New NADPH Oxidase 2 Inhibitors Display Potent Activity against Oxidative Stress by Targeting p22phox-p47phox Interactions.

NADPH oxidase (NOX2) is responsible for reactive oxygen species (ROS) production in neutrophils and has been recognized as a key mediator in inflammatory and cardiovascular pathologies. Nevertheless, there is a lack of specific NOX2 pharmacological inhibitors. In medicinal chemistry, heterocyclic compounds are essential scaffolds for drug design, and among them, indole is a very versatile pharmacophore. We tested the hypothesis that indole heteroaryl-acrylonitrile derivatives may serve as NOX2 inhibitors by evaluating the capacity of 19 of these molecules to inhibit NOX2-derived ROS production in human neutrophils (HL-60 cells). Of these compounds, C6 and C14 exhibited concentration-dependent inhibition of NOX2 (IC50~1 µM). These molecules also reduced NOX2-derived oxidative stress in cardiomyocytes and prevented cardiac damage induced by ischemia-reperfusion. Compound C6 significantly reduced the membrane translocation of p47phox, a cytosolic subunit that is required for NOX2 activation. Molecular docking analyses of the binding modes of these molecules with p47phox indicated that C6 and C14 interact with specific residues in the inner part of the groove of p47phox, the binding cavity for p22phox. This combination of methods showed that novel indole heteroaryl acrylonitriles represent interesting lead compounds for developing specific and potent NOX2 inhibitors.

Metabolic regulation of the proteasome under hypoxia by Poldip2 controls fibrotic signaling in vascular smooth muscle cells.

The polymerase delta interacting protein 2 (Poldip2) is a nuclear-encoded mitochondrial protein required for oxidative metabolism. Under hypoxia, Poldip2 expression is repressed by an unknown mechanism. Therefore, low levels of Poldip2 are required to maintain glycolytic metabolism. The Cellular Communication Network Factor 2 (CCN2, Connective tissue growth factor, CTGF) is a profibrogenic molecule highly expressed in cancer and vascular inflammation in advanced atherosclerosis. Because CCN2 is upregulated under hypoxia and is associated with glycolytic metabolism, we hypothesize that Poldip2 downregulation is responsible for the upregulation of profibrotic signaling under hypoxia. Here, we report that Poldip2 is repressed under hypoxia by a mechanism that requires the activation of the enhancer of zeste homolog 2 repressive complex (EZH2) downstream from the Cyclin-Dependent Kinase 2 (CDK2). Importantly, we found that Poldip2 repression is required for CCN2 expression downstream of metabolic inhibition of the ubiquitin-proteasome system (UPS)-dependent stabilization of the serum response factor. Pharmacological or gene expression inhibition of CDK2 under hypoxia reverses Poldip2 downregulation, the inhibition of the UPS, and the expression of CCN2, collagen, and fibronectin. Thus, our findings connect cell cycle regulation and proteasome activity to mitochondrial function and fibrotic responses under hypoxia.

Maresin 1, a Proresolving Lipid Mediator, Ameliorates Liver Ischemia-Reperfusion Injury and Stimulates Hepatocyte Proliferation in Sprague-Dawley Rats.

Maresin-1 (MaR1) is a specialized pro-resolving mediator, derived from omega-3 fatty acids, whose functions are to decrease the pro-inflammatory and oxidative mediators, and also to stimulate cell division. We investigated the hepatoprotective actions of MaR1 in a rat model of liver ischemia-reperfusion (IR) injury. MaR1 (4 ng/gr body weight) was administered prior to ischemia (1 h) and reperfusion (3 h), and controls received isovolumetric vehicle solution. To analyze liver function, transaminases levels and tissue architecture were assayed, and serum cytokines TNF-α, IL-6, and IL-10, mitotic activity index, and differential levels of NF-κB and Nrf-2 transcription factors, were analyzed. Transaminase, TNF-α levels, and cytoarchitecture were normalized with the administration of MaR1 and associated with changes in NF-κB. IL-6, mitotic activity index, and nuclear translocation of Nrf-2 increased in the MaR1-IR group, which would be associated with hepatoprotection and cell proliferation. Taken together, these results suggest that MaR1 alleviated IR liver injury, facilitated by the activation of hepatocyte cell division, increased IL-6 cytokine levels, and the nuclear localization of Nrf-2, with a decrease of NF-κB activity. All of them were related to an improvement of liver injury parameters. These results open the possibility of MaR1 as a potential therapeutic tool in IR and other hepatic pathologies.

Propolis as an Adjuvant in the Healing of Human Diabetic Foot Wounds Receiving Care in the Diagnostic and Treatment Centre from the Regional Hospital of Talca.

OBJECTIVE: Diabetic foot wounds are a relevant diabetes complication and a major health problem. It has been described that propolis has health benefits due to its anti-inflammatory, antioxidant, and support in the healing process. The current study assessed the effect of propolis as an adjuvant in the healing of human diabetic foot ulcers. This was evaluated in a randomized placebo-controlled study of subjects receiving care in the Diagnostic and Treatment Centre from the Regional Hospital of Talca, Chile. RESEARCH DESIGN AND METHODS: Randomized subjects received ambulatory healing treatment for diabetes foot wounds with propolis spray (3%), which was applied to cover the entire wound surface each time it was dressed from week 0 until cicatrization or 8 weeks as a maximum. Two serum samples were taken (day 0 and end of the study) for cytokine and oxidative stress analyses. Also, macro- and microscopy were analyzed in the process of wound healing. RESULTS: The study comprised 31 subjects with type 2 diabetes in treatment for diabetic foot wounds in the Diagnostic and Treatment Centre from the Regional Hospital of Talca. Propolis promotes a reduction of the wound's area by an average of 4 cm2, related to an increase in the connective tissue deposit compared to the control. Also, propolis increased the glutathione (GSH) and GSH/glutathione disulfide (GSSG) ratio (p < 0.02), depleted tumor necrosis factor- (TNF-) α, and increased interleukin- (IL-) 10 levels. Topical propolis did not modify the biochemical parameters in the serum of the studied subjects. CONCLUSIONS: The topical use of propolis turned out to be an interesting therapeutic strategy as an adjuvant in the care of diabetes foot wounds due to its ability to improve and promote healing based on its anti-inflammatory and antioxidant profile. This trial is registered with NCT03649243.

NOX Inhibition Improves ß-Adrenergic Stimulated Contractility and Intracellular Calcium Handling in the Aged Rat Heart.

Cardiac aging is characterized by alterations in contractility and intracellular calcium ([Ca2+]i) homeostasis. It has been suggested that oxidative stress may be involved in this process. We and others have reported that in cardiomyopathies the NADPH oxidase (NOX)-derived superoxide is increased, with a negative impact on [Ca2+]i and contractility. We tested the hypothesis that in the aged heart, [Ca2+]i handling and contractility are disturbed by NOX-derived superoxide. For this we used adults (≈5 month-old) and aged (20⁻24 month-old) rats. Contractility was evaluated in isolated hearts, challenged with isoproterenol. To assess [Ca2+]i, isolated cardiac myocytes were field-stimulated and [Ca2+]i was monitored with fura-2. Cardiac concentration-response to isoproterenol was depressed in aged compared to adults hearts (p < 0.005), but was restored by NOX inhibitors apocynin and VAS2870. In isolated cardiomyocytes, apocynin increased the amplitude of [Ca2+]i in aged myocytes (p < 0.05). Time-50 [Ca2+]i decay was increased in aged myocytes (p < 0.05) and reduced towards normal by NOX inhibition. In addition, we found that myofilaments Ca2+ sensitivity was reduced in aged myocytes (p < 0.05), and was further reduced by apocynin. NOX2 expression along with NADPH oxidase activity was increased in aged hearts. Phospholamban phosphorylation (Ser16/Thr17) after isoproterenol treatment was reduced in aged hearts compared to adults and was restored by apocynin treatment (p < 0.05). In conclusion, ß-adrenergic-induced contractility was depressed in aged hearts, and NOX inhibition restored back to normal. Moreover, altered Ca2+ handling in aged myocytes was also improved by NOX inhibition. These results suggest a NOX-dependent effect in aged myocytes at the level of Ca2+ handling proteins and myofilaments.