Efficacy of Medical Ozone for Treatment of Chronic Musculoskeletal Pain with Abnormal Mitochondrial Redox State: Prospective Randomized Clinical Trial.

BACKGROUND: Chronic primary musculoskeletal pain is multifaceted and 20% of the adult population lives with severe chronic pain and experience symptoms such as intense pain, depression, weakness, sleep problems, decreased quality of life and decreased emotional well-being. OBJECTIVES: This paper studies the efficacy of trigger point injections with ozone compared to standard steroid injection or combination therapy for the treatment of chronic musculoskeletal pain in patients with abnormal mitochondrial redox state. STUDY DESIGN: This is a prospective randomized clinical study conducted with 51 patients experiencing chronic musculoskeletal pain. SETTING: Medical Research Institute Hospital, Alexandria University. METHODS: By computer-generated random numbers the 51 patients were divided into 3 groups. Group A (17 patients) received ozone injection, group B (17 patients) received betamethasone injection and group C (17 patients) received combined ozone and betamethasone injections. The groups were compared based on the intensity of pain and correction of mitochondrial redox state of the patients. RESULTS: Three days after intervention, the visual analog scale (VAS) scores reported by patients were lower in group A compared to group B (with a mean difference 1.27, 95% confidence interval (CI) of 0.15-2.39 (P < 0.02). One and 3 weeks after intervention, VAS scores of patients were lower in groups A and C compared to group B. At one week the mean difference between A and B was 1.2, with a 95% CI of 0.15-2.25 (P < 0.02) and the mean difference between C and B was 1.73 with a 95% CI of 0.69-2.78 (P < 0.001). At 3 weeks the mean difference between A and B was 1.5 with a 95% CI of 0.2-2.87 (P < 0.01) and the mean difference between C and B was 2.27 with a 95% CI of 0.93-3.60 (P < 0.0001). The reduced/oxidized glutathione ratio after intervention was higher in groups A and C compared to group B (P > 0.008). The mitochondrial copy number was higher in group A compared to group B (P < 0.002). LIMITATION: This study didn't allow for the comparison of the experimental groups with a placebo or control group for musculoskeletal pain conditions in orderto establish the role of an abnormal mitochondrial redox state on the pathogenesis of patients from an ethical view. CONCLUSIONS: Ozone therapy or combined ozone and betamethasone treatment are  effective techniques for management of pain since it produced a significant reduction of muscle pain and increase of the pain free interval experienced by patients. Ozone therapy causes pain improvement which increases with time and it improves muscle oxygenation and mitochondrial function. TRIAL REGISTRATION: This study was approved by the Ethics Committee of Medical Research Institute (IORH: IOR 00088812) and was registered at the Pan African Clinical Trial Registry (www.pactr.org) under the identification number PACTR201908620943471. The registration this experiment started on 07/08/2019. This study's protocol followed the CONSORT guidelines and was performed under the relevant guidelines.

Sodium nitroprusside modulates oxidative and nitrosative processes in Lycopersicum esculentum L. under drought stress.

KEY MESSAGE: Sodium nitroprusside mediates drought stress responses in tomatoes by modulating nitrosative and oxidative pathways, highlighting the interplay between nitric oxide, hydrogen sulfide, and antioxidant systems for enhanced drought tolerance. While nitric oxide (NO), a signalling molecule, enhances plant tolerance to abiotic stresses, its precise contribution to improving tomato tolerance to drought stress (DS) through modulating oxide-nitrosative processes is not yet fully understood. We aimed to examine the interaction of NO and nitrosative signaling, revealing how sodium nitroprusside (SNP) could mitigate the effects of DS on tomatoes. DS-seedlings endured 12% polyethylene glycol (PEG) in a 10% nutrient solution (NS) for 2 days, then transitioned to half-strength NS for 10 days alongside control plants. DS reduced total plant dry weight, chlorophyll a and b, Fv/Fm, leaf water potential (ΨI), and relative water content, but improved hydrogen peroxide (H2O2), proline, and NO content. The SNP reduced the DS-induced H2O2 generation by reducing thiol (-SH) and the carbonyl (-CO) groups. SNP increased not only NO but also the activity of L-cysteine desulfhydrase (L-DES), leading to the generation of H2S. Decreases in S-nitrosoglutathione reductase (GSNOR) and NADPH oxidase (NOX) suggest a potential regulatory mechanism in which S-nitrosylation [formation of S-nitrosothiol (SNO)] may influence protein function and signaling pathways during DS. Moreover, SNP improved ascorbate (AsA) and glutathione (GSH) and reduced oxidized glutathione (GSSG) levels in tomato plants under drought. Furthermore, the interaction of NO and H2S, mediated by L-DES activity, may serve as a vital cross-talk mechanism impacting plant responses to DS. Understanding these signaling interactions is crucial for developing innovative drought-tolerance strategies in crops.

The inhibitor of the redox activity of APE1/REF-1, APX2009, reduces the malignant phenotype of breast cancer cells.

Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/REF-1) is a multifunctional protein acting on cellular signaling pathways, including DNA repair and redox activities. APE1/REF-1 has emerged as a target for cancer therapy, and its role in breast cancer models would reveal new strategies for cancer therapy. APX2009 is a specific APE1/REF-1 redox inhibitor whose anticancer properties have not been described in breast cancer cells. Here, we investigated the effect of the APX2009 treatment in the breast cancer cell lines MDA-MB-231 and MCF-7. Breast cancer cell lines were cultured, and WST1 and colony formation assays were performed to evaluate cell proliferation. Annexin V-FITC/7-AAD and LDH-Glo™ assays were performed to evaluate cell death. The wound healing assay and Matrigel transwell assay were performed after APX2009 treatment to evaluate the cellular migration and invasion processes, respectively. Our findings demonstrated that APX2009 treatment decreased breast cancer cell proliferative, migratory, and invasive properties. Furthermore, it induced apoptosis in both cell lines. Our study is the first to show the effects of APX2009 treatment on apoptosis in a breast cancer cell. Therefore, this study suggested that APX2009 treatment is a promising anticancer molecule for breast cancer.

Exogenous erythropoietin increases hematological status, fat oxidation, and aerobic performance in males following prolonged strenuous training.

This study investigated the effects of EPO on hemoglobin (Hgb) and hematocrit (Hct), time trial (TT) performance, substrate oxidation, and skeletal muscle phenotype throughout 28 days of strenuous exercise. Eight males completed this longitudinal controlled exercise and feeding study using EPO (50 IU/kg body mass) 3×/week for 28 days. Hgb, Hct, and TT performance were assessed PRE and on Days 7, 14, 21, and 27 of EPO. Rested/fasted muscle obtained PRE and POST EPO were analyzed for gene expression, protein signaling, fiber type, and capillarization. Substrate oxidation and glucose turnover were assessed during 90-min of treadmill load carriage (LC; 30% body mass; 55 ± 5% V̇O2peak) exercise using indirect calorimetry, and 6-6-[2H2]-glucose PRE and POST. Hgb and Hct increased, and TT performance improved on Days 21 and 27 compared to PRE (p < 0.05). Energy expenditure, fat oxidation, and metabolic clearance rate during LC increased (p < 0.05) from PRE to POST. Myofiber type, protein markers of mitochondrial biogenesis, and capillarization were unchanged PRE to POST. Transcriptional regulation of mitochondrial activity and fat metabolism increased from PRE to POST (p < 0.05). These data indicate EPO administration during 28 days of strenuous exercise can enhance aerobic performance through improved oxygen carrying capacity, whole-body and skeletal muscle fat metabolism.

Vascular Response of Triiodothyronine on Isolated Aortic Rings: Contribution of Redox Mechanisms. / Resposta Vascular da Triiodotironina sobre Anéis de Aortas Isoladas: Contribuição de Mecanismos Redox.

BACKGROUND: Vascular dysfunction constitutes the etiology of many diseases, such as myocardial infarction and hypertension, with the disruption of redox homeostasis playing a role in the imbalance of the vasomotor control mechanism. Our group previously has shown that thyroid hormones exert protective effects on the aortic tissue of infarcted rats by improving angiogenesis signaling. OBJECTIVE: Investigate the role of triiodothyronine (T3) on vascular response, exploring its effects on isolated aortas and whether there is an involvement of vascular redox mechanisms. METHODS: Isolated aortic rings (intact- and denuded-endothelium) precontracted with phenylephrine were incubated with T3 (10-8, 10-7, 10-6, 10-5, and 10-4 M), and tension was recorded using a force-displacement transducer coupled with an acquisition system. To assess the involvement of oxidative stress, aortic rings were preincubated with T3 and subsequently submitted to an in vitro reactive oxygen species (ROS) generation system. The level of significance adopted in the statistical analysis was 5%. RESULTS: T3 (10-4 M) promoted vasorelaxation of phenylephrine precontracted aortic rings in both intact- and denuded-endothelium conditions. Aortic rings preincubated in the presence of T3 (10-4 M) also showed decreased vasoconstriction elicited by phenylephrine (1 µM) in intact-endothelium preparations. Moreover, T3 (10-4 M) vasorelaxation effect persisted in aortic rings preincubated with NG-nitro-L-arginine methylester (L-NAME, 10 µM), a nonspecific NO synthase (NOS) inhibitor. Finally, T3 (10-4 M) exhibited, in vitro, an antioxidant role by reducing NADPH oxidase activity and increasing SOD activity in the aorta's homogenates. CONCLUSION: T3 exerts dependent- and independent-endothelium vasodilation effects, which may be related to its role in maintaining redox homeostasis.

FUNDAMENTO: A disfunção vascular constitui a etiologia de diversas doenças, incluindo infarto do miocárdio e hipertensão, diante da ruptura da homeostase oxi-redutiva ("redox"), desempenhando um papel no desequilíbrio do mecanismo de controle vasomotor. Nosso grupo demonstrou anteriormente que os hormônios tireoidianos melhoram a sinalização da angiogênese, exercendo efeitos protetores sobre o tecido aórtico de ratos infartados. OBJETIVOS: Investigar o papel da triiodotironina (T3) na resposta vascular, explorando seus efeitos em aortas isoladas e a presença de mecanismos redox vasculares. MÉTODOS: Anéis aórticos isolados (endotélio intacto e desnudado) pré-contraídos com fenilefrina foram incubados com T3 (10-8, 10-7, 10-6, 10-5 e 10-4 M) e a tensão foi registrada usando um transdutor de deslocamento de força acoplado a um sistema de coleta. Para avaliar o envolvimento do estresse oxidativo, os anéis aórticos foram pré-incubados com T3 e posteriormente submetidos a um sistema de geração de espécies reativas de oxigênio (ROS) in vitro. O nível de significância adotado na análise estatística foi de 5%. RESULTADOS: A T3 (10-4 M) promoveu o vasorrelaxamento dos anéis aórticos pré-contraídos com fenilefrina em endotélio intacto e desnudado. Os anéis aórticos pré-incubados na presença de T3 (10-4 M) também mostraram diminuição da vasoconstrição provocada pela fenilefrina (1 µM) em preparações de endotélio intacto. Além disso, o efeito vasorrelaxante da T3 (10-4 M) persistiu em anéis aórticos pré-incubados com éster metílico de NG-nitro-L-arginina (L-NAME, 10 µM), um inibidor inespecífico da NO sintase (NOS). Por fim, a T3 (10-4 M) exibiu, in vitro, um papel antioxidante ao reduzir a atividade da NADPH oxidase e aumentar a atividade da SOD nos homogenatos aórticos. CONCLUSÃO: A T3 exerce efeitos dependentes e independentes de endotélio, o que pode estar relacionado ao seu papel na manutenção da homeostase redox.

PM2.5 toxicity in blood impairs cardiac redox balance and promotes mitochondrial dysfunction in rat heart that further aggravates ischemia reperfusion injury by modulating PI3K/AKT/mTOR/NF-kB signaling axis.

According to the pathophysiological mechanisms linking particulate matter (PM2.5) exposure and cardiovascular diseases, PM2.5 may directly translocate into the blood stream and remote target organs and thereby induce cardiovascular effects. The toxicity of PM2.5 is known to induce oxidative stress in pulmonary tissue, but its impact on the redox state in heart (distant organ) is unknown and how it modulates the cardiac response to ischemia reperfusion (IR) remains unclear. In the present study, we evaluated the toxic effect of PM2.5 on cardiac physiology in the presence and absence of IR after introducing PM2.5 into the blood. Female Wistar rats were injected with diesel particulate matter (DPM) via i.p & i.v routes at a concentration of 10 µg/ml. The toxic impact of PM2.5 not only adversely affects the cardiac ultra-structure (leading to nuclear infiltration, edema, irregularities in heart muscle and nuclear infiltration), but also altered the cellular redox balance, elevated inflammation and promoted the upregulation of proapoptotic mediator genes at the basal level of myocardium. The results showed alterations in cardiac ultrastructure, elevated oxidative stress and significant redox imbalance, increased inflammation and proapoptotic mediators at the basal level of myocardium. Moreover, the cardioprotective pro survival signaling axis was declined along with an increased NF-kB activation at the basal level. IR inflicted further injury with deterioration of cardiac hemodynamic indices (Heart rate [HR], Left ventricular developed pressure [LVDP], Left ventricular end-diastolic pressure [LVEDP] and rate pressure product [RPP]) along with prominent inactivation of signaling pathways. Furthermore, the levels of GSH/GSSG, NADH/NAD, NADPH/NADP were significantly low along with increased lipid peroxidation in mitochondria of PM2.5 treated IR rat hearts. This observation was supported by downregulation of glutaredoxin and peroxiredoxin genes in the myocardium. Similarly the presence of oxidative stress inducing metals was found at a higher concentration in cardiac mitochondria. Thus, the toxic impact of PM2.5 in heart augment the IR associated pathological changes by altering the physiological response, initiating cellular metabolic alterations in mitochondria and modifying the signaling molecules.

Synergistic Anti-Inflammatory Activity of Lipid-Free Apolipoprotein (apo) A-I and CIGB-258 in Acute-Phase Zebrafish via Stabilization of the apoA-I Structure to Enhance Anti-Glycation and Antioxidant Activities.

CIGB-258, a 3 kDa peptide from heat shock protein 60, exhibits synergistic anti-inflammatory activity with apolipoprotein A-I (apoA-I) in reconstituted high-density lipoproteins (rHDLs) via stabilization of the rHDL structure. This study explored the interactions between CIGB-258 and apoA-I in the lipid-free state to assess their synergistic effects in the structural and functional enhancement of apoA-I and HDL. A co-treatment of lipid-free apoA-I and CIGB-258 inhibited the cupric ion-mediated oxidation of low-density lipoprotein (LDL) and a lowering of oxidized species in the dose-responsive manner of CIGB-258. The co-presence of CIGB-258 caused a blue shift in the wavelength of maximum fluorescence (WMF) of apoA-I with protection from proteolytic degradation. The addition of apoA-I:CIGB-258, with a molar ratio of 1:0.1, 1:0.5, and 1:1, to HDL2 and HDL3 remarkably enhanced the antioxidant ability against LDL oxidation up to two-fold higher than HDL alone. HDL-associated paraoxonase activities were elevated up to 28% by the co-addition of apoA-I and CIGB-258, which is linked to the suppression of Cu2+-mediated HDL oxidation with the slowest electromobility. Isothermal denaturation by a urea treatment showed that the co-presence of CIGB-258 attenuated the exposure of intrinsic tryptophan (Trp) and increased the mid-points of denaturation from 2.33 M for apoA-I alone to 2.57 M for an apoA-I:CIGB-258 mixture with a molar ratio of 1:0.5. The addition of CIGB-258 to apoA-I protected the carboxymethyllysine (CML)-facilitated glycation of apoA-I with the prevention of Trp exposure. A co-treatment of apoA-I and CIGB-258 synergistically safeguarded zebrafish embryos from acute death by CML-toxicity, suppressing oxidative stress and apoptosis. In adult zebrafish, the co-treatment of apoA-I+CIGB-258 exerted the highest anti-inflammatory activity with a higher recovery of swimming ability and survivability than apoA-I alone or CIGB-258 alone. A co-injection of apoA-I and CIGB-258 led to the lowest infiltration of neutrophils and interleukin (IL)-6 generation in hepatic tissue, with the lowest serum triglyceride, aspartate transaminase, and alanine transaminase levels in plasma. In conclusion, the co-presence of CIGB-258 ameliorated the beneficial functionalities of apoA-I, such as antioxidant and anti-glycation activities, by enhancing the structural stabilization and protection of apoA-I. The combination of apoA-I and CIGB-258 synergistically enforced the anti-inflammatory effect against CML toxicity in embryos and adult zebrafish.

Immunoglobulin Superfamily Containing Leucine-Rich Repeat (ISLR) Serves as a Redox Sensor That Modulates Antioxidant Capacity by Suppressing Pyruvate Kinase Isozyme M2 Activity.

Cells defend against oxidative stress by enhancing antioxidant capacity, including stress-activated metabolic alterations, but the underlying intracellular signaling mechanisms remain unclear. This paper reports that immunoglobulin superfamily containing leucine-rich repeat (ISLR) functions as a redox sensor that responds to reactive oxygen species (ROS) stimulation and modulates the antioxidant capacity by suppressing pyruvate kinase isozyme M2 (PKM2) activity. Following oxidative stress, ISLR perceives ROS stimulation through its cysteine residue 19, and rapidly degrades in the autophagy-lysosome pathway. The downregulated ISLR enhances the antioxidant capacity by promoting the tetramerization of PKM2, and then enhancing the pyruvate kinase activity, PKM2-mediated glycolysis is crucial to the ISLR-mediated antioxidant capacity. In addition, our results demonstrated that, in triple-negative breast cancer, cisplatin treatment reduced the level of ISLR, and PKM2 inhibition sensitizes tumors to cisplatin by enhancing ROS production; and argued that PKM2 inhibition can synergize with cisplatin to limit tumor growth. Our results demonstrate a molecular mechanism by which cells respond to oxidative stress and modulate the redox balance.

The Effect of Orally Administered Multi-Strain Probiotic Formulation (Lactobacillus, Bifidobacterium) on the Phagocytic Activity and Oxidative Metabolism of Peripheral Blood Granulocytes and Monocytes in Lambs.

Probiotic feed additives have attracted considerable research interest in recent years because the effectiveness of probiotics can differ across microbial strains and the supplemented macroorganisms. The present study was conducted on 16 lambs divided equally into two groups (C-control and E-experimental). The examined lambs were aged 11 days at the beginning of the experiment and 40 days at the end of the experiment. The diet of group E lambs was supplemented with a multi-strain probiotic formulation (Lactobacillus plantarum AMT14, Lactobacillus plantarum AMT4, Lactobacillus rhamnosus AMT15, and Bifidobacterium animalis AMT30), whereas group C lambs did not receive the probiotic additive. At the beginning of the experiment (day 0) and on experimental days 15 and 30, blood was sampled from the jugular vein to determine and compare: phagocytic activity (Phagotest) and oxidative metabolism (Phagoburst) of peripheral blood granulocytes and monocytes by flow cytometry. An analysis of the phagocytic activity of granulocytes and monocytes revealed significantly higher levels of phagocytic activity (expressed as the percentage of phagocytic cells and mean fluorescence intensity) in lambs that were administered the multi-strain probiotic formulation compared with lambs in the control group. The probiotic feed additive also exerted a positive effect on the oxidative metabolism of both granulocytes and monocytes (expressed as the percentage of oxidative metabolism and mean fluorescence intensity) after stimulation with Escherichia coli bacteria and with PMA (4-phorbol-12-ß-myristate-13-acetate). These findings suggest that the tested probiotic formulation may have a positive effect on the immune status of lambs.

Effect of Garlic Extract on the Erythrocyte as a Simple Model Cell.

Garlic is known to have diverse effects on mammalian cells, being cytotoxic, especially to cancer cells, but also protect against oxidative stress. Mammalian erythrocyte is a simple cell devoid of intracellular organelles, protein synthesis ability, and most signaling pathways. Therefore, examination of the effects of garlic on erythrocytes allows for revealing primary events in the cellular action of garlic extract. In this study, human erythrocytes or erythrocyte membranes were exposed to garlic extract at various dilutions. Hemoglobin oxidation to methemoglobin, increased binding of hemoglobin to the membrane, and formation of Heinz bodies were observed. Garlic extract depleted acid-soluble thiols, especially glutathione, and induced a prooxidative shift in the cellular glutathione redox potential. The extract increased the osmotic fragility of erythrocytes, induced hemolysis, and inhibited hemolysis in isotonic ammonium chloride, indicative of decreased membrane permeability for Cl- and increased the membrane fluidity. Fluorescent probes indicated an increased level of reactive oxygen species and induction of lipid peroxidation, but these results should be interpreted with care since the extract alone induced oxidation of the probes (dichlorodihydrofluorescein diacetate and BODIPY C11). These results demonstrate that garlic extract induces oxidative changes in the erythrocyte, first of all, thiol and hemoglobin oxidation.

Graphene and Natural Products: A Review of Antioxidant Properties in Graphene Oxide Reduction.

This review article addresses the antioxidant properties of different natural products, including ascorbic acid, gallic acid, oxalic acid, L-glutathione (GSH), bacteriorhodopsin, green tea polyphenols, glucose, hydroxycinnamic acid, ethanoic acid, betanin, and L-glutathione, in the reduction of graphene oxide (rGO). rGO can cause damage to cells, including oxidative stress and inflammation, limiting its application in different sectors that use graphene, such as technologies used in medicine and dentistry. The natural substances reviewed have properties that help reduce this damage, neutralizing free radicals and maintaining cellular integrity. This survey demonstrates that the combination of these antioxidant compounds can be an effective strategy to minimize the harmful effects of rGO and promote cellular health.

Metallofullerenol alleviates alcoholic liver damage via ROS clearance under static magnetic and electric fields.

Alcoholic liver disease (ALD) is a common chronic redox disease caused by increased alcohol consumption. Abstinence is a major challenge for people with alcohol dependence, and approved drugs have limited efficacy. Therefore, this study aimed to explore a new treatment strategy for ALD using ferroferric oxide endohedral fullerenol (Fe3O4@C60(OH)n) in combination with static magnetic and electric fields (sBE). The primary hepatocytes of 8-9-week-old female BALB/c mice were used to evaluate the efficacy of the proposed combination treatment. A mouse chronic binge ethanol feeding model was established to determine the alleviatory effect of Fe3O4@C60(OH)n on liver injury under sBE exposure. Furthermore, the ability of Fe3O4@C60(OH)n to eliminate •OH was evaluated. Alcohol-induced hepatocyte and mitochondrial damage were reversed in vitro. Additionally, the combination therapy reduced liver damage, alleviated oxidative stress by improving antioxidant levels, and effectively inhibited liver lipid accumulation in animal experiments. Here, we used a combination of magnetic derivatives of fullerenol and sBE to further improve the ROS clearance rate, thereby alleviating ALD. The developed combination treatment may effectively improve alcohol-induced liver damage and maintain redox balance without apparent toxicity, thereby enhancing therapy aimed at ALD and other redox diseases.

Glycated LDL generates reactive species that damage cell components, oxidize hemoglobin and alter surface morphology in human erythrocytes.

Low-density lipoprotein (LDL) transports cholesterol to various tissues via the blood. Glycation of LDL occurs during hyperglycemic condition which is characterised by persistently high blood glucose level. Circulating erythrocytes can come in direct contact with glycated LDL (G-LDL). The objective of this study was to investigate the effect of G-LDL on human erythrocytes, specifically on hemoglobin, intracellular generation of reactive species and the antioxidant defence system. Isolated erythrocytes were incubated with G-LDL (3 and 6 mg/ml) and native LDL (6 mg/ml) at 37 °C for 24 h. Native LDL and G-LDL untreated erythrocytes were similarly incubated at 37 °C and served as control. G-LDL treatment increased hemolysis compared to control and native LDL-treated erythrocytes. Incubation of erythrocytes with G-LDL led to an increase in protein oxidation and lipid peroxidation while greatly decreasing the total sulfhydryl content. It also significantly enhanced hemoglobin oxidation, heme degradation, and the release of free iron moiety. Treatment with G-LDL led to an appreciable increase in the production of reactive oxygen and nitrogen species. The antioxidant power and activities of major antioxidant enzymes were drastically reduced, while critical membrane-bound enzymes were inhibited. The surface morphology of G-LDL-treated erythrocytes was altered leading to the formation of echinocytes. Importantly, treatment of erythrocytes with native LDL did not significantly affect the above-mentioned parameters and values were similar to the corresponding controls. Thus, G-LDL is cytotoxic to human erythrocytes and causes oxidative damage to cell components. This can reduce the oxygen-transporting ability of blood and also result in red cell senescence and anemia.

Inhibition of QDPR synergistically modulates intracellular tetrahydrobiopterin profiles in cooperation with methotrexate.

Tetrahydrobiopterin (BH4) is an essential cofactor for dopamine and serotonin synthesis in monoaminergic neurons, phenylalanine metabolism in hepatocytes, and nitric oxide synthesis in endothelial and immune cells. BH4 is consumed as a cofactor or is readily oxidized by autooxidation. Quinonoid dihydropteridine reductase (QDPR) is an enzyme that reduces quinonoid dihydrobiopterin (qBH2) back to BH4, and we have previously demonstrated the significance of QDPR in maintaining BH4 in vivo using Qdpr-KO mice. In addition to the levels of BH4 in the cells, the ratios of oxidized to reduced forms of BH4 are supposed to be important for regulating nitric oxide synthase (NOS) via the so-called uncoupling of NOS. However, previous studies were limited due to the absence of specific and high-affinity inhibitors against QDPR. Here, we performed a high-throughput screening for a QDPR inhibitor and identified Compound 9b with an IC50 of 0.72 µM. To understand the inhibition mechanism, we performed kinetic analyses and molecular dynamics simulations. Treatment with 9b combined with methotrexate (MTX), an inhibitor of another BH4-reducing enzyme, dihydrofolate reductase (DHFR), significantly oxidized intracellular redox states in HepG2, Jurkat, SH-SY5Y, and PC12D cells. Collectively, these findings suggest that 9b may enhance the anticancer and anti-autoimmune effects of MTX.

Redox balance and immunity of piglets pre- and post-E. coli challenge after treatment with hemp or fish oil, and vitamin E.

This study investigated the influence of polyunsaturated fatty acid composition and vitamin E supplementation on oxidative status and immune responses in weanling piglets pre- and post-E. coli challenge. Suckling piglets (n = 24) were randomly selected from two litters for an oral supplementation (1 mL/day) with fish oil or hemp oil and vitamin E supplementation (60 mg natural vitamin E/mL oil) from day 10 to 28 of age. At day 29 and 30 of age, each piglet was orally inoculated with 6.7 × 108 and 3.96 × 108 CFU of F4 and F18 E. coli, respectively. Blood was sampled from all piglets on day 28 before E. coli challenge and on day 35 of age to investigate immunological and oxidative stress markers in plasma. One week after weaning and exposure to E. coli, a general reduction in the α-tocopherol concentration and activity of GPX1 was obtained. Vitamin E supplementation lowered the extent of lipid peroxidation and improved the antioxidative status and immune responses after E. coli challenge. Hemp oil had the greatest effect on antioxidant enzyme activity. Provision of hemp oil and vitamin E to suckling piglets may reduce the incidence of post-weaning diarrhea.

Redox homeostasis in human renal cells that had been treated with amphotericin B in combination with selected 1,3,4-thiadiazole derivatives.

BACKGROUND: The use of amphotericin B (AmB) in the therapy of systemic mycosis is associated with strong side effects, including nephrotoxicity, and hepatotoxicity. Therefore, agents that can reduce the toxic effects of AmB while acting synergistically as antifungal agents are currently being sought. 1,3,4-thiadiazole derivatives are promising compounds that have an antifungal activity and act synergically with AmB. Such combinations might allow the dose of AmB, which is essential for preventing patients from having serious side effects, to be decreased. This might result from the antioxidant properties of 1,3,4-thiadiazoles. Thus, the aim of the study was to investigate redox homeostasis in human renal proximal tubule epithelial cells (RPTEC) after they had been treated with AmB in combination with 1,3,4-thiadiazole derivatives. METHODS: Cellular redox homeostasis was assessed by investigating the total antioxidant capacity (TAC) of cells, the malondialdehyde (MDA) concentration, and the activity of antioxidant enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). TAC was measured using an ABTS method. The MDA concentration, and the activity of SOD, GPX, and CAT were determined spectrophotometrically using commercially available assays. Additionally, the antioxidant defense system-related gene expression profile was determined using oligonucleotide microarrays (HG-U133A 2.0). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to confirm the microarray results. RESULTS: Amphotericin B and selected 1,3,4-thiadiazole derivatives had a significant effect on the total antioxidant capacity of the RPTEC cells, and the activity of the antioxidant enzymes. We also revealed that the effect of thiadiazoles on the SOD and CAT activities is dependent on the treatment of RPTEC cells with AmB. At the transcriptional level, the expression of several genes was affected by the studied compounds and their combinations. CONCLUSIONS: The results confirmed that thiadiazoles can stimulate the RPTEC cells to defend against the oxidative stress that is generated by AmB. In addition, together with the previously demonstrated synergistic antifungal activity, and low nephrotoxicity, these compounds have the potential to be used in new therapeutic strategies in the treatment of fungal infections.

The effect of dietary eugenol nano-emulsion supplementation on growth performance, serum metabolites, redox homeostasis, immunity, and pro-inflammatory responses of growing rabbits under heat stress.

Background: Heat stress (HS) is a main abiotic stress factor for the health and welfare of animals. Recently, the use of nano-emulsion essential oils exhibited a promising approach to mitigate the detrimental impacts of abiotic and biotic stresses, ultimately contributing to the global aim of sustainable livestock production. Aim: The current study was piloted to assess the impact of eugenol nano-emulsion (EUGN) supplementation on growth performance, serum metabolites, redox homeostasis, immune response, and pro-inflammatory reactions in growing rabbits exposed to HS. Methods: A total of 100 male weaning rabbits aged 35 days were divided into 4 treatments. Rabbits were fed the diet with EUGN at different concentrations: 0 (control group; EUGN0), 50 (EUGN50), 100 (EUGN100), and 150 (EUGN150) mg/kg diet for 8 weeks under summer conditions. Results: Dietary EUGN levels significantly improved (p < 0.05) the body weight, body weight gain, carcass weights, and improved feed conversion ratio of rabbits. EUGN supplementation significantly increased Hb, platelets, and red blood cells , while the mean corpuscular hemoglobin and eosinophils were significantly decreased compared to the control one. Compared with EUGN0 stressed rabbits, all EUGN-experimental groups had a reduction in levels of total glycerides (p < 0.01), uric acid, total bilirubin, direct bilirubin, and gamma-glutamyl transpeptidase (p < 0.01). Total antioxidant capacity and glutathione peroxidase were significantly improved by EUGN treatment when compared to the control one (p < 0.01), while the EUGN100 exhibited the greatest levels of catalase. Lipid peroxidation (malondialdehyde) was significantly decreased in EUGN-treated groups. All pro-inflammatory cytokines serum interleukin 4, Interleukin 1ß, and tumor necrosis factor alpha were considerably decreased after dietary EUGN supplementation (p < 0.05). The serum concentrations of immunoglobulins (IgG and IgM) were significantly improved in rabbits of the EUGN150 group. Conclusion: This study shows that EUGN can be used as a novel feed additive to enhance the growth performance, immune variables, and antioxidants, and reduce the inflammatory response of growing rabbits exposed to thermal stress.

Oxidized glutathione reverts carbapenem resistance in blaNDM-1-carrying Escherichia coli.

The emergence of drug-resistant Enterobacteriaceae carrying plasmid-mediated ß-lactamase genes has become a significant threat to public health. Organisms in the Enterobacteriaceae family containing New Delhi metallo-ß-lactamase­1 (NDM-1) and its variants, which are capable of hydrolyzing nearly all ß-lactam antibacterial agents, including carbapenems, are referred to as superbugs and distributed worldwide. Despite efforts over the past decade, the discovery of an NDM-1 inhibitor that can reach the clinic remains a challenge. Here, we identified oxidized glutathione (GSSG) as a metabolic biomarker for blaNDM-1 using a non-targeted metabolomics approach and demonstrated that GSSG supplementation could restore carbapenem susceptibility in Escherichia coli carrying blaNDM-1 in vitro and in vivo. We showed that exogenous GSSG promotes the bactericidal effects of carbapenems by interfering with intracellular redox homeostasis and inhibiting the expression of NDM-1 in drug-resistant E. coli. This study establishes a metabolomics-based strategy to potentiate metabolism-dependent antibiotic efficacy for the treatment of antibiotic-resistant bacteria.

Gallic acid mitigates intestinal inflammation and loss of tight junction protein expression using a 2D-Caco-2 and RAW 264.7 co-culture model.

A 2D-intestinal epithelial Caco-2/RAW 264.7 macrophage co-culture model was developed to demonstrate the relative efficacy of different phenolic acids to mitigate changes in Caco-2 epithelial cell redox state initiated both directly by autoxidation products, H2O2, and indirectly through cell communication events originating from cytokine stimulated macrophage. An inducer cocktail (lipopolysaccharide + interferon gamma) was used to activate RAW 264.7 cells in the 2D- Caco-2/RAW co-culture and intracellular changes in Caco-2 cell redox signaling occurred in response to positive changes (p < 0.05) in inflammatory biomarkers derived in macrophage that included IL-6, TNF-α, nitric oxide and peroxynitrite, respectively. Phenolic acids varied in relative capacity to reduce NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in cocktail inflamed induced macrophage. This response in addition to the relative predisposition of gallic acid (GA) to undergo autoxidation to generate H2O2 activity (p < 0.05), culminated in downstream cell signaling in Caco-2 nuclear factor erythroid 2-related factor (Nrf2) activity (increase 26.9 %), altered monolayer integrity (increase 33.7 %), and release of interleukin 8 (IL-8) (decrease 80.5 %) (p < 0.05). It can be concluded that the co-culture model described herein was useful to assess the importance of communication between cytokine stimulated macrophage and intestinal cells. Moreover, the relative unique efficacy of GA, compared to other phenolic acids tested to protect against activated macrophage induced changes related to intestinal dysfunction were particularly relevant to epithelial redox signaling, intestinal permeability and regulation of tight junction proteins. This study concludes that phenolic acids are not equal in the capacity to protect against intestinal cell dysfunction despite some indication of biological activity.

KN-93 promotes HDAC4 nucleus translocation to promote fatty acid oxidation in myocardial infarction.

Myocardial infarction (MI) is a potentially fatal disease that causes a significant number of deaths worldwide. The strategy of increasing fatty acid oxidation in myocytes is considered a therapeutic avenue to accelerate metabolism to meet energy demands. We conducted the study aiming to investigate the effect of KN-93, which induces histone deacetylase (HDAC)4 shuttling to the nucleus, on fatty acid oxidation and the expression of related genes. A mouse model of myocardial infarction was induced by isoprenaline administration. Heart damage was assessed by the detection of cardiac injury markers. The level of fatty acid oxidation level was evaluated by testing the expression of related genes. Both immunofluorescence and immunoblotting in the cytosol or nucleus were utilized to observe the distribution of HDAC4. The interaction between HDAC4 and specificity protein (SP)1 was confirmed by co-immunoprecipitation. The acetylation level of SP1 was tested after KN-93 treatment and HDAC4 inhibitor. Oxygen consumption rate and immunoblotting experiments were used to determine whether the effect of KN-93 on increasing fatty acid oxidation is through HDAC4 and SP1. Administration of KN-93 significantly reduced cardiac injury in myocardial infarction and promoted fatty acid oxidation both in vitro and in vivo. KN-93 was shown to mediate nuclear translocation of HDAC4. HDAC4 was found to interact with SP1 and reduce SP1 acetylation. HDAC4 or SP1 inhibitors attenuated the effect of KN-93 on fatty acid oxidation. In conclusion, KN-93 promotes HDAC4 translocation to the nucleus, thereby potentially enhancing fatty acid oxidation by SP1.