Neuroprotective Effects of Probucol against Rotenone-Induced Toxicity via Suppression of Reactive Oxygen Species Production in SH-SY5Y Cells.

Probucol is a hyperlipidemic drug with antioxidant properties. It has been reported to prevent mitochondrial dysfunction, reduce oxidative stress, and suppress neurotoxicity in neurodegenerative disease models, including Parkinson's disease models. However, the molecular mechanisms underlying the neuroprotective effects of probucol have been not examined yet. Thus, in this study, we investigated whether probucol can alleviate the effects of a mitochondrial complex I inhibitor, rotenone, on a human neuroblastoma cell line (SH-SY5Y). We evaluated the cell viability and cytotoxicity and apoptosis rates of SH-SY5Y cells treated with rotenone and probucol or edaravone, a known free-radical scavenger. Subsequently, mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) levels in the cells were evaluated to determine the effects of probucol on mitochondrial function. We found that rotenone caused cytotoxicity, cell apoptosis, and mitochondrial dysfunction, enhanced ROS generation, and impaired MMP. However, probucol could inhibit this rotenone-induced decrease in cell viability, MMP loss, intracellular ROS generation, and apoptosis. These results suggest that probucol exerts neuroprotective effects via MMP stabilization and the inhibition of ROS generation. Additionally, this effect of probucol was equal to or greater than and more persistent than that of edaravone. Thus, we believe probucol may be a promising drug for the treatment of neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases.

Polymer-Based Nanoparticles with Probucol and Lithocholic Acid: A Novel Therapeutic Approach for Oxidative Stress-Induced Retinopathies.

Oxidative stress is pivotal in retinal disease progression, causing dysfunction in various retinal components. An effective antioxidant, such as probucol (PB), is vital to counteract oxidative stress and emerges as a potential candidate for treating retinal degeneration. However, the challenges associated with delivering lipophilic drugs such as PB to the posterior segment of the eye, specifically targeting photoreceptor cells, necessitate innovative solutions. This study uses formulation-based spray dry encapsulation technology to develop polymer-based PB-lithocholic acid (LCA) nanoparticles and assesses their efficacy in the 661W photoreceptor-like cell line. Incorporating LCA enhances nanoparticles' biological efficacy without compromising PB stability. In vitro studies demonstrate that PB-LCA nanoparticles prevent reactive oxygen species (ROS)-induced oxidative stress by improving cellular viability through the nuclear erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. These findings propose PB-LCA nanoparticles as a promising therapeutic strategy for oxidative stress-induced retinopathies.

Probucol protects against brain damage caused by intra-neural pyroptosis in rats with vascular dementia through inhibition of the Syk/Ros pathway.

BACKGROUND: Neuronal injury in chronic cerebral hypoperfusion (CCH) is the main pathogenic factor of vascular dementia (VD). Clinically, there isn't a drug specifically for VD; instead, the majority of medications used to treat Alzheimer's disease (AD) are also used to treat VD. Based on the proven anti-inflammatory and antioxidant effects of Probucol, we hypothesized that it may have therapeutic effects on VD, but more research is required to determine its exact mechanism of action. METHODS: In vivo experiment: We used SD rats and most commonly used bilateral carotid artery occlusion (2-VO) in VD for modeling. After successful modeling, SD rats were given Probucol 3.5 mg/kg/day for 8 weeks to evaluate the therapeutic effect. In vitro experiment: BV-2 microglia of rats were cultured and divided into Control group and Probucol group. Each group was treated with hypoxia-hypoglycemia, hypoxia-hypoglycemia hydrogen peroxide and hypoxia-hypoglycemia hydrogen peroxide Syk inhibitor respectively. RESULTS: The results of immunofluorescence and Western blot showed that Probucol could significantly improve the cognitive impairment induced by CCH, and the neuronal damage was also attenuated. On the one hand, the underlying mechanism of Probucol was to reduce oxidative stress and cell apoptosis of hippocampal neurons by inhibiting the expression of phosphorylated spleen tyrosine kinase (P-Syk); On the other hand, it exerted a protective effect by reducing NLRP3-dependent cell pyroptosis and inhibiting neuroinflammation induced by microglia activation. CONCLUSION: Probucol could reduce oxidative stress and cell apoptosis by inhibiting the Syk/ROS signaling pathway, thereby improving CCH-induced cognitive impairment in vitro and in vivo.

Probucol-bile acid based nanoparticles protect auditory cells from oxidative stress: an in vitro study.

Aim: Excessive free radicals contribute to oxidative stress and mitochondrial dysfunction in sensorineural hearing loss (SNHL). The antioxidant probucol holds promise, but its limited bioavailability and inner ear barriers hinder effective SNHL treatment. Methodology: We addressed this by developing probucol-loaded nanoparticles with polymers and lithocholic acid and tested them on House Ear Institute-Organ of Corti cells. Results: Probucol-based nanoparticles effectively reduced oxidative stress-induced apoptosis, enhanced cellular viability, improved probucol uptake and promoted mitochondrial function. Additionally, they demonstrated the capacity to reduce reactive oxygen species through the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 pathway. Conclusion: This innovative nanoparticle system holds the potential to prevent oxidative stress-related hearing impairment, providing an effective solution for SNHL.

Hearing loss affects millions of people worldwide, and its prevalence is expected to double by 2050. Current treatments have limitations, pushing researchers to explore new options. Oxidative stress is a key player in hearing loss and is known to damage inner ear hair cells. While antioxidants, known for their protective effects, hold promise, delivering them effectively to the inner ear is challenging. Scientists have been testing nanoparticles loaded with the antioxidant probucol to fight hearing loss. In this study, these particles protected inner ear cells in cell studies, offering potential hope for preventing hearing problems. This research is a significant step toward finding better treatments for hearing loss.

Collagenase Type I and Probucol-Loaded Nanoparticles Penetrate the Extracellular Matrix to Target Hepatic Stellate Cells for Hepatic Fibrosis Therapy.

Hepatic fibrosis is a common pathological process in chronic liver diseases, characterized by excessive reactive oxygen species (ROS), activated hepatic stellate cells (HSCs), and massive synthesis of extracellular matrix (ECM), which are important factors in the development of liver cirrhosis, liver failure, and liver cancer. During the development of hepatic fibrosis, ECM collagen produced by activated HSCs significantly hinders medication delivery to targeted cells and reduces the efficiency of pharmacological therapy. In this study, we designed a multifunctional hyaluronic acid polymeric nanoparticle (HA@PRB/COL NPs) based on autophagy inhibitor probucol (PRB) and collagenase type I (COL) modification, which could enhance ECM degradation and accurately target HSCs through specificity binding CD44 receptor in hepatic fibrosis therapy. Upon encountering excessive collagen I-deposition formed barrier, HA@PRB/COL NPs performed the nanodrill-like function to effectively degrade pericellular collagen I, leading to greater ECM penetration and prominent HSCs internalization capacity of delivered PRB. In mouse hepatic fibrosis model, HA@PRB/COL NPs were efficiently delivered to HSCs through binding CD44 receptor to achieve efficient accumulation in fibrotic liver. Further, we showed that HA@PRB/COL NPs executed the optimal anti-fibrotic activity by inhibiting autophagy and activation of HSCs. In conclusion, our novel dual-functional co-delivery system with degrading fibrotic ECM collagen and targeting activated HSCs exhibits great potentials in the treatment of hepatic fibrosis in clinic. STATEMENT OF SIGNIFICANCE: The excess release of extracellular matrix (ECM) such as collagen in hepatic fibrosis hinders medication delivery and decreases the efficiency of pharmacological drugs. We aimed to develop a nano-delivery carrier system with protein hydrolyzed surfaces and further encapsulated an autophagy inhibitor (PRB) to enhance fibrosis-related ECM degradation-penetration and hepatic stellate cells (HSCs) targeting in hepatic fibrosis niche (HA@PRB/COL NPs). The COL of HA@PRB/COL NPs successfully worked as a scavenger to promote the digestion of the ECM collagen I barrier for deeper penetration into fibroid liver tissue. It also accurately targeted HSCs through specifically binding to the CD44 receptor and subsequently released PRB to inhibit autolysosome and ROS generation, thus preventing HSCs activation. Our HA@PRB/COL NPs system provided a promising therapeutic strategy for hepatic fibrosis in a clinic setting.

Probucol attenuates high glucose-induced Müller cell damage through enhancing the Nrf2/p62 signaling pathway.

PURPOSE: This study investigated the protective effect of probucol on Müller cells exposed to high glucose conditions and examined potential mechanisms of action. METHODS: Primary human retinal Müller cells were incubated with high glucose (HG, 35 mM) in the present or absence of different concentrations of probucol for 24 h. Cell viability was determined using the CCK-8 method. Mitochondrial membrane potential (MMP) was measured using JC-1 staining and cell cycle by flow cytometry. The expression of nuclear factor E2-related factor 2 (Nrf2), glutamate-cysteine ligase catalytic subunit, and p62 was quantified using quantitative polymerase chain reaction and western blot. RESULTS: We found that HG inhibited cell proliferation, arrested cell cycle, and increased MMP in human Müller cells. Probucol activated the Nrf2/p62 pathway and upregulated the anti-apoptotic protein, Bcl2, and attenuated HG-mediated damage in Müller cells. CONCLUSIONS: Our results suggest that probucol may protect Müller cells from HG-induced damage through enhancing the Nrf2/p62 signaling pathway.

Probucol neuroprotection against manganese-induced damage in adult Wistar rat brain slices.

Manganese (Mn) is an abundant element used for commercial purposes and is essential for the proper function of biological systems. Chronic exposure to high Mn concentrations causes Manganism, a Parkinson's-like neurological disorder. The pathophysiological mechanism of Manganism remains unknown; however, it involves mitochondrial dysfunction and oxidative stress. This study assessed the neuroprotective effect of probucol, a hypolipidemic agent with anti-inflammatory and antioxidant properties, on cell viability and oxidative stress in slices of the cerebral cortex and striatum from adult male Wistar rats. Brain structure slices were kept separately and incubated with manganese chloride (MnCl2) and probucol to evaluate the cell viability and oxidative parameters. Probucol prevented Mn toxicity in the cerebral cortex and striatum, as evidenced by the preservation of cell viability observed with probucol (10 and 30 µM) pre-treatment, as well as the prevention of mitochondrial complex I inhibition in the striatum (30 µM). These findings support the protective antioxidant action of probucol, attributed to its ability to prevent cell death and mitigate Mn-induced mitochondrial dysfunction.

Pharmaceutical characterization of probucol bile acid-lithocholic acid nanoparticles to prevent chronic hearing related and similar cellular oxidative stress pathologies.

Background: Sensorineural hearing loss has been associated with oxidative stress. However, an antioxidant that passes effectively through the ear remains elusive. Method: Probucol (PB)-based nanoparticles were formed using a spray-drying encapsulation technique, characterized and tested in vitro. Results: Uniform, spherical nanoparticles were produced. The addition of lithocholic acid to PB formulations did not affect drug content or production yield, but it did modify capsule size, surface tension, electrokinetic stability and drug release. Cell viability, bioenergetics and inflammatory profiles were improved when auditory cells were exposed to PB-based nanoparticles, which showed antioxidant properties (p < 0.05). Conclusion: PB-based nanoparticles can potentially protect the auditory cell line from oxidative stress and could be used in future in vivo studies as a potential new therapeutic agent for sensorineural hearing loss.

Oxidative stress is an imbalance of cellular processes in which the production of free radicals outweighs the cellular defense mechanism. The association of oxidative stress with the pathophysiology of sensorineural hearing loss (SHL) is well established. SHL development is associated with chronic damage in the structure of the inner ear or auditory nerve. Therefore, potent antioxidants such as probucol could be one way to prevent or treat SHL. However, due to its isolated position, SHL is challenging to treat, imposing a desperate need for refining existing therapeutic methods; one way to do this is by optimizing the formulation using nanoparticles. We aimed to design a novel, stable formulation of PB using polymers and excipients to develop nanoparticles and examine the efficiency of these formulations on the HEI-OC1 stress cell line. We found that the prepared nanoparticle is robust and stable and protects HEI-OC1 from cellular toxicity and oxidative stress. It could be a novel therapeutic agent to treat or prevent SHL.

Novel Probucol Analogue, 4,4'-Diselanediylbis (2,6-Di-tert-Butylphenol), Prevents Oxidative Glutamate Neurotoxicity In Vitro and Confers Neuroprotection in a Rodent Model of Ischemic Stroke.

Oxidative glutamate toxicity is regarded as one of the injurious mechanisms associated with ischemic stroke, which represents a major health problem and requires improved pharmacological treatments. We designed and synthesized two new probucol analogues [2,6-di-tert-butyl-4-selenocyanatophenol (C1) and 4,4'-diselanediylbis (2,6-di-tert-butylphenol) (C2)] and investigated their effects against glutamate-induced neuronal oxidative toxicity in vitro in cultured HT22 cells, compared with their parental compound (probucol). In addition, C2, which exhibited the lowest toxicity, was investigated in an in vivo rodent model of ischemic stroke. Glutamate caused concentration- and time-dependent cytotoxicity in HT22 neuronal cells, which was preceded by increased levels of oxidants and depletion of the antioxidant glutathione. The analogues (C1 and C2), but not probucol, significantly decreased the levels of oxidants (including mitochondrial superoxide anion and lipid reactive oxygen species (ROS)) and protected against glutamate-induced cytotoxicity. In the in vivo model of ischemic stroke, which was based on central injections of the vasoconstrictor agent endothelin-1 (800 pmol/site), C2 (20 or 50 mg/kg/day, intraperitoneally, for 4 consecutive days after stroke) displayed significant beneficial effects against ischemic injury in vivo, improving rats' motor-related behavioral skills and decreasing stroke-related striatal gliosis. This is the first study to design, synthesize, and present a probucol analogue (C2) with in vivo beneficial effects against ischemic stroke. This novel compound, which was able to mitigate glutamate-induced oxidative toxicity in vitro, represents a promising neuroprotective drug.

Probucol treatment after traumatic brain injury activates BDNF/TrkB pathway, promotes neuroregeneration and ameliorates functional deficits in mice.

BACKGROUND AND PURPOSE: Traumatic brain injury (TBI) is a major cause of mortality and morbidity worldwide, yet pharmacotherapies for TBI are currently lacking. Neuroregeneration is important in brain repair and functional recovery. In this study, probucol, a cholesterol-lowering drug with established safety profiles, was examined for its therapeutic effects and neuroregenerative actions in TBI. EXPERIMENTAL APPROACH: Male mice were subjected to the controlled cortical impact model of TBI, followed by daily administration of probucol. Neurological and cognitive functions were evaluated. Histological analyses of the neocortex and hippocampus were performed to detect the lesion, dendritic degeneration (microtubule-associated protein 2), synaptic density (synaptophysin), neurogenesis (doublecortin), brain-derived neurotrophic factor (BDNF) and tropomyosin receptor kinase B (TrkB) activation. Involvement of BDNF/TrkB pathway in probucol-mediated effects was examined in primary cultures of cortical neurons. KEY RESULTS: Probucol reduced brain lesion volume, enhanced the recovery of body symmetry, improved motor function and attenuated memory dysfunction after TBI. Meanwhile, probucol promoted post-injury dendritic growth and synaptogenesis and increased hippocampal proliferating neuronal progenitor cells, along with the formation as well as the survival of newborn neurons. Moreover, probucol enhances BDNF expression and TrkB activation. In vitro, probucol promoted neurite outgrowth, which was inhibited by a selective TrkB antagonist ANA-12. CONCLUSIONS AND IMPLICATIONS: Probucol enhanced functional restoration and ameliorated cognitive impairment after TBI by promoting post-injury neuronal remodelling and neurogenesis. Increased activation of BDNF/TrkB pathway by probucol, at least in part, contributed to the neuroregenerative effects of probucol. Together, it may be promising to repurpose probucol for TBI.

An AI-guided screen identifies probucol as an enhancer of mitophagy through modulation of lipid droplets.

Failures in mitophagy, a process by which damaged mitochondria are cleared, results in neurodegeneration, while enhancing mitophagy promotes the survival of dopaminergic neurons. Using an artificial intelligence platform, we employed a natural language processing approach to evaluate the semantic similarity of candidate molecules to a set of well-established mitophagy enhancers. Top candidates were screened in a cell-based mitochondrial clearance assay. Probucol, a lipid-lowering drug, was validated across several orthogonal mitophagy assays. In vivo, probucol improved survival, locomotor function, and dopaminergic neuron loss in zebrafish and fly models of mitochondrial damage. Probucol functioned independently of PINK1/Parkin, but its effects on mitophagy and in vivo depended on ABCA1, which negatively regulated mitophagy following mitochondrial damage. Autophagosome and lysosomal markers were elevated by probucol treatment in addition to increased contact between lipid droplets (LDs) and mitochondria. Conversely, LD expansion, which occurs following mitochondrial damage, was suppressed by probucol and probucol-mediated mitophagy enhancement required LDs. Probucol-mediated LD dynamics changes may prime the cell for a more efficient mitophagic response to mitochondrial damage.

Research and Progress of Probucol in Nonalcoholic Fatty Liver Disease.

With the development of the social economy over the last 30 years, non-alcoholic fatty liver disease (NAFLD) is affected by unhealthy living habits and eating styles and has gradually become an increasingly serious public health problem. It is very important to investigate the pathogenesis and treatment of NAFLD for the development of human health. Probucol is an antioxidant with a bis-phenol structure. Although probucol is a clinically used cholesterol-lowering and antiatherosclerosis drug, its mechanism has not been elucidated in detail. This paper reviews the chemical structure, pharmacokinetics and pharmacological research of probucol. Meanwhile, this paper reviews the mechanism of probucol in NAFLD. We also analyzed and summarized the experimental models and clinical trials of probucol in NAFLD. Although current therapeutic strategies for NAFLD are not effective, we hope that through further research on probucol, we will be able to find suitable treatments to solve this problem in the future.

Probucol suppresses osteoclastogenesis via activating Nrf2 signaling and ameliorates ovariectomy-induced bone loss.

Osteoporosis is a systemic and endocrine bone disorder distinguished by declined bone mineral density, compromised bone strength, and destruction of trabecular structure. The abnormally excessive osteoclastogenesis and bone erosion play imperative roles in the progression of osteoporosis. However, treatment of osteoporosis is far from satisfactory due to poor adherence to existing medications and adverse reactions, there is an urgent to develop novel therapies for osteoporosis. Probucol, a synthetic compound with two characteristic phenolic rings, owns anti-inflammatory and antioxidant properties. Accumulating evidence have indicated that intracellular reactive oxygen species (ROS) is closely related to osteoclastogenesis. Hence, we investigated the potential effects of probucol on osteoclastogenesis in vivo and in vitro. In this study, TRAP staining and bone slice resorption assay showed that probucol suppressed RANKL-induced osteoclast formation and function. The mRNA and protein levels of osteoclastogenesis marker genes were reduced by probucol in a concentration-dependent manner. Besides, probucol suppressed osteoclast differentiation by inhibiting ROS production, MAPKs and NF-κB signaling pathways, while Nrf2 silencing reversed the inhibitory effect of probucol on osteoclast formation and function. Consistent with the above findings, in vivo experiments demonstrated that probucol visibly alleviated bone loss caused by estrogen deficiency. In brief, these results showed the potential of anti-oxidant compound probucol in the treatment of osteoporosis, highlighting Nrf2 as a promising target in osteoclast-related disease.

Inhibition of the cholesterol transporter ABCA1 by probucol decreases capacitation and tyrosine phosphorylation of dog spermatozoa, and is dose dependent.

The ATP binding cassette (ABC) transporter molecule ABCA1 participates in the cholesterol transport within and through cell membranes. We recently demonstrated that in dog spermatozoa, capacitation could be decreased with probucol (PRO), an ABCA1 specific antagonist. In this study, a dose-effect relationship of PRO on dog sperm capacitation, tyrosine phosphorylation and cholesterol efflux from the sperm plasma membrane was investigated. A total of 16 ejaculates from dogs of different breeds, aged 2-4 years were used. Sperm motility and membrane integrity in the main fraction was determined by CASA. Samples were stained with a boron dipyrromethene difluoride (BODIPY) fluorophore (P9672, Sigma- Aldrich, A) diluted in DMSO at a final concentration of 0.4 µM. All samples were divided into 5 aliquots, with 0, 100, 250, 500 and 1000 µM of PRO. After incubation at 37 °C for 2 h, PI was added and flow cytometry performed. All aliquots were examined for capacitation and acrosome reaction by using the CTC assay and tyrosine phosphorylation (TP). Membrane integrity was measured in all aliquots to investigate the effect of PRO on cell membranes. Membrane integrity did not differ between controls (0 µM), and 100, 250 and 500 µM PRO, but decreased with 1000 µM PRO (p < 0.05). Increasing PRO concentration decreased the percentage alive cells with cholesterol efflux per PRO group (0 µM: 77.8 ± 10.6%, 100 µM: 63.7 ± 11.7%, 250 µM: 52.1 ± 12.9%, 500 µM: 37.7 ± 11.6%, 1000 µM: 33.1 ± 14.4%; p < 0.05), decreased head and entire tail phosphorylated cells (0 µM: 34.6%, 1000 µM: 5.1% p < 0.05); and decreased the percentage capacitated cells (maximum with PRO 500 µM: capacitated vs. control: 54.2 ± 17% vs 25 ± 7.7%, p < 0.05). Conclusion: PRO decreased the cholesterol efflux, and decreased tyrosine phosphorylation and capacitation in a dose-dependent manner. This suggests a strong involvement of the ABCA1 transporter in different functional aspects of sperm capacitation in dogs.

Probucol promotes osteoblasts differentiation and prevents osteoporosis development through reducing oxidative stress.

Probucol (PBC) is a potent cholesterol-lowering drug and has been studied extensively for its powerful antioxidative stress. The purpose of this study is to investigate the role of PBC in ovariectomized rat model and to explore the mechanism of osteogenic differentiation of MC3TE-E1 Cells. RT-qPCR and Immunofluorescence were used to measure the expression level of SOD2, SIRT1, intracellular oxidative stress levels and osteogenic markers proteins. Moreover, CCK-8 assay was conducted to detect cell viability. Alizarin red staining and alkaline phosphatase staining were applied to examine osteogenic function and calcium deposits. The ovariectomized rat model was set up successfully and HE staining were employed to examine femoral trabeculae tissue. Our results showed that PBC suppressed MC3TE-E1 resist oxidative stress to promote osteogenic differentiation. Additionally, it was confirmed that PBC promoted osteogenic differentiation of MC3TE-E1 through inhibiting oxidative stress. Further study indicated that PBC exerted its beneficial function by suppressing oxidative stress-mediated alter bone metabolism to alleviate osteoporosis in vivo. Our research suggested that the PBC-modulated oxidative stress inhibition is responsible for activation of the process of osteogenic differentiation, providing a novel insight into the treatment of osteoporosis.

Efficacy of probucol on cognitive function in Alzheimer's disease: study protocol for a double-blind, placebo-controlled, randomised phase II trial (PIA study).

INTRODUCTION: Preclinical, clinical and epidemiological studies support the hypothesis that aberrant systemic metabolism of amyloid beta (Aß) in the peripheral circulation is causally related to the development of Alzheimer's disease (AD). Specifically, recent studies suggest that increased plasma concentrations of lipoprotein-Aß compromise the brain microvasculature, resulting in extravasation and retention of the lipoprotein-Aß moiety. The latter results in an inflammatory response and neurodegeneration ensues. Probucol, a historic cholesterol-lowering drug, has been shown in murine models to suppress lipoprotein-Aß secretion, concomitant with maintaining blood-brain-barrier function, suppressing neurovascular inflammation and supporting cognitive function. This protocol details the probucol in Alzheimer's study, a drug intervention trial investigating if probucol has potential to attenuate cognitive decline, delay brain atrophy and reduce cerebral amyloid burden in patients with mild-to-moderate AD. METHODS AND ANALYSIS: The study is a phase II, randomised, placebo-controlled, double-blind single-site clinical trial held in Perth, Australia. The target sample is 314 participants with mild-to-moderate AD. Participants will be recruited and randomised (1:1) to a 104-week intervention consisting of placebo induction for 2 weeks followed by 102 weeks of probucol (Lorelco) or placebo. The primary outcome is changed in cognitive performance determined via the Alzheimer's Disease Assessment Scales-Cognitive Subscale test between baseline and 104 weeks. Secondary outcomes measures will be the change in brain structure and function, cerebral amyloid load, quality of life, and the safety and tolerability of Lorelco, after a 104week intervention. ETHICS AND DISSEMINATION: The study has been approved by the Bellberry Limited Human Research Ethics Committee (approval number: HREC2019-11-1063; Version 4, 6 October 2021). Informed consent will be obtained from participants prior to any study procedures being performed. The investigator group will disseminate study findings through peer-reviewed publications, key conferences and local stakeholder events. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry (ACTRN12621000726853).

Protective Effects of Probucol on Different Brain Cells Exposed to Manganese.

Manganese (Mn) is an essential metal for many functions in the body. However, in excess, it can be neurotoxic and cause a Parkinson-like syndrome, known as manganism. Here, we aimed to identify a protective effect of probucol, a lipid-lowering agent with anti-inflammatory and antioxidant properties, against Mn-induced toxicity in human neuroblastoma (SH-SY5Y) and glioblastoma (C6) cell lines. The cells were incubated with increasing concentrations of Mn followed by probucol addition 1, 3, 6, and/or 24 h to assess the metal toxic doses and measure the protective effect of probucol against Mn-induced oxidative damage. Longer exposition to Mn showed decreased SH-SY5Y cellular viability in concentrations higher than 100 µM, and probucol was able to prevent this effect. The C6 cells were more sensitive to the Mn deleterious actions, decreasing the cell viability after 6 h of 500 µM Mn exposure. In addition, probucol prevents the complex I and II of the mitochondrial respiratory chain (MRC) inhibition caused by Mn and decreased the intracellular ROS production. Taken together, our results showed that Mn toxicity affects differently both cell lines and probucol has a protective effect against the oxidative imbalance in the central nervous system.

A Novel Diselenide-Probucol-Analogue Protects Against Methylmercury-Induced Toxicity in HT22 Cells by Upregulating Peroxide Detoxification Systems: a Comparison with Diphenyl Diselenide.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant whose mechanisms of action involve oxidation of endogenous nucleophilic groups (mainly thiols and selenols), depletion of antioxidant defenses, and disruption of neurotransmitter homeostasis. Diphenyl diselenide-(PhSe)2-a model diaryl diselenide, has been reported to display significant protective effects against MeHg-induced neurotoxicity under both in vitro and in vivo experimental conditions. In this study, we compared the protective effects of (PhSe)2 with those of RC513 (4,4'-diselanediylbis(2,6-di-tert-butylphenol), a novel diselenide-probucol-analog) against MeHg-induced toxicity in the neuronal (hippocampal) cell line HT22. Although both (PhSe)2 and RC513 significantly mitigated MeHg- and tert-butylhydroperoxide (t-BuOOH)-cytotoxicity, the probucol analog exhibited superior protective effects, which were observed earlier and at lower concentrations compared to (PhSe)2. RC513 treatment (at either 0.5 µM or 2 µM) significantly increased glutathione peroxidase (GPx) activity, which has been reported to counteract MeHg-toxicity. (PhSe)2 was also able to increase GPx activity, but only at 2 µM. Although both compounds increased the Gpx1 transcripts at 6 h after treatments, only RC513 was able to increase mRNA levels of Prx2, Prx3, Prx5, and Txn2, which are also involved in peroxide detoxification. RC513 (at 2 µM) significantly increased GPx-1 protein expression in HT22 cells, although (PhSe)2 displayed a minor (nonsignificant) effect in this parameter. In agreement, RC513 induced a faster and superior capability to cope with exogenously-added peroxide (t-BuOOH). In summary, when compared to the prototypical organic diaryl diselenide [(PhSe)2], RC513 displayed superior protective properties against MeHg-toxicity in vitro; this was paralleled by a more pronounced upregulation of defenses related to detoxification of peroxides, which are well-known MeHg-derived intermediate oxidant species.

Prevention of ferroptosis in acute scenarios: an in vitro study with classic and novel anti-ferroptotic compounds.

Ferroptosis, an iron-dependent form of cell death, has critical roles in diverse pathologies. Data on the temporal events mediating the prevention of ferroptosis are lacking. Focused on temporal aspects of cytotoxicity/protection, we investigated the effects of classic (Fer-1) and novel [2,6-di-tert-butyl-4-(2-thienylthio)phenol (C1) and 2,6-di-tert-butyl-4-(2-thienylselano)phenol (C2)] anti-ferroptotic agents against RSL3-, BSO- or glutamate-induced ferroptosis in cultured HT22 neuronal cell line, comparing their effects with those of the antioxidants trolox, ebselen and probucol. Glutamate (5 mM), BSO (25 µM) and RSL3 (50 nM) decreased approximately 40% of cell viability at 24 h. At these concentrations, none of these agents changed cell viability at 6 h after treatments; RSL3 increased lipoperoxidation from 6 h, although BSO and glutamate only did so at 12 h after treatments. At similar conditions, BSO and glutamate (but not RSL3) decreased GSH levels at 6 h after treatments. Fer-1, C1 and C2 exhibited similar protective effects against glutamate-, BSO- and RSL3-cytotoxicity, but this protection was limited when the protective agents were delivered to cells at time-points characterized by increased lipoperoxidation (but not glutathione depletion). Compared to Fer-1, C1 and C2, the anti-ferroptotic effects of trolox, ebselen and probucol were minor. Cytoprotective effects were not associated with direct antioxidant efficacies. These results indicate that the temporal window is central in affecting the efficacies of anti-ferroptotic drugs in acute scenarios; ferroptosis prevention is improbable when significant rates of lipoperoxidation were already achieved. C1 and C2 displayed remarkable cytoprotective effects, representing a promising new class of compounds to treat ferroptosis-related pathologies.

Role of oxidative stress versus lipids in monocrotaline-induced pulmonary hypertension and right heart failure.

Pulmonary hypertension (PH) is a global health issue with a prevalence of 10% in ages >65 years. Right heart failure (RHF) is the main cause of death in PH. We have previously shown that monocrotaline (MCT)-induced PH and RHF are due to an increase in oxidative stress. In this study, probucol (PROB), a strong antioxidant with a lipid-lowering property, versus lovastatin (LOV), a strong lipid-lowering drug with some antioxidant effects, were evaluated for their effects on the MCT-induced RHF. Rats were treated (I.P.) with PROB (10 mg/kg ×12) or LOV (4 mg/kg ×12), daily 6 days before and 6 days after a single MCT injection (60 mg/kg). Serial echocardiography was performed and at 4-week post-MCT, lung wet-to-dry weight, hemodynamics, RV glutathione peroxidase (GSHPx), superoxide dismutase (SOD), catalase, lipid peroxidation, and myocardial as well as plasma lipids were examined. MCT increased RV systolic and diastolic pressures, wall thickness, RV end diastolic diameter, mortality, and decreased ejection fraction as well as pulmonary artery acceleration time. These changes were mitigated by PROB while LOV had no effect. Furthermore, PROB prevented lipid peroxidation, lowered lipids, and increased GSHPx and SOD in RV myocardium. LOV did decrease the lipids but had no effect on antioxidants and lipid peroxidation. A reduction in oxidative stress and not the lipid-lowering effect of PROB may explain the prevention of MCT-induced PH, RHF, and mortality. Thus targeting of oxidative stress as an adjuvant therapy is suggested.