A dual-acting aldose reductase inhibitor impedes oxidative and carbonyl stress in tissues of fructose- and streptozotocin-induced rats: comparison with antioxidant stobadine.

Inhibiting aldose reductase (ALR2, AR) as well as maintaining a concomitant antioxidant (AO) activity via dual-acting agents may be a rational approach to prevent cellular glucotoxicity and at least delay the progression of diabetes mellitus (DM). This study was aimed at evaluating the dual-acting AR inhibitor (ARI) cemtirestat (CMTI) on tissue oxidative stress (OS) and carbonyl stress (CS) biomarkers in rats exposed to fructose alone (F) or fructose plus streptozotocin (D; type-2 diabetic). D and F rats were either untreated or treated daily with low- or high-dose CMTI, ARI drug epalrestat (EPA) or antioxidant stobadine (STB) for 14 weeks. Malondialdehyde (MDA), glutathione S-transferase (GST), nitric oxide synthase (NOS), and catalase (CAT) were increased in the sciatic nerve of F and D. These increases were attenuated by low doses of CMTI and STB in D, but exacerbated by low-dose EPA and high-dose CMTI in F. STB and CMTI and to a lesser extent EPA improved MDA, protein-carbonyl, GST and CAT in the hearts and lungs of F and D. CMTI and STB were more effective than EPA in improving the increased MDA and protein-carbonyl levels in the kidneys of F and especially D. CMTI ameliorated renal GST inhibition in D. In the lungs, hearts, and kidneys of F and D, the GSH to GSSG ratio decreased and caspase-3 activity increased, but partially resolved with treatments. In conclusion, CMTI with ARI/AO activity may be advantageous in overcoming OS, CS, and their undesirable consequences, with low dose efficacy and limited toxicity, compared to ARI or antioxidant alone.

Graphene- and MXene-based materials for neuroscience: diagnostic and therapeutic applications.

MXenes and graphene are two-dimensional materials that have gained increasing attention in neuroscience, particularly in sensing, theranostics, and biomedical engineering. Various composites of graphene and MXenes with fascinating thermal, optical, magnetic, mechanical, and electrical properties have been introduced to develop advanced nanosystems for diagnostic and therapeutic applications, as exemplified in the case of biosensors for neurotransmitter detection. These biosensors display high sensitivity, selectivity, and stability, making them promising tools for neuroscience research. MXenes have been employed to create high-resolution neural interfaces for neuroelectronic devices, develop neuro-receptor-mediated synapse devices, and stimulate the electrophysiological maturation of neural circuits. On the other hand, graphene/derivatives exhibit therapeutic applicability in neuroscience, as exemplified in the case of graphene oxide for targeted delivery of therapeutic agents to the brain. While MXenes and graphene have potential benefits in neuroscience, there are also challenges/limitations associated with their use, such as toxicity, environmental impacts, and limited understanding of their properties. In addition, large-scale production and commercialization as well as optimization of reaction/synthesis conditions and clinical translation studies are very important aspects. Thus, it is important to consider the use of these materials in neuroscience research and conduct further research to obtain an in-depth understanding of their properties and potential applications. By addressing issues related to biocompatibility, long-term stability, targeted delivery, electrical interfaces, scalability, and cost-effectiveness, MXenes and graphene have the potential to greatly advance the field of neuroscience and pave the way for innovative diagnostic and therapeutic approaches for neurological disorders. Herein, recent advances in therapeutic and diagnostic applications of graphene- and MXene-based materials in neuroscience are discussed, focusing on important challenges and future prospects.

Monoacylglycerol Lipase Inhibition Prevents Short-Term Mitochondrial Dysfunction and Oxidative Damage in Rat Brain Synaptosomal/Mitochondrial Fractions and Cortical Slices: Role of Cannabinoid Receptors.

Inhibition of enzymes responsible for endocannabinoid hydrolysis represents an invaluable emerging tool for the potential treatment of neurodegenerative disorders. Monoacylglycerol lipase (MAGL) is the enzyme responsible for degrading 2-arachydonoylglycerol (2-AG), the most abundant endocannabinoid in the central nervous system (CNS). Here, we tested the effects of the selective MAGL inhibitor JZL184 on the 3-nitropropinic acid (3-NP)-induced short-term loss of mitochondrial reductive capacity/viability and oxidative damage in rat brain synaptosomal/mitochondrial fractions and cortical slices. In synaptosomes, while 3-NP decreased mitochondrial function and increased lipid peroxidation, JZL184 attenuated both markers. The protective effects evoked by JZL184 on the 3-NP-induced mitochondrial dysfunction were primarily mediated by activation of cannabinoid receptor 2 (CB2R), as evidenced by their inhibition by the selective CB2R inverse agonist JTE907. The cannabinoid receptor 1 (CB1R) also participated in this effect in a lesser extent, as evidenced by the CB1R antagonist/inverse agonist AM281. In contrast, activation of CB1R, but not CB2R, was responsible for the protective effects of JZL184 on the 3-NP-iduced lipid peroxidation. Protective effects of JZL184 were confirmed in other toxic models involving excitotoxicity and oxidative damage as internal controls. In cortical slices, JZL184 ameliorated the 3-NP-induced loss of mitochondrial function, the increase in lipid peroxidation, and the inhibition of succinate dehydrogenase (mitochondrial complex II) activity, and these effects were independent on CB1R and CB2R, as evidenced by the lack of effects of AM281 and JTE907, respectively. Our novel results provide experimental evidence that the differential protective effects exerted by JZL184 on the early toxic effects induced by 3-NP in brain synaptosomes and cortical slices involve MAGL inhibition, and possibly the subsequent accumulation of 2-AG. These effects involve pro-energetic and redox modulatory mechanisms that may be either dependent or independent of cannabinoid receptors' activation.

Cemtirestat, an aldose reductase inhibitor and antioxidant compound, induces ocular defense against oxidative and inflammatory stress in rat models for glycotoxicity.

Fructose, endogenously produced as a consequence of activation of the polyol pathway under hyperglycemic conditions, contribute to formation of advanced glycoxidation end products (AGEs) and carbonyl stress. Oxidative stress is increased in diabetes (DM) due to AGEs formation and the utilization of NADPH by aldo-keto reductase, AKR1B1(AR), the first enzyme in polyol pathway. Since inhibition of AR is an attractive approach for the management of diabetic eye diseases, we aimed to compare the effects of a novel AR inhibitor (ARI)/antioxidant (AO) compound cemtirestat on eye tissues with the effects of ARI drug epalrestat and AO agent stobadine in rat model for glycotoxicity. One group of rats was fed high fructose (10% drinking water; 14 weeks), while type-2 DM was induced in the other group of rats with fructose plus streptozotocin (40 mg/kg-bw/day). Diabetic (D) and nondiabetic fructose-fed rats (F) were either untreated or treated with two different doses of cemtirestat (2.5 and 7.5 mg/kg-bw/day), epalrestat (25 and 50 mg/kg-bw/day), or stobadine (25 and 50 mg/kg-bw/day) for 14 weeks. Cemtirestat, epalrestat, and stobadine elaviate the increase in TNF-α, IL-1ß, NF-ƙB, and caspase-3 in retina, lens, cornea, and sclera of F and D rats. Both glycotoxicity models resulted in a decrease in GSH to GSSG ratio and a change in glutathione S-transferase activity in eye tissues, but these alterations were improved especially with cemtirestat and stobadine. Lens D-sorbitol of D rats increased more than that of F rats, this increase was only attenuated by cemtirestat and epalrestat. Epalrestat was more effective than cemtirestat and stobadine in inhibiting the increase of vascular endothelial growth factor (VEGF) in the retina of F and D rats. Cemtirestat and stobadine but not epalrestat decreased high level of Nε-(carboxymethyl)lysine in the lens and retina of F and D rats. Cemtirestat is a potential therapeutic in protecting the rat eye against glycotoxicity insults.

Alpha-Mangostin Alleviates the Short-term 6-Hydroxydopamine-Induced Neurotoxicity and Oxidative Damage in Rat Cortical Slices and in Caenorhabditis elegans.

The development, at the experimental level, of therapeutic strategies based on natural products to attenuate neurological alterations in degenerative disorders has gained attention. Antioxidant molecules exhibit both anti-inflammatory and neuroprotective properties. Alpha-mangostin (α-Man) is a natural xanthonoid isolated from the mangosteen tree with demonstrated antioxidant and cytoprotective properties. In this study, we investigated the antioxidant and protective properties of α-Man, both ex vivo and in vivo. We assessed the mitochondrial reductant capacity and oxidative damage to lipids in rat cortical slices, and several endpoints characteristic of physiological stress in the nematode, Caenorhabditis elegans (C. elegans), upon exposure to the parkinsonian neurotoxin, 6-hydroxydopamine (6-OHDA). In rat cortical slices, α-Man (25 and 50 µM) reduced the 6-OHDA (100 µM)-induced oxidative damage to lipid levels, but failed to reverse loss in cell viability. In wild-type (N2) C. elegans, α-Man (5-100 µM) protected against 6-OHDA (25 mM)-induced decrease in survival when administered either as pre- or post-treatment. Protective effects of α-Man were also observed on survival in the VC1772 strain (skn-1 KO-) exposed to 6-OHDA, though the extent of the protection was lesser than in the wild-type N2 strain. However, α-Man (5-50 µM) failed to attenuate the 6-OHDA-induced motor alterations in the N2 strain. The loss of lifespan induced by 6-OHDA in the N2 strain was fully reversed by high concentrations of α-Man. In addition, while 6-OHDA decreased the expression of glutathione S-transferase in the CL2166 C. elegans strain, α-Man preserved and stimulated the expression of this protein. α-Man (25 µM) also prevented 6-OHDA-induced dopaminergic neurodegeneration in the BZ555 C. elegans strain. Altogether, our novel results suggest that α-Man affords partial protection against several, but not all, short-term toxic effects induced by 6-OHDA in cortical slices and in a skn-1-dependent manner in C. elegans.

Thallium Induces Antiproliferative and Cytotoxic Activity in Glioblastoma C6 and U373 Cell Cultures via Apoptosis and Changes in Cell Cycle.

Thallium (Tl+) is a heavy metal that causes toxicity in several organs, including the brain. Its cytotoxic profile, combined with its affinity for tumor cells when used as a radioligand for labeling these cells, suggests its potential use as antitumor therapy. In this study, glioblastoma cell lines C6 (from rat) and U373 (from human) were exposed to increased concentrations of thallium(I) acetate (5, 10, 50, 100, or 200 µM) and several toxic endpoints were evaluated, including loss of confluence and morphological changes, loss of cell viability, changes in cell cycle, and apoptosis. Tl+ was detected in cells exposed to thallium(I) acetate, demonstrating efficient uptake mechanism. Confluence in both cell lines decreased in a concentration-dependent manner (50-200 µM), while morphological changes (cell shrinkage and decreased cell volume) were more evident at exposures to higher Tl+ concentrations. For both parameters, the effects of Tl+ were more prominent in C6 cells compared to U373 cells. The same trend was observed for cell viability, with Tl+ affecting this parameter in C6 cells at low concentrations, whereas U373 cells showed greater resistance, with significant changes observed only at the higher concentrations. C6 and U373 cells treated with Tl+ also showed morphological characteristics corresponding to apoptosis. The cytotoxic effects of Tl+ were also assessed in neural and astrocytic primary cultures from the whole rat brain. Primary neural and astrocytic cultures were less sensitive than C6 and U373 cells, showing changes in cell viability at 50 and 100 µM concentrations, respectively. Cell cycle in both brain tumor cell lines was altered by Tl+ in G1/G2 and S phases. In addition, when combined with temozolamide (500 µM), Tl+ elicited cell cycle alterations, increasing SubG1 population. Combined, our novel results characterize and validate the cytotoxic and antiproliferative effects of Tl+ in glioblastoma cells.

On the Biomedical Properties of Endocannabinoid Degradation and Reuptake Inhibitors: Pre-clinical and Clinical Evidence.

The endocannabinoid system (ECS) is composed of endogenous cannabinoids; components involved in their synthesis, transport, and degradation; and an expansive variety of cannabinoid receptors. Hypofunction or deregulation of the ECS is related to pathological conditions. Consequently, endogenous enhancement of endocannabinoid levels and/or regulation of their metabolism represent promising therapeutic approaches. Several major strategies have been suggested for the modulation of the ECS: (1) blocking endocannabinoids degradation, (2) inhibition of endocannabinoid cellular uptake, and (3) pharmacological modulation of cannabinoid receptors as potential therapeutic targets. Here, we focused in this review on degradation/reuptake inhibitors over cannabinoid receptor modulators in order to provide an updated synopsis of contemporary evidence advancing mechanisms of endocannabinoids as pharmacological tools with therapeutic properties for the treatment of several disorders. For this purpose, we revisited the available literature and reported the latest advances regarding the biomedical properties of fatty acid amide hydrolase and monoacylglycerol lipase inhibitors in pre-clinical and clinical studies. We also highlighted anandamide and 2-arachidonoylglycerol reuptake inhibitors with promising results in pre-clinical studies using in vitro and animal models as an outlook for future research in clinical trials.

Platinum nanoparticles Protect Against Lipopolysaccharide-Induced Inflammation in Microglial BV-2 Cells via Decreased Oxidative Damage and Increased Phagocytosis.

Neuroinflammation and oxidative stress cooperate to compromise the function of the central nervous system (CNS). Colloidal platinum nanoparticles (Pt NPs) are ideal candidates for reducing the deleterious effects of neuroinflammation since they act as free radical scavengers. Here we evaluated the effects of Pt NPs on several markers of lipopolysaccharide (LPS)-induced inflammation in cultured BV-2 microglial cells. BV-2 cells were treated with increased dilutions (1-100 ppm) of Colloidal Pt and/or LPS (1-10 µg/mL) at different exposure times. Three different protocols of exposure were used combining Pt NPs and LPS: (a) conditioning-protective effect (pre-post-treat), (b) therapeutic effect (co-treat) and (c) conditioning-therapeutic effect (pre-co-treat). After exposure to LPS for 24 h, cells were used for assessment of cell viability, reactive oxygen species (ROS) generation, lactate dehydrogenase (LDH) activity, apoptosis and caspase-3 levels, cell proliferation, mitochondrial membrane potential, inducible nitric oxide (iNOS) activity, pro-inflammatory cytokine (IL-1ß, TNF-α and IL-6) levels, and phagocytic activity. Low concentrations (below or equal to 10 ppm) of Colloidal Pt prevented or ameliorated the LPS-induced increase in ROS formation, loss of mitochondrial membrane potential, induction of apoptosis, increase in LDH release, increase in pro-inflammatory cytokines and iNOS, inhibition of phagocytosis linked to microglial persistence in the M1 phase phenotype, loss of cell adhesion, differentiation and/or proliferation, as well as loss of cell viability. These protective effects were evident when cells were preconditioned with Pt NPs prior to LPS treatment. Collectively, the findings demonstrate that at low concentrations, Pt NPs can regulate the function and phenotype of BV-2 cells, activating protective mechanisms to maintain the microglial homeostasis and reduce inflammatory events triggered by the inflammatory insults induced by LPS. These preventive/protective effects on the LPS pro-inflammatory model are linked to the antioxidant properties and phagocytic activity of these NPs.

Development of Novel Indole-Based Bifunctional Aldose Reductase Inhibitors/Antioxidants as Promising Drugs for the Treatment of Diabetic Complications.

Aldose reductase (AR, ALR2), the first enzyme of the polyol pathway, is implicated in the pathophysiology of diabetic complications. Aldose reductase inhibitors (ARIs) thus present a promising therapeutic approach to treat a wide array of diabetic complications. Moreover, a therapeutic potential of ARIs in the treatment of chronic inflammation-related pathologies and several genetic metabolic disorders has been recently indicated. Substituted indoles are an interesting group of compounds with a plethora of biological activities. This article reviews a series of indole-based bifunctional aldose reductase inhibitors/antioxidants (ARIs/AOs) developed during recent years. Experimental results obtained in in vitro, ex vivo, and in vivo models of diabetic complications are presented. Structure-activity relationships with respect to carboxymethyl pharmacophore regioisomerization and core scaffold modification are discussed along with the criteria of 'drug-likeness". Novel promising structures of putative multifunctional ARIs/AOs are designed.

S-allylcysteine inhibits chondrocyte inflammation to reduce human osteoarthritis via targeting RAGE, TLR4, JNK, and Nrf2 signaling: comparison with colchicine.

The discovery of new pharmacological agents is needed to control the progression of osteoarthritis (OA), characterized by joint cartilage damage. Human OA chondrocyte (OAC) cultures were either applied to S-allylcysteine (SAC), a sulfur-containing amino acid derivative, or colchicine, an ancient anti-inflammatory therapeutic, for 24 h. SAC or colchicine did not change viability at 1 nM-10 µM but inhibited p-JNK/pan-JNK. While SAC seems to be more effective, both agents inhibited reactive oxygen species (ROS), 3-nitrotyrosine (3-NT), lipid hydroperoxides (LPO), advanced lipoxidation end-products (ALEs as 4-hydroxy-2-nonenal, HNE), advanced glycation end-products (AGEs), and increased glutathione peroxidase (GPx) and type-II-collagen (COL2). IL-1ß, IL-6, and osteopontin (OPN) were more strongly inhibited by SAC than by colchicine. In contrast, TNF-α was inhibited only by SAC, and COX2 was only inhibited by colchicine. Casp-1/ICE, GM-CSF, receptor for advanced glycation end-products (RAGE), and toll-like receptors (TLR4) were inhibited by both agents, but bone morphogenetic protein 7 (BMP7) was partially inhibited by SAC and induced by colchicine. Nuclear factor erythroid 2-related factor 2 (Nrf2) was induced by SAC; in contrast, it was inhibited by colchicine. Although they exert opposite effects on TNF-α, COX2, BMP7, and Nrf2, SAC and colchicine exhibit anti-osteoarthritic properties in OAC by modulating redox-sensitive inflammatory signaling.

Protective Effects of Novel Substituted Triazinoindole Inhibitors of Aldose Reductase and Epalrestat in Neuron-like PC12 Cells and BV2 Rodent Microglial Cells Exposed to Toxic Models of Oxidative Stress: Comparison with the Pyridoindole Antioxidant Stobadine.

Aldose reductase (AR) catalyzes the conversion of glucose to sorbitol in a NADPH-dependent reaction, thereby increasing the production of reactive oxygen species (ROS). Since AR activation is linked to redox dysregulation and cell damage in neurodegenerative diseases, AR inhibitors (ARIs) constitute promising therapeutic tools for the treatment of these disorders. Among these compounds, the novel substituted triazinoindole derivatives cemtirestat (CMTI) and COTI, as well as the clinically employed epalrestat (EPA) and the pyridoindole-antioxidant stobadine (STB), were tested in both PC12 cells and BV2 microglia exposed to four different neurotoxic models. These include (1) oxidative stress with hydrogen peroxide (H2O2), (2) mitochondrial complex IV inhibition with NaN3, (3) endoplasmic reticulum-stress and lipotoxicity induced by palmitic acid/bovine serum albumin (PAM/BSA), and (4) advanced carbonyl compound lipotoxicity by 4-hydroxynonenal (4-HNE). All toxic compounds decreased cell viability and increased ROS formation in both PC12 and BV2 cells in a concentration-dependent manner (1-1000 µM; NaN3 < H2O2≈PAM/BSA < 4-HNE). In PC12 cells, EPA increased cell viability in all toxic models only at 1 µM, whereas CMTI restored baseline viability in all toxic models. COTI afforded protection against lipotoxicity, while STB only prevented H2O2-induced toxicity. Except for the 4-HNE model, EPA prevented ROS generation in all other toxic models, whereas CMTI, COTI, and STB prevented ROS production in all toxic models. In BV2 cells, EPA and CMTI restored baseline cell viability in all toxic models tested, while COTI and STB did not prevent the loss of viability in the NaN3 model. All ARIs and STB efficiently prevented ROS formation in all toxic models in a concentration-independent manner. The differential protective effects evoked by the novel ARIs and STB on the toxic models tested herein provide novel and relevant comparative evidence for the design of specific therapeutic strategies against neurodegenerative events associated with neurological disorders.

Redox-active phytoconstituents ameliorate cell damage and inflammation in rat hippocampal neurons exposed to hyperglycemia+Aß1-42 peptide.

Alzheimer's disease (AD) is the most common dementia causing progressive loss of memory and compromised cognitive functions. Although the neurotoxic mechanisms underlying AD have yet to be fully elucidated, hyperglycemia seems to trigger oxidative and inflammatory responses in the brain of afflicted patients. Removal of free radicals reduces the neurotoxic effects of hyperglycemia in AD models. In this study we investigated the neuroprotective effects of the antioxidant phytoconstituents oleuropein (OLE), rutin (RUT), luteolin (LUT) and S-allylcysteine (SAC) in an experimental model combining the exposure to high glucose (HG, mimicking chronic hyperglycemia) plus amyloid-ß peptide 1-42 (Aß1-42, mimicking AD) in primary hippocampal neurons. Cells were pre-treated with OLE, RUT, LUT or SAC (10-1000 nM), and then co-treated with high glucose (GLU, 150 mM) for 24 h plus 500 nM oligomeric Aß1-42 for 24 h more. Cell viability and reactive oxygen species (ROS) formation were assessed as indices of survival/toxicity and oxidative stress, respectively. Activity/expression of antioxidant enzymes, toxic adducts, inflammatory molecules, mitochondrial membrane potential (ΔΨm) and the pattern of amyloid aggregation were also assessed. The GLU + Aß1-42 treatment significantly decreased cell viability, increased ROS formation, reduced superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase activities, augmented Advanced Glycation End Products- and 4-hydroxynonenal-adducts generation, increased 3-nitrotyrosine and inflammatory outcomes such as inducible nitric oxide synthase, interleukin 1ß and Tumor Necrosis Factor α, decreased MMP and augmented amyloid aggregation. All phytoconstituents reduced in a differential manner all toxic endpoints, with SAC showing the highest efficacy in preventing loss of cell viability and oxidative damage, whereas RUT was most efficacious in mitigating inflammatory endpoints. Combined, the results of this study suggest that protection afforded by these compounds against GLU + Aß1-42-induced cell damage in hippocampal neurons is attributable to their properties as redox modulators, which might act through a concerted mechanism oriented to reduce oxidative stress and neuroinflammation.

Bacopa Monnieri Protects the Directly Affected Organ as Well as Distant Organs Against I/R Injury by Modulating Anti-Inflammatory and Anti-Nitrosative Pathways in A Rat Model for Infra-Renal Aortic Occlusion.

OBJECTIVE: To investigate the protective effect and underlying mechanisms of B. monnieri, a medicinal plant, on kidney and skeletal muscle injury induced by infra-renal abdominal aorta clamping for 2-hours (ischemia) and following removal of the clamp (reperfusion, 2-hours). METHODS: Rats were divided into four groups (n = 6): (I) animals given only saline (sham-control); (II) animals given B. monnieri extract for 10-days (300 mg/kg/day) (Bacopa-treated sham); (III) animals subjected to ischemia/reperfusion (I/R); (IV) animals given B. monnieri extract and then subjected to I/R. Kidneys and lower extremity muscles were examined for GPx, CAT, iNOS, 3-NT, IL-1ß and TNF-α. Apoptosis and injury were evaluated by TUNEL and H&E staining, respectively. RESULTS: I/R resulted in TUNEL positive cells, periarterial edema and glomerular capillary dilatation, decreased GPx activity, unchanged CAT, iNOS, 3-NT, IL-1ß and TNF-α in kidney. B. monnieri minimized renal remote reperfusion injury, and Group IV showed a lower degree of renal histopathology score when compared to the others. B. monnieri mitigated muscle I/R injury, decreased muscle hypertrophy, myofibril abnormalities and apoptosis. Muscle 3-NT and cytokine levels were increased by I/R, and B. monnieri inhibited iNOS and 3-NT both in sham-control and I/R groups. Muscle GPx unaffected by I/R or B. monnieri, but CAT was inhibited only in B. monnieri-treated I/R group. Muscle iNOS, 3-NT, IL-1ß, TNF-α levels and CAT activity of B. monnieri-treated I/R rats were lower than those in sham-control or Bacopa-treated sham. CONCLUSIONS: B. monnieri can protect the directly affected organ as well as distant organs against I/R injury by modulating anti-inflammatory and anti-nitrosative pathways.

Combatting Nitrosative Stress and Inflammation with Novel Substituted Triazinoindole Inhibitors of Aldose Reductase in PC12 Cells Exposed to 6-Hydroxydopamine Plus High Glucose.

Cellular redox dysregulation produced by aldose reductase (AR) in the presence of high blood sugar is a mechanism involved in neurodegeneration commonly observed in diabetes mellitus (DM) and Parkinson's disease (PD); therefore, AR is a key target for treatment of both diseases. The substituted triazinoindole derivatives 2-(3-thioxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl) acetic acid (cemtirestat or CMTI) and 2-(3-oxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl) acetic acid (COTI) are well-known AR inhibitors (ARIs). The neuroprotective properties of CMTI, COTI, the clinically used epalrestat (EPA), and the pyridoindole antioxidants stobadine and SMe1EC2 were all tested in the neurotoxic models produced by hyperglycemic glucotoxicity (HG, 75 mM D-glucose, 72 h), 6-hydroxydopamine (6-OHDA), and HG+6-OHDA models in PC12 cells. Cell viability decreased in all toxic models, increased by 1-5 µM EPA, and decreased by COTI at ≥ 2.5 µM. In the HG model alone, where compounds were present in the medium for 24 h after a continuous 24-h exposure to HG, cell viability was improved by 100 nM-5 µM EPA, 1-10 µM ARIs, and the antioxidants studied, but decreased by EPA at ≥ 10 µM. In the 6-OHDA model alone, where cells were treated with compounds for 24 h and further exposed to 100 µM 6-OHDA (8 h), only the antioxidants protected cell viability. In the HG+6-OHDA model, where cells were treated with all compounds (1 nM to 50 µM) for 48 h and exposed to 75 mM glucose for 24 h followed by incubation with 6-OHDA for 8 h, cell viability was protected by 100 nM-10 µM ARIs and 100-500 nM EPA, but not by antioxidants. All ARIs inhibited the HG+6-OHDA-induced increase in iNOS, IL-1ß, TNF-α, 3-NT, and total oxidant status at 1-50 µM, while increased SOD, CAT, GPx, and total antioxidant status at 1-10 µM. EPA and CMTI also reduced the HG+6-OHDA-induced increase in the cellular levels of nuclear factor kB (NF-KB). The neuroprotective potential of the novel ARIs and the pyridoindole antioxidants studied constitutes a promising tool for the development of therapeutic strategies against DM-induced and PD-related neurodegeneration.

URB597 Prevents the Short-Term Excitotoxic Cell Damage in Rat Cortical Slices: Role of Cannabinoid 1 Receptors.

Endocannabinoid-based therapies constitute an emerging tool for the potential treatment of neurodegenerative disorders, requiring characterization at the experimental level. The effects of URB597, an inhibitor of the fatty acid amide hydrolase (FAAH), were tested against the quinolinic acid (QUIN)-induced early toxic effects in rat cortical slices, and compared with those effects exerted by the endocannabinoid anandamide (AEA). URB597 prevented the QUIN-induced loss of mitochondrial function/cell viability and lipid peroxidation, while reduced necrosis, and to a lesser extent, apoptosis. The protective effects of URB597 were mediated by activation of cannabinoid receptor 1 (CB1r), as evidenced by their inhibition by the selective CB1r antagonist AM281. Similar effects were observed when testing AEA against QUIN toxicity. Our findings demonstrate the neuroprotective properties of URB597 during the early stages of excitotoxic damage to cortical tissue, suggesting that these properties are mediated by FAAH inhibition, and might be linked to the protective effects of AEA, or the combination of endocannabinoids.

TLR4, RAGE, and p-JNK/JNK mediated inflammatory aggression in osteoathritic human chondrocytes are counteracted by redox-sensitive phenolic olive compounds: Comparison with ibuprofen.

Osteoarthritic chondrocytes show an over-activity of inflammatory catabolic mediators, and olive products have attracted attention because they were discovered to have some benefits on osteoarthritis patients. We investigated the mechanisms of action of olive leaf polyphenolic compounds in osteoarthritic chondrocytes (OACs) using a standardized leaf extract, ZeyEX, and its main phenolic component, oleuropein, also compared with anti-inflammatory drug ibuprofen. OACs, isolated from joint-cartilages of Grade 4 OA patients, were found to express COMP and MMP-9 throughout their culture period. ZeyEX, oleuropein, and ibuprofen increased cell viability at concentrations of 1-100 nM, did not change at 500 nM-50 µM, but inhibited at ≥100 µM. The adherence profile of OACs increased with 1 µM of ibuprofen or ZeyEX and 10 nM-1 µM oleuropein. Although the markers for oxidative and nitrosative stresses (ROS and 3-NT) generally inhibited by three agents, the inhibitory effect of ZeyEX on 3-NT emerged dramatically (1 nM-10 µM). Lipid-hydroperoxides and HNE-adducts were also inhibited by each agent, but AGE-adducts unchanged by oleuropein while reduced by ZeyEX and ibuprofen. Inflammatory biomarkers, IL-1ß, IL-6, Casp-1/ICE, and TNF-α, were inhibited by three agents, however osteopontin and GM-CSF by only ZeyEX and ibuprofen. A decreased COMP, TLR4, and RAGE expression levels were observed by three agents, but only the effects of ZeyEX was concentration-dependent. In particular, ZeyEX and oleuropein improved COL2, inhibited p-JNK/JNK, and increased GPx. COX2 was only inhibited by ibuprofen. The results indicate that polyphenolic-olive compounds counteract redox-sensitive inflammatory aggressions in osteoarthritic chondrocytes that may stop the progression of pathology and allow regeneration.

Cannabinoid-profiled agents improve cell survival via reduction of oxidative stress and inflammation, and Nrf2 activation in a toxic model combining hyperglycemia+Aß1-42 peptide in rat hippocampal neurons.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder linked to various converging toxic mechanisms. Evidence suggests that hyperglycemia induces oxidative stress, mitochondrial dysfunction, inflammation and excitotoxicity, all of which play important roles in the onset and progression of AD pathogenesis. The endocannabinoid system (ECS) orchestrates major physiological responses, including neuronal plasticity, neuroprotection, and redox homeostasis, to name a few. The multi-targeted effectiveness of the ECS emerges as a potential approach to treat AD. Here we characterized the protective properties of the endocannabinoids arachidonylethanolamide (AEA) and 2-arachidonoylglycerol (2-AG), the synthetic cannabinoids CP 55-940 and WIN 55,212-2, and the fatty acid amide hydrolase (FAAH) inhibitor URB597, on a combined hyperglycemia + oligomeric amyloid ß peptide (Aß1-42) neurotoxic model in primary hippocampal neurons which exhibit several AD features. Cells were treated with cannabinoid agents at increased concentrations (1 nM-1 µM) for 6 h, and then co-treated with 150 mM glucose (GLU, 24 h), followed by incubation with 500 nM Aß1-42 (24 h). Cell viability/survival, reactive oxygen species (ROS) levels, antioxidant enzyme (SOD, CAT, GPx and GRx) activities, biological products of oxidative damage (AGE and HNE adducts) and nitrosative stress (3-NT), several endpoints of inflammation (iNOS, IL-1ß and TNF-α), amyloid quantification, mitochondrial membrane potential, and the involvement of the Nrf2 pathway, were all evaluated. The combined high glucose + amyloid beta 1-42 (GLU + Aß1-42) condition decreased cell viability and mitochondrial membrane potential, while augmenting oxidative damage and inflammation. All agents tested preserved cell viability and stimulated mitochondrial membrane potential, while reducing all the evaluated toxic endpoints in a differential manner, with URB597 showing the highest efficacy. The neuroprotective efficacy of all cannabinoid agents, except for URB597, led to partial recruitment of specific antioxidant activity and Nrf2 pathway regulation. Our results support the neuroprotective potential of these agents at low concentrations against the damaging effects of GLU + Aß1-42, affording new potential modalities for the design of AD therapies.

Antioxidant Mechanisms in the Neuroprotective Action of Cemtirestat: Studies in Chemical Models, Liposomes and Rat Brain Cortical Slices.

Neuroprotective action of the novel aldose reductase (AR) inhibitor cemtirestat (CMT), 2-(3-thioxo-2H-[1,2,4]triazino[5,6-b]indol-5(3H)-yl)acetic acid, was recently proved in experimental rat models of diabetes. The in vivo results indicated that the antioxidant activity of this compound might have participated on its effects. The aim of this study was to explore in a greater detail the putative antioxidant mechanisms potentially involved in CMT mediated neuroprotection. Antioxidant efficacy per se of CMT was proved by a ferric reducing antioxidant power (FRAP) test and CMT was found to scavenge reactive oxygen species (ROS) generated in water phase chemically with decreasing efficacy as follows ROO > H2O2 > O2-. Studies in liposomes revealed the ability of CMT to inhibit lipid peroxidation more efficiently than melatonin, yet less effectively than Trolox. In the rat brain cortical slices, CMT reduced the loss of cell viability/mitochondrial function induced by quinolinic acid (QUIN), and inhibited lipid peroxidation. In addition, CMT normalized the GSH/GSSG ratio which could be explained, at least partially, by the ability of this compound to release free GSH from the pool of endogenously bound disulfides. Neuronal cell damage induced by QUIN or H2O2 was reduced by CMT as proved by significant drop in propidium iodide incorporation into cells. On balance then, our results corroborated the notion of a multifunctional action of CMT as a drug combining AR inhibition with direct antioxidant and ROS scavenging activity. Moreover, the ability of CMT to restore thiol-disulfide homeostasis was proved.

Thallium Toxicity in Caenorhabditis elegans: Involvement of the SKN-1 Pathway and Protection by S-Allylcysteine.

Monovalent thallium (Tl+) is a cation that can exert complex neurotoxic patterns in the brain by mechanisms that have yet to be completely characterized. To learn more about Tl+ toxicity, it is necessary to investigate its major effects in vivo and its ability to trigger specific signaling pathways (such as the antioxidant SKN-1 pathway) in different biological models. Caenorhabditis elegans (C. elegans) is a nematode constituting a simple in vivo biological model with a well-characterized nervous system, and high genetic homology to mammalian systems. In this study, both wild-type (N2) and skn-1 knockout (KO) mutant C. elegans strains subjected to acute and chronic exposures to Tl+ [2.5-35 µM] were evaluated for physiological stress (survival, longevity, and worm size), motor alterations (body bends), and biochemical changes (glutathione S-transferase regulation in a gst-4 fluorescence strain). While survival was affected by Tl+ in N2 and skn-1 KO (worms lacking the orthologue of mammalian Nrf2) strains in a similar manner, the longevity was more prominently decreased in the skn-1 KO strain compared with the wild-type strain. Moreover, chronic exposure led to a greater compromise in the longevity in both strains compared with acute exposure. Tl+ also induced motor alterations in both skn-1 KO and wild-type strains, as well as changes in worm size in wild-type worms. In addition, preconditioning nematodes with the well-known antioxidant S-allylcysteine (SAC) reversed the Tl+-induced decrease in survival in the N2 strain. GST fluorescent expression was also decreased by the metal in the nematode, and recovered by SAC. Our results describe and validate, for the first time, features of the toxic pattern induced by Tl+ in an in vivo biological model established with C. elegans, supporting an altered redox component in Tl+ toxicity, as previously described in mammal models. We demonstrate that the presence of the orthologous SKN-1 pathway is required for worms in evoking an efficient antioxidant defense. Therefore, the nematode represents an optimal model to reproduce mammalian Tl+ toxicity, where toxic mechanisms and novel therapeutic approaches of clinical value may be successfully pursued.