Superoxide Release Research Articles

Effects of uric acid on oxidative stress in vascular smooth muscle cells.

Hyperuricemia during hypertension is associated with aberrant vascular functions and increased oxidative stress, which affects endothelial dysfunction. Nevertheless, the molecular mechanisms underlying the effects of uric acid on vascular smooth muscle cells (VSMCs) through oxidative stress remain unclear. The aim of the present study was to investigate the dose- and time-dependent effects of uric acid on oxidative stress and p53 protein expression in VSMCs. VSMCs were incubated with various concentrations of uric acid (0-50 mg/dl) for different time periods (1-24 h). Thiobarbituric acid reactive substances (TBARs), protein carbonylation and nitric oxide (NO) levels were determined using appropriate assay kits. Superoxide anion release was detected using the Görlach method. Western blotting was performed to determine the protein expression levels of p53. The findings demonstrated that the application of uric acid led to an increase in protein carbonylation and superoxide anion levels while causing a decrease in NO levels. Conversely, no significant effect was observed on TBARS levels. Additionally, it was observed that high concentrations of uric acid suppressed p53 expression at 6, 12 and 24 h. The present study provided evidence that the influence of uric acid on oxidative stress was more closely associated with time than dose; however, not all effects observed were strictly time-dependent.

MicroRNA 29a alleviates mitochondrial stress in diet-induced NAFLD by inhibiting the MAVS pathway

Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder characterized by fat accumulation in the liver. This leads to aggravated hepatocyte inflammation due to impaired mitochondrial function, mitochondrial double-stranded RNA (mt-dsRNA) release, elevated oxidative stress, and reactive oxygen species (ROS) production.MicroRNA-29a (miR-29a) is used to reduce hepatic fibrosis in cases of cholestatic liver damage and lessen the severity of non-alcoholic steatohepatitis in animal studies by influencing mitochondrial protein balance. However, the effectiveness of miR-29a in diminishing mt-dsRNA-induced exacerbation of NAFLD remains poorly understood, particularly in the context of a Western diet (WD). Our results have found that mice with increased miR-29a levels and fed a WD showed notably decreased serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol, and low-density lipoprotein cholesterol levels. They also experienced less weight gain and lower final body and liver weights. In addition, overexpression of miR-29a reduced the severity of fibrosis, alleviated hepatic oxidative stress, misfolded protein aggregates, and the release of mt-dsRNA. Moreover, miR-29a attenuated the innate immune mitochondrial antiviral-signaling protein (MAVS) pathway response. In vitro, the research using HepG2 cells confirmed that miR-29a reduces MAVS expression and decreases the release of mt-dsRNA and superoxide initiated by palmitic acid (PA). Analysis of luciferase activity further established that the specific binding of miR-29a to the 3′UTR of MAVS led to a repression of its expression. In conclusion, these groundbreaking findings underscore the potential of miR-29a in improving the treatment of NAFLD and liver steatofibrosis by inhibiting the MAVS signaling pathway.

Abstract We092: Naltrindole Attenuates Superoxide Release in Polymorphonuclear Leukocytes by a Novel Mechanism devoid of Delta opioid receptor antagonism Related to Reduced Intracellular Calcium Levels

Introduction: Naltrindole (NTI), known as a delta opioid receptor antagonist, inhibited phorbol 12-myristate 13-acetate (PMA) induced superoxide (SO) release in polymorphonuclear leukocytes (PMNs). PMNs do not express opioid receptors. We propose NTI reduces PMN SO by reducing intracellular calcium (Ca 2+ ). To test this hypothesis, we did experiments using KB-R7943 (KB) as a positive control, known to reduce intracellular Ca 2+ via inhibition of the reverse mode Na + /Ca 2+ exchanger. Naloxone (NX), a broad-spectrum opioid receptor antagonist, was used as a negative control. We predict that NTI will diminish PMA-induced PMN SO release similar to KB, while NX will have no effect, and that there will be no difference in cell viability compared to vehicle controls. Methods: Male Sprague Dawley (SD) rat PMNs (5x10 6 ) were incubated for 15 min at 37 o C in the presence or absence (dH 2 O vehicle control) of NTI (10 - 200 µM), KB (5-20 µM), and NX (100-200 µM). PMN SO release was measured by the change in absorbance at 550 nm over 420 sec via ferricytochrome c reduction after PMA stimulation (100 nM). The cell viability was determined microscopically by 0.2% trypan blue exclusion at the end of PMN SO release assay. Data were analyzed using ANOVA Fisher’s PLSD post-hoc test. Results: NTI significantly decreased PMN SO release at 200 μM (n=10, 0.196±0.06, p<0.05) compared to vehicle control (n=22, 0.731±0.07) or NX 100/200 μM (n=3, 0.593±0.06), and NTI at 100 μM (n=7, 0.461±0.08, p<0.05) versus vehicle control. Likewise, KB significantly decreased PMN SO release at 20 μM (n=7, 0.507±0.07), 10 μM (n=7, 0.552±0.07), and 5 μM (n=5, 0.615±0.11) compared to vehicle control (n=10, 0.908±0.07). Cell viability was statistically similar (80-90±5%) among all study groups. Conclusion: NTI (10-200 μM) and KB (5-20 µM) exhibited concentration-dependent effects in reducing PMN SO release without compromising cell viability. The results suggest that the novel effect of NTI maybe mediated through a reduction in intracellular Ca 2+ and independent of delta opioid receptor antagonism. These results are consistent with the effects of NTI, KB and NX in myocardial ischemia/reperfusion injuries. Future studies will assess the effects of NTI on ROS attenuation in human umbilical vein endothelial cells.

Abstract Tu145: Exploring Naltrindole’s hypercontracture attenuating effects during myocardial ischemia via a novel mechanism that produces cardioprotective effects in myocardial ischemia/reperfusion injury

Introduction: Previously, naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5µM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. Additionally, we recently showed that NTI 100-200µM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. Hypothesis: We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca 2+ ). Testing this hypothesis, we conducted isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca 2+ via inhibition of reverse mode Na + /Ca 2+ exchanger and, therefore, should attenuate myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). We predict that both NTI and KB will improve LVEDP and decrease infarct size compared to vehicle (dH 2 O) control and naloxone (NX) used as a negative control. Methods: Hearts were isolated from anesthetized male Sprague Dawley (SD) rats (~300g). We then infused NTI 1.25-5µM, KB 10-20µM, or NX 10µM for 5 min into perfused hearts, just prior to global I(30min)/R(45min). Cardiac left ventricular function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in the MIR assay. Data were analyzed using ANOVA and Fishers post-hoc analysis, with p<0.05 considered statistically significant. Results: MIR assay: NTI 5µM, 2.5µM, 1.25µM and KB 20 µM reduced infarct size (4.8±3%, n=5, p<0.05), (6.8±0.8%, n=6, p<0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p<0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10µM controls (18±3%, n=3). NTI 5 µM and KB 20 µM had significantly improved LVEDP to (32.4±11%, n=5, p<0.05) and (22.8±4%, n=5, p<0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10µM controls (54.2±8%, n=3) Conclusion: Both NTI (2.5µM, 5µM) and KB (20µM) reduced infarct size and improved LVEDP. The novel MOA of NTI may be mediated via a reduction in intracellular Ca 2+ during myocardial ischemia. The infarct reducing effect of NTI can benefit patients undergoing elective PCI, CABG, and transplant procedures.

Signaling by Neutrophil G Protein-Coupled Receptors that Regulate the Release of Superoxide Anions.

In human peripheral blood, the neutrophil granulocytes (neutrophils) are the most-abundant white blood cells. These professional phagocytes are rapidly recruited from the bloodstream to inflamed tissues by chemotactic factors that signal danger. Neutrophils, which express many receptors that are members of the large family of G protein-coupled receptors (GPCRs), are critical for the elimination of pathogens and inflammatory insults, as well as for the resolution of inflammation leading to tissue repair. Danger-signaling molecular patterns such as the N-formylated peptides that are formed during bacterial and mitochondrial protein synthesis and recognized by formyl peptide receptors (FPRs) and free fatty acids recognized by free fatty acid receptors (FFARs) regulate neutrophil functions. Short peptides and short chain fatty acids activate FPR1 and FFA2R, respectively, while longer peptides and fatty acids activate FPR2 and GPR84, respectively. The activation profiles of these receptors include the release of reactive oxygen species (ROS) generated by the NADPH oxidase. Activation of the oxidase and the production of ROS are processes that are regulated by proinflammatory mediators, including TNFα and GM-CSF. The receptors have signaling and functional similarities, although there are also important differences, not only between the two closely related neutrophil FPRs, but also between the FPRs and the FFARs. In neutrophils, these receptors never walk alone, and additional mechanistic insights into the regulation of the GPCRs and the novel regulatory mechanisms underlying the activation of NADPH oxidase advance our understanding of the role of receptor transactivation in the regulation of inflammatory reactions.

Real-time monitoring of cellular superoxide anion release in THP-1 cells using a catalytically amplified superoxide dismutase-based microbiosensor.

Reactive oxygen species (ROS) including the superoxide anion (O2•-) are typically studied in cell cultures using fluorescent dyes, which provide only discrete single-point measurements. These methods lack the capabilities for assessing O2•- kinetics and release in a quantitative manner over long monitoring times. Herein, we present the fabrication and application of an electrochemical biosensor that enables real-time continuous monitoring of O2•- release in cell cultures for extended periods (> 8h) using an O2•- specific microelectrode. To achieve the sensitivity and selectivity requirements for cellular sensing, we developed a biohybrid system consisting of superoxide dismutase (SOD) and Ti3C2Tx MXenes, deposited on a gold microwire electrode (AuME) as O2•- specific materials with catalytic amplification through the synergistic action of the enzyme and the biomimetic MXenes-based structure. The biosensor demonstrated a sensitivity of 18.35 nA/μM with a linear range from 147 to 930nM in a cell culture medium. To demonstrate its robustness and practicality, we applied the biosensor to monitor O2•- levels in human leukemia monocytic THP-1 cells upon stimulation with lipopolysaccharide (LPS). Using this strategy, we successfully monitored LPS-induced O2•- in THP-1 cells, as well as the quenching effect induced by the ROS scavenger N-acetyl-L-cysteine (NAC). The biosensor is generally useful for exploring the role of oxidative stress and longitudinally monitoring O2•- release in cell cultures, enabling studies of biochemical processes and associated oxidative stress mechanisms in cellular and other biological environments.

Arginine-specific gingipains (RgpA/RgpB) knockdown modulates neutrophil machinery

ABSTRACT Background Gingipains are important virulence factors present in Porphyromonas gingivalis. Arginine-specific gingipains (RgpA and RgpB) are critically associated with increased proteolytic activity and immune system dysfunction, including neutrophilic activity. In this study, we assessed the impact of gingipains (RgpA and RgpB) on neutrophil function. Methods Peripheral blood samples were obtained; neutrophils were isolated and incubated with P. gingivalis A7436, W50, and the double RgpA/RgpB double knockout mutant E8 at MOI 20 for 2 hours. Neutrophil viability was assessed by Sytox staining. Phagocytic capacity and apoptosis were measured by flow cytometry. Superoxide release was measured by superoxide dismutase and cytochrome c reduction assay. Gene expression of TLR2, p47-phox, p67-phox, and P2 × 7was measured by qPCR. Inflammatory cytokine and chemokine production was measured by IL-1β, IL-8, RANTES, and TNF-α in cell supernatants. Results Neutrophil TLR2 gene expression was reduced in the absence of RgpA/RgpB (p < 0.05), while superoxide production was not significantly impacted. RgpA/RgpB−/− significantly impaired neutrophil phagocytic function (p < 0.05) and increased TNF-α production when compared with the wild-type control (p < 0.05). Neutrophil apoptosis was not altered when exposed to RgpA/RgpB−/− E8 (p > 0.05). Conclusion These data suggest that arginine-specific gingipains (RgpA/RgpB) can modulate neutrophil responses against P. gingivalis infection.

Naltrindole attenuates irreversible Hypercontracture during Myocardial Ischemia by a Novel Mechanism and elicits robust cardioprotective effects in Myocardial Ischemia/Reperfusion Injury

Introduction: Previous studies have shown that naltrindole (NTI) produced robust cardioprotective effects (e.g.~80% reduction of infarct size) in both ex-vivo (2.5-5μM) and in-vivo (3.75-7.5 mg/kg) rat myocardial ischemia-reperfusion (MIR) injury models. We recently showed that NTI 100-200μM attenuated phorbol 12-myristate 13-acetate (PMA) induced polymorphonuclear leukocytes (PMNs) superoxide (SO) release by a novel mechanism of action (MOA) devoid of opioid receptors. We hypothesize NTI’s novel MOA is a reduction of intracellular calcium (Ca2+). To test this hypothesis, we conducted PMN SO release and isolated perfused MIR assays using KB-R7943(KB). KB is known to reduce intracellular Ca2+ via inhibition of reverse mode Na+/Ca2+ exchanger and therefore should attenuate PMN SO release, myocardial ischemic hypercontracture, infarct size, and left ventricular end diastolic pressure (LVEDP). Methods: PMNs and hearts isolated from anesthetized male Sprague Dawley (SD) rats (~400g SO release assay, ~300g for perfused hearts). PMN SO release was stimulated using PMA (100nM) ± NTI 10-200 μM, KB 5-20 μM, or vehicle (0.5% DMSO; controls) and measured spectrophotometrically as a change in absorbance @ 550nm over 390 secs and cell viability was determined using 0.2% Trypan blue exclusion. We then infused NTI 1.25-5μM, KB 10-20μM, or naloxone (NX; negative control) 10μM for 5 min into perfused hearts, just prior to global I (30min)/R(45min). Cardiac LV function was measured via a pressure transducer and infarct size using 1% triphenyltetrazolium chloride staining in MIR assay. Data were analyzed using ANOVA and Fischers post-hoc analysis, with p&lt;0.05 considered statistically significant. Results: PMN SO release assay: KB 10 μM, KB 20 μM, NTI 100 μM (~3.75 mg/kg), and NTI 200 μM(~7.5 mg/kg) significantly attenuated PMA induced PMN SO release by ~40% (KB 10μM, n=7, p&lt;0.05), ~50% ((KB 20μM, n=7, p&lt;0.05), ~30% (NTI 100 μM, n=7, p&lt;0.05), 75% (NTI 200μM, n=7, p&lt;0.05) respectively when compared to untreated control (n=10). MIR assay: NTI 5μM, 2.5μM, 1.25μM and KB 20 μM reduced infarct size (4.8±3%, n=5, p&lt;0.05), (6.8±0.8%, n=6, p&lt;0.05), (12.2±3%, n=5), and (2.9±2%, n=5, p&lt;0.05) respectively compared to vehicle (14.1±2%, n=9) and NX 10μM controls (18±3%, n=3). NTI 5 μM and KB 20 μM had significantly improved LVEDP to (32.4±11%, n=5, p&lt;0.05) and (22.8±4%, n=5, p&lt;0.05) respectively compared to vehicle control (72.4±6%, n=9) and NX 10μM controls(54.2±8%, n=3) Conclusion: NTI and KB showed concentration-dependent effects on reducing PMN SO release. Both NTI (2.5μM, 5μM) and KB (20μM) reduced infarct size and improved LVEDP. The novel MOA of NTI is most likely mediated via a reduction in intracellular Ca2+ and subsequent hypercontracture during myocardial ischemia. The infarct reducing effect of NTI can benefit patients who will undergo elective PCI, CABG, and transplants. Funding: This study was funded by the Philadelphia College of Osteopathic Medicine, Department of Biomedical Sciences, Division of Research, and the Center for Chronic Diseases of Aging and Young Therapeutics, LLC. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Neutrophil response to Porphyromonas gingivalis is modulated by low-level laser application.

Neutrophil response is critical in inflammatory regulation and immune response to bacterial infections. During periodontal disease, pathogenic bacteria lead to exaggerated neutrophil responses. We hypothesized that low-level laser application (LLLT), therapeutic strategy for dampening inflammatory processes, will regulate neutrophil activity in response to periodontopathogens. The impact of LLLT on neutrophil responses was measured by light delivered at wavelength of 850 nm. The direct effect of LLLT on P. gingivalis A7436 was determined by flow cytometry using LIVE/DEADTM Cell Vitality kit. The phagocytosis of P. gingivalis A7436 by human neutrophils was measured using flow cytometry. Superoxide generation was measured by cytochrome-C-reduction in the presence of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP; 1 mM). Cytokine release by neutrophils was measured by multiplex immunoassay. The phagocytosis of P. gingivalis by primary human neutrophils was significantly reduced in response to LLLT (p < 0.05). While LLLT led to increased superoxide production in neutrophils that were not challenged by P. gingivalis, it dampened the increased superoxide and IL-6 release by the neutrophils in response to P. gingivalis. LLLT did not directly affect the viability of P. gingivalis. These results suggested that LLLT can provide therapeutic strategy in periodontal disease, regulating the neutrophil response to P. gingivalis.

Mineralocorticoid receptor promotes cardiac macrophage inflammaging

Inflammaging, a pro-inflammatory status that characterizes aging and primarily involving macrophages, is a master driver of age-related diseases. Mineralocorticoid receptor (MR) activation in macrophages critically regulates inflammatory and fibrotic processes. However, macrophage-specific mechanisms and the role of the macrophage MR for the regulation of inflammation and fibrotic remodeling in the aging heart have not yet been elucidated. Transcriptome profiling of cardiac macrophages from male/female young (4 months-old), middle (12 months-old) and old (18 and 24 months-old) mice revealed that myeloid cell-restricted MR deficiency prevents macrophage differentiation toward a pro-inflammatory phenotype. Pathway enrichment analysis showed that several biological processes related to inflammation and cell metabolism were modulated by the MR in aged macrophages. Further, transcriptome analysis of aged cardiac fibroblasts revealed that macrophage MR deficiency reduced the activation of pathways related to inflammation and upregulation of ZBTB16, a transcription factor involved in fibrosis. Phenotypic characterization of macrophages showed a progressive replacement of the TIMD4+MHC-IIneg/low macrophage population by TIMD4+MHC-IIint/high and TIMD4–MHC-IIint/high macrophages in the aging heart. By integrating cell sorting and transwell experiments with TIMD4+/TIMD4–macrophages and fibroblasts from old MRflox/MRLysMCre hearts, we showed that the inflammatory crosstalk between TIMD4– macrophages and fibroblasts may imply the macrophage MR and the release of mitochondrial superoxide anions. Macrophage MR deficiency reduced the expansion of the TIMD4– macrophage population and the emergence of fibrotic niches in the aging heart, thereby protecting against cardiac inflammation, fibrosis, and dysfunction. This study highlights the MR as an important mediator of cardiac macrophage inflammaging and age-related fibrotic remodeling.

CD11B/CD18 SIALYLATION REGULATES NEUTROPHIL TRAFFICKING AND INFLAMMATORY FUNCTION IN THE INTESTINE

Abstract Pathogen-triggered neutrophil (PMN) recruitment is critical for innate immunity, but excessive PMN influx and associated mucosal tissue damage is implicated in the pathogenesis of numerous human inflammatory disorders including inflammatory bowel disease (IBD). Critically in both ulcerative colitis and Crohn’s disease, disease flares requiring medical intervention are associated with migration of large numbers of PMNs across colonic epithelium resulting in mucosal injury/ulceration. Recent studies have shown that specific PMN glycans can be targeted to reduce transepithelial migration (TEpM) the critical final step in PMN intestinal trafficking. However the role of Sialic acid (Sia), the most abundant PMN expressed terminal glycan, in regulating PMN migration and inflammatory function in the gut is not well understood. Using sophisticated models of human and murine PMN intestinal migration, we show that blocking sialidase-mediated removal of alpha 2-3 linked sialic acid from CD11b/CD18 inhibits PMN TEpM in vitro and in vivo.Measurement of cytochrome C reduction revealed that inhibition of sialidases reduced bacterial peptide (fMLF) stimulated superoxide release by human and murine PMN. Flow cytometry analyses revealed that sialidase inhibition decreased fMLF mediated PMN degranulation and fMLF mediated CD11b/CD18 conformational activation. Highlighting the functional importance of individual Sia residues, inhibition of alpha 2-3 sialidase activity with 3’ Sialyllactose (3’SL) was found to inhibit PMN intestinal trafficking in vitro and in vivo and reduce PMN degranulation responses. Immunoblotting studies showed that sialidase inhibition deactivates spleen tyrosine kinase (Syk) and p38 MAP kinase, key intracellular mediators that signal downstream of PMN CD11b/CD18. Finally, inhibition of sialidase activity reduced adhesion of PMN to ICAM-1 (a major physiological substrate of CD11b/CD18) demonstrating that endogenous PMN sialidases regulate binding interactions between migrating PMN and intestinal epithelial receptors. Taken together data show that upon activation PMN sialidases remove alpha 2-3 Sia from CD11b/CD18 which leads to integrin activation and subsequent outside in signaling mediated by Syk to drive PMN inflammatory effector functions including migration, degranulation, and superoxide release. As such targeting CD11b/CD18 sialylation represents a promising new therapeutic strategy for reducing dysregulated PMN influx and associated mucosal tissue damage in IBD and other inflammatory disorders.

How a natural antibiotic uses oxidative stress to kill oxidant-resistant bacteria.

Natural products that possess antibiotic and antitumor qualities are often suspected of working through oxidative mechanisms. In this study, two quinone-based small molecules were compared. Menadione, a classic redox-cycling compound, was confirmed to generate high levels of reactive oxygen species inside Escherichia coli. It inactivated iron-cofactored enzymes and blocked growth. However, despite the substantial levels of oxidants that it produced, it was unable to generate significant DNA damage and was not lethal. Streptonigrin, in contrast, was poorer at redox cycling and did not inactivate enzymes or block growth; however, even in low doses, it damaged DNA and killed cells. Its activity required iron and oxygen, and in vitro experiments indicated that its quinone moiety transferred electrons through the adjacent iron atom to oxygen. Additionally, in vitro experiments revealed that streptonigrin was able to damage DNA without inhibition by catalase, indicating that hydrogen peroxide was not involved. We infer that streptonigrin can reduce bound oxygen directly to a ferryl species, which then oxidizes the adjacent DNA, without release of superoxide or hydrogen peroxide intermediates. This scheme allows streptonigrin to kill a bacterial cell without interference by scavenging enzymes. Moreover, its minimal redox-cycling behavior avoids alerting either the OxyR or the SoxRS systems, which otherwise would block killing. This example highlights qualities that may be important in the design of oxidative drugs. These results also cast doubt on proposals that bacteria can be killed by stressors that merely stimulate intracellular O2- and H2O2 formation.

Eucalyptus-derived essential oils alleviate microbes and modulate inflammation by suppressing superoxide and elastase release

The Eucalyptus tree, belonging to the myrtle family, grows all over the world for its pharmaceutical and industrial benefits. In this article, we present a comparative analysis of the chemical composition of the hydrodistilled oils obtained from three different Eucalyptus species growing in Egypt viz. E. citriodora, E. camaldulensis, and E. ficifolia. Gas Chromatography-Mass Spectrometric guided analysis resulted in the identification of a total of 20 metabolites in E. citriodora oil with citronellal (54.9%) and citronellol (25.4%) being the most dominant components. β-cymene (12.7%) and 1,8-cineole (11.7%) were the major volatile constituents identified in E. camaldulensis oil, while trans-β-ocimene (22.4%), 1,8-cineole (13.5%), and L-trans-pinocarveol (12.5%) were the dominating components in the oil of E. ficifolia. The essential oils of the studied species were evaluated for their in vitro anti-inflammatory, antiviral including anti-SARS-CoV-2 (severe acute respiratory syndrome corona virus 2), antibacterial, and antifungal activities. E. citriodora oil displayed the highest inhibitory activity on the release of the superoxide radical (32%) and elastase enzyme (31%) in human neutrophils, while E. ficifolia oil had enhancing effects on elastase. The latter showed significant antiviral effects against hepatitis A, herpes simplex, and coxsackie viruses with IC50 values at 2.1, 2.5, and 5.6 μg/mL, respectively. Moderate antibacterial and antifungal activities were observed for Eucalyptus oils with Staphylococcus aureus being the most susceptible bacterial strain. E. ficifolia oil, similarly, displayed the best antibacterial activity with minimum inhibitory concentration (MIC) value at ca. 25 μg/mL (for S. aureus). On the contrary, E. camaldulensis oil was the most active against Candida albicans with an MIC value at 45 μg/mL. In silico studies were performed with a number of macromolecular drug targets for confirming the biological activities of the identified compounds and for interpreting their ADME (absorption-distribution-metabolism-elimination) parameters.

Unprecedented anti-inflammatory biscembranoids with 3-hydroperoxy-3-methylcyclohex-1-ene moiety isolated from aquaculture Sarcophyton trocheliophorum

Recent advancements in the discovery of natural products have made significant progress in rapidly identifying known compounds from complex extracts using advanced spectroscopic technologies. Among these methods, one powerful approach called MS/MS molecular networking (MN) organizes chemical similarity based on MS/MS fragmentation data, serving as a valuable complement to traditional dereplication strategies. In our ongoing research focused on lead discovery from a sustainable medicinal source, an aquaculture soft coral Sarcophyton trocheliophorum, the MN profiling approach revealed the chemical diversity of biscembranoid analogs. By analyzing the MN pattern, biscembranoid derivatives with unique MS/MS fragments were prioritized for further investigation. Two novel biscembranoids, sarcotroxide A (1) and B (2), were successfully obtained through targeted isolation. These novel biscembranes contained an unprecedented 3-hydroperoxy-3-methylcyclohex-1-ene moiety compared with all biscembranoid analogs. The chemical structures of the two new biscembranoids were confirmed through spectroscopic analyses and in silico calculations. Their anti-inflammatory properties were assessed against the generation of superoxide anion (O2•−) and elastase release in activated human neutrophils. Sarcotroxides A (1) and B (2) exhibited anti-inflammatory activity by inhibiting O2•− accumulation (with 43.67% and 61.62% inhibition) and elastase release (with 38.21% and 61.62% inhibition) at 10 µM. These findings validated the potential of these biscembranoids from the cultured S. trocheliophorum as promising candidates for the future development of anti-inflammatory agents specifically designed to target neutrophils.

Abstract 17814: Naltrindole Mitigates Rigor During Myocardial Ischemia by a Novel Mechanism and Shows Translational Cardioprotection in Myocardial Ischemia-Reperfusion Injury Models

Introduction: Naltrindole (NTI) was cardioprotective in both ex-vivo (5μM) and in-vivo (7.5mg/kg) rat myocardial ischemia-reperfusion (MIR) models. Recently, we showed that NTI 50-200μM (~2-7.5mg/kg, in-vivo) attenuated phorbol ester-induced PMN superoxide release by a novel mechanism devoid of opioid receptors. We hypothesize that NTI exerts its cardioprotective effects (and therefore, its effects on the infarct size) in a dose-dependent manner. Methods: Ex-vivo studies were done with Sprague Dawley male rats using a Langendorff apparatus. Excised hearts were pretreated with Krebs buffer (controls), NTI 1.25μM, NTI 2.5μM, or NTI 5μM for 5 min, followed by 30 min of global ischemia (I) and a 45 min reperfusion (R) period. In-vivo studies were performed with Sprague Dawley female rats which received infusions (NTI 0.2-1.0 mg/kg, 3.75 mg/kg, or 7.5 mg/kg) through the jugular vein (0.4mL/min) over 5 mins before regional I was induced by occluding the LAD artery for 30 min followed by a 3 hr R period. Cardiac function was measured in both studies through a pressure transducer placed into the left ventricle. Results: Ex-vivo: NTI 5μM, 2.5μM and 1.25μM reduced infarct size by 66% (4.8±1%, n=5, p&lt;0.05), 52% (6.8±0.8%, n=6, p&lt;0.05), and 13% (12.2±3%, n=5) respectively compared to control (14.1±1%, n=7). NTI 5μM (1358±300 mmHg) had significantly improved dP/dt max at the end of R compared to untreated controls (576±163, p&lt;0.05). In-vivo: High Dose (7.5 mg/kg x 5 min = 37.5 mg/kg [total dose]), Intermediate Dose (3.75 mg/kg x 5 min = 18.75 mg/kg [total dose]), and Low Dose (0.2-1.0 mg/kg x 5 min = 1.0-5.0 mg/kg [total dose]) NTI reduced infarct size by 81% (11±4%, n=5, p&lt;0.05), 55% (26±7%, n=5, p&lt;0.05), and 4% (55±4%, n=18) respectively compared to untreated controls (57±2%, n=15). DP/dt max was similar among all study groups at the end of R. Conclusions: Both ex-vivo and in-vivo MIR models showed a dose-dependent effect on reducing infarct size. This effect is most likely correlated with a reduction in rigor during myocardial ischemia. Future in-vivo studies will test 2 mg/kg and 7.5 mg/kg in rat and porcine MIR respectively. The infarct reducing effect of NTI might benefit patients who will opt for elective PCI, CABG, and transplant.

Direct real-time measurements of superoxide release from skeletal muscles in rat limbs and human blood platelets using an implantable Cytochrome C microbiosensor

Oxidative stress and excessive accumulation of the superoxide (O2.−) anion are at the genesis of many pathological conditions and the onset of several diseases. The real time monitoring of (O2.−) release is important to assess the extent of oxidative stress in these conditions. Herein, we present the design, fabrication and characterization of a robust (O2.−) biosensor using a simple and straightforward procedure involving deposition of a uniform layer of L-Cysteine on a gold wire electrode to which Cytochrome C (Cyt c) was conjugated. The immobilized layers, studied using conductive Atomic Force Microscopy (c-AFM) revealed a stable and uniformly distributed redox protein on the gold surface, visualized as conductivity and surface topographical plots. The biosensor enabled detection of (O2.−) at an applied potential of 0.15 V with a sensitivity of 42.4 nA/μM and a detection limit of 2.4 nM. Utility of the biosensor was demonstrated in measurements of real time (O2.−) release in activated human blood platelets and skeletal rat limb muscles following ischemia reperfusion injury (IRI), confirming the biosensor's stability and robustness for measurements in complex biological systems. The results demonstrate the ability of these biosensors to monitor real time release of (O2.−) and estimate the extent of oxidative injury in models that could easily be translated to human pathologies.

Pitfalls of Mitochondrial Redox Signaling Research.

Redox signaling from mitochondria (mt) to the cytosol and plasma membrane (PM) has been scarcely reported, such as in the case of hypoxic cell adaptation or (2-oxo-) 2-keto-isocaproate (KIC) β-like-oxidation stimulating insulin secretion in pancreatic β-cells. Mutual redox state influence between mitochondrial major compartments, the matrix and the intracristal space, and the cytosol is therefore derived theoretically in this article to predict possible conditions, when mt-to-cytosol and mt-to-PM signals may occur, as well as conditions in which the cytosolic redox signaling is not overwhelmed by the mitochondrial antioxidant capacity. Possible peroxiredoxin 3 participation in mt-to-cytosol redox signaling is discussed, as well as another specific case, whereby mitochondrial superoxide release is diminished, whereas the matrix MnSOD is activated. As a result, the enhanced conversion to H2O2 allows H2O2 diffusion into the cytosol, where it could be a predominant component of the H2O2 release. In both of these ways, mt-to-cytosol and mt-to-PM signals may be realized. Finally, the use of redox-sensitive probes is discussed, which disturb redox equilibria, and hence add a surplus redox-buffering to the compartment, where they are localized. Specifically, when attempts to quantify net H2O2 fluxes are to be made, this should be taken into account.

A novel PPARβ/FFA1 dual agonist Y8 promotes diabetic wound healing

BackgroundDiabetes ulcer is one of the leading causes of disability and death in diabetics. Y8 [(2-(2-fluoro-4-((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methoxy) phenoxy)acetic acid)], a dual agonist of peroxisome proliferation activated receptorβ (PPARβ) and free fatty acid receptor 1 (FFA1/FFAR1/GPR40), a new compound molecule with the potential for diabetes ulcer treatment. ObjectiveTo research the effect of the dual target agonist Y8 and its mechanism of action in the treatment of diabetic ulcers. MethodsWe have established a wound model in diabetic mice. After treatment with Y8, wound healing was evaluated by tissue pathology, reactive oxygen species (ROS) levels, and gene expression testing. Under high sugar conditions, the mechanism of Y8 affecting fibroblasts' proliferation and keratinocytes' migration is further studied. ResultsWe found that Y8 accelerated wound healing and shortened healing time in diabetic mice. Granulation tissue generation and extracellular matrix (ECM) deposition were significantly increased in Y8-treated mice. Mechanistically, Y8 promotes keratinocyte proliferation by activating PPARβ and migration of keratinocytes by triggering FFA1 in vitro. In addition, Y8 also decreased ROS levels in fibroblasts in vitro and in vivo by activating PPARβ, reducing their release of superoxide anions. ConclusionOur results suggest that PPARβ/FFA1 dual agonist Y8 has the effect of promoting the healing of diabetic ulcer wounds in vivo and in vitro, and its therapeutic effect is better than that of single-target agonists.

Aminocoumarin derivatives grafted on graphene oxide - new antimicrobial agents to combat the resistance of Mycobacterium tuberculosis and ESKAPE pathogens

Two hybrid materials, based on 7-amino-4-(trifluoromethyl)coumarin and 7-amino-4-methyl-coumarin that attach covalently to graphene oxide (GO) have been synthesized in two steps: i) increasing the number of carboxyl groups required for functionalization with aminocoumarin derivatives by derivatization of the hydroxyls groups with chloroacetic acid, transforming GO into GO-COOH material; ii) activation of carboxylic groups using the carbodiimide-promoted reaction. The obtained composites were characterized by numerous methods that highlighted their successful obtaining. The antimicrobial activity was evaluated on resistant Mycobacterium tuberculosis strains, as well as on ESKAPE pathogens ((Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) in planktonic and biofilm growth states. The biocompatibility of the materials has been assayed using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay as well as by measuring the level of nitric oxide/total reactive oxygen species and superoxide release in treated cells. The results have shown that materials exhibited improved inhibitory activity against resistant M. tuberculosis strains in comparison with GO, but also against planktonic and adherent strains. Also, the tested composites have been proved to be biocompatible on the MRC-5 fibroblast cells, demonstrating their promising potential to develop novel agents effective against multidrug resistant pathogens, including M. tuberculosis, as well as by the activation of a pro-inflammatory and of an oxidative response in the mammalian cells.

Abstract P2173: Naltrindole Offers Potent Cardioprotection From Ischemia Reperfusion Injury; Opioid Antagonism Or Calcium Handling?

Coronary Artery Disease accounts for approximately 25% of deaths annually, and remains theleading cause of mortality inthe United States. Myocardial ischemic (MI) damage is the initialinjury, characterized by irreversible hyper-contracture of cardiac muscle. Currently, there is nopharmacologic treatment tomitigate MI damage that may occur inthe 250,000CABGs,215,000percutaneous coronary interventions (PCI), and heart transplantation. We havepreviously reported that naltrindole (NTI) dose-dependently reduces infarct size upto82% ininvivomyocardial ischemia-reperfusion(MI/R) studies whengivenprior toregional ischemia. Werecently have shownthat NTI elicits this robust infarct size reducing effect by anovelmechanism since NTI alsoreduces polymorphonuclear leukocyte (PMNs) superoxide (SO)release at concentrations corresponding tothe blood concentrationinthe invivoMI/R study(8mg/kg = ~200uM; 4mg/kg = ~100uM). PMNs donot express opioid receptors, and NTI isknowntobe adeltaopioid receptor antagonist.Inour ex vivoLangendorff rat MI/R model, male SD rats (~300g) were anesthetized, heartswere removed, perfused with 37°C Krebs’ buffer and apressure transducer was placed intheleft ventricle (LV). Baseline recordings were made during the first 10minand the hearts werepretreated with NTI (5uM, n=8), naloxone (NX; panopioid receptor antagonist; 10uM, n=6)and binaltorphimine (BNI; kappaopioid receptor antagonist, 5uM, n=7) or controls (n=12) for 5mins prior toglobal I(30min)/R(45min).Ischemic PeakPressure (IPP) incontrolI/R heartswas39±3mmHg comparedtopre-ischemicLVEDP of9±1mmHg atbaseline(n=12,p&lt;0.01),resultinginsubstantialinfarctatthe endof45minR (32±4%).IPP wasreduced byNTI (18±3mmHg/s)comparedtoallgroups(p&lt;0.05).FinaldP/dt maxandinfarctsize were mostimprovedwith NTI (1830±90mmHg/s, 7±2%)comparedtocontrol(777±142mmHg/s, 32±4%,p&lt;0.01).Cardiacfunctionandinfarctsize did notimprovewith BNI orNX. The resultssuggested that NTI exerted robust cardioprotectionwith significant reductionininfarct sizeindependent of deltaor kappaopioid receptor which led toinvestigating its possible underlyingmechanism of action.