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Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

The use of antioxidants could thus prove an effective medication to prevent or facilitate recovery from oxidative stress-induced sensorineural hearing loss (SNHL). One promising strategy to prevent SNHL is developing probucol (PB)-based nanoparticles using encapsulation technology and administering them to the inner ear via the established intratympanic route. The preclinical, clinical and epidemiological studies support that PB is a proven antioxidant that could effectively prevent oxidative stress in different study models. Such findings suggest its applicability in preventing oxidative stress within the inner ear and its associated neural cells. However, several hurdles, such as overcoming the blood-labyrinth barrier, ensuring sustained release, minimising systemic side effects and optimising targeted delivery in the intricate inner ear structures, must be overcome to efficiently deliver PB to the inner ear. This review explores the background and pathogenesis of hearing loss, the potential of PB in treating oxidative stress and its cellular mechanisms, and the obstacles linked to inner ear drug delivery for effectively introducing PB to the inner ear.

Targeted drug delivery is an ongoing aspect of scientific research that is expanding through the design of micro- and nanoparticles. In this paper, we focus on spray dried microparticles as carriers for a repurposed lipophilic antioxidant (probucol). We characterise the microparticles and quantify probucol prior to assessing cytotoxicity on both control and cisplatin treated hair cells (known as House Ear Institute-Organ of Corti 1; HEI-OC1). The addition of water-soluble polymers to 2% β-cyclodextrin resulted in a stable probucol formulation. Ursodeoxycholic acid (UDCA) used as formulation excipient increases probucol miscibility and microparticle drug content. Formulation characterisations reveals spray drying results in spherical UDCA-drug microparticles with a mean size distribution of ∼5-12 μm. Probucol microparticles show stable short-term storage conditions accounting for only ∼10% loss over seven days. By mimicking cell culture conditions, both UDCA-probucol (67%) and probucol only (82%) microparticles show drug release in the initial two hours. Furthermore, probucol formulations with or without UDCA preserve cell viability and reduce cisplatin-induced oxidative stress. Mitochondrial bioenergetics results in lower basal respiration and non-mitochondrial respiration, with higher maximal respiration, spare capacity, ATP production and proton leak within cisplatin challenged UDCA-probucol groups. Overall, we present a facile method for incorporating lipophilic antioxidant carriers in polymer-based particles that are tolerated by HEI-OC1 cells and show stable drug release, sufficient in reducing cisplatin-induced reactive oxygen species accumulation., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Al-Salami H has been and is currently receiving funding from Beijing Nat-Med Biotechnology Co. Ltd. and Glanis PTY Ltd. All other authors have no completing interest to declare., (Copyright © 2024 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.)

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.

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.

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 .

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no competing interest., (Copyright © 2023 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

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.

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., (© 2023. The Author(s).)

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 (MnCl 2 ) 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., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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., (© 2023 The Authors. British Journal of Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

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.

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.

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Moskal et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (© 2022. The Author(s).)

Introduction: Preclinical, clinical and epidemiological studies support the hypothesis that aberrant systemic metabolism of amyloid beta (Aβ) 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-Aβ compromise the brain microvasculature, resulting in extravasation and retention of the lipoprotein-Aβ 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-Aβ 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)., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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.

Background: In the present study, we aimed to investigate the effects of probucol on aging-related hippocampus-dependent cognitive impairment and explore the potential mechanisms., Methods: D-galactose (100 mg/kg, once daily for 6 weeks) was subcutaneously injected to induce aging in mice. Then the mice were administered with probucol or vehicle once a day for 2 weeks. The hippocampus-related cognition was evaluated with Morris water maze test, novel object recognition test, and contextual fear conditioning test. Moreover, synaptic plasticity was assessed, and RNA-sequencing was applied to further explore the molecular mechanisms., Results: Aging mice induced by D-galactose showed conspicuous learning and memory impairment, which was significantly ameliorated by probucol. Meanwhile, probucol enhanced the spine density and dendritic branches, improved long-term potentiation, and increased the expression of PSD95 of aging mice. Probucol regulated 70 differentially expressed genes compared to D-galactose group, of which 38 genes were upregulated and 32 genes were downregulated. At last, RNA-sequencing results were verified by quantitative reverse transcription-polymerase chain reaction., Conclusions: Probucol improved learning and memory in aging mice through enhancing synaptic plasticity and regulating gene expression, indicating the potential application of probucol to prevent and treat aging-related disorders., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., (© 2021 The Authors. Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

The development of high-end targeted drugs and vaccines against modern pandemic infections, such as COVID-19, can take a too long time that lets the epidemic spin up and harms society. However, the countermeasures must be applied against the infection in this period until the targeted drugs became available. In this regard, the non-specific, broad-spectrum anti-viral means could be considered as a compromise allowing overcoming the period of trial. One way to enhance the ability to resist the infection is to activate the nonspecific immunity using a suitable driving-up agent, such as plant polysaccharides, particularly our drug Panavir isolated from the potato shoots. Earlier, we have shown the noticeable anti-viral and anti-bacterial activity of Panavir. Here we demonstrate the pro-inflammation activity of Panavir, which four-to-eight times intensified the ATP and MIF secretion by HL-60 cells. This effect was mediated by the active phagocytosis of the Panavir particles by the cells. We hypothesized the physiological basis of the Panavir proinflammatory activity is mediated by the indol-containing compounds (auxins) present in Panavir and acting as a plant analog of serotonin., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Osteoarthritis (OA) is a chronic joint disease characterized by cholesterol accumulation in chondrocytes, cartilage degeneration, as well as extracellular matrix (ECM) destruction, and joint dysfunction. Curcumin, a chemical that can reduce cholesterol levels in OA patients, also can inhibit the progression of OA. However, a high concentration of curcumin may also trigger apoptosis in normal chondrocytes. Besides curcumin, probucol that is found can also effectively decrease the cholesterol level in OA patients. Considering that high cholesterol is a risk factor of OA, it is speculated that the combination treatment of curcumin and probucol may be effective in the prevention of OA. To investigate the possible effects of such two chemicals on OA pathophysiology, chondrocyte apoptosis and autophagy behavior under inflammatory cytokine stress were studied, and specifically, the PI3K-Akt-mTOR signaling pathway was studied. Methods . Cell proliferation, colony formation, and EdU assay were performed to identify the cytotoxicity of curcumin and probucol on chondrocytes. Transwell assay was conducted to evaluate chondrocyte migration under TNF- α inflammation stress. Immunofluorescence, JC-1, flow cytometry, RT-PCR, and western blot were used to investigate the signal variations related to autophagy and apoptosis in chondrocytes and cartilage. A histological study was carried out on OA cartilage. Glycosaminoglycan (GAG) release was determined to evaluate the ECM degradation under stress. Results . Compared with a single intervention with curcumin or probucol, a combined treatment of these two chemicals is more effective in terms of protecting chondrocytes from stress injury induced by inflammatory cytokines. The promoted protection may be attributed to the inhibition of apoptosis and the blockage of the autophagy-related PI3K/Akt/mTOR pathway. Such results were also verified in vitro by immunofluorescence staining of OA chondrocytes and in vivo by immunohistochemistry staining of cartilage. Besides, in vivo studies also showed that when applied in combination, curcumin and probucol could block the PI3K-AKT-mTOR signaling pathway; promote COL-II expression; suppress P62, MMP-3, and MMP-13 expression; and inhibit TNF- α -stimulated cartilage degradation. Moreover, the combined medication could help reduce the release of ECM GAGs in OA cartilage and alleviate the severity of OA. Conclusion . A combined treatment of curcumin and probucol could be used to protect chondrocytes from inflammatory cytokine stress via inhibition of the autophagy-related PI3K/Akt/mTOR pathway both in vitro and in vivo, which might be of potential pharmaceutical value for OA prevention and therapy., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2021 Guangtao Han et al.)

Atherosclerosis is a chronic inflammatory, metabolic, and epigenetic disease, which leads to the life-threatening coronary artery disease. Emerging studies from bench to bedside have demonstrated the pivotal role of low-density lipoprotein (LDL) oxidation in the initiation and progression of atherosclerosis. This article hereby reviews oxidation mechanism of LDL, and the pro-atherogenic and biomarker role of oxidized LDL in atherosclerosis. We also review the pharmacological effects of several representative natural products (vitamin E, resveratrol, quercetin, probucol, tanshinone IIA, epigallocatechin gallate, and Lycopene) in protecting against LDL oxidation and atherosclerosis. Clinical and basic research supports the beneficial effects of these natural products in inhibiting LDL oxidation and preventing atherosclerosis, but the data are still controversial. This may be related to factors such as the population and the dosage and time of taking natural products involved in different studies. Understanding the mechanism of LDL oxidation and effect of oxidized LDL help researchers to find novel therapies against atherosclerosis., (© 2020 John Wiley & Sons, Ltd.)

This study was to explore the effect and mechanism of Probucol on STZ-induced erectile dysfunction in diabetic rats. Thirty SD male rats aged 12 weeks were given intraperitoneal injection of STZ after fasting for 12 hr. Diabetic rats were haphazardly partitioned under two assemblies and administered 0 or 500 mg/kg probucol by oral gavage to 12 weeks. Control group was intraperitoneally injected with physiological saline, and saline was administered by oral gavage daily. Intracorporeal pressure was used to evaluate erectile function. Levels of proteins were detected using immunohistochemistry and Western blotting. α-SMA and vWF were detected using immunofluorescence staining. After treatment, erectile function in probucol group was significantly improved. Endoplasmic reticulum stress-related proteins were expressed higher in DM group than in sham group, while expression of these proteins decreased significantly in probucol group. However, α-SMA and vWF were expressed at lower levels in DM group than in sham group, and probucol treatment reversed this phenomenon. Finally, Bax and Caspase3 were expressed at higher levels and Bcl-2 was expressed at lower levels in DM group, while the opposite result was obtained in probucol group. In conclusions, probucol improves erectile function by reducing endothelial dysfunction and inhibiting PERK/ATF4/CHOP pathway in STZ-induced diabetic rats., (© 2021 Wiley-VCH GmbH.)

Background: Symmetrical dimethylarginine (ADMA) endogenously inhibits nitric oxide synthase (NOS) and strongly indicates oxidant stress, whose formation primarily derived from type 1 protein arginine N-methyltransferase (PRMT1) and whose metabolism was governed by type 1 dimethylarginine dimethylaminohydrolase (DDAH1). This study aimed to evaluate participation of the PRMT1-ADMA-DDAH1 metabolism axis in the kidneys of type 2 diabetes model rats and human subjects, and the effect of probucol on this axis and renal function., Methods: A total of 30 rats were randomly assigned to a normal group (NC, n=10), diabetic group (DM, n=10), and a diabetics under probucol treatment group (PM, n=10). Throughout 8 weeks of probucol treatment, plasma NOS, the malondialdehyde (MDA), superoxide dismutase (SOD), nitric oxide (NO), and catalase (CAT) activity were evaluated by chemical colorimetric approach. ADMA concentration was evaluated with an enzyme-linked immunosorbent assay (ELISA) and analysis of expression of PRMT1 and DDAH1 in kidneys with reverse transcription-polymerase chain reaction (RT-PCR), immunohistochemistry (IHC) and western blotting were performed., Results: The expression of DDAH1 in the kidney, and the plasma NOS, NO, SOD, and CAT activities in diabetic group were lower, while MDA and the expression of PRMT1 and ADMA were higher in contrast to the control group. In diabetics rats receiving probucol, the expressions of DDAH1 and ADMA were downregulated, whereas that of PRMT1 was upregulated. Probucol inhibited the indexes of oxidative stress and improved the kidney function in both diabetic rats and humans., Conclusions: We found that the expression of the PRMT1-ADMA-DDAH1 axis was altered in the kidneys of diabetic rats. Moreover, results indicated that probucol therapy regulates expression at both ends of this axis, which may preserve renal function by reducing oxidant stress. Therefore, probucol may partially restore expression of the PRMT1-ADMA-DDAH1 axis in diabetic kidneys, immigrate oxidant stress, and enhance renal function.

The elevated homocysteine level is an independent risk factor for atherosclerosis, which is characterized as a chronic inflammatory disease associated with oxidative stress. We have confirmed that homocysteine can stimulate the production of C-reactive protein (CRP) in rat aortic smooth muscle cells (RASMCs). In the present study, we investigated the role of probucol in homocysteine-induced CRP expression in cultured RASMCs and high-methionine-diet-induced hyperhomocysteinemic rats. The results showed that probucol decreased homocysteine-induced CRP mRNA and protein expression in RASMCs in a concentration-dependent manner. In addition, the animal experiment showed that probucol not only inhibited CRP expression in the vessel wall but also reduced the circulating CRP level in hyperhomocysteinemic rats. Further investigations revealed that probucol markedly increased heme oxygenase-1 activity, suppressed nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, diminished superoxide anion generation, and decreased p38 phosphorylation in RASMCs and hyperhomocysteinemic rat aorta. These data demonstrate that probucol can inhibit homocysteine-induced CRP generation by interfering with the NADPH oxidase/p38 signal pathway in RASMCs, which will provide new evidence for the anti-inflammatory and anti-atherosclerotic effects of probucol., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

High-fat pancreatitis and hyperlipidemia refer to disorders of blood lipid metabolism caused by abnormally elevated blood lipids, and are risk factors for high-risk diseases such as atherosclerosis, coronary heart disease, and cerebral infarction. Hyperlipidemia is also a common disease that is common in modern people, and has a tendency to become younger. In this paper, probucol is made into a self-assembled probucol loaded nanosuspensions (SPN) using molecular selfassembly technology for research on improving its oral absorption. The main research contents include: preparation, prescription screening and characterization of physicochemical properties of SPN nanosuspensions; study of SPN intestinal cell uptake and in vivo dynamic behavior; research on the mechanism of SPN improving oral absorption of probucol and its gastrointestinal Preliminary Evaluation of Physiological Safety. And by using the method of intraperitoneal injection of SPN to interfere with the retrograde bile duct injection of SPN in the hyperlipidemia model, to make hyperlipidemia combined with severe acute pancreatitis to observe the severity of pancreatitis and lung injury; discuss the protective effect of SPN on hyperlipidemia combined with severe acute pancreatitis with lung injury and its mechanism.

This study explored the effects of probucol on myocardial injury, oxidative stress, and Cav-3 and Smad3 expression in myocardial tissues by establishing VMC rat models, in order to provide a basis for exploring the mechanism of probucol in treatment of VMC. Sixty rats were randomly divided into control group, model group, probucollowdose group, andprobucol highdose group, with 15 in each group. Except for the control group, rats in each group were intraperitoneally injected coxsackievirus B3 diluent (0.2 ml) to replicate VMC models every 4 days. The results showed that Caspase-3 and Caspase-9, myocardial enzymes, cTn I, and MDA levels in the model group significantly increased (P < 0.05), while the SOD level significantly decreased (P < 0.05); and after probucol treatment, Caspase-3 and Caspase-9, myocardial enzymes, cTn I and MDA levels significantly decreased (P < 0.05), and the SOD level significantly increased (P < 0.05). Compared with the control group, there was an increase in myocardial fibers with significant lesions in the model group, and the pathological scores and the mRNA and protein expression levels of Cav-3 and Smad3 in myocardial cells significantly increased (P < 0.05). Compared with the control group, the myocardial tissue lesions were improved in the probucol low dose group and highdose group, and the pathological scores and the mRNA and protein expression levels of Cav-3 and Smad3 in myocardial cells were significantly reduced (P < 0.05). In conclusion, probucol can significantly improve the pathological damage of myocardial tissue in VMC rats, and its mechanism may be related to improving the expression of myocardium-related proteins Caspase-3 and Caspase-9, inhibiting oxidative stress response, and down-regulating Cav-3 and Smad3 gene expression in myocardial tissue of VMC rats., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Probucol, a hypocholesterolemic compound, is neuroprotective in several models of neurodegenerative diseases but has serious adverse effects in vivo. We now describe the design and synthesis of two new probucol analogues that protect against glutamate-induced oxidative cell death, also known as ferroptosis, in cultured mouse hippocampal (HT22) cells and in primary cortical neurons, while probucol did not show any protective effect. Treatment with both compounds did not affect glutathione depletion but still significantly decreased glutamate-induced production of oxidants, mitochondrial superoxide generation, and mitochondrial hyperpolarization in HT22 cells. Both compounds increase glutathione peroxidase (GPx) 1 levels and GPx activity, also exhibiting protection against RSL3, a GPx4 inactivator. These two compounds are therefore potent activators of GPx activity making further studies of their neuroprotective activity in vivo worthwhile.

Experimental evidence has shown that probucol, a hypocholesterolemic agent, is also able to increase glutathione peroxidase (GPx) activity. However, there is a lack of knowledge about the mechanism(s) involved in this event. In this study, in vitro experiments with purified GPx1 from bovine erythrocytes and cultured SH-SY5Y neuroblastoma cells, as well as in silico studies with GPx1, were performed in order to elucidate mechanisms mediating the stimulatory effect of probucol on GPx activity and to investigate the relevance of this event in terms of susceptibility against peroxide-induced cytotoxicity. In vitro experiments with purified GPx1 showed a direct stimulatory effect of probucol on the activity of GPx1, which was related to an increase in V max with no changes in K M . Probucol also increased GPx activity in cultured SH-SY5Y neuroblastoma cells, while the levels of GPx1 expression were not changed, corroborating the results found with the purified enzyme. In addition, probucol rendered SH-SY5Y cells more resistant to hydroperoxide-induced cytotoxicity, and this event was abolished in GPx1 knocked-down cells. In silico studies with GPx1 pointed to a potential binding site for probucol at the close vicinity of the GSH pocket. Collectively, the results presented herein indicate that GPx1 plays a central role in the probucol-induced protective effects against peroxide toxicity. This highlights a novel target (GPx1) and a new mechanism of action (direct activation) for an "old drug." The relevance of such results for in vivo conditions deserves further investigation.

Background: Intracavernous injection of mesenchymal stem cells (MSCs) is a promising method for diabetic mellitus-induced erectile dysfunction (DMED), but short survival time of MSCs in cavernous is a fatal defect for therapy. This study investigated therapeutic efficiency and potential mechanism of probucol combined with MSCs., Methods: In vivo study, a total of forty-eight 10-week-old male Sprague-Dawley (SD) rats were used. Twelve rats received intraperitoneal injection of PBS as the sham group; the rest received intraperitoneal injection of 60 mg/kg streptozotocin to establish DM models. DM rats were randomly divided into three groups: received intracavernosal (IC) injection of either PBS (DM group), MSCs (M group), or administrated probucol after intracavernosal injection of MSCs (P + M group). Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated for histologic examination and Western blotting. In in vitro experiment, H 2 O 2 was used to create oxidative stress environment to detect changes in cell viability. CCK8 was used to measure cell viability of MSCs treated with or without probucol. Intracellular ROS changes were detected by flow cytometry. Autophagy and apoptosis were detected by Western blotting and confocal microscopy., Results: Recovery of erectile function was observed in the P + M group. The combination therapy decreased fibrosis and increased endothelial function compared with MSC therapy alone. Western blotting results confirmed the increased expression of Nrf2 and HO-1 in cavernous body. H 2 O 2 induced high oxidative stress and reduced cell viability in vitro, which was gradually reversed with increased concentration of probucol. H 2 O 2 reduced Nrf2 expression, which was reversed by probucol's intervention. Furthermore, the expression of Bax, Caspase3, and Cleaved-Caspase3 decreased, and the expression of Bcl-2 increased in a dose-dependent manner because of probucol's intervention. In addition, Beclin1 and LC3II both increased in a dose-dependent manner. Meanwhile, the expression of P62 decreased. In the study of autophagy flux, we found probucol did not block it., Conclusion: Probucol enhanced therapeutic efficiency of MSCs in DMED by prolonging their survival time, which mediated through improving the transplanted microenvironment of MSCs, increasing self-antioxidant ability of MSCs, strengthening protective autophagy, and inhibiting apoptosis of MSCs via Nrf2 pathway. Schematic model showing combined probucol and MSCs to improve DMED. Probucol increases self-antioxidant ability of MSCs, strengthening protective autophagy and inhibiting apoptosis via Nrf2/HO-1 and Nrf2/autophagy pathways.

Autophagy and apoptosis have been regarded as important processes in the development of diabetic erectile dysfunction (DMED). Probucol is considered to have anti-apoptotic effects, but its relationship with autophagy has not been reported. The aim of this study was to investigate the effects and mechanisms of probucol on erectile function. Thirty Sprague-Dawley (SD) male rats (12 weeks old) were fasted for 12 h. Twenty SD rats were injected with a single intraperitoneal injection of 60 mg kg -1 streptozotocin (STZ). Ten rats were given vehicle only and used as a sham group. After 72 h, 20 STZ-treated rats with random blood glucose concentrations consistently greater than 16.7 mmol l -1 were used as successfully established diabetic rats. The diabetic rats were divided randomly into two groups and treated with a daily gavage of probucol at a dose of 0 or 500 mg kg -1 for 12 weeks. After treatment, the intracavernous pressure (ICP) was used to measure erectile function upon electrical stimulation of the cavernous nerve. After euthanasia, penile tissue was examined using immunohistochemistry and Western blot to assess the protein levels of B-cell lymphoma-2 (Bcl-2), BCL2-associated X (Bax), microtubule-associated protein light chain 3-II (LC3-II), mammalian target of rapamycin (mTOR), and sequestosome 1 (P62). Caspase-3 activity was measured to determine apoptosis using a caspase-3 assay kit. After 12 weeks of treatment, the erectile function of the probucol group was significantly better than that of the DM group (P < 0.05). Bax and LC3-II protein expression and caspase-3 activity were significantly lower in the probucol group than those in the DM group (all P < 0.05), while Bcl-2, mTOR, and P62 protein expression levels were significantly higher than those in the DM group (all P < 0.05). We demonstrated that probucol inhibited apoptosis and autophagy in STZ-induced diabetic rats., Competing Interests: None

Probucol (PB) is a drug that exhibits significant hydrophobicity and substantial intra and inter individual variability in oral absorption, with a miniature bioavailability and complex three compartmental pharmacokinetic modelling due to its high lipid affinity, low stability and high octanol to water partition coefficient. Multiple attempts to formulate PB have not produced satisfactory stable matrices, drug-release profile or rheological flow properties for optimum manufacturing conditions, and with positive and none toxic biological effects. Lithocholic acid (LCA) has recently shown to optimise formulation and cell uptake of drugs. Hence, the aim of this study was to design new PB delivery system, using LCA, and examine its morphology, rheology, stability, and cellular effects. PB was formulated with LCA and sodium alginate (PB-LCA-SA) using various microencapsulation methodologies, and best formulation was investigated in vitro and ex vivo. Using our Ionic Gelation Vibrational Jet flow technology, PB-LCA-SA microcapsules showed good stability and significantly enhanced cell viability, cellular respiration, and reduced inflammation suggesting potential LCA applications in PB delivery and biological effects.

Diabetic retinopathy (DR) is a sight-threatening complication of diabetes. This study investigated the therapeutic effect of probucol in a mouse model of diabetic retinopathy. C57BL/6 mice were rendered diabetic through Streptozotocin (STZ) intraperitoneal injection. Mice were treated with probucol (150 mg/kg, gavage administration) or vehicle (DMSO) for 12 weeks. Optical coherence tomography (OCT), fundus photography (FP), and fundus fluorescein angiography (FFA) were conducted to evaluate retinal structure and damage. Eyes were collected for histology, reactive oxygen species (ROS) assay, apoptotic cells count, and western blot. After STZ injection, all mice developed hyperglycemia. Compared with the retina of the control group, the retina of diabetic mice showed enhanced arterial reflex and beaded vein dilatation. Besides, reduced inner and middle retinal thickness and significantly fewer nuclei were found in diabetic retina. Moreover, the diabetic retina also presented increased ROS generation and more TUNEL-positive cells. Probucol treatment prevented diabetes-induced lesions. In addition, the treatment also upregulated Nrf2 expression in diabetic retina. It was suggested that probucol attenuated diabetes-induced retinal neuronal degeneration via upregulating the Nrf2 signaling pathway possibly. Probucol may be repurposed for DR management., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Heng-Wei Liu et al.)

The antilipidemic drug, probucol (PB), has demonstrated potential applications in Type 2 diabetes (T2D) through its protective effects on pancreatic β-cells. PB has poor solubility and bioavailability, and despite attempts to improve its oral delivery, none has shown dramatic improvements in absorption or antidiabetic effects. Preliminary data has shown potential benefits from bile acid co-encapsulation with PB. One bile acid has shown best potential improvement of PB oral delivery (ursodeoxycholic acid, UDCA). This study aimed to examine PB and UDCA microcapsules (with UDCA microcapsules serving as control) in terms of the microcapsules' morphology, biological effects ex vivo, and their hypoglycemic and antilipidemic and anti-inflammatory effects in vivo. PBUDCA and UDCA microcapsules were examined in vitro (formulation studies), ex vivo and in vivo. PBUDCA microcapsules exerted positive effects on β-cells viability at hyperglycemic state, and brought about hypoglycemic and anti-inflammatory effects on the prediabetic mice. In conclusion, PBUDCA co-encapsulation have showed beneficial therapeutic impact of dual antioxidant-bile acid effects in diabetes treatment.

Background: The ATP binding cassette (ABC) transporters participate in the cholesterol and phospholipid transport within and through cell membranes of many cells including spermatozoa. Cholesterol efflux is important for capacitation of spermatozoa. ABCA1 expression has been assessed in canine spermatozoa previously but its role in capacitation still has to be determined. The aim of the study was to test whether inhibition of ABCA1 (1) decreases capacitation in ejaculated and epididymal canine sperm samples and (2) decreases cholesterol efflux in the same samples. Twenty-one ejaculates and sperm from 22 epididymal tails were collected from healthy dogs. Motility was measured by CASA and viability assessed after staining with SYBR-14/PI. Samples from ejaculated sperm and sperm from epididymal tails were aliquoted. One part was incubated with the ABCA1 inhibitor probucol, the other served as a negative control. In all samples, capacitation was evaluated by chlortetracyclin (CTC) assay and cholesterol was measured by cholesterol efflux assay and colorimetric enzymatic assay., Results: In ejaculated sperm, blockade of ABCA1 with 100 µM of probucol/mL of sample resulted in a significantly higher percentage of uncapacitated and acrosome reacted spermatozoa (P < 0.001 and P = 0.031), capacitation was significantly decreased (35% in probucol samples vs 54.2% in controls, P < 0.001). In probucol inhibited sperm samples from epididymal tails, the percentage of capacitated spermatozoa did not differ between groups but the percentage of acrosome reacted spermatozoa increased significantly (P = 0.014). The cholesterol measurement revealed significantly lower cholesterol concentration in the probucol group when compared to the controls (P = 0.035), however only in ejaculated sperm samples., Conclusions: CTC assay and cholesterol measurement revealed significant differences between groups; we conclude that inhibition of ABCA1 significantly decreased capacitation and cholesterol efflux in ejaculated canine spermatozoa. The inhibition was not complete but ABCA1 is supposed to contribute to capacitation in canine ejaculated spermatozoa. ABCA1 is probably not important for capacitation of epididymal spermatozoa but might exert other functions during spermatozoa ripening.

Background and Purpose: Abdominal aortic aneurysm (AAA) is a degenerative disease with irreversible and progressive dilation of the artery. But there are few options for efficacious treatment except for traditional surgery. Probucol has been widely applied to treat hyperlipidaemia and atherosclerosis in clinic, but whether it can protect against AAA remains unknown. In this study, the protective effects of probucol against AAA and its related mechanisms were explored., Experimental Approach: The model of AAA was induced in mice by periaortic application of elastase (40 min) to the abdominal aorta. Probucol at different doses was administered by daily gavage, starting on the same day as AAA was induced, for 14 days. In vitro, cultures of rat vascular smooth muscle cells (VSMCs) were stimulated with TNF-α. Haem oxygenase (HO)-1 siRNA and HO-1 plasmid were used to regulate the expression or activity of HO-1 in the VSMCs and to clarify the effects of HO-1., Key Results: Probucol dose-dependently prevented the development of AAA, reflected by decreased incidence of AAA, diameter of aortic dilation, elastin degradation, and infiltration of inflammatory cells. Probucol also protected VSMCs from oxidative injury and enhanced elastin biosynthesis. This anti-inflammatory effects of probucol on VSMCs were significantly decreased when HO-1 was inhibited by siRNA., Conclusion and Implications: Probucol protected against AAA through inhibiting the degradation of elastin induced by inflammation and oxidation and by facilitating the biosynthesis of elastin. HO-1 played a crucial role in the anti-inflammatory effects of probucol in VSMCs., (© 2019 The British Pharmacological Society.)

Aim: Probucol is a controversial drug to inhibit ATP-binding cassette transporter A1 (ABCA1) and to exhibit some positive clinical effects such as regression of xanthomas. It reportedly rescues female infertility in scavenger receptor BI-deficient mice. Here, we investigated the effect of probucol on propagation in HDL-deficient mice as alternative models for impaired HDL-mediated cholesterol delivery., Methods: Propagation of ABCA1-deficient (Abca1-/-) mice and lecithin: cholesterol acyltransferase (LCAT)-deficient (Lcat-/-) mice were quantitatively observed under the probucol treatment., Results: Abca1-/- and Lcat-/- mice appear with negligible plasma HDL concentration. Upon backcrossing Abc1+/- with the Abc1-/- mice and cross-breeding between Abc1+/- mice, the numbers of Abc1-/- weaned pups were reduced to 54.7% and to 57.1% from those expected by Mendelian genetics, respectively. Similarly, Lcat-/- weaned pups decreased to 67.7% and to 35.9% but only in the male. Probucol severely reduced plasma HDL-cholesterol to 5% in the wild-type mice, but showed no effects on their propagation. Probucol corrected the deflections of the genotype distribution in the weaned pups recovery in the LCAT-deficient mice propagation but not in the ABCA1-deficient mice while plasma HDL was kept negligible. Probucol had no effect on cholesterol content in the steroidogenic organs of the HDL-deficient mice, while it somewhat increased plasma corticosterone and expression of adrenal cortex HMG-CoA reductase, StAR, cytochrome P450scc, and VKORC1 indicating increase in the synthesis of cholesterol and steroid hormones and in vitamin K turn-over. However, no evident mechanistic background was indicated., Conclusions: Probucol corrected deflection of genotype distribution in propagation of the LCAT-deficient mice but not the ABCA1-deficient mice at the weaning stage, apparently not through normalization of hypoalphalipoproteinemia.

Purpose: To explore the protective effect of probucol on human retinal Müller cells cultured in high glucose., Methods: Primary Müller cells from human retinas were cultured in complete DMEM. Third-generation Müller cells were identified using glutamine synthetase (GS) antibody and randomly divided into three groups: normoglycemia (NG, 5.5 mmol/L); hyperglycemia (HG, 30 mmol/L); and hyperglycemia (30 mmol/L) with probucol (10 μmol/L; HGPB). After a 24-h intervention, cell proliferation, apoptosis, and cellular reactive oxygen species (ROS) were measured with a CCK-8 kit, flow cytometry, and DCFH-DA probe, respectively. Kelch-like ECH-associated protein 1 (Keap1), NF-E2-related factor 2 (Nrf2), and glutamate cysteine ligase catalytic subunit (GCLC) protein expression were detected by immunofluorescence staining., Results: For NG, HG, and HGPB, optical density (OD) values for cell proliferation were 0.98 ± 0.23, 0.58 ± 0.11, and 0.73 ± 0.11; apoptotic rates were 2.79 ± 0.52%, 7.70 ± 0.44%, and 4.00 ± 0.95%; and intracellular ROS were 20.89 ± 5.14, 55.17 ± 14.07, and 26.28 ± 4.73, respectively. Compared to NG, OD was markedly decreased (P < 0.01), apoptosis was increased (P < 0.001), and intracellular ROS level was significantly higher than in HG (P < 0.01). Compared to HG, OD was markedly increased (P < 0.01), apoptosis was meaningfully decreased (P < 0.01), and intracellular ROS level was significantly lower than in HGPB (P < 0.01). GS, Keap1, Nrf2, and GCLC had positive expression., Conclusions: Probucol could inhibit intracellular ROS generation, promote proliferation, and decrease apoptosis of human retinal Müller cells cultured in high glucose. This might also be associated with Keap1/Nrf2/ARE oxidative stress signaling pathway activation.

Oxidative stress plays a vital role in the initiation and progression of age-related neurodegenerative diseases. Ameliorating oxidative damage is therefore considered as a beneficial strategy for the treatment of age-related neurodegenerative disorders. Probucol (Prob), a lipid-lowering prototype agent, was reported to treat cardiovascular diseases, chronic kidney disease and diabetes mellitus. However, whether Prob has an effect on age-related neurodegenerative diseases remains unknown. In the study, it was found that Prob ameliorated D-galactose (D-gal) induced cognitive deficits and neuronal loss in the hippocampal CA1 region. Moreover, Prob alleviated ROS and MDA levels by elevating SOD, GSH-PX and HO-1 mRNA and protein expressions, and improving plasmic and cerebral SOD and GSH-PX activities in D-gal treated mice. Furthermore, Prob promoted the dissociation of Keap1/Nrf2 complex leading to the accumulation of Nrf2 in nucleus, implying that the improved anti-oxidant property of Prob is mediated by Keap1/Nrf2 pathway. The study firstly demonstrates the favorable effects of Prob against cognitive impairments in a senescent mouse model, rendering this compound a promising agent for the treatment or prevention of age-related neurodegenerative disease.

Antioxidants play a vital role in scavenging reactive oxygen species (ROS) produced by the reduction of molecular oxygen from various cellular mechanisms. Under oxidative stress, an increase in the levels of ROS overwhelms the antioxidant response, causing oxidative damage to biological molecules, and leading to the development of various diseases. Drug compounds with potent antioxidant properties are typically poorly water soluble and highly hydrophobic. An extreme case is Probucol (PB), a potent antioxidant with reported water solubility of 5 ng/ml, and oral bioavailiability of <10%. In this study, PB was loaded in mesoporous silica at various drug loadings to understand the changes to the physical properties of the loaded drug, and it's in vitro drug release. Further in vitro studies were conducted in endothelial and microglia cell models to compare the free radical scavening efficiency of ascorbic acid, PB, and PB release from mesoporous silica particles. Out of the three different mesostructured particles studied, the maximum loading of PB was achieved for large pore mesoporous particles (SBA-15) at 50 wt% drug loading, before complete pore filling was observed. For all materials, loadings above complete pore filling resulted in the recrystallization of PB on the external surface. In vitro drug release measurements showed a rapid dissolution rate at low drug loadings compared to a bimodal release profile of amorphous and crystalline drug at higher drug loadings. PB loaded in mesoporous particle was shown to enhance the antioxidant response to extracellular ROS in the endothelial cell line model, and to intracellular ROS in the microglia cell model. Our results indicate that the antioxidant properties of PB can be significantly improved by using mesoporous silica as a delivery vehicle., (Copyright © 2019. Published by Elsevier B.V.)

The pathogenesis of Alzheimer's disease (AD) is characterized by overproduction, impaired clearance, and deposition of amyloid-β peptides (Aβ) and connected to cholesterol homeostasis. Since the blood-brain barrier (BBB) is involved in these processes, we investigated effects of the retinoid X receptor agonist, bexarotene (Bex), and the peroxisome proliferator-activated receptor α agonist and antioxidant, astaxanthin (Asx), on pathways of cellular cholesterol metabolism, amyloid precursor protein processing/Aβ production and transfer at the BBB in vitro using primary porcine brain capillary endothelial cells (pBCEC), and in 3xTg AD mice. Asx/Bex downregulated transcription/activity of amyloidogenic BACE1 and reduced Aβ oligomers and ~80 kDa intracellular 6E10-reactive APP/Aβ species, while upregulating non-amyloidogenic ADAM10 and soluble (s)APPα production in pBCEC. Asx/Bex enhanced Aβ clearance to the apical/plasma compartment of the in vitro BBB model. Asx/Bex increased expression levels of ABCA1, LRP1, and/or APOA-I. Asx/Bex promoted cholesterol efflux, partly via PPARα/RXR activation, while cholesterol biosynthesis/esterification was suppressed. Silencing of LRP-1 or inhibition of ABCA1 by probucol reversed Asx/Bex-mediated effects on levels of APP/Aβ species in pBCEC. Murine (m)BCEC isolated from 3xTg AD mice treated with Bex revealed elevated expression of APOE and ABCA1. Asx/Bex reduced BACE1 and increased LRP-1 expression in mBCEC from 3xTg AD mice when compared to vehicle-treated or non-Tg treated mice. In parallel, Asx/Bex reduced levels of Aβ oligomers in mBCEC and Aβ species in brain soluble and insoluble fractions of 3xTg AD mice. Our results suggest that both agonists exert beneficial effects at the BBB by balancing cholesterol homeostasis and enhancing clearance of Aβ from cerebrovascular endothelial cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Pulmonary fibrosis is a progressive fibrotic lung disease with a paucity of therapeutic options. Here we investigated the potential roles of probucol, a cholesterol-lowering drug with potent anti-oxidation properties, on pulmonary epithelial-mesenchymal transition (EMT) and fibrosis. We found that bleomycin-induced lung fibrosis was associated with increased transforming growth factor (TGF)-β1, α-smooth muscle actin (α-SMA) and decreased E-cadherin expression in lung tissues, indicating EMT formation. Bleomycin treatment resulted in an induction of oxidative stress in lung tissues. Probucol treatment attenuated bleomycin-induced TGF-β1 production, EMT and pulmonary fibrosis, meanwhile it suppressed bleomycin-induced oxidative stress. Bleomycin treatment resulted in decreases in protein expressions of Sirtuin 3 (SIRT3) in the lung, which were restored by ROS scavenger NAC and probucol treatment, suggesting that probucol might restore SIRT3 expression by suppressing bleomycin-induced oxidative stress. In the mouse alveolar type II epithelial cell line MLE-12, probucol treatment leads to an increase in SIRT3 expression in bleomycin-treated AT-II cells, which might contribute to the inhibitory effect of probucol on EMT through suppressing hypoxia inducible factor (HIF)-1α/TGF-β1 pathway. In addition, probucol inhibited bleomycin-induced macrophage infiltration in the lung. Bleomycin decreased SIRT3 protein expression, whereas increased HIF-1α activation and TGF-β1 release in the mouse macrophage cell line RAW264.7, which were attenuated by probucol treatment. Taken together, the present study suggests that probucol may ameliorate EMT and lung fibrosis through restoration of SIRT3 expression. The data obtained in this study provides proof for the idea that probucol may be a potential therapeutic option for the treatment of pulmonary fibrosis., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The effect of the lipid-lowering agent probucol in inflammatory hyperalgesia and leukocyte recruitment was evaluated in a model of subacute inflammation by Complete Freund's adjuvant (CFA). As CFA induces long-lasting nociception characterized by peripheral and spinal cord inflammation, the anti-inflammatory activity of probucol was assessed at both foci. Probucol at 0.3-3 mg/kg was administrated per oral daily starting 24 h after CFA intraplantar injection. Mechanical and thermal hyperalgesia induced by CFA were determined using an electronic anesthesiometer and hot plate apparatus, respectively. Post-treatment with probucol at 3 mg/kg inhibited CFA-induced hyperalgesia over the course of 7 days as well as paw edema. Overt pain-like behaviors, which were determined by the number of flinches and time spent licking paw immediately following CFA injection, were also reduced by probucol at 3 mg/kg administered as a pre-treatment. To investigate the mechanisms underlying the analgesic effect of probucol, neutrophil recruitment to paw was assessed by myeloperoxidase activity, cytokine production, Cox-2 expression, and NF-κB activation in both paw and spinal cord by ELISA. Iba-1, GFAP, and substance P protein expression and nuclear localization of phosphorylated NF-κB were evaluated in the spinal cord by immunofluorescence. Probucol at 3 mg/kg attenuated neutrophil recruitment, cytokine levels, and NF-κB activation as well microglia and astrocyte activation, and substance P staining in the spinal cord. Taken together, the results suggest that probucol exerts its analgesic and anti-inflammatory activity in an experimental model of persistent inflammation by targeting the NF-κB pathway in peripheral and spinal cord foci.