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Neurotoxicity, Mitochondria, Annexin A5, Apoptosis, NF-E2-related factor 2, What&,rsquo s Known L-glutamate induces neurodegeneration by increasing oxidative stress. Annexin A5 exhibits anti-apoptotic and membrane protective roles in eukaryotic cells. What&,rsquo s New Annexin A5 reduced L-glutamate-induced cell death. Annexin A5 blocked L-glutamate-induced mitochondrial dysfunction. Annexin A5 reversed L-glutamate-induced Bax expression. Annexin A5 increased nuclear factor-2 expression. IntroductionL-glutamate (L-Glu) serves as the primary excitatory amino acid in the human central nervous system, where it plays essential roles in diverse physiological functions by activating its metabotropic and ionotropic receptors. 1, However, elevated levels of L-Glu are neurotoxic, leading to cell death by damaging cellular components. This neurotoxicity is linked to neuronal loss in various neurological conditions, including cerebrovascular accident, physical injury, Alzheimer&,rsquo s disease (AD), Parkinsonian syndrome, and Huntington&,rsquo s disease, 2, making it crucial to find protective strategies to combat these injuries.The research has proposed several molecular mechanisms for L-Glu-induced neurotoxicity. Oxidative stress (OS), Ca2+ homeostasis dysregulation, mitochondrial damage, and apoptosis are amongst these mechanisms. 3, In vitro studies have shown that high L-Glu triggers OS in neurons by dysregulating cysteine glutamate antiporters and reducing cellular glutathione (GSH) levels, 4, resulting in the buildup of reactive oxygen species (ROS) and OS. OS induces Ca2+ ion influx, resulting in high cellular Ca2+ concentration, which activates apoptosis by activating caspases and inducing mitochondrial dysfunction. 5, Therefore, antioxidant agents such as vitamin E and plant-based antioxidants can mitigate L-Glu neurotoxicity. 6, Annexin A5 (ANXA5) is a calcium-binding protein that exhibits antioxidant and anti-apoptotic characteristics. 7, Recent studies have shown that this protein can prevent further cellular membrane damage by binding to damaged membrane phospholipids 8, and act as a neuroprotective agent. 9, Additionally, ANXA5 can prevent neuronal cell death by inhibiting inflammatory signaling pathways such as NF-&,kappa B and reducing ROS production. 10, However, the possible protective role of ANXA5 against glutamate-induced damage has not been investigated yet. The neuroblastoma-derived SH-SY5Y cell line is a well-known neuronal cellular model and serves as an appropriate tool for studying neurotoxicity. 11, , 12, This cellular model is frequently used to study L-Glu-induced neurotoxicity. 2, This study aims to evaluate the protective effects of a recombinant ANXA5 against L-Glu-induced toxicity in SH-SY5Y cells.Materials and MethodsThis study was conducted in the Diagnostic Laboratory Sciences and Technology Research Center of Shiraz University of Medical Sciences between April and December 2024. This research received approval from the Ethics Committee at Shiraz University of Medical Sciences (IR.SUMS.REC.1403.152). Expression and Purification of ANXA5 Protein A pET28a plasmid harboring a codon-optimized human ANXA5-coding sequence with a His-tag sequence at its N-terminal domain, supplied by ShineGene Bio-Technologies Company (Shanghai, China), was used to overexpress ANXA5 protein in competent BL21 ( DE3) cells (Invitrogen), as previously described. 9, The bacteria were transformed with the plasmid and cultured in LB broth (Difco, BD, UK) containing 70 &,mu g/mL kanamycin (Sigma-Aldrich, USA) at 37 &,#730 C overnight. The expression of ANXA5 was examined in the presence (induced condition) and absence (non-induced) of 0.1 mM isopropyl &,beta -D-1-thiogalactopyranoside (IPTG CINNAclone, IRAN) at 25 &,#730 C for 20 hours. This temperature was the optimum temperature for the expression of ANXA5, according to our previous study. 9, The cell pellet was harvested from the bacterial culture and lysed by ultrasonication in lysis buffer (300 mM NaCl, 50 mM NaH2PO4, pH 8.0, 2 M urea, 15 mM imidazole, 10% glycerol, 1% Triton, and 5% isopropanol). The supernatant was collected, and ANXA5 was purified using Ni-NTA chromatography (QIAGEN, Netherlands). The purified protein was filtered through a sterile syringe filter (0.22 &,mu m, Membrane Solutions, USA) to remove contaminants. The protein concentration was measured using the Bradford method with bovine serum albumin (Sigma-Aldrich, USA) as a standard protein, as previously described. 13, The purity of the recombinant protein was confirmed using SDS-PAGE. SH-SY5Y Cell Culture SH-SY5Y cells were purchased from the Pasteur Institute (Tehran, Iran) and cultured in Dulbecco&,rsquo s Modified Eagle Medium (DMEM) (Invitrogen Inc., USA) with high glucose concentration (4.5 g/L) containing 10% Fetal Bovine Serum (FBS, Gibco), 100 units/mL penicillin, and 100 &,mu g/mL streptomycin. Cultures were maintained in a humidified incubator at 37 &,deg C with 5% CO2. MTT Assay Cell viability was measured using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT Sigma-Aldrich, USA) assay. In brief, the SH-SY5Y cells (1&,times 104 cells) were cultured in each well of a 96-well plate. After 24 hours, cells were treated with L-Glu (0-300 mM) and ANXA5 (0-5 &,mu g/mL) for 24 hours. The cell viability was then evaluated using the MTT assay. Briefly, the cells were incubated with MTT solution (5 g/L) for 4 hours at 37 &,deg C. Dimethyl sulfoxide was added, and the absorbance was measured at 545 nm using a microplate reader. 14, The results were expressed as percentages relative to the control group, and calculations were performed using Prism 10.2.3 software. The results were expressed as a percentage of the control group. Mitochondrial Membrane Potential Assay Rhodamine 123 (Rh123) staining was used to determine mitochondrial membrane potential (MMP) dissipation. This method is based on the accumulation of Rh123 in the mitochondria with an intact membrane and high MMP. 15, , 16, In brief, SH-SY5Y cells (3&,times 105) were seeded in each well of 6-well plates. The cells were exposed to L-Glu (165 mM) alone or in combination with ANXA5 (2.5 &,micro g/mL) for 24 hours. After completing the treatment, the cells were washed with phosphate buffer saline (PBS). Then, Rh 123 solution (2 &,micro g/mL) was added to the cells in the dark. The cells were incubated for 30 min at 37 &,deg C and washed with PBS buffer. The stained cells were stimulated at a wavelength of 490 nm, and the resulting fluorescent light emitted at 520 nm was recorded in the FL-1 channel of a FACS Calibur (BD). 15, Real-time PCR Real-time PCR was used to investigate the effects of the treatments on the expression of Bax, Bcl-2, and Nrf-2 genes. The cells were treated with L-Glu (165 mM) or ANXA5 (2.5 and 5 &,mu g/mL) alone or in combination for 24 hours. RNA was extracted using TRIzol (CINNA clone, Iran). cDNA synthesis was performed using the Sinnaclon kit (Sinnagene, Iran). Real-time PCR was conducted using cDNA and specific primers (table 1,). The Rotor-Gene Q system (QIAGEN, USA) was used. PCR conditions included a denaturation step at 95 &,deg C for 30 sec, followed by 40 cycles of denaturation at 95 &,deg C for 30 sec, annealing at 58 &,deg C for 30 sec, and extension at 72 &,deg C for 30 secs. A final extension step was conducted at 72 &,ordm C for 30 sec. The TATA binding protein ( TBP) gene was employed as a reference housekeeping gene, 17, and fold change was calculated using the 2&,minus &,Delta &,Delta CT method.PrimersSequencesPrimersTBP (F)5&,prime -GTGCCCGAAACGCCGAAT-3&,prime TBP (F)TBP (R)5&,prime -GTCTGGACTGTTCTTCACTCTTGG-3&,prime TBP (R)Bax (F)5&,prime -CCGAGAGGTCTTTTTCCGAG-3&,prime Bax (F)Bax (R) 5&,prime -AAGTCCAATGTCCAGCCCA-3&,prime Bax (R) Bcl-2 (F)5&,prime -GCGACTCCTGATTCATTGGG-3&,prime Bcl-2 (F)Bcl-2 (R)5&,prime -CTACTTCCTCTGTGATGTTGTATT-3&,prime Bcl-2 (R)Nrf-2(F)5&,prime -AGATGACAATGAGGTTTCTTCGG-3&,prime Nrf-2(F)Nrf-2(R)5&,prime -AGTTTGGCTTCTGGACTTGG-3&,prime Nrf-2(R)TBP, TATA binding protein F, forward primer R, reverse primer Bax, BCL2 Associated X Bcl-2, B-cell leukemia/lymphoma 2 protein Nrf-2, NF-E2-Related Factor 2 |