Chloroquine (CQ) and hydroxychloroquine (HCQ) have been proposed as treatments for COVID-19. These drugs have been studied for many decades, primarily in the context of their use as antimalarials, where they induce oxidative stress-killing of the malarial parasite. Less appreciated, however, is evidence showing that CQ/HCQ causes systemic oxidative stress. In vitro and observational data suggest that CQ/HCQ can be repurposed as potential antiviral medications. This review focuses on the potential health concerns of CQ/HCQ induced by oxidative stress, particularly in the hyperinflammatory stage of COVID-19 disease. The pathophysiological role of oxidative stress in acute respiratory distress syndrome (ARDS) has been well-documented. Additional oxidative stress caused by CQ/HCQ during ARDS could be problematic. In vitro data showing that CQ forms a complex with free-heme that promotes lipid peroxidation of phospholipid bilayers are also relevant to COVID-19. Free-heme induced oxidative stress is implicated as a systemic activator of coagulation, which is increasingly recognized as a contributor to COVID-19 morbidity. This review will also provide a brief overview of CQ/HCQ pharmacology with an emphasis on how these drugs alter proton fluxes in subcellular organelles. CQ/HCQ-induced alterations in proton fluxes influence the type and chemical reactivity of reactive oxygen species (ROS).

Dectin-1/CLEC7A is a pattern recognition receptor that recognizes β-1,3 glucans, and its stimulation initiates signaling events characterized by the production of inflammatory cytokines from human dendritic cells (DCs) required for antifungal immunity. β-glucans differ greatly in size, structure, and ability to activate effector immune responses from DC; as such, small particulate β-glucans are thought to be poor activators of innate immunity. We show that β-glucan particle size is a critical factor contributing to the secretion of cytokines from human DC; large β-glucan-stimulated DC generate significantly more IL-1β, IL-6, and IL-23 compared to those stimulated with the smaller β-glucans. In marked contrast, the secretion of TSLP and CCL22 were found to be insensitive to β-glucan particle size. Furthermore, we show that the capacity to induce phagocytosis, and the relative IL-1β production determined by β-glucan size, regulates the composition of the cytokine milieu generated from DC. This suggests that β-glucan particle size is critically important in orchestrating the nature of the immune response to fungi.

The transcription factor nuclear factor κB (NF-κB) regulates multiple immediate-early gene expressions involved in immune and inflammatory responses and cellular defenses. Ischemia-reperfusion induces many immediate- early gene expressions, but little is known about the NF-κB activation in myocardium during ischemia and reperfusion. This study demonstrated that ischemia alone rapidly induced NF-κB activation in the myocardium of isolated working rat hearts. Electrophoretic mobility shift assay showed that NF-κB binding activity significantly increased in the nucleus after 5 min of ischemia and remained elevated for up to 30 min. Western blot analysis suggested that the levels of inhibitory IκBα protein in the cytoplasm became markedly decreased at 4, 5, 7.5, and 10 min of ischemia but were gradually restored following 10 min of ischemia. Reduction of IκBα protein in the cytoplasm by ischemia resulted in NF-κB translocation to the nucleus. Northern blot hybridization showed that IκBα mRNA levels were not significantly elevated during myocardial ischemia. Pyrrolidine dithiocarbamate, an antioxidant, significantly inhibited the loss of IκBα protein from the cytoplasm and prevented NF-κB binding activity in the nucleus. Reperfusion following short periods of ischemia augmented NF-κB binding activity in the nucleus induced by ischemia. The results suggest that early activation of NF-κB induced by ischemia in the myocardium could be a signal mechanism for controlling and regulating immediate-early gene expression during ischemia-reperfusion.

Stimulation of β-adrenergic receptors (βARs) causes apoptosis in adult rat ventricular myocytes (ARVMs). The role of reactive oxygen species (ROS) in mediating βAR-stimulated apoptosis is not known. Stimulation of βARs with norepinephrine (10 μmol/L) in the presence of prazosin (100 nmol/L) for 24 hours increased the number of apoptotic myocytes as determined by TUNEL staining by 3.6-fold. The superoxide dismutase/catalase mimetics Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP; 10 μmol/L) and Euk-134 decreased βAR-stimulated apoptosis by 89±6% and 76±10%, respectively. Infection with an adenovirus expressing catalase decreased βAR-stimulated apoptosis by 82±15%. The mitochondrial permeability transition pore inhibitor bongkrekic acid (50 μmol/L) decreased βAR-stimulated apoptosis by 76±8%, and the caspase inhibitor zVAD-fmk (25 μmol/L) decreased βAR-stimulated apoptosis by 62±11%. βAR-stimulated cytochrome c release was inhibited by MnTMPyP. βAR stimulation caused c-Jun NH2-terminal kinase (JNK) activation, which was abolished by MnTMPyP. Transfection with an adenovirus expressing dominant-negative JNK inhibited βAR-stimulated apoptosis by 81±12%, and the JNK inhibitor SP600125 inhibited both βAR-stimulated apoptosis and cytochrome c release. Thus, βAR-stimulated apoptosis in ARVMs involves ROS/JNK-dependent activation of the mitochondrial death pathway.

Increased osteopontin (OPN) expression in the heart, specifically in myocytes, associates with increased myocyte apoptosis and myocardial dysfunction. Recently, we provided evidence that OPN interacts with CD44 receptor, and induces myocyte apoptosis via the involvement of endoplasmic reticulum stress and mitochondrial death pathways. Here we tested the hypothesis that OPN induces oxidative stress in myocytes and the heart via the involvement of mitochondria and NADPH oxidase-4 (NOX-4). Treatment of adult rat ventricular myocytes (ARVMs) with OPN (20 nM) increased oxidative stress as analyzed by protein carbonylation, and intracellular reactive oxygen species (ROS) levels as analyzed by ROS detection kit and dichlorohydrofluorescein diacetate staining. Pretreatment with NAC (antioxidant), apocynin (NOX inhibitor), MnTBAP (superoxide dismutase mimetic), and mitochondrial KATP channel blockers (glibenclamide and 5-hydroxydecanoate) decreased OPN-stimulated ROS production, cytosolic cytochrome c levels, and apoptosis. OPN increased NOX-4 expression, while decreasing SOD-2 expression. OPN decreased mitochondrial membrane potential as measured by JC-1 staining, and induced mitochondrial abnormalities including swelling and reorganization of cristae as observed using transmission electron microscopy. OPN increased expression of BIK, a pro-apoptotic protein involved in reorganization of mitochondrial cristae. Expression of dominant-negative BIK decreased OPN-stimulated apoptosis. In vivo, OPN expression in cardiac myocyte-specific manner associated with increased protein carbonylation, and expression of NOX-4 and BIK. Thus, OPN induces oxidative stress via the involvement of mitochondria and NOX-4. It may affect mitochondrial morphology and integrity, at least in part, via the involvement of BIK.

The molecular mechanism responsible for degenerative process in the nigrostriatal dopaminergic system in Parkinson’s disease (PD) remains unknown. One major advance in this field has been the discovery of several genes associated to familial PD, including alpha synuclein, parkin, LRRK2, etc., thereby providing important insight toward basic research approaches. There is an consensus in neurodegenerative research that mitochondria dysfunction, protein degradation dysfunction, aggregation of alpha synuclein to neurotoxic oligomers, oxidative and endoplasmic reticulum stress, and neuroinflammation are involved in degeneration of the neuromelanin-containing dopaminergic neurons that are lost in the disease. An update of the mechanisms relating to neurotoxins that are used to produce preclinical models of Parkinson´s disease is presented. 6-Hydroxydopamine, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, and rotenone have been the most wisely used neurotoxins to delve into mechanisms involved in the loss of dopaminergic neurons containing neuromelanin. Neurotoxins generated from dopamine oxidation during neuromelanin formation are likewise reviewed, as this pathway replicates neurotoxin-induced cellular oxidative stress, inactivation of key proteins related to mitochondria and protein degradation dysfunction, and formation of neurotoxic aggregates of alpha synuclein. This survey of neurotoxin modeling—highlighting newer technologies and implicating a variety of processes and pathways related to mechanisms attending PD—is focused on research studies from 2012 to 2014.

It has been long noted that cardiac cell apoptosis provoked by excessive production of nitric oxide (NO) plays important roles in the pathogenesis of variant cardiac diseases. Attenuation of NO-induced injury would be an alternative therapeutic approach for the development of cardiac disorders. This study investigated the effects of α-lipoic acid (LA) on the injury induced by sodium nitroprusside (SNP), a widely used NO donor, in rat cardiomyoblast H9c2 cells. SNP challenge significantly decreased cell viability and increased apoptosis, as evidenced by morphological abnormalities, nuclear condensation and decline of mitochondrial potential (δ. Ψm). These changes induced by SNP were significantly attenuated by LA pretreatment. Furthermore, LA pretreatment prevented the SNP-triggered suppression of Akt and Gsk-3β activation. Blockade of Akt activation with triciribin (API) completely abolished the cytoprotection of LA against SNP challenge. In addition, LA moderately increased intracellular ROS production. Interestingly, inhibition of ROS with N-acetylcysteine abrogated Akt/Gsk-3β activation and the LA-induced cytoprotection following SNP stimulation. Taken together, the results indicate that LA protected the SNP-induced injury in cardiac H9c2 cells through, at least in part, the activation of Akt/Gsk-3β signaling in a ROS-dependent mechanism.

Background: Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth. Methods: To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for α-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth. Results: LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth. Conclusion: Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Glutamate, an excitatory neurotransmitter in the central nervous system, plays an important role in neurological disorders. Previous studies have shown that excess glutamate can cause oxidative stress in a hippocampal HT-22 cell line. 7,8-Dihydroxyflavone (7,8-DHF), a member of the flavonoid family, is a selective tyrosine kinase receptor B (TrkB) agonist that has neurotrophic effects in various neurological diseases such as stroke and Parkinson's disease. In this study, we found that there is no TrkB receptor in HT-22 cells. Despite this, our data demonstrate that 7,8-DHF still protects against glutamate-induced toxicity in HT-22 cells in a concentration-dependent manner, indicating that 7,8-DHF prevents cell death through other mechanisms rather than TrkB receptors in this cell model. We further show that 7,8-DHF increases cellular glutathione levels and reduces reactive oxygen species (ROS) production caused by glutamate in HT-22 cells. Finally, our data demonstrate that 7,8-DHF protects against hydrogen peroxide and menadione-induced cell death, suggesting that 7,8-DHF has an antioxidant effect. In summary, although 7,8-DHF is considered as a selective TrkB agonist, our results demonstrate that 7,8-DHF can still confer neuroprotection against glutamate-induced toxicity in HT-22 cells via its antioxidant activity.

Microglia are the resident innate immune cells that are critical for innate and adaptive immune responses within the CNS. They recognize and are activated by pathogen-associated molecular patterns (PAMPs) present on the surface of pathogens. β-glucans, the major PAMP present within fungal cell walls, are recognized by Dectin-1, which mediates numerous intracellular events invoked by β-glucans in various immune cells. Previously, we showed that Dectin-1 mediates phagocytosis of β-glucan and subsequent superoxide production in microglia. Here, we report that the guanine nucleotide exchange factor Vav1 as well as phosphoinositide-3 kinase (PI3K) are downstream mediators of what is now recognized as the Dectin-1 signaling pathway. Both Vav1 and PI3K are activated upon stimulation of microglia with β-glucans, and the two proteins are required for phagocytosis of the glucan particles and for subsequent superoxide production. We also show that Vav1 functions upstream of PI3K and is required for activation of PI3K. Together, our results provide an important insight into the mechanistic aspects of microglial activation in response to β-glucans.