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Bilirubin Oxidation End Products (Boxes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway

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Bilirubin Oxidation End Products (Boxes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway Hindawi Oxidative Medicine and Cellular Longevity Volume 2021, Article ID 8869908, 11 pages https://doi.org/10.1155/2021/8869908 Research Article Bilirubin Oxidation End Products (BOXes) Induce Neuronal Oxidative Stress Involving the Nrf2 Pathway Yinzhong Lu ,1,2 Wenyi Zhang,1 Bing Zhang,1 Stefan H. Heinemann,3 Toshinori Hoshi,4 Shangwei Hou ,1,2 and Guangming Zhang 1 1Department of Anesthesiology, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China 2Hongqiao International Institute of Medicine, Tongren Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200336, China 3Center for Molecular Biomedicine, Department of Biophysics, Friedrich Schiller University Jena & Jena University Hospital, Hans- Knöll-Str. 2, D-07745 Jena, Germany 4Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA Correspondence should be addressed to Shangwei Hou; [email protected] and Guangming Zhang; [email protected] Received 9 October 2020; Revised 4 June 2021; Accepted 22 June 2021; Published 31 July 2021 Academic Editor: Cinzia Signorini Copyright © 2021 Yinzhong Lu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Delayed ischemic neurological deficit (DIND) is a severe complication after subarachnoid hemorrhage (SAH). Previous studies have suggested that bilirubin oxidation end products (BOXes) are probably associated with the DIND after SAH, but there is a lack of direct evidence yet even on cellular levels. In the present study, we aim to explore the potential role of BOXes and the involved mechanisms in neuronal function. We synthesized high-purity (>97%) BOX A and BOX B isomers. The pharmacokinetics showed they are permeable to the blood-brain barrier. Exposure of a moderate concentration (10 or 30 μM) of BOX A or BOX B to isolated primary cortical neurons increased the production of reactive oxygen species. In the human neuroblastoma SH-SY5Y cells, BOX A and BOX B decreased the mitochondrial membrane potential and enhanced nuclear accumulation of the protein Nrf2 implicated in oxidative injury repair. In addition, both chemicals increased the mRNA and protein expression levels of multiple antioxidant response genes including Hmox1, Gsta3, Blvrb, Gclm, and Srxn1, indicating that the antioxidant response element (ARE) transcriptional cascade driven by Nrf2 is activated. In conclusion, we demonstrated that primary cortical neurons and neuroblastoma cells undergo an adaptive response against BOX A- and BOX B-mediated oxidative stress by activation of multiple antioxidant responses, in part through the Nrf2 pathway, which provides in-depth insights into the pathophysiological mechanism of DIND after SAH or other neurological dysfunctions related to cerebral hemorrhage. 1. Introduction initial impact of SAH and have had their aneurysm effectively treated [2]. However, the etiology and pathophysiology of Subarachnoid hemorrhage (SAH) is a serious cerebrovascu- SAH and DNID remain incompletely understood. lar complication with complex underlying mechanisms Accumulating evidence has shown that oxidative stress, inflicting brain perfusion and function. Despite great prog- acute and subsequent consisting immunological response, ress in the understanding of SAH pathophysiology and man- and other factors collectively cause the severe adverse effects agement of ruptured aneurysms, SAH remains a severe and of SAH [3, 4]. Erythrocyte lysis, a major initiator, is the significant health problem [1]. SAH causes early brain injury, release of heme and its subsequent degradation cascade, which may be followed after 7 to 14 days by delayed ischemic which collectively exert a strong effect on the affected neu- neurological deficit (DIND). DIND is the leading cause of rons and their electrical excitability [5–7]. Various heme morbidity and mortality in the patients who survive the and heme degradation products (HHDPs) were identified 2 Oxidative Medicine and Cellular Longevity in the cerebrospinal fluid (CSF) from the patients after SAH mm column with gradient elution using mobile phase A con- or other types of brain hemorrhage [8–11] and serum from taining 0.1% formic acid in water and mobile phase B (0.1% hepatic failure patients [12]. formic acid in acetonitrile) at a flow rate of 500 μL/min. Notably, an array of bilirubin oxidation end products The gradient run was as follows: 10% solvent B (0.01–0.30 (BOXes) with small molecular weight were identified [11, min), from 10% to 98% B (0.30–1.30 min), 98% B (1.30– 13, 14] and detected in the CSF of a patient with DIND after 1.80 min), from 98%B to 10% B (1.80–1.90 min), and 10%B SAH [8, 9, 15]. Several studies using crude extraction or (1.90–2.40 min). Detection was performed by MS/MS using synthesized BOXes have shown that BOXes constrict mouse an API4000 electrospray ionization (ESI) mass spectropho- cortical blood vessels and damage the normal contractile of tometer (Applied Biosystems, API 4000 triple quadrupole vascular smooth-muscle cells [16, 17], suggesting that BOXes mass spectrometer, AB SCIEX, Concord, Ontario, Canada). are involved in the delayed cerebral vasospasm, a potential The masses for tolbutamide were precursor ion m/z 271.2 cause of DIND. Our previous study showed that a crude and product ion m/z 155.1, for BOX A, precursor ion m/z BOXes mixture regulates the blood tone by modulation of 179.2 and product ion m/z 162.2, and for BOX B, precursor 2+ + Ca - and voltage-gated K channels (Slo BKCa) [18]. How- ion m/z 179.2 and product ion m/z 162.3. ever, the direct impact of BOXes on neuronal cells remains For pharmacological kinetics study, a single dose of 12 to be examined. mg/kg body weight BOX A or BOX B (powder dissolved in In this study, we hypothesized that BOX A or BOX B are 5% DMSO : 95% saline (pH = 11) [20, 21] with final concen- important functional regulators of neurons during the pro- tration 2.4 mg/mL) was injected in vein (i.v.) with 5 mL/kg gression of DIND after SAH. Therefore, we synthesized body weight in male ICR mice (n =3) obtained from Beijing BOX A and BOX B following the reported protocol [14, 19] Vital River Laboratory Animal Technology Co., Ltd. (Beijing, and examined their biological effects on cultured primary China). After 30 min, the plasma concentrations (ng/mL) cortical neurons and neuronal cell lines to elucidate the and the brain concentrations (ng/g) were measured. For underlying molecular mechanisms. preparations of mouse plasma and brain samples, 100 μL cal- ibration curve samples in single, QC samples in duplicate, 2. Materials and Methods and mouse plasma samples were mixed with 300 μL acetoni- trile containing Internal standard (50 ng/mL of propranolol, 2.1. Reagent and Antibodies. 2′,7′-Dichlorofluorescein diace- 200 ng/mL of tolbutamide, and 50 ng/mL of diclofenac) in tate (DCF-DA) and dimethyl sulfoxide (DMSO) were Eppendorf tubes. After the mixture was vortexed for 1 min, purchased from Sigma-Aldrich, the antibody raised against then centrifuged for 10 min, transfer 50 μL supernatant to a HO-1 (1 : 500), BLVRB (1 : 500), and Gclm (1 : 1000) from Pro- 96- well plate with 100 μL pure water, shaking for 10 min, teintech Group, Inc. (Chicago, IL, USA); Nrf2 (WB, 1 : 1000, IF, and finally inject 10 μL into LC-MS/MS system. For brain 1 : 200) and Gsta3 (1 : 500) from Abcam (Cambridge, MA, samples, brain samples were added with 5 folds (w/v) phos- USA); SRXN1 (1 : 500) from Bioss (Beijing, China); Caspase 3 phate buffer (100 mM, pH 7.4) in terms of the weight of (WB, 1 : 1000) from Abmart (Shanghai, China); and β-actin samples, then homogenated. Subsequent procedures were (WB, 1 : 2000) from OriGene Technologies, Inc. (Rockville, performed as described for plasma. The brain concentration MD, USA). Alexa Fluor 546 anti-mouse secondary Ab (IF:1 : to the plasma concentration was calculated as B/P value. The 2000, A-11030), goat anti-mouse IgG (H + L) peroxidase animal study including the use of embryos for primary neu- conjugated (WB: 1 : 20000, 31430), and goat anti-rabbit IgG ron cultures was carried out in accordance with regulations (H + L) peroxidase conjugated (WB: 1 : 20000, 31460) were pur- for animal experimentation and were approved by the chased from Thermo Fisher Scientific(Waltham,MA,USA). Animal Committee of Tongren Hospital affiliated to Shang- hai Jiao Tong University School of Medicine. 2.2. BOXes Synthesis and B/P Study. BOX A and BOX B were For other assays in this study, the ready-for-use BOX A synthesized as shown in Figure 1(a). Compound 1 (bromoci- or BOX B solution was prepared as 1,000 times in DMSO traconic anhydride) reacts with triphenylphosphoylide in as the final concentration in administered cells and stored toluene with reflux. Because the two carbonyl reaction sites at -20°C in the dark. Therefore, the final concentration of are different, two products can be obtained: 3 is the primary DMSO in all treatments was 0.1%. product, 2 is the by-product, and two of which can be sepa- rated by silica gel column chromatography. Compounds 2 2.3. Primary Cortical Neurons and Neuroblastoma Cell Lines. and 3 have the reactivity of palladium-catalyzed coupling Mouse primary cortical neurons (PCNs) were cultured as reaction and amine acetate substitution reaction (the reac- previously described [22] with minor modifications. Briefly, tion order is not required). Then, compounds 6 and 7 were E17.5 ~ E18.5 embryos of either sex were dissected from hydrolyzed into carboxylic acid intermediates by alkali pregnant C57BL/6J mice for cortex dissection in cold hydrolysis, and then the target products BOX A and BOX B Dulbecco’s Modified Eagle Medium (DMEM).
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