Extracellular Superoxide Dismutase Research Articles

Innexin hemichannel activation by Microplitis bicoloratus ecSOD monopolymer reduces ROS

The extracellular superoxide dismutases (ecSODs) secreted by Microplitis bicoloratus reduce the reactive oxygen species (ROS) stimulated by the Microplitis bicoloratus bracovirus. Here, we demonstrate that the bacterial transferase hexapeptide (hexapep) motif and bacterial-immunoglobulin-like (BIg-like) domain of ecSODs bind to the cell membrane and transiently open hemichannels, facilitating ROS reductions. RNAi-mediated ecSOD silencing invivo elevated ROS in host hemocytes, impairing parasitoid larva development. Invitro, the ecSOD-monopolymer needed to be membrane bound to open hemichannels. Furthermore, the hexapep motif in the beta-sandwich of ecSOD49 and ecSOD58, and BIg-like domain in the signal peptides of ecSOD67 were required for cell membrane binding. Hexapep motif and BIg-like domain deletions induced ecSODs loss of adhesion and ROS reduction failure. The hexapep motif and BIg-like domain mediated ecSOD binding via upregulating innexins and stabilizing the opened hemichannels. Our findings reveal a mechanism through which ecSOD reduces ROS, which may aid in developing anti-redox therapy.

Non-Thyroidal Illness in Chronic Renal Failure: Triiodothyronine Levels and Modulation of Extra-Cellular Superoxide Dismutase (ec-SOD).

Oxidative stress (OS) is implicated in several chronic diseases. Extra-cellular superoxide dismutase (ec-SOD) catalyses the dismutation of superoxide anions with a protective role in endothelial cells. In chronic kidney disease (CKD), OS and thyroid dysfunction (low fT3 syndrome) are frequently present, but their relationship has not yet been investigated. This cohort study evaluated ec-SOD activity in CKD patients during haemodialysis, divided into "acute haemodialytic patients" (AH, 1-3 months of treatment) and "chronic haemodialytic patients" (CH, treated for a longer period). We also evaluated plasmatic total antioxidant capacity (TAC) and its relationships with thyroid hormones. Two basal samples ("basal 1", obtained 3 days after the last dialysis; and "basal 2", obtained 2 days after the last dialysis) were collected. On the same day of basal 2, a sample was collected 5 and 10 min after the standard heparin dose and at the end of the procedure. The ec-SOD values were significantly higher in CH vs. AH in all determinations. Moreover, the same patients had lower TAC values. When the CH patients were divided into two subgroups according to fT3 levels (normal or low), we found significantly lower ec-SOD values in the group with low fT3 in the basal, 5, and 10 min samples. A significant correlation was also observed between fT3 and ec-SOD in the basal 1 samples. These data, confirming OS and low fT3 syndrome in patients with CKD, suggest that low fT3 concentrations can influence ec-SOD activity and could therefore potentially contribute to endothelial oxidative damage in these patients.

A Novel Extracellular Catalase Produced by the Antarctic Filamentous Fungus Penicillium Rubens III11-2

Catalase (CAT) is an enzyme involved in the first line of cellular antioxidant defense. It plays a key role in the protection of a wide range of Antarctic organisms against cold stress. Extracellular catalase is very rare and data on it are extremely scarce. The aim of the present study was to select an efficient producer of extracellular catalase from amongst Antarctic filamentous fungi. Sixty-two Antarctic filamentous fungal strains were investigated for their potential ability to synthesize intracellular and extracellular CAT. The Antarctic strain Penicillium rubens III11-2 was selected as the best producer of extracellular catalase. New information on the involvement of the extracellular antioxidant enzymes superoxide dismutase and CAT in the response of filamentous fungi against low-temperature stress was obtained. An efficient scheme for the purification of CAT from culture fluid was developed. An enzyme preparation with high specific activity (513 U/mg protein) was obtained with a yield of 19.97% and a purification rate of 98.4-fold. The purified enzyme exhibited maximal enzymatic activity in the temperature range of 5–40 °C and temperature stability between 0 and 30 °C, therefore being characterized as temperature sensitive. To our knowledge, this is the first purified extracellular cold active catalase preparation from Antarctic filamentous fungi.

SOD3 regulates FLT1 to affect bone metabolism by promoting osteogenesis and inhibiting adipogenesis through PI3K/AKT and MAPK pathways

Osteoporosis is a chronic disease that seriously affects the quality of life and longevity of the elderly, so exploring the mechanism of osteoporosis is crucial for drug development and treatment. Bone marrow mesenchymal stem cells are stem cells with multiple differentiation potentials in bone marrow, and changing their differentiation direction can change bone mass. As an extracellular superoxide dismutase, Superoxide Dismutase 3 (SOD3) has been proved to play an important role in multiple organs, but the detailed mechanism of action in bone metabolism is still unclear.In this study, the results of clinical serum samples ELISA and single cell sequencing chip analysis proved that the expression of SOD3 was positively correlated with bone mass, and SOD3 was mainly expressed in osteoblasts and adipocytes and rarely expressed in osteoblasts in BMSCs. In vitro experiments showed that SOD3 can promote osteogenesis and inhibit adipogenesis. Compared with WT mice, the mice that were knocked out of SOD3 had a significant decrease in bone mineral density and significant changes in related parameters. The results of HE and IHC staining suggested that knocking out SOD3 would lead to fat accumulation in the bone marrow cavity and weakened osteogenesis. Both in vitro and in vivo experiments indicated that SOD3 affects bone metabolism by promoting osteogenesis and inhibiting adipogenesis. The results of transcriptome sequencing and revalidation showed that SOD3 can affect the expression of FLT1. Through in vitro experiments, we proved that FLT1 can also promote osteogenesis and inhibit adipogenesis. In addition, through the repeated experiments, the interaction between the two molecules (SOD3 and FLT1) was verified again. Finally, it was verified by WB that SOD3 regulates FLT1 to affect bone metabolism through PI3K/AKT and MAPK pathways.

Simulating immunosuppressive mechanism of Microplitis bicoloratus bracovirus coordinately fights Spodoptera frugiperda.

Parasitoid wasps control pests via a precise attack leading to the death of the pest. However, parasitoid larvae exhibit self-protection strategies against bracovirus-induced reactive oxygen species impairment. This has a detrimental effect on pest control. Here, we report a strategy for simulating Microplitis bicoloratus bracovirus using Mix-T dsRNA targeting 14 genes associated with transcription, translation, cell-cell communication, and humoral signaling pathways in the host, and from wasp extracellular superoxide dismutases. We implemented either one-time feeding to the younger instar larvae or spraying once on the corn leaves, to effectively control the invading pest Spodoptera frugiperda. This highlights the conserved principle of "biological pest control," as elucidated by the triple interaction of parasitoid-bracovirus-host in a cooperation strategy of bracovirus against its pest host.

Highly efficient expression of human extracellular superoxide dismutase (rhEcSOD) with ultraviolet-B-induced damage-resistance activity in transgenic silkworm cocoons.

Extracellular superoxide dismutase (EcSOD) protects tissues from oxidative stress, and thus is considered as a therapeutic agent for many diseases such as atherosclerosis, hypertension, and cancer. However, cost-effective production of bioactive recombinant human EcSOD (rhEcSOD) remains a challenge. Herein, we developed an efficient strategy for producing active rhEcSOD by transgenic silkworms. rhEcSOD was successfully synthesized as homodimers and homotetramers in the middle silk gland and spun into the cocoons with a concentration of 9.48±0.21mg/g. Purification of rhEcSOD from the cocoons could be conveniently achieved with a purity of 99.50% and a yield of 3.5±0.5mg/g. Additionally, N-glycosylation at the only site of N89 in rhEcSOD with 10 types were identified. The purified rhEcSOD gained the potent enzymatic activity of 4162±293U/mg after Cu/Zn ions incorporation. More importantly, rhEcSOD was capable of penetrating and accumulating in the nuclei of cells to maintain cell morphology and attenuate ultraviolet B-induced cell apoptosis by eliminating reactive oxygen species and inhibiting the C-Jun N-terminal kinase signaling pathway. These results demonstrated that the transgenic silkworm could successfully produce rhEcSOD with enzymatic and biological activities for biomedical applications.

Extracellular Superoxide Dismutase Attenuates Hepatic Oxidative Stress in Nonalcoholic Fatty Liver Disease through the Adenosine Monophosphate-Activated Protein Kinase Activation.

Oxidative stress is key in type 2 diabetes-associated nonalcoholic fatty liver disease (NAFLD). We explored whether extracellular superoxide dismutase (EC-SOD) activates adenosine monophosphate-activated protein kinase (AMPK) to enhance antioxidant synthesis and lipid metabolism in NAFLD. Human recombinant EC-SOD (hEC-SOD) was administered to 8-week-old male C57BLKS/J db/db mice through intraperitoneal injection once a week for 8 weeks. Target molecules involved in oxidative stress and lipid metabolism were investigated. hEC-SOD improved insulin resistance and systemic and hepatic oxidative stress characterized by increases in urinary 8-hydroxy-deoxyguanosine and 8-isoprostane levels in db/db mice and a decrease in DHE expression in the liver, respectively. Hepatic SOD3 expression in db/db mice was reversed by hEC-SOD, which improved hepatic steatosis, inflammation with M2 polarization, apoptosis, autophagy, fibrosis and lipid metabolism in db/db mice, as reflected by the changes in serum and hepatic markers, monocyte chemoattractant protein-1, tumor necrosis factor-α, TUNEL-positive cells, Bcl-2/BAX ratio, beclin1 and LC3-II/LC3-1. At the molecular level, hEC-SOD increased phosphorylated-AMPK related to CaMKKß, activation of peroxisome proliferative-activated receptor-gamma coactivator (PGC)-1α and dephosphorylation of forkhead box O (FoxO)1 and their subsequent downstream signaling. In HepG2Cs cells using AMPKα1 and AMPKα2 siRNA, hEC-SOD demonstrated a protective effect via the direct activation of both AMPK-PGC-1α and AMPK-FoxO1. EC-SOD might be a potential therapeutic agent for NAFLD through the activation of AMPK-PGC-1α and AMPK-FoxO1 signaling in hepatocytes, which modulates lipid metabolism, leading to anti-inflammatory, antioxidative and antiapoptotic effects and improving autophagy in the liver.

Redox imbalance in patients with heart failure and ICD/CRT-D intervention. Can it be an underappreciated and overlooked arrhythmogenic factor? A first preliminary clinical study.

Introduction: Redox imbalance and oxidative stress are involved in the pathogenesis of arrhythmias. They also play a significant role in pathogenesis of heart failure (HF). In patients with HFand implanted cardioverter-defibrillator (ICD) or cardiac resynchronization therapy defibrillator (CRT-D), the direct current shocks may be responsible for additional redox disturbances and additionally increase arrhythmia risk. However, the precise role of oxidative stress in potentially fatal arrhythmias and shock induction remains unclear. Methods: 36 patients with diagnosed HF and implanted ICD/CRT-D were included in this study. Patients were qualified to the study group in case of registered ventricular arrhythmia and adequate ICD/CRT-D intervention. The control group consisted of patients without arrhythmia with elective replacement indicator (ERI) status. Activity of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH) in erythrocyte (RBC), SOD, GPx activity and reactive oxygen/nitrogen species (ROS/RNS) concentration in plasma were determined. The values were correlated with glucose, TSH, uric acid, Mg and ion concentrations. Results: In the perishock period, we found a significant decrease in RBC and extracellular (EC) SOD and RBC CAT activity (p = 0.0110, p = 0.0055 and p = 0.0002, respectively). EC GPx activity was also lower (p = 0.0313). In all patients, a decrease in the concentration of all forms of glutathione was observed compared to the ERI group. Important association between ROS/RNS and GSH, Mg, TSH and uric acid was shown. A relationship between the activity of GSH and antioxidant enzymes was found. Furthermore, an association between oxidative stress and ionic imbalance has also been demonstrated. The patients had an unchanged de Haan antioxidant ratio and glutathione redox potential. Conclusion: Here we show significant redox disturbances in patients with HF and ICD/CRT-D interventions. Oxidative stress may be an additional risk factor for the development of arrhythmia in patients with HF. The detailed role of oxidative stress in ventricular arrhythmias requires further research already undertaken by our team.

Abstract 15747: AMPK Cysteine Oxidation by SOD3-Derived H 2 O 2 Mediates Exercise-Induced Restoration of Skeletal Muscle Angiogenesis in Type2 Diabetes

Background: Exercise restores decreased capillary density in skeletal muscle (SKM) (capillary rarefaction) in type2 diabetes (T2D) that has eNOS uncoupling/excess O 2 - , which is required for treatment of diabetic peripheral vascular disease (PAD) via unknown mechanisms. AMP-activated protein kinase (AMPK) is a key angiogenic/metabolic kinase mainly regulated by phosphorylation and is required for baseline, but not exercise-induced capillary density in SKM under healthy conditions. AMPK senses H 2 O 2 signal to increase its activity via oxidative modification of Cys299/304 in vitro. In addition, extracellular SOD (SOD3) that catalyzes the dismutation of O 2 - to H 2 O 2 is increased in response to exercise. However, the role of AMPK and SOD3 in exercise-induced SKM angiogenesis in T2D has not been reported. Oxidation of Cysteine residues to generate cysteine sulfenic acid (Cys-OH) is a key event in H 2 O 2 -mediated signaling. Results: Here we show that reduced CD31+ capillary density in SKM of high fat diet-induced T2D mice was restored (2.4-fold) by voluntary wheel exercise training (2 weeks) with enhanced SOD3 activity (224%) and extracellular H 2 O 2 levels (3.2-fold). Of note, exercise in T2D mice increased AMPK activity (by p-ACC) via inducing CysOH formation of AMPK (3.6-fold), but not p-AMPK induction, which was inhibited by SOD3 -/- /T2D mice that reduced exercise-induced H 2 O 2 . To demonstrate functional significance of AMPK Cys oxidation, we generated CRISPR/Cas9-genome editing “redox-dead” AMPK-Cys299/304Ala (AMPK-C/A) knock-in (KI) mutant mice and found that AMPK-C/A KI mice showed significant inhibition of exercise-induced angiogenesis (44%) via reducing angiogenic PGC1α and VEGF protein expression in SKM of T2D mice. In vitro, C2C12 skeletal muscles overexpressing SOD3 treated with palmitate (mimic T2D with exercise) induced AMPK-CysOH formation and VEGF secretion. Its conditioned media promoted angiogenic responses such as EC migration (1.8-fold) and tube formation (1.7-fold), which was inhibited by C2C12 cells overexpressing AMPK C/A mutant. Conclusion: Novel exercise-induced AMPK CysOH formation at Cys299/304 restores angiogenesis in SKM of T2D via SOD3-mediated H 2 O 2 , which may contribute to revascularization in diabetic PAD.

Extracellular SOD modulates canonical TNFα signaling and α5β1 integrin transactivation in vascular smooth muscle cells

TNFα activates NADPH oxidase 1 (Nox1) in vascular smooth muscle cells (VSMCs). The extracellular superoxide anion (O2•-) produced is essential for the pro-inflammatory effects of the cytokine but the specific contributions of O2•- to signal transduction remain obscure. Extracellular superoxide dismutase (ecSOD, SOD3 gene) is a secreted protein that binds to cell surface heparin sulfate proteoglycans or to Fibulin-5 (Fib-5, FBLN5 gene), an extracellular matrix protein that also associates with elastin and integrins. ecSOD converts O2•- to hydrogen peroxide (H2O2) which prevents NO• inactivation, limits generation of hydroxyl radical (OH•), and creates high local concentrations of H2O2. We hypothesized that ecSOD modifies TNFα signaling in VSMCs. Knockdown of ecSOD (siSOD3) suppressed downstream TNFα signals including MAPK (JNK and ERK phosphorylation) and NF-κB activation (luciferase reporter and IκB phosphorylation), interleukin-6 (IL-6) secretion, iNOS and VCAM expression, and proliferation (Sulforhodamine B assay, PCNA western blot). These effects were associated with significant reductions in the expression of both Type1 and 2 TNFα receptors. Reduced Fib-5 expression (siFBLN5) similarly impaired NF-κB activation by TNFα, but potentiated FAK phosphorylation at Y925. siSOD3 also increased both resting and TNFα-induced phosphorylation of FAK and of glycogen synthase kinase-3β (GSK3β), a downstream target of integrin linked kinase (ILK). These effects were dependent upon α5β1 integrins and siSOD3 increased resting sulfenylation (oxidation) of both integrin subunits, while preventing TNFα-induced increases in sulfenylation. To determine how ecSOD modified TNFα-induced inflammation in intact blood vessels, mesenteric arteries from VSMC-specific ecSOD knockout (KO) mice were exposed to TNFα (10 ng/ml) in culture for 48 h. Relaxation to acetylcholine and sodium nitroprusside was impaired in WT but not ecSOD KO vessels. Thus, ecSOD association with Fib-5 supports pro-inflammatory TNFα signaling while tonically inhibiting α5β1 integrin activation.

Interleukin-1β triggers muscle-derived extracellular superoxide dismutase expression and protects muscles from doxorubicin-induced atrophy.

Doxorubicin, a conventional chemotherapeutic agent prescribed for cancer, causes skeletal muscle atrophy and adversely affects mobility and strength. Given that doxorubicin-induced muscle atrophy is attributable primarily to oxidative stress, its effects could be mitigated by antioxidant-focused therapies; however, these protective therapeutic targets remain ambiguous. The aim of this study was to demonstrate that doxorubicin triggers severe muscle atrophy via upregulation of oxidative stress (4-hydroxynonenal and malondialdehyde) and atrogenes (atrogin-1/MAFbx and muscle RING finger-1) in association with decreased expression of the antioxidant enzyme extracellular superoxide dismutase (EcSOD), in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein elevated EcSOD levels on the cellular membrane of cultured myotubes, consequently inhibiting doxorubicin-induced oxidative stress and myotube atrophy. Furthermore, doxorubicin treatment reduced interleukin-1β (IL-1β) mRNA expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment increased EcSOD protein expression on the myotube membrane. Notably, transient IL-1β treatment of cultured myotubes and local administration in mouse skeletal muscle attenuated doxorubicin-induced muscle atrophy, which was associated with increased EcSOD expression. Collectively, these findings reveal that the regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a potential therapeutic approach to counteract the muscle atrophy mediated by doxorubicin and oxidative stress. KEY POINTS: Doxorubicin, a commonly prescribed chemotherapeutic agent for patients with cancer, induces severe muscle atrophy owing to increased expression of oxidative stress; however, protective therapeutic targets are poorly understood. Doxorubicin induced muscle atrophy owing to increased expression of oxidative stress and atrogenes in association with decreased protein expression of extracellular superoxide dismutase (EcSOD) in cultured C2C12myotubes and mouse skeletal muscle. Supplementation with EcSOD recombinant protein increased EcSOD levels on the cellular membrane of cultured myotubes, resulting in inhibition of doxorubicin-induced oxidative stress and myotube atrophy. Doxorubicin treatment decreased interleukin-1β (IL-1β) expression in cultured myotubes and skeletal muscle, whereas transient IL-1β treatment in vivo and in vitro increased EcSOD protein expression and attenuated doxorubicin-induced muscle atrophy. These findings reveal that regulation of skeletal muscle EcSOD via maintenance of IL-1β signalling is a possible therapeutic approach for muscle atrophy mediated by doxorubicin and oxidative stress.

Study on the Properties and Synergistic Antioxidant Effects of Novel Bifunctional Fusion Proteins Expressed Using the UTuT6 System.

Important antioxidant enzymes, glutathione peroxidase (GPx) and superoxide dismutase (SOD), are involved in maintaining redox balance. They can protect each other and result in more efficiently removing excessive reactive oxygen species (ROS), protecting cells against injury, and maintaining the normal metabolism of ROS. In this study, human cytosolic GPx (hGPx1) and human phospholipid hydroperoxide GPx (hGPx4) genes were integrated into the same open reading frame with human extracellular SOD active site (SOD3-72P) genes, respectively, and several novel fusion proteins were obtained by using the UTuT6 expression system for the first time. Among them, Se-hGPx1UAG-L4-SOD3-72P is the bifunctional fusion protein with the highest GPx activity and the best anti-hydrogen peroxide inactivation ability thus far. The Se-hGPx4UAG-L3-SOD3-72P fusion protein exhibits the strongest alkali and high temperature resistance and a greater protective effect against lipoprotein peroxidation damage. Se-hGPx1UAG-L4-SOD3-72P and Se-hGPx4UAG-L3-SOD3-72P fusion proteins both have good synergistic and antioxidant abilities in H2O2-induced RBCs and liver damage models. We believe that this research will help with the development of novel bifunctional fusion proteins and the investigation of the synergistic and catalytic mechanisms of GPx and SOD, which are important in creating novel protein therapeutics.

Mesenchymal stem cells in synovial fluid increase in number in response to synovitis and display more tissue-reparative phenotypes in osteoarthritis

BackgroundSynovial fluid mesenchymal stem cells (SF-MSCs) originate in the synovium and contribute to the endogenous repair of damaged intra-articular tissues. Here, we clarified the relationship between their numbers and joint structural changes during osteoarthritis (OA) progression and investigated whether SF-MSCs had phenotypes favorable for tissue repair, even in an OA environment.MethodsPartial medial meniscectomy (pMx) and sham surgery were performed on both knees of rats. SF and knee joints were collected from intact rats and from rats at 2, 4, and 6 weeks after surgery. SF was cultured for 1 week to calculate the numbers of colony-forming cells and colony areas. Joint structural changes were evaluated histologically to investigate their correlation with the numbers and areas of colonies. RNA sequencing was performed for SF-MSCs from intact knees and knees 4 weeks after the pMx and sham surgery.ResultsColony-forming cell numbers and colony areas were greater in the pMx group than in the intact and sham groups and peaked at 2 and 4 weeks, respectively. Synovitis scores showed the strongest correlation with colony numbers (R = 0.583) and areas (R = 0.456). RNA sequencing revealed higher expression of genes related to extracellular matrix binding, TGF-β signaling, and superoxide dismutase activity in SF-MSCs in the pMx group than in the sham group.ConclusionThe number of SF-MSCs was most closely correlated with the severity of synovitis in this rat OA model. Tissue-reparative gene expression patterns were observed in SF-MSCs from OA knees, but not from knees without intra-articular tissue damage.

Lauric acid provides neuroprotection against oxidative stress in mouse model of hyperglycaemic stroke

During ischemic stroke, higher glucose level linked worse outcomes were reported even in patients without pre-existing diabetes. Evidence suggest that such worse stroke outcomes were mainly due to production of reactive, toxic glucose metabolites that expands oxidative damage inside the brain. As a consequence of high oxidative stress, microvasculature structures and tight junctions compromised their functionally, infarct volume expands and brain edema exacerbates. In a mouse model of ischemic stroke with induced acute hyperglycaemia, Lauric acid (LA) as a natural saturated fatty acid demonstrated neuroprotection by attenuating infarct volume and brain edema. In addition, in the ipsilateral hyperglycaemic brain, the LA significantly increased the expression of tight junction representative protein (occludin) as well as anti-oxidative markers; Manganese superoxide dismutase (Mn) SOD, Extracellular superoxide dismutase (Ec-SOD) and nuclear factor-erythroid factor 2-related factor 2 (Nrf2) in the ipsilateral region against hyperglycemic ischemic stroke. LA treated animals showed a significant reduction in the production of lipid peroxidation products (4-HNE) in the microvascular structures, maintained the blood brain barrier (BBB) integrity. LA linked neuroprotective outcomes were further confirmed by behavioral tests, where functional outcomes and motor coordination were improved significantly. Furthermore, LA treatment enhanced food intake, decreased mortality rate, and net body weight loss. Conclusively, LA modulated ischemic insult exacerbated by hyperglycemia and provided neuroprotection.

Disruption of extracellular redox balance drives persistent lung fibrosis and impairs fibrosis resolution

Lung fibrosis is a devastating outcome of various diffuse parenchymal lung diseases. Despite rigorous research efforts, the mechanisms that propagate its progressive and nonresolving nature remain enigmatic. Oxidative stress has been implicated in the pathogenesis of lung fibrosis. However, the role of extracellular redox state in disease progression and resolution remains largely unexplored. Here, we show that compartmentalized control over extracellular reactive oxygen species (ROS) by aerosolized delivery of recombinant extracellular superoxide dismutase (ECSOD) suppresses an established bleomycin-induced fibrotic process in mice. Further analysis of publicly available microarray, RNA-seq and single-cell RNAseq datasets reveals a significant decrease in ECSOD expression in fibrotic lung tissues that can be spontaneously restored during fibrosis resolution. Therefore, we investigate the effect of siRNA-mediated ECSOD depletion during the established fibrotic phase on the self-limiting nature of the bleomycin mouse model. Our results demonstrate that in vivo knockdown of ECSOD in mouse fibrotic lungs impairs fibrosis resolution. Mechanistically, we demonstrate that transforming growth factor (TGF)-β1 downregulates endogenous ECSOD expression, leading to the accumulation of extracellular superoxide via Smad-mediated signaling and the activation of additional stores of latent TGF-β1. In addition, depletion of endogenous ECSOD during the fibrotic phase in the bleomycin model induces an apoptosis-resistant phenotype in lung fibroblasts through unrestricted Akt signaling. Taken together, our data strongly support the critical role of extracellular redox state in fibrosis persistence and resolution. Based on these findings, we propose that compartment-specific control over extracellular ROS may be a potential therapeutic strategy for managing fibrotic lung disorders.

Molecular dynamics and simulation analysis against superoxide dismutase (SOD) target of Micrococcus luteus with secondary metabolites from Bacillus licheniformis recognized by genome mining approach

Micrococcus luteus, also known as M. luteus, is a bacterium that inhabits mucous membranes, human skin, and various environmental sources. It is commonly linked to infections, especially among individuals who have compromised immune systems. M. luteus is capable of synthesizing the enzyme superoxide dismutase (SOD) as a component of its protective response to reactive oxygen species (ROS). This enzyme serves as a promising target for drug development in various diseases. The current study utilized a subtractive genomics approach to identify potential therapeutic targets from M. luteus. Additionally, genome mining was employed to identify and characterize the biosynthetic gene clusters (BGCs) responsible for the production of secondary metabolites in Bacillus licheniformis (B. licheniformis), a bacterium known for its production of therapeutically relevant secondary metabolites. Subtractive genomics resulted in identification of important extracellular protein SOD as a drug target that plays a crucial role in shielding cells from damage caused by ROS. Genome mining resulted in identification of five potential ligands (secondary metabolites) from B. licheniformis such as, Bacillibactin (BAC), Paenibactin (PAE), Fengycin (FEN), Surfactin (SUR) and Lichenysin (LIC). Molecular docking was used to predict and analyze the binding interactions between these five ligands and target protein SOD. The resulting protein–ligand complexes were further analyzed for their motions and interactions of atoms and molecules over 250 ns using molecular dynamics (MD) simulation analysis. The analysis of MD simulations suggests, Bacillibactin as the probable candidate to arrest the activities of SOD. All the five compounds reported in this study were found to act by directly/indirectly interacting with ROS molecules, such as superoxide radicals (O2–) and hydrogen peroxide (H2O2), and transforming them into less reactive species. This antioxidant activity contributes to its protective effects against oxidative stress-induced damage in cells making them likely candidate for various applications, including in the development of antioxidant-based therapies, nutraceuticals, and functional foods.

Use of Electron Paramagnetic Resonance (EPR) to Evaluate Redox Status in a Preclinical Model of Acute Lung Injury.

Patients with hyper- vs. hypo-inflammatory subphenotypes of acute respiratory distress syndrome (ARDS) exhibit different clinical outcomes. Inflammation increases the production of reactive oxygen species (ROS) and increased ROS contributes to the severity of illness. Our long-term goal is to develop electron paramagnetic resonance (EPR) imaging of lungs in vivo to precisely measure superoxide production in ARDS in real time. As a first step, this requires the development of in vivo EPR methods for quantifying superoxide generation in the lung during injury, and testing if such superoxide measurements can differentiate between susceptible and protected mouse strains. In WT mice, mice lacking total body extracellular superoxide dismutase (EC-SOD) (KO), or mice overexpressing lung EC-SOD (Tg), lung injury was induced with intraperitoneal (IP) lipopolysaccharide (LPS) (10mg/kg). At 24h after LPS treatment, mice were injected with the cyclic hydroxylamines 1-hydroxy-3-carboxy-2,2,5,5-tetramethylpyrrolidine hydrochloride (CPH) or 4-acetoxymethoxycarbonyl-1-hydroxy-2,2,5,5-tetramethylpyrrolidine-3-carboxylic acid (DCP-AM-H) probes to detect, respectively, cellular and mitochondrial ROS - specifically superoxide. Several probe delivery strategies were tested. Lung tissue was collected up to one hour after probe administration and assayed by EPR. As measured by X-band EPR, cellular and mitochondrial superoxide increased in the lungs of LPS-treated mice compared to control. Lung cellular superoxide was increased in EC-SOD KO mice and decreased in EC-SOD Tg mice compared to WT. We also validated an intratracheal (IT) delivery method, which enhanced the lung signal for both spin probes compared to IP administration. We have developed protocols for delivering EPR spin probes in vivo, allowing detection of cellular and mitochondrial superoxide in lung injury by EPR. Superoxide measurements by EPR could differentiate mice with and without lung injury, as well as mouse strains with different disease susceptibilities. We expect these protocols to capture real-time superoxide production and enable evaluation of lung EPR imaging as a potential clinical tool for subphenotyping ARDS patients based on redox status.

Sex-Dependent Impairment of Endothelium-Dependent Relaxation in Aorta of Mice with Overexpression of Hyaluronan in Tunica Media.

Diabetic macroangiopathy is characterized by increased extracellular matrix deposition, including excessive hyaluronan accumulation, vessel thickening and stiffness, and endothelial dysfunction in large arteries. We hypothesized that the overexpression of hyaluronan in the tunica media also led to endothelial cell (EC) dysfunction. To address this hypothesis, we investigated the following in the aortas of mice with excessive hyaluronan accumulation in the tunica media (HAS-2) and wild-type mice: EC dysfunction via myograph studies, nitric oxide (NO) bioavailability via diaminofluorescence, superoxide formation via dihydroethidium fluorescence, and the distances between ECs via stereological methods. EC dysfunction, characterized by blunted relaxations in response to acetylcholine and decreased NO bioavailability, was found in the aortas of male HAS-2 mice, while it was unaltered in the aortas of female HAS-2 mice. Superoxide levels increased and extracellular superoxide dismutase (ecSOD) expression decreased in the aortas of male and female HAS-2 mice. The EC-EC distances and LDL receptor expression were markedly increased in the HAS-2 aortas of male mice. Our findings suggest hyaluronan increases oxidative stress in the vascular wall and that together with increased EC distance, it is associated with a sex-specific decrease in NO levels and endothelial dysfunction in the aorta of male HAS-2 transgenic mice.

N-Acetyl Cysteine Restores the Diminished Activity of the Antioxidant Enzymatic System Caused by SARS-CoV-2 Infection: Preliminary Findings.

SARS-CoV-2 infects type II pneumocytes and disrupts redox homeostasis by overproducing reactive oxygen species (ROS). N-acetyl cysteine (NAC) is a precursor of the synthesis of glutathione (GSH) and it restores the loss of redox homeostasis associated to viral infections. The aim of the study is to evaluate the effect of the treatment with NAC on the enzymatic antioxidant system in serum from patients infected by SARS-CoV-2. We evaluated the enzymatic activities of thioredoxin reductase (TrxR), glutathione peroxidase (GPx), -S-transferase (GST), and reductase (GR) by spectrophotometry and the concentrations of the glutathione (GSH), total antioxidant capacity (TAC), thiols, nitrites (NO2-), and lipid peroxidation (LPO) in serum. The activity of the extracellular super oxide dismutase (ecSOD) was determined by native polyacrylamide gels, and 3-nitrotyrosine (3-NT) was measured by ELISA. A decrease in the activities of the ecSOD, TrxR, GPx, GST GR, (p = 0 ≤ 0.1), and the GSH, TAC, thiols, and NO2- (p ≤ 0.001) concentrations and an increase in LPO and 3-NT (p = 0.001) concentrations were found in COVID-19 patients vs. healthy subjects. The treatment with NAC as an adjuvant therapy may contribute to a reduction in the OS associated to the infection by SARS-CoV-2 through the generation of GSH. GSH promotes the metabolic pathways that depend on it, thus contributing to an increase in TAC and to restore redox homeostasis.

Hyperbaric Oxygen Therapy Reduces Oxidative Stress and Inflammation, and Increases Growth Factors Favouring the Healing Process of Diabetic Wounds.

Hyperbaric oxygen therapy (HBOT) is the clinical application of oxygen at pressures higher than atmospheric pressure. HBOT has been effectively used to manage diverse clinical pathologies, such as non-healing diabetic ulcers. The aim of the present study was to analyse the effects of HBOT on the plasma oxidative and inflammation biomarkers and growth factors in patients with chronic diabetic wounds. The participants received 20 HBOT sessions (five sessions/week), and blood samples were obtained at sessions 1, 5 and 20, before and 2 h after the HBOT. An additional (control) blood sample was collected 28 days after wound recovery. No significant differences were evident in haematological parameters, whereas the biochemical parameters progressively decreased, which was significant for creatine phosphokinase (CPK) and aspartate aminotransferase (AST). The pro-inflammatory mediators, tumour necrosis factor alpha (TNF-α) and interleukin 1β (IL-1β), progressively decreased throughout the treatments. Biomarkers of oxidative stress--plasma protein levels of catalase, extracellular superoxide dismutase, myeloperoxidase, xanthine oxidase, malondialdehyde (MDA) levels and protein carbonyls--were reduced in accordance with wound healing. Plasma levels of growth factors--platelet-derived growth factor (PDFG), transforming growth factor β (TGF-β) and hypoxia-inducible factor 1-alpha (HIF-1α)-- were increased as a consequence of HBOT and reduced 28 days after complete wound healing, whereas matrix metallopeptidase 9 (MMP9) progressively decreased with the HBOT. In conclusion, HBOT reduced oxidative and pro-inflammatory mediators, and may participate in activating healing, angiogenesis and vascular tone regulation by increasing the release of growth factors.