer and Cancer Stem-Like Cells.

Differential redox homeostasis in normal and malignant cells suggests that pro-oxidant-induced upregulation of cellular reactive oxygen species (ROS) should selectively target cancer cells without compromising the viability of untransformed cells. Consequently, a pro-oxidant deviation well-tolerated by nonmalignant cells might rapidly reach a cell-death threshold in malignant cells already at a high setpoint of constitutive oxidative stress. To test this hypothesis, we took advantage of a selected number of amine-pyridine-based Fe(II) complexes that operate as efficient and robust oxidation catalysts of organic substrates upon reaction with peroxides. Five of these Fe(II)-complexes and the corresponding aminopyridine ligands were selected to evaluate their anticancer properties. We found that the iron complexes failed to display any relevant activity, while the corresponding ligands exhibited significant antiproliferative activity. Among the ligands, none of which were hemolytic, compounds 1, 2 and 5 were cytotoxic in the low micromolar range against a panel of molecularly diverse human cancer cell lines. Importantly, the cytotoxic activity profile of some compounds remained unaltered in epithelial-to-mesenchymal (EMT)-induced stable populations of cancer stem-like cells, which acquired resistance to the well-known ROS inducer doxorubicin. Compounds 1, 2 and 5 inhibited the clonogenicity of cancer cells and induced apoptotic cell death accompanied by caspase 3/7 activation. Flow cytometry analyses indicated that ligands were strong inducers of oxidative stress, leading to a 7-fold increase in intracellular ROS levels. ROS induction was associated with their ability to bind intracellular iron and generate active coordination complexes inside of cells. In contrast, extracellular complexation of iron inhibited the activity of the ligands. Iron complexes showed a high proficiency to cleave DNA through oxidative-dependent mechanisms, suggesting a likely mechanism of cytotoxicity. In summary, we report that, upon chelation of intracellular iron, the pro-oxidant activity of amine-pyrimidine-based iron complexes efficiently kills cancer and cancer stem-like cells, thus providing functional evidence for an efficient family of redox-directed anti-cancer metallodrugs.

Hydrogen-rich saline inhibits NLRP3 inflammasome activation and attenuates experimental acute pancreatitis in mice.

Increasing evidence has demonstrated that reactive oxygen species (ROS) induces oxidative stress and plays a crucial role in the pathogenesis of acute pancreatitis (AP). Hydrogen-rich saline (HRS), a well-known ROS scavenger, has been shown to possess therapeutic benefit on AP in many animal experiments. Recent findings have indicated that the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome, an intracellular multiprotein complex required for the maturation of interleukin- (IL-) 1β, may probably be a potential target of HRS in the treatment of AP. Therefore, in this study, we evaluated the activation of NLRP3 inflammasome and meanwhile assessed the degree of oxidative stress and inflammatory cascades, as well as the histological alterations in mice suffering from cerulein-induced AP after the treatment of HRS. The results showed that the activation of NLRP3 inflammasome in AP mice was substantially inhibited following the administration of HRS, which was paralleled with the decreased NF-κB activity and cytokines production, attenuated oxidative stress and the amelioration of pancreatic tissue damage. In conclusion, our study has, for the first time, revealed that inhibition of the activation of NLRP3 inflammasome probably contributed to the therapeutic potential of HRS in AP.

Uric acid induces NADPH oxidase-independent neutrophil extracellular trap formation

Neutrophil extracellular traps (NETs) are composed of extracellular DNA fibers with antimicrobial peptides that capture and kill microbes. NETs play a critical role in innate host defense and in autoimmune and inflammatory diseases. While the mechanism of NET formation remains unclear, reactive oxygen species (ROS) produced via activation of NADPH oxidase (Nox) are known to be an important requirement. In this study, we investigated the effect of uric acid (UA) on NET formation. UA, a well-known ROS scavenger, was found to suppress Nox-dependent ROS release in a dose-dependent manner. Low concentrations of UA significantly inhibited Nox-dependent NET formation. However, high concentrations of UA unexpectedly induced, rather than inhibited, NET formation. NETs were directly induced by UA alone in a Nox-independent manner, as revealed by experiments using control neutrophils treated with ROS inhibitors or neutrophils of patients with chronic granulomatous disease who have a congenital defect in ROS production. Furthermore, we found that UA-induced NET formation was partially mediated by NF-κB activation. Our study is the first to demonstrate the novel function of UA in NET formation and may provide insight into the management of patients with hyperuricemia.

Reduction of Early Reperfusion Injury With the Mitochondria-Targeting Peptide Bendavia

We recently showed that Bendavia, a novel mitochondria-targeting peptide, reduced infarction and no-reflow across several experimental models. The purpose of this study was to determine the therapeutic timing and mechanism of action that underlie Bendavia's cytoprotective property. In rabbits exposed to in vivo ischemia/reperfusion (30/180 min), Bendavia administered 20 minutes prior to reperfusion (0.05 mg/kg/h, intravenously) reduced myocardial infarct size by ∼50% when administered for either 1 or 3 hours of reperfusion. However, when Bendavia perfusion began just 10 minutes after the onset of reperfusion, the protection against infarction and no-reflow was completely lost, indicating that the mechanism of protection is occurring early in reperfusion. Experiments in isolated mouse liver mitochondria found no discernible effect of Bendavia on blocking the permeability transition pore, and studies in isolated heart mitochondria showed no effect of Bendavia on respiratory rates. As Bendavia significantly lowered reactive oxygen species (ROS) levels in isolated heart mitochondria, the ROS-scavenging capacity of Bendavia was compared to well-known ROS scavengers using in vitro (cell-free) systems that enzymatically generate ROS. Across doses ranging from 1 nmol/L to 1 mmol/L, Bendavia showed no discernible ROS-scavenging properties, clearly differentiating itself from prototypical scavengers. In conclusion, Bendavia is a promising candidate to reduce cardiac injury when present at the onset of reperfusion but not after reperfusion has already commenced. Given that both infarction and no-reflow are related to increased cellular ROS, Bendavia's protective mechanism of action likely involves reduced ROS generation (as opposed to augmented scavenging) by endothelial and myocyte mitochondria.