Protective Effect of Apelin-13 on D-Glutamic Acid-Induced Excitotoxicity in SH-SY5Y Cell Line: An In-Vitro Study

Protective Effect of Apelin-13 on D-Glutamic Acid-Induced Excitotoxicity in SH-SY5Y Cell Line: An In-Vitro Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Effect of Apelin-13 on D-Glutamic Acid-Induced Excitotoxicity in SH-SY5Y Cell Line: An In-Vitro Study Kadriye Yağmur Oruç, Aykut Oruç, Gökhan Ağtürk, Karolin Yanar, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4736431/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Excitotoxicity, resulting from excessive accumulation of glutamate in the extracellular space, leads to neuronal cell death. This study investigates the protective effects of Apelin-13 on D-glutamic acid-induced excitotoxicity in SH-SY5Y human neuroblastoma cells, an in vitro model for neurodegenerative diseases. Unlike the commonly studied L-glutamic acid, this research focuses on D-glutamic acid to understand its specific impacts. SH-SY5Y cells were treated with varying concentrations of D-glutamic acid and Apelin-13, followed by analyses at 12 and 24 hours to evaluate cell viability, oxidative stress markers, and inflammatory cytokine levels. Cell viability assays revealed significant cytotoxic effects of D-glutamic acid at doses of 10 mM and 20 mM, reducing viability by over 50%. However, Apelin-13 treatment mitigated these effects, especially at 2 µg/mL, enhancing cell viability and reducing inflammatory cytokine levels (IL-1β and TNF-α). Apelin-13 also increased anti-inflammatory cytokine levels (IL-10 and TGF-β1) and brain-derived neurotrophic factor (BDNF), indicating its neuroprotective role. Oxidative stress markers, including ROS, AGE, AOPP, DT, and T-SH, were significantly elevated by D-glutamic acid but effectively reduced by Apelin-13. The neuroprotective mechanisms of Apelin-13 involve modulation of cAMP/PKA and MAPK signaling pathways, enhancing BDNF synthesis and suppressing oxidative stress and inflammatory responses. This study is the first to demonstrate the effects of D-glutamic acid on SH-SY5Y cells. It highlights Apelin-13’s potential as a therapeutic agent against excitotoxicity-induced neuronal damage, emphasizing its ability to modulate key molecular pathways involved in inflammation and oxidative stress. Further in vivo studies are warranted to explore the long-term neuroprotective effects of Apelin-13 in treating neurodegenerative diseases. apelin-13 excitotoxicity D-glutamic acid neuroinflammation SH-SY5Y cell line Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Excitotoxicity is defined by the excessive buildup of glutamate or other excitatory amino acids in the extracellular space, leading to the heightened activation of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) subtype, in the adult central nervous system (CNS) [ 1 , 2 ]. This excessive activation causes a sustained influx of calcium (Ca 2+ ) through receptor channels, leading to neuronal cell death. Elevated levels of glutamate in the CNS increase intracellular Ca 2+ concentrations, triggering apoptotic stimuli within sensitive organelles like mitochondria and the endoplasmic reticulum (ER). This results in the production of toxic radicals, disruption of cellular energy production, and ultimately, cell death through acute necrosis and/or delayed apoptosis [ 3 ]. Additionally, glutamate exposure can activate extrasynaptic NMDA Receptors (NMDARs), halt cAMP response element-binding protein (CREB) activity, cause mitochondrial membrane potential loss, and lead to cell death [ 4 ]. Apelin, an endogenous neuropeptide widely distributed throughout various physiological systems, particularly prevalent within the nervous system, undergoes intricate post-translational modifications, resulting in the generation of diverse mature apelin active peptides, with apelin-13 exhibiting the most robust biological activity [ 5 ]. Apelin is a natural ligand for the G protein-coupled apelin receptor (APJ) [ 5 ]. The apelin/APJ system has been shown to possess neuroprotective properties, including anti-inflammatory, anti-oxidative stress, anti-apoptotic effects, autophagy regulation, and inhibition of excitotoxicity, suggesting its potential as a therapeutic target for neurological disorders [ 6 , 7 , 8 ]. Glutamate excitotoxicity, neuroinflammation, and oxidative stress collectively manifest as a pathogenic “triad” in various brain disorders, leading to cellular demise and perturbations in network dynamics [ 9 ]. Neuroinflammation is characterized by the activation of neuronal cells, leading to dysregulation of anti-inflammatory/proinflammatory cytokine ratios due to the excessive secretion of proinflammatory cytokines. These cytokines influence glutamate homeostasis by upregulating glutamate receptors, enhancing glutamatergic neurotransmission, and exacerbating excitotoxicity [ 10 ]. Oxidative stress, resulting from an imbalance in the redox state of cells due to excessive production of reactive oxygen species (ROS), can lead to oxidative damage to cellular macromolecules and contribute to various neurodegenerative diseases [ 11 ]. Imbalances in the oxidant/antioxidant relationship and excessive ROS production increase the levels of molecules such as advanced glycation end products (AGE), advanced oxidation protein products (AOPP), and kynurenine (KYN), dityrosine (DT), inducing DNA methylation and creating toxic effects on neurons [ 12 , 13 ]. High ROS levels activate inflammatory pathways such as nuclear factor-kappa beta (NF-κB) and mitogen-activated protein kinase (MAPK), causing cellular damage and decreasing Brain-Derived Neurotrophic Factor (BDNF) expression in neurons [ 14 ]. Apelin-13 increases intracellular cyclic Adenosin Monophosphate (cAMP) levels, activating the protein kinase A (PKA) pathway, which suppresses inflammatory signaling pathways and increases BDNF synthesis [ 15 – 17 ]. Additionally, Apelin-13 binds to the APJ receptor, initiating MAPK activation through G-protein coupled receptor (GPCR) signaling pathways, triggering phospholipase C (PLC) activation [ 18 ]. PLC stimulates the production of inositol triphosphate (IP3) and diacylglycerol (DAG), leading to intracellular Ca + 2 release and protein kinase C (PKC) activation. These signals activate the Extracellular signal-Regulated Kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPK pathways, regulating BDNF gene expression [ 15 – 17 ]. Total thiol levels reduce oxidative stress by enhancing cellular antioxidant defense capacity [ 19 ]. However, the increase in ROS levels due to mitochondrial dysfunction and the direct interaction of ROS with thiol groups (-SH) leads to the oxidation of free thiol groups [ 20 , 21 ]. The increase in disulfide bonds (R-S-S-R) in thiol groups causes protein structural degradation and amino acid side chain oxidation, leading to carbonylation, decreased -SH reactivity, and peroxidation [ 21 ]. As a result of this process, total thiol levels have been reported to decrease in neurodegenerative diseases [ 20 , 21 ]. Interleukin-1β (IL-1β) and Tumor necrosis Factor-α (TNF-α) have been shown to play a role in glutamate-mediated excitotoxicity in several studies [ 22 , 23 ]. These cytokines are secreted by Human Neuroblastoma cell line (SH-SY5Y cell line) in response to NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-induced excitotoxicity, triggering inflammatory responses that lead to cellular damage, neurodegeneration, apoptosis, and further inflammation [ 24 , 25 ]. Apelin-13, known for its anti-inflammatory effects on neurons, can reduce inflammation and neuronal damage by suppressing the production of IL-1β and TNF-α [ 18 , 26 ]. Apelin-13 binds to the APJ receptor, enhancing adenylate cyclase activity and cAMP production, which leads to PKA activation and suppression of inflammatory pathways such as NF-κB [ 27 ]. Anti-inflammatory cytokines play a crucial role in regulating inflammatory responses and protecting cells from the damaging effects of inflammation. Transforming growth factor β (TGF-β) is a prominent anti-inflammatory cytokine, existing in three isoforms: TGF-β1, TGF-β2, and TGF-β3 [ 28 ]. Among these, TGF-β1 is the most abundant subtype in mammals, with significant anti-inflammatory and neuroprotective effects [ 29 ]. TGF-β1 has been shown to stimulate the regrowth of damaged neurons after axonal injury [ 30 ]. Although its role in neural plasticity and memory in humans is not well understood, TGF-β1’s contribution to learning and memory mechanisms has been demonstrated in rodents [ 31 – 33 ]. Evidence suggests that TGF-β1 may have a protective role in neurodegenerative and neuroinflammatory diseases such as Alzheimer’s and multiple sclerosis [ 34 , 35 ]. Interleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), serves as an immunomodulator in the anti-inflammatory process [ 36 ]. IL-10 possesses numerous immunoregulatory effects crucial for resolving inflammation and can inhibit the production of various inflammatory cytokines such as TNF-α, IL-1β, Interleukin-6 (IL-6), and Interferon-gamma (IFN-γ) [ 37 ]. It is produced by nearly all leukocytes, including T cell subsets, monocytes, macrophages, neutrophils, eosinophils, mast cells, dendritic cells (DCs), B cells, and natural killer (NK) cells, as well as by keratinocytes, epithelial cells, and Nestin + neuroblasts [ 36 , 38 ]. In the central nervous system, IL-10 receptors (IL-10R) are expressed by microglia, astrocytes, oligodendrocytes, and even neurons under both physiological and pathological conditions, initiating the Janus kinase (Jak)/tyrosine kinase (Tyk) signaling pathway in neurons [ 39 – 41 ]. BDNF is a neurotrophic factor critical for maintaining neuronal health and enhancing synaptic plasticity in the central nervous system. BDNF supports the growth, differentiation, and survival of neurons while also contributing to the strengthening of synaptic connections [ 42 , 43 ]. Environmental factors such as stress and toxicity can affect BDNF levels; some studies report that BDNF levels increase with stress, while others indicate a decrease [ 44 , 45 ]. A reduction in BDNF levels can negatively impact neuronal health and function, and is associated with neurodegenerative diseases [ 46 ]. High doses of glutamate increase intracellular Ca 2+ levels through the overactivation of NMDA and AMPA receptors, leading to mitochondrial dysfunction and increased ROS production. This process exacerbates neuronal stress and toxicity, suppressing BDNF production and negatively affecting neuronal health [ 47 , 48 ]. This study uses D-glutamic acid instead of the more commonly studied L-glutamic acid. D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are stereospecific enzymes that metabolize D-amino acids (D-AAs). DDO specifically acts on acidic D-AAs like D-aspartate, D-glutamate, and NMDA, converting them into imino acids while reducing flavin adenine dinucleotide (FAD) [ 49 ]. Subsequently, FAD is reoxidized in the presence of oxygen, producing hydrogen peroxide (H 2 O 2 ). High levels of H 2 O 2 cause oxidative stress and trigger events such as mitochondrial dysfunction, pro-inflammatory cytokine activation, and apoptotic and autophagic cell death [ 50 ]. Additionally, the accumulation of D-AAs has been associated with immune activation, and cellular exposure to D-AAs is suggested to induce inflammation and cell death through H 2 O 2 and NF-κB activation [ 51 ]. Aim The aim of this in vitro study is to evaluate the protective effect of apelin-13 in a glutamic acid-induced excitotoxicity model in SH-SY5Y cells and to determine the underlying protective mechanisms. Specifically, the study aims to assess the changes in levels of BDNF, IL-10, IL-1β, TNF-α, TGF-β1, and ROS as well as KYN, DT, AGEs, AOPP, and T-SH following apelin-13 treatment. The findings of this study may shed light on the neuroprotective effects of apelin-13, elucidate its mechanisms of action, and identify potential novel targets for the treatment of neurodegenerative diseases. 2. Materials and Methods 2.1. Cell Culture The study was conducted in the research laboratories of Istanbul University Cerrahpaşa Faculty of Medicine and Haliç University Faculty of Medicine. SH-SY5Y cells used in our study were obtained from the American Type Culture Collection (SH-SY5Y (ATCC CRL-2266) ATCC; Rockville, USA) as the 5th and 7th passages. All cell culture procedures were performed in a sterile laminar flow hood. After thawing the cells stored in cryotubes in a liquid nitrogen tank at -196°C, they were transferred to 25 cm 2 flasks. The cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% antibiotics (Penicillin/Streptomycin, Gibco, Thermo Fisher Scientific). The cells were incubated at 37°C with 5% CO 2 . Following the incubation period, SH-SY5Y cells were checked under a ZEISS Primo Vert (Inverted Microscope Carl Zeiss Microscopy GmbH 07745 Jena, Germany) microscope, and when the cell density reached approximately 85–90%, trypsinization was performed using Trypsin-EDTA (Gibco, Thermo Fisher Scientific). 2.2. Cell Treatments with D-Glutamic Acid and Apelin-13, Cell Viability Test The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a common method used to measure cellular metabolic activity and thus cell viability. This test is based on the ability of mitochondrial dehydrogenases in living cells to convert MTT into formazan crystals [ 52 ]. 1 mg of D-Glutamic acid (Sigma, CAS:6893-26-1, Saint Louis, USA) was dissolved in 1 mL of dimethyl-sulphoxide (DMSO) at room temperature (25°C) and adjusted to pH 7.5. Four experimental groups were created: Control, D-Glutamic acid, Apelin-13, and D-Glutamic acid + Apelin-13. Cells in the control group received no treatment. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, Invitrogen) assay was performed on SH-SY5Y cells at 12th and 24th hours to determine cytotoxicity values in groups treated with D-Glutamic acid, Apelin-13 (Cayman Chemicals, Item 13523, Michigan, USA), and D-Glutamic acid + Apelin-13. Cells were seeded in 96-well plates at a density of 1x105 cells for MTT assay. Samples were read at 540, 570, and 590 nanometer (nm) wavelengths using a spectrophotometer for MTT analysis. D-Glutamic acid concentrations of 10 millimolar (mM) and 20 mM, resulting in 80% cell viability, were selected for treatments [ 53 , 54 ]. Apelin-13 concentrations of 1, 2, and 5 micrograms per milliliter (µg/ml), resulting in 80% cell viability, were selected for treatments [ 55 , 56 ]. In the D-Glutamic acid + Apelin-13 group, cells were first treated with D-Glutamic acid at concentrations of 10 mM and 20 mM, followed by treatment with Apelin-13 at concentrations of 1, 2, and 5 µg/ml for 10 mM D-Glutamic acid, and 1, 2, and 5 µg/ml for 20 mM D-Glutamic acid. Subsequently, all samples were collected to analyze their status at 12th and 24th hours. 2.3. Biochemical Antioxidant Status Analyses Cells collected for biochemical analysis were lysed using the Radio-Immunoprecipitation Assay (RIPA) buffer protocol as described in the literature [ 57 ]. 2.3.1. The levels of Dityrosine, Kynurenine, and Advanced Glycation End Products: The levels of DT, KYN, and AGEs were measured spectrofluorometrically described by Sadowska and Bartosz et al. [ 58 ]. The concentrations were expressed as fluorescence units (FU) per milligram of protein. 2.3.2. Advanced Oxidation Protein Products: The spectrophotometric method developed by Hanasand et al. was used to evaluate AOPP levels [ 59 ]. The levels of AOPP were expressed as micromoles per liter of Chloramine-T equivalents. 2.3.3. Total-Thiol: The T-SH levels in cell lysates were measured using 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) following the method reported by Sedlak and Lindsay [ 60 ]. The concentrations were expressed as nmol/mg protein. 2.3.4. Total protein content: Total protein content was measured by Bicinchoninic acid (BCA) assay described by Smith et al. [ 61 ]. 2.4. Enzyme-Linked Immunosorbent Assay (ELISA) analyses: The levels of BDNF, IL-10, IL-1β, TNF- α, TGF-β1, and ROS in the supernatants of SH-SY5Y cells subjected to various treatments were quantified using respective ELISA kits (Brain-Derived Neurotrophic Factor BDNF Cat. No. E1302Hu, interleukin-10 IL-10 Cat. No. E0102Hu, interleukin-1β IL-1β Cat. No. E0143Hu, tumor necrosis factor-alpha TNF-α Cat. No. E0082Hu, transforming growth factor-beta1 TGF-β1 Cat. No. E0134Hu; BT LAB, Shanghai, China. Human reactive oxygen species ROS Cat. No. EK710415; AFG Bioscience, Northbrook, USA), in accordance with the manufacturer's instructions. 2.5. Imaging Culture plates were carefully placed on a ZEISS Primo Vert (Inverted Microscope Carl Zeiss Microscopy GmbH 07745 Jena, Germany) microscope stage. The area where the cells were located was focused with a 40x lens. Photographs of cells were taken using imaging software (Borox, SCMOS, China). 2.6. Statistical Analyses Statistical analyses were conducted using GraphPad Prism Version 10.2.0 (GraphPad Software, Inc., San Diego, CA, USA). The Shapiro-Wilk Test was used to assess the normality of data distribution, and the Levene Test was used to assess homogeneity. Data were found to be normally distributed and homogenous. Therefore, group comparisons were made using One-way ANOVA test followed by post hoc Tukey test. Results are presented as mean ± standard deviation for ELISA and manual biochemical analyses, and as minimum, maximum, median, and percentage for MTT test. Values with p < 0.05 were considered statistically significant. 3. Results 3.1. MTT Tests D-Glutamic acid treated groups showed a significant decrease (p < 0.001) in comparison with the D-CTL-12h group at 12 hours in the D-40mM-12h, D-60mM-12h, and D-80mM-12h groups, while no statistically significant difference was observed in the other groups. In comparison with the D-CTL-24h group at 24 hours, a significant decrease (p < 0.001) was observed in the D-40mM-24h, D-60mM-24h, and D-80mM-24h groups, and a slight significant decrease (p < 0.05) was observed in the D-20mM-24h group, while no statistically significant difference was observed among the other groups. For the Apelin-13 treated groups, a significant decrease (p < 0.001) was observed in the A-10µg/mL-12h group compared to the A-CTL-12h group at 12 hours, while no statistically significant difference was observed in the other groups. At 24 hours, a slight significant decrease (p < 0.05) was observed in the A-2µg/mL-24h group, a significant decrease (p < 0.01) was observed in the A-5µg/mL-24h group, and a significant decrease (p < 0.001) was observed in the A-10µg/mL-24h group compared to the A-CTL-24h group. In the D-Glutamic acid + Apelin-13 treated groups, significant differences were observed in the D-20mM + A2µg/mL-12h, D-20mM + A5µg/mL-12h, D-40mM + A1µg/mL-12h, D-40mM + A2µg/mL-12h, and D-40mM + A5µg/mL-12h groups compared to the CTL group at 12 hours (p < 0.01, p < 0.001, p < 0.01, p < 0.05, p < 0.01, respectively), while no statistically significant difference was observed in the other groups. At 24 hours, significant statistical differences were observed in the D-40mM + A2µg/mL-24h and D-40mM + A5µg/mL-24h groups compared to the CTL group (p < 0.01, p < 0.05, respectively), while no statistically significant difference was observed among the other groups. Relevant graphics and images are in Figure.1 and Figure.2. 3.2. ELISA Tests 3.2.1. ROS: At 12 hours, a significant increase (p < 0.001) in ROS levels was observed in the D-10mM group compared to the CTL group and between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 12 hours, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups compared to the CTL group (p < 0.001, p < 0.01, p < 0.001, p < 0.001, p < 0.001, p < 0.001, respectively). Additionally, a significant decrease (p < 0.001) was observed in the D-10mM + A2µg/ml group compared to the D-10mM group, while a significant decrease (p 0.001) was observed in the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups. At 24 hours, a significant increase (p < 0.001) in ROS levels was observed in the D-10mM group compared to the CTL group and between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 24 hours, significant increases were observed in the D-10mM + A1µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups compared to the CTL group (p < 0.001), and a slight significant increase (p < 0.05) was observed in the D-10mM + A5µg/ml group. Additionally, a significant decrease (p < 0.001) was observed in the D-10mM + A2µg/ml group compared to the D-10mM group, and a significant decrease (p 0.001). Relevant graphics are in Figure.3. 3.2.2. IL-1β At 12 hours, a significant increase (p < 0.01) was observed in IL-1β levels when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 12 hours, no statistically significant difference was found when compared to the CTL group. However, a significant decrease (p < 0.01) was observed in the D-10mM + A2µg/ml group compared to the D-10mM group, and a significant decrease was observed when comparing the D-20mM group to the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups (p < 0.001, p < 0.01, p < 0.05, respectively). At 24 hours, a statistically significant increase (p < 0.001) in IL-1β levels was observed when comparing the CTL group with the D-10mM group and between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 24 hours, statistically significant increases (p < 0.001) were found in the D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups compared to the CTL group. Significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, and D-10mM + A5µg/ml groups compared to the D-10mM group (p < 0.001, p < 0.001, p < 0.01, respectively). When comparing the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups with the D-20mM group, significant decreases were observed (p < 0.001, p < 0.01, p < 0.001, respectively). Relevant graphics are in Figure.3. 3.2.3. TNF-α At 12 hours, a significant increase (p < 0.01) was observed when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 12 hours, statistically significant increases were observed in the D-20mM + A1µg/ml and D-20mM + A5µg/ml groups compared to the CTL group (p < 0.001, p < 0.001), while no statistically significant difference was found in the other groups. However, no statistically significant difference was found when comparing the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups to the D-10mM group. When compared to the D-20mM group, a statistically significant decrease was observed only in the D-20mM + A1µg/ml group (p < 0.001). At 24 hours, a significant increase (p < 0.001) was observed when comparing the CTL group with the D-10mM group, and a slight significant increase (p < 0.01) was observed when comparing the D-10mM group with the D-20mM group. For the D-Glutamic acid + Apelin-13 treated groups at 24 hours, a significant increase (p < 0.01) was observed in the D-10mM + A1µg/ml group and a slight increase (p < 0.05) was observed in the D-10mM + A2µg/ml group compared to the CTL group. In all other groups, a statistically significant increase was observed (p < 0.001). When comparing the D-10mM + A1µg/ml, D-10mM + A2µg/ml, and D-10mM + A5µg/ml groups to the D-10mM group, a significant decrease was observed in all groups (p < 0.001). When comparing the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups to the D-20mM group, a significant decrease was observed in all groups (p < 0.001). Relevant graphics are in Figure.3. 3.2.4. IL-10 At 12 hours, a significant decrease (p < 0.001) was observed when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 12 hours, no statistically significant difference was found when compared to the CTL group. However, when compared to the D-10mM group, a significant increase (p < 0.001) was observed in all groups, and when comparing the D-20mM group with the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups, a significant increase (p < 0.001) was observed in all groups. At 24 hours, a significant decrease (p < 0.001) was observed when comparing the CTL group with the D-10mM group and between the D-10mM and D-20mM groups. For the D-Glutamic acid + Apelin-13 treated groups at 24 hours, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, and D-10mM + A5µg/ml groups compared to the CTL group (p < 0.01, p < 0.001, p < 0.01), while no significant difference was observed in the other groups. When comparing the D-10mM + A1µg/ml, D-10mM + A2µg/ml, and D-10mM + A5µg/ml groups to the D-10mM group, a significant increase was observed in all groups (p < 0.001). When comparing the D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups to the D-20mM group, a significant increase was observed in all groups (p < 0.001). Relevant graphics are in Figure.4. 3.2.5. TGF-β1 At 12 hours, there was a significant decrease (p < 0.001) compared to the CTL group when compared to the D-10 mM group, and there was no statistically significant difference in TGF-β1 levels between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid + Apelin-13, only the D-10mM + A2µg/ml group showed a statistically significant slight increase (p < 0.05) compared to the CTL group. However, when compared to the D-10 mM group, all groups showed a statistically significant increase (p < 0.001). When comparing the D-20 mM group with its respective D-Glutamic acid + Apelin-13 treated groups (D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml), all groups showed a statistically significant increase (p < 0.001). At 24 hours, there was a significant decrease (p < 0.001) compared to the CTL group when compared to the D-10 mM group, and there was no statistically significant difference in TGF-β1 levels between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid + Apelin-13, only the D-10mM + A2µg/ml group showed a statistically significant slight increase (p < 0.05) compared to the CTL group. When compared to the D-10 mM group, all groups showed a statistically significant increase (p < 0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid + Apelin-13 treated groups (D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml), all groups showed a statistically significant increase (p < 0.001). Relevant graphics are in Figure.4. 3.2.6. BDNF At 12 hours, there was a significant decrease (p < 0.001) in BDNF levels compared to both the D-CTL and D-10 mM groups, and no statistically significant difference was observed between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid + Apelin-13, there was a significant increase (p < 0.001) in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, and D-10mM + A5µg/ml groups compared to the CTL group. Additionally, there was a statistically significant but less pronounced increase in the D-20mM + A2µg/ml and D-20mM + A5µg/ml groups (p < 0.05). Furthermore, when compared to the D-10 mM group, all groups showed a statistically significant increase (p < 0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid + Apelin-13 treated groups (D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml), all groups showed a statistically significant increase (p < 0.001). At 24 hours, there was a significant decrease (p < 0.001) compared to the CTL group and no statistically significant difference between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid + Apelin-13, there was a statistically significant increase (p < 0.001) in all groups compared to the CTL group. When compared to the D-10 mM group, all groups showed a statistically significant increase (p < 0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid + Apelin-13 treated groups (D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml), all groups showed a statistically significant increase (p < 0.001). Relevant graphics are in Figure.4. 3.3. Oxidative Stress Markers 3.3.1. AGE At the 12th hour, a significant increase in AGE levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. It was noted that the increase in AGE levels could accompany D-glutamic acid-mediated excitotoxicity, and this increase could be concentration-dependent on D-glutamic acid. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, and D-20mM + A1µg/ml groups (p < 0.001, p < 0.001, and p < 0.001, respectively), while no statistically significant difference was observed in other groups. However, significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p > 0.001), and in the D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml groups when compared to the D-20 mM group (p > 0.001). This suggests that Apelin-13 may have a protective effect against the increase in AGE levels in 10mM and 20 mM D-glutamic acid-mediated excitotoxicity. At the 24th hour, a significant increase in AGE levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. It was observed that AGE levels increased both depending on D-glutamic acid concentration and time in excitotoxicity. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, and D-20mM + A5µg/ml groups (p < 0.001), a slightly significant increase in the D-20mM + A2µg/ml group (p < 0.01), and a significant increase in the D-20mM + A2µg/ml group (p 0.001), and when compared to the D-20 mM group, significant decreases were observed in all groups (p > 0.001). Relevant graphics are in Figure.5. 3.3.2. AOPP At the 12th hour, a significant increase in AOPP levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, and D-20mM + A1µg/ml groups (p < 0.001), while statistically significant differences were not observed in other groups. However, significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p > 0.001), and in the D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml groups when compared to the D-20 mM group (p > 0.001). At the 24th hour, a significant statistical increase (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group, and a slightly significant increase (p < 0.05) was observed when comparing the D-10 mM group with the D-20mM group in AOPP levels. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, and D-20mM + A5µg/ml groups (p < 0.001), a slightly significant increase in the D-20mM + A2µg/ml group (p < 0.01), and a slightly significant increase in the D-20mM + A5-µg/ml group (p 0.001), and in all groups when compared to the D-20 mM group (p > 0.001). Relevant graphics are in Figure.5. 3.3.3. DT At the 12th hour, a significant increase in DT levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, and D-20mM + A5µg/ml groups (p < 0.001 for all), while no statistically significant difference was observed in other groups. However, significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p > 0.001), and in the D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml groups when compared to the D-20 mM group (p > 0.001). At the 24th hour, a significant statistical increase (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group, while no significant difference was found when comparing the D-10 mM group with the D-20mM group in DT levels. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM + A1µg/ml, D-20mM + A1µg/ml, and D-20mM + A5µg/ml groups (p < 0.001 for all), a slightly significant increase in the D-10mM + A5µg/ml group (p < 0.01), and significant differences were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p < 0.001, p < 0.001, p 0.001). Relevant graphics are in Figure.5. 3.3.4. KYN At the 12th hour, a significant increase in KYN levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups (p < 0.001 for all), while no statistically significant difference was observed in the D-10mM + A2µg/ml group. However, significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p > 0.001), and in the D-20mM + A1µg/ml, D-20mM + A2µg/ml, D-20mM + A5µg/ml groups when compared to the D-20 mM group (p > 0.001). At the 24th hour, a significant increase in KYN levels (p < 0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant differences were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups (p < 0.001, p < 0.001, p < 0.001, p < 0.01, p < 0.001 respectively). Significant decreases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml groups when compared to the D-10 mM group (p < 0.001, p < 0.001, p 0.001). Relevant graphics are in Figure.5. 3.3.5. T-SH At the 12th hour, there was a significant increase in T-SH antioxidant levels (p < 0.001) when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups (p < 0.001 for all), while no statistically significant difference was observed in the D-10mM + A2µg/ml group. However, when compared to the D-10 mM group, there was a slightly significant decrease in the D-10mM + A1µg/ml group (p < 0.05), and a highly significant decrease in the D-10mM + A2µg/ml group (p 0.001). At the 24th hour, there was a statistically significant increase in T-SH levels (p < 0.001) when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid + Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM + A1µg/ml, D-10mM + A2µg/ml, D-10mM + A5µg/ml, D-20mM + A1µg/ml, D-20mM + A2µg/ml, and D-20mM + A5µg/ml groups (p < 0.001 for all). When compared to the D-10 mM group, all groups showed a statistically significant decrease (p 0.001). Relevant graphics are in Figure.5. 4. Discussion In this study, the protective effect of Apelin-13 against D-glutamic acid-induced excitotoxicity in the SH-SY5Y cell line was investigated. The obtained results support that Apelin-13 has a protective effect against excitotoxicity; however, this protective effect is dose- and time-dependent. 4.1. MTT Tests According to the MTT test results, D-glutamic acid was observed to have a significant cytotoxic effect on SH-SY5Y cells. D-glutamic acid, applied at doses ranging from 10 mM to 80 mM, was examined for its effect on cell viability at both 12-hour and 24-hour durations. At the 12-hour treatment duration, a low dose (10 mM) of D-glutamic acid caused a significant decrease in cell viability (p < 0.01), while higher doses (20 mM, 40 mM, 60 mM, and 80 mM) resulted in highly significant reductions (p < 0.001). Similarly, Zhang and Xu [ 62 ] reported that glutamate induces cell death through oxidative stress and inflammation by increasing intracellular calcium entry via NMDA receptors. At the 24-hour treatment duration, the cytotoxic effects of D-glutamic acid became more pronounced. Similarly, Lewerenz et al. noted that the long-term effects of glutamate lead to mitochondrial dysfunction and oxidative damage in neuronal cells, highlighting the significant role of chronic excitotoxicity in neurodegenerative diseases [ 8 ]. Based on the MTT test results, 10 mM and 20 mM doses of D-glutamic acid were chosen to induce acute and chronic excitotoxicity in this study, as these doses caused significant cytotoxic effects in SH-SY5Y cells without exceeding 50% cell loss. Previous studies have reported toxic effects of L-glutamic acid in SH-SY5Y cells at doses ranging from 8 mM to 80 mM, with Palanivel V et al. identifying 40 mM as the ideal dose [ 53 , 63 , 64 ]. In another study conducted on rats, we demonstrated D-glutamic acid-mediated excitotoxicity and suggested it occurs through increased active GSK-3β levels and oxidative stress [ 65 ]. However, no study has been conducted on SH-SY5Y cells. Therefore, this study is the first to demonstrate that D-glutamic acid induces excitotoxicity in SH-SY5Y cells and shows effects at lower doses compared to L-glutamic acid. Apelin-13 was applied at various doses (1 µg/mL, 2 µg/mL, 5 µg/mL, and 10 µg/mL), and its effects on cell viability were evaluated over both 12-hour and 24-hour durations. During the 12-hour treatment duration, a high dose (10 µg/mL) of Apelin-13 resulted in a highly significant decrease in cell viability (p < 0.001), with no significant difference observed at other doses. Furthermore, during the 24-hour treatment duration, the toxic effects of high-dose Apelin-13 became more pronounced. In the A-5µg/mL-24h group, a slight decrease (p < 0.05) was observed, while a highly significant decrease (p < 0.001) was observed in the A-10µg/mL-24h group. This suggests that Apelin-13 may exhibit toxic effects at high doses over short durations. Other studies have highlighted that high doses of Apelin may cause toxic effects and that high serum Apelin levels indicate poor prognosis in glioblastoma multiforme [ 66 ]. The doses of 2 µg/mL and 5 µg/mL of Apelin-13 were found to have minimal toxic effects on cell viability, indicating these doses are ideal for supporting cellular viability. The combination of D-glutamic acid and Apelin-13 was effective in preserving cell viability. During the 12-hour treatment duration, combinations of D-glutamic acid (10 mM and 20 mM) and Apelin-13 (1 µg/mL, 2 µg/mL, and 5 µg/mL) provided significant protection for cell viability. Studies have shown that Apelin reduces ER stress-mediated oxidative stress and neuroinflammation via AMPK inhibition, and inhibits cleaved caspase-1, IL-1β and TNF-α, MPO, and ROS production, and programmed cell death in neurons [ 18 , 26 ]. Additionally, other studies report that Apelin-13 increases BDNF levels, reduces neuronal damage through AKT and ERK1/2 pathway activation, and exhibits neuroprotective effects via increased BDNF expression through cAMP/PKA pathways [15 However, at the 24-hour treatment duration, certain doses of Apelin-13 did not mitigate the toxic effects of D-glutamic acid as expected. Specifically, significant decreases in cell viability (p < 0.01) were observed in the D-20mM + A1µg/mL-24h and D-20mM + A5µg/mL-24h groups. This indicates that the most effective protective dose of Apelin-13 against acute and chronic toxicity induced by 10 mM and 20 mM D-glutamic acid is 2 µg/mL. 4.2. Proinflammatory Cytokines D-glutamic acid application at both 10 mM and 20 mM doses caused a significant increase in IL-1β levels at the 12th hour (p < 0.01). Similarly, Nopparat C et al. reported that glutamate triggers IL-1β production by increasing intracellular calcium loading through NMDA receptors [ 67 ]. In the groups treated with Apelin-13, especially at the 2 µg/mL dose, a significant reduction in IL-1β levels was observed (p < 0.01). Luo H et al. similarly demonstrated that Apelin-13 provides anti-inflammatory effects in neuronal cells by suppressing the production of inflammatory cytokines [ 15 ]. The combination of Apelin-13 with 20 mM D-glutamic acid also significantly reduced IL-1β levels (p < 0.001, p < 0.01, p < 0.05). These findings indicate that Apelin-13 modulates IL-1β production, particularly at the 2 µg/mL dose, controlling inflammatory responses. At the 24-hour duration of D-glutamic acid administration, more pronounced increases in IL-1β levels were observed (p < 0.001). Leverenz J et al. similarly reported that prolonged exposure to glutamate could increase IL-1β levels, exacerbating cell damage [ 8 ]. In the groups combined with Apelin-13, significant reductions in IL-1β levels were observed at the 24-hour treatment duration. The 1 µg/mL and 2 µg/mL doses of Apelin-13 significantly reduced IL-1β levels (p < 0.001). TNF-α levels also significantly increased following D-glutamic acid administration. At the 12th hour, the D-10 mM group showed a significant increase in TNF-α levels (p < 0.01), with no significant difference observed between the D-10 mM and D-20 mM groups. TNF-α is a cytokine that promotes inflammatory responses and cell death, and it has been reported that glutamate enhances this process through NMDA receptors [ 68 ]. Additionally, Jara et al. indicated that TNF-α and glutamate work synergistically on NMDA receptor activation, with TNF-α enhancing calcium influx dependent on NMDA receptors, facilitating glutamate-mediated neurotoxicity through ERK activation [ 69 ]. In the groups treated with Apelin-13, significant reductions in TNF-α levels were observed. Combinations of 20 mM D-glutamic acid and Apelin-13 (1 µg/mL and 5 µg/mL) resulted in significant decreases in TNF-α levels (p < 0.001). The 2 µg/mL dose of Apelin-13 effectively reduced D-glutamic acid-induced inflammation. During the 24-hour treatment duration, more pronounced increases in TNF-α levels were observed. Significant increases in TNF-α levels were noted between the D-10 mM and D-20 mM groups (p < 0.001). Literature indicates that prolonged application of glutamate can enhance inflammatory responses, leading to cell damage [ 45 ]. In the groups combined with Apelin-13, significant reductions in TNF-α levels were observed during the 24-hour treatment duration. The 1 µg/mL and 2 µg/mL doses of Apelin-13 significantly reduced TNF-α levels (p < 0.001). The suppressive effect of Apelin-13 on the production of inflammatory cytokines has also been demonstrated by Yuan et al., showing that Apelin-13 inhibits TNF-α through APJ receptors via the inhibition of Nicotinamide-Adenine Dinucleotide Phosphate Hydrogen (NADPH) oxidase (NOX-4), with its effect increasing over time [ 70 , 71 ]. 4.3. Oxidative Stress Markers An imbalance in the oxidant/antioxidant relationship and excessive production of ROS can increase the levels of molecules such as AGE, AOPP, and DT, inducing DNA methylation and creating toxic effects on neurons [ 8 , 72 ]. D-glutamic acid application resulted in significant oxidative stress in SH-SY5Y cells, leading to notable increases in ROS, AGE, AOPP, and DT levels. It has been found that D-amino acids accumulate in age-related diseases such as Alzheimer’s disease, chronic kidney disease, and cataracts, suggesting that D-amino acids may play a role in aging and neurodegeneration processes [ 73 ]. In this study, D-glutamic acid application led to significant increases in ROS levels at both 12-hour and 24-hour treatment durations (p < 0.001). ROS enhance inflammatory responses by causing oxidative damage to cellular structures and increase the production of IL-1β and TNF-α [ 74 ]. The 1 µg/mL and 2 µg/mL doses of Apelin-13 significantly reduced ROS levels, mitigating cellular damage. Zhang et al. reported that Apelin-13 controls ROS production through the MAPKs and Phosphoinositide 3-Kinase (PI3K)/AKT pathways and reduces oxidative damage [ 75 ]. AGE levels showed significant increases following D-glutamic acid application (p < 0.001). AGEs form through the non-enzymatic reaction of reduced sugars with lysine or arginine amino groups of proteins and through glycoxidation reactions under oxidative stress. Their interactions with the receptor for advanced glycation end-products (RAGE) trigger inflammatory events and are particularly effective in neurodegenerative diseases such as Alzheimer’s disease, traumatic brain injury (TBI), and amyotrophic lateral sclerosis (ALS) [ 76 ]. Apelin-13 was effective in reducing AGE levels at certain doses. Similarly, Wen et al. demonstrated that Apelin-13 modulates cellular stress responses by inhibiting AGE production [ 77 ]. AOPP levels showed significant increases following D-glutamic acid application (p < 0.001). AOPP is a marker indicating the oxidative modification of proteins and is considered an important indicator of cellular damage [ 78 ]. Apelin-13 reduced AOPP levels by mitigating oxidative protein modifications induced by D-glutamic acid. This suggests that Apelin-13 may alleviate the harmful effects of oxidative stress by supporting intracellular antioxidant defense mechanisms. Similarly, Kamińska et al. reported that Apelin-13 reduces AOPP levels, preserving cellular functions and preventing oxidative damage [ 79 ]. DT levels showed significant increases following D-glutamic acid application (p < 0.001). Tyrosine is one of the primary targets of protein oxidation. DT, a tyrosine dimer formed through reactive oxygen species, metal-catalyzed oxidation, ultraviolet irradiation, and peroxidases, is considered a good indicator of protein oxidation [ 80 ]. It was observed that Apelin-13, when combined with D-glutamic acid, reduced DT levels. This indicates that Apelin-13 contributes to the reduction of intracellular oxidative stress by preventing protein oxidation. KYN levels showed significant increases following D-glutamic acid application (p < 0.001). KYN is the primary catabolic pathway of the essential amino acid tryptophan and affects glutamatergic activity in various ways, including direct effects on ionotropic and metabotropic glutamate receptors or vesicular glutamate transport. Additionally, high doses of KYN contribute to neuronal damage by producing highly reactive free radicals through both direct and glutamatergic pathways [ 81 , 82 ]. In this study, it was observed that Apelin-13 was also effective in reducing KYN levels. This suggests that Apelin-13 may reduce KYN production by modulating intracellular metabolic pathways and reducing oxidative stress responses. The effects observed in this study are consistent with Muneer A’s study examining the effects of KYN on inflammatory and oxidative stress responses [ 83 ]. The findings are also in line with Schwarcz et al.’s report that KYN is associated with neuroinflammation and that Apelin-13 may provide neuroprotective effects by modulating this metabolite [ 84 ]. Kwak et al. demonstrated that Apelin-13 reduces KYN production, preventing cellular damage and protecting neurons [ 85 ]. Adding D-glutamic acid at 10 mM and 20 mM concentrations to SH-SY5Y cells led to significant decreases in T-SH levels in the D-10mM and D-20mM groups at both 12-hour and 24-hour time points compared to the control (p < 0.001 in both groups). In a study by Shivasharan et al., 2g/kg of monosodium glutamate (MSG) was administered intraperitoneally for 7 days to induce glutamate-related neurotoxicity in rats, and only the neurotoxicity group showed reduced T-SH levels [ 87 ]. Hamza et al. also reported that high-dose oral MSG (17.5 mg/kg/day) administration for 30 days reduced thiol levels in rats [ 88 ]. These findings may explain the decrease in T-SH levels due to oxidative stress in SH-SY5Y cells treated with D-glutamic acid in this study. However, Apelin-13 treatment at doses of 1 µg/mL, 2 µg/mL, and 5 µg/mL resulted in significantly elevated T-SH levels, indicating its protective effect. Specifically, Apelin-13 at 1 µg/mL and 5 µg/mL doses showed significant increases in T-SH levels at both 12 and 24 hours, with 2 µg/mL being the most effective dose. This finding aligns with Topcu et al., who reported that Apelin-13 reduces oxidative stress and toxicity by increasing T-SH levels. In their study, Apelin-13 mitigated cisplatin-induced renal toxicity by boosting T-SH levels and reducing oxidative stress [ 89 ]. These results suggest that Apelin-13, by boosting T-SH levels at various doses and time points, particularly at 2 µg/mL, may offer protective benefits against oxidative stress in neuronal cells. Anti-inflammatory Cytokines In this study, the application of D-glutamic acid resulted in significant decreases in IL-10 and TGF-β1 levels at both 12 and 24 hours (p < 0.001). Previous studies have shown that glutamate suppresses anti-inflammatory responses in neurons by both activating NMDA receptors and disrupting glial cell function [ 90 ]. However, the similar effect of D-glutamic acid on SH-SY5Y cells has not been demonstrated before, making this study the first to show such an effect. Apelin-13 plays a crucial role in modulating inflammatory responses and has been effective in reducing D-glutamic acid-induced inflammation and cell death at all doses, especially at 2 µg/mL, at both 12 and 24 hours [ 15 , 16 , 18 ]. It has been shown that Apelin-13 increases adenylate cyclase activation and cAMP production by binding to APJ receptors, and through these pathways, it reduces monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) chemokines, thereby increasing IL-10 [ 91 ]. Similarly, while it has been demonstrated that Apelin-13 increases TGF-β1 levels in renal tubular cells, there is insufficient research on its effects in neurons [ 92 ]. Thus, this study is the first to show the effects of Apelin-13 on TGF-β1 levels in SH-SY5Y cells. 4.4. BDNF In this study, D-glutamic acid caused a significant decrease in BDNF levels in SH-SY5Y cells. At doses of 10 mM and 20 mM, significant reductions in BDNF levels were observed at both 12-hour and 24-hour treatment durations. When co-administered with D-glutamic acid, Apelin-13 resulted in significant increases in BDNF levels. At 12 hours, low doses of Apelin-13 (1 µg/mL and 2 µg/mL) led to highly significant increases in BDNF levels compared to the D-10 mM group. In the 24-hour treatment duration, Apelin-13 at all doses, particularly at 5 µg/mL, caused more pronounced increases in BDNF levels (p < 0.001). This finding indicates that Apelin-13 supports neuronal health in the short term and mitigates the effects of D-glutamic acid-induced cellular toxicity by increasing BDNF production. Similar studies in the field support our findings [ 15 – 17 ]. 5. Conclusion This study evaluated the excitotoxic effects of D-glutamic acid on SH-SY5Y cells and the protective effects of Apelin-13 against this toxicity. The study demonstrated that D-glutamic acid at doses of 10 mM and 20 mM caused significant cytotoxic effects on SH-SY5Y cells when applied for 12 and 24 hours. Particularly, at a dose of 20 mM with 24-hour application, cell viability dropped below 50%. This is the first study to show that D-glutamic acid induces excitotoxicity in SH-SY5Y cells, filling an important gap in the literature. Apelin-13, at specific doses (1–10 µg/mL), alleviated D-glutamic acid-induced excitotoxicity, increased cell viability, and modulated inflammatory responses. Apelin-13 provided the most effective protection at a dose of 2 µg/mL, reducing levels of pro-inflammatory cytokines such as IL-1β and TNF-α while increasing levels of anti-inflammatory cytokines such as IL-10 and TGF-β1 to control inflammation. Additionally, Apelin-13 was observed to reduce levels of oxidative stress markers such as ROS, AGE, AOPP, and DT, and to support neuronal health and synaptic plasticity by increasing BDNF and T-SH levels. These findings suggest that Apelin-13 should be considered a protective agent against neurodegenerative processes and excitotoxicity and holds promising potential for future research. Future studies should more comprehensively investigate the effects of Apelin-13 on different cell lines and in vivo models. Additionally, the dose-response relationship and long-term effects of Apelin-13 should be thoroughly evaluated to explore its potential uses in the treatment of neurodegenerative diseases. Declarations Funding: This study was supported by Istanbul University-Cerrahpaşa Scientific Research Projects Unit (Project grant number: TSA-2023-37359). Competing Interests: The authors have no relevant financial or non-financial interests to disclose. Author Contributions: All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Kadriye Yağmur Oruç, Aykut Oruç, Gökhan Ağtürk and Karolin Yanar. The first draft of the manuscript was written by Kadriye Yağmur Oruç, Aykut Oruç and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Conceptualization: Kadriye Yağmur Oruç, Hakkı Oktay Seymen Methodology: Kadriye Yağmur Oruç, Gökhan Ağtürk, Karolin Yanar Formal analysis and investigation: Kadriye Yağmur Oruç, Aykut Oruç, Gökhan Ağtürk, Karolin Yanar Writing - original draft preparation: Kadriye Yağmur Oruç, Aykut Oruç Writing - review and editing: Kadriye Yağmur Oruç, Aykut Oruç, Hakkı Oktay Seymen Supervision: Kadriye Yağmur Oruç, Aykut Oruç, Hakkı Oktay Seymen Data Availability Statement: The raw data supporting this study will be available by authors to any qualified researcher upon request. Ethics Approval: This is an in-vitro study. The Istanbul University-Cerrahpaşa Research Local Ethics Committee has confirmed that no ethical approval is required. References Neves D, Salazar IL, Almeida RD, Silva RM (2023) Molecular mechanisms of ischemia and glutamate excitotoxicity. Life Sci 328:121814. https://doi.org/10.1016/j.lfs.2023.121814 Lai TW, Zhang S, Wang YT (2014) Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115:157-88. https://doi.org/10.1016/j.pneurobio.2013.11.006 Dutta R, Trapp BD (2011) Mechanisms of neuronal dysfunction and degeneration in multiple sclerosis. Prog Neurobiol 93(1):1-12. https://doi.org/10.1016/j.pneurobio.2010.09.005 Wang J, Wang F, Mai D, Qu S (2020) Molecular Mechanisms of Glutamate Toxicity in Parkinson's Disease. Front Neurosci 14:585584. https://doi.org/10.3389/fnins.2020.585584 Li A, Zhao Q, Chen L, Li Z (2023) Apelin/APJ system: an emerging therapeutic target for neurological diseases. Mol Biol Rep 50(2):1639-1653. https://doi.org/10.1007/s11033-022-08075-9 Zhang Y, Jiang W, Sun W et al (2023) Neuroprotective Roles of Apelin-13 in Neurological Diseases. Neurochem Res 48(6):1648-1662. https://doi.org/10.1007/s11064-023-03869-0 Zhou JX, Shuai NN, Wang B, Jin X, Kuang X, Tian SW (2021) Neuroprotective gain of Apelin/APJ system. Neuropeptides 87:102131. https://doi.org/10.1016/j.npep.2021.102131 Lewerenz J, Maher P (2015) Chronic Glutamate Toxicity in Neurodegenerative Diseases-What is the Evidence?. Front Neurosci 9:469. https://doi.org/10.3389/fnins.2015.00469 Ambrogini P, Torquato P, Bartolini D, et al (2019) Excitotoxicity, neuroinflammation and oxidant stress as molecular bases of epileptogenesis and epilepsy-derived neurodegeneration: The role of vitamin E. Biochim Biophys Acta Mol Basis Dis 1865(6):1098-1112. https://doi.org/10.1016/j.bbadis.2019.01.026 Iovino L, Tremblay ME, Civiero L (2020) Glutamate-induced excitotoxicity in Parkinson's disease: The role of glial cells. J Pharmacol Sci 144(3):151-164. https://doi.org/10.1016/j.jphs.2020.07.011 Kim GH, Kim JE, Rhie SJ, Yoon S (2015) The Role of Oxidative Stress in Neurodegenerative Diseases. Exp Neurobiol 24(4):325-340. https://doi.org/10.5607/en.2015.24.4.325 Lambeth JD (2007) Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med 43(3):332-347. https://doi.org/10.1016/j.freeradbiomed.2007.03.027 Osredkar J, Gosar D, Maček J et al (2019) Urinary Markers of Oxidative Stress in Children with Autism Spectrum Disorder (ASD). Antioxidants (Basel) 8(6):187. https://doi.org/10.3390/antiox8060187 Hachem LD, Mothe AJ, Tator CH (2015) Effect of BDNF and Other Potential Survival Factors in Models of In Vitro Oxidative Stress on Adult Spinal Cord-Derived Neural Stem/Progenitor Cells. Biores Open Access 4(1):146-159. https://doi.org/10.1089/biores.2014.0058 Luo H, Xiang Y, Qu X et al (2019) Apelin-13 Suppresses Neuroinflammation Against Cognitive Deficit in a Streptozotocin-Induced Rat Model of Alzheimer's Disease Through Activation of BDNF-TrkB Signaling Pathway. Front Pharmacol 10:395. https://doi.org/10.3389/fphar.2019.00395 Saral S, Topçu A, Alkanat M et al (2021) Apelin-13 activates the hippocampal BDNF/TrkB signaling pathway and suppresses neuroinflammation in male rats with cisplatin-induced cognitive dysfunction. Behav Brain Res 408:113290. https://doi.org/10.1016/j.bbr.2021.113290 Dai TT, Wang B, Xiao ZY, You Y, Tian SW (2018) Apelin-13 Upregulates BDNF Against Chronic Stress-induced Depression-like Phenotypes by Ameliorating HPA Axis and Hippocampal Glucocorticoid Receptor Dysfunctions. Neuroscience 390:151-159. https://doi.org/10.1016/j.neuroscience.2018.08.018 Khaksari M, Aboutaleb N, Nasirinezhad F, Vakili A, Madjd Z (2012) Apelin-13 protects the brain against ischemic reperfusion injury and cerebral edema in a transient model of focal cerebral ischemia. J Mol Neurosci 48(1):201-208. https://doi.org/10.1007/s12031-012-9808-3 Brancaccio M, Mennitti C, Cesaro A et al (2020) Dietary Thiols: A Potential Supporting Strategy against Oxidative Stress in Heart Failure and Muscular Damage during Sports Activity. Int J Environ Res Public Health 17(24):9424. https://doi.org/10.3390/ijerph17249424 Peng JJ, Lin SH, Liu YT, Lin HC, Li TN, Yao CK (2019) A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophilamotor system. Elife 8:e47372. https://doi.org/10.7554/eLife.47372 Cremers CM, Jakob U (2013) Oxidant sensing by reversible disulfide bond formation. J Biol Chem 288(37):26489-26496. https://doi.org/10.1074/jbc.R113.462929 Ye L, Huang Y, Zhao L et al (2013) IL-1β and TNF-α induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase. J Neurochem 125(6):897-908. https://doi.org/10.1111/jnc.12263 Leal MC, Casabona JC, Puntel M, Pitossi FJ (2013) Interleukin-1β and tumor necrosis factor-α: reliable targets for protective therapies in Parkinson's Disease?. Front Cell Neurosci 7:53. https://doi.org/10.3389/fncel.2013.00053 Kania J, Barańska A, Guzdek A (2003) Cytokine action and oxidative stress response in differentiated neuroblastoma SH-SY5Y cells. Acta Biochim Pol 50(3):659-666. Bernardino L, Xapelli S, Silva AP et al (2005) Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures. J Neurosci 25(29):6734-6744. https://doi.org/10.1523/JNEUROSCI.1510-05.2005 Xu W, Li T, Gao L et al (2019) Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. J Neuroinflammation 16(1):247. https://doi.org/10.1186/s12974-019-1620-3 Zhang H, Chen S, Zeng M et al (2018) Apelin-13 Administration Protects Against LPS-Induced Acute Lung Injury by Inhibiting NF-κB Pathway and NLRP3 Inflammasome Activation. Cell Physiol Biochem 49(5):1918-1932. https://doi.org/10.1159/000493653 Dobaczewski M, Chen W, Frangogiannis NG (2011) Transforming growth factor (TGF)-β signaling in cardiac remodeling. J Mol Cell Cardiol 51(4):600-606. https://doi.org/10.1016/j.yjmcc.2010.10.033 Harder T, Guttek K, Philipsen L, Simeoni L, Schraven B, Reinhold D (2014) Selective targeting of transforming growth factor-beta1 into TCR/CD28 signalling plasma membrane domains silences T cell activation. Cell Commun Signal 12:74. https://doi.org/10.1186/s12964-014-0074-6 Diniz LP, Tortelli V, Matias I et al (2017) Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against Aβ Oligomers in Alzheimer's Disease Model. J Neurosci 37(28):6797-6809. https://doi.org/10.1523/JNEUROSCI.3351-16.2017 Shen WX, Chen JH, Lu JH, Peng YP, Qiu YH (2014) TGF-β1 protection against Aβ1-42-induced neuroinflammation and neurodegeneration in rats. Int J Mol Sci 15(12):22092-22108. https://doi.org/10.3390/ijms151222092 Chen JH, Ke KF, Lu JH, Qiu YH, Peng YP (2015) Protection of TGF-β1 against neuroinflammation and neurodegeneration in Aβ1-42-induced Alzheimer's disease model rats. PLoS One 10(2):e0116549. https://doi.org/10.1371/journal.pone.0116549 Raschick M, Richter A, Fischer L et al (2023) Plasma concentrations of anti-inflammatory cytokine TGF-β are associated with hippocampal structure related to explicit memory performance in older adults. J Neural Transm (Vienna) 130(8):989-1002. https://doi.org/10.1007/s00702-023-02638-1 Martínez-Canabal A (2015) Potential neuroprotective role of transforming growth factor β1 (TGFβ1) in the brain. Int J Neurosci 125(1):1-9. https://doi.org/10.3109/00207454.2014.903947 Cao BB, Zhang XX, Du CY, Liu Z, Qiu YH, Peng YP (2020) TGF-β1 Provides Neuroprotection via Inhibition of Microglial Activation in 3-Acetylpyridine-Induced Cerebellar Ataxia Model Rats. Front Neurosci 14:187. https://doi.org/10.3389/fnins.2020.00187 Porro C, Cianciulli A, Panaro MA (2020) The Regulatory Role of IL-10 in Neurodegenerative Diseases. Biomolecules 10(7):1017. https://doi.org/10.3390/biom10071017 Batista ML Jr, Lopes RD, Seelaender MC, Lopes AC (2010) Anti-inflammatory effect of physical training in heart failure: role of TNF-alpha and IL-10. Arq Bras Cardiol 95(2):279. Pereira L, Font-Nieves M, Van den Haute C, Baekelandt V, Planas AM, Pozas E (2015) IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity. Front Cell Neurosci 9:57. https://doi.org/10.3389/fncel.2015.00057 Moore KW, de Waal Malefyt R, Coffman RL, O'Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683-765. https://doi.org/10.1146/annurev.immunol.19.1.683 Xin J, Wainwright DA, Mesnard NA, Serpe CJ, Sanders VM, Jones KJ (2011) IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection. Brain Behav Immun 25(5):820-829. https://doi.org/10.1016/j.bbi.2010.08.004 Cannella B, Raine CS (2004) Multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation. Ann Neurol 55(1):46-57. https://doi.org/10.1002/ana.10764 Huang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677-736. https://doi.org/10.1146/annurev.neuro.24.1.677 Park H, Poo MM (2013) Neurotrophin regulation of neural circuit development and function. Nat Rev Neurosci 14(1):7-23. https://doi.org/10.1038/nrn3379 Duman RS, Deyama S, Fogaça MV (2021) Role of BDNF in the pathophysiology and treatment of depression: Activity-dependent effects distinguish rapid-acting antidepressants. Eur J Neurosci 53(1):126-139. https://doi.org/10.1111/ejn.14630 Correia AS, Cardoso A, Vale N (2023) BDNF Unveiled: Exploring Its Role in Major Depression Disorder Serotonergic Imbalance and Associated Stress Conditions. Pharmaceutics 15(8):2081. https://doi.org/10.3390/pharmaceutics15082081 Miao Z, Wang Y, Sun Z (2020) The Relationships Between Stress, Mental Disorders, and Epigenetic Regulation of BDNF. Int J Mol Sci 21(4):1375. https://doi.org/10.3390/ijms21041375 Martin JL, Finsterwald C (2011) Cooperation between BDNF and glutamate in the regulation of synaptic transmission and neuronal development. Commun Integr Biol 4(1):14-16. https://doi.org/10.4161/cib.4.1.13761 Gulyaeva NV (2017) Interplay between Brain BDNF and Glutamatergic Systems: A Brief State of the Evidence and Association with the Pathogenesis of Depression. Biochemistry (Mosc) 82(3):301-307. https://doi.org/10.1134/S0006297917030087 Katane M, Homma H (2010) D-aspartate oxidase: the sole catabolic enzyme acting on free D-aspartate in mammals. Chem Biodivers 7(6):1435-1449. https://doi.org/10.1002/cbdv.200900250 Sacchi S, Cappelletti P, Murtas G (2018) Biochemical Properties of Human D-amino Acid Oxidase Variants and Their Potential Significance in Pathologies. Front Mol Biosci 5:55. https://doi.org/10.3389/fmolb.2018.00055 Yap SH, Lee CS, Furusho A et al (2022) Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV. AIDS 36(7):911-921. https://doi.org/10.1097/QAD.0000000000003207 Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1-2):55-63. https://doi.org/10.1016/0022-1759(83)90303-4 Palanivel V, Gupta V, Mirshahvaladi SSO et al (2022) Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions. Cells 11(22):3665. https://doi.org/10.3390/cells11223665 Çetindağ Çiltaş A, Gündoğdu S, Yulak F (2022) Levetiracetam Protects Against Glutamate-Induced Excitotoxicity in SH-SY5Y Cell Line. Int J Nat Sci 6(2), 142-150. https://doi.org/10.47947/ijnls.1187054 Samandari-Bahraseman MR, Elyasi L (2021) Apelin-13 protects human neuroblastoma SH-SY5Y cells against amyloid-beta induced neurotoxicity: Involvement of anti oxidant and anti apoptotic properties. J Basic Clin Physiol Pharmacol 33(5):599-605. https://doi.org/10.1515/jbcpp-2020-0294 Jiang Y, Liu H, Ji B et al (2018) Apelin‑13 attenuates ER stress‑associated apoptosis induced by MPP+ in SH‑SY5Y cells. Int J Mol Med 42(3):1732-1740. https://doi.org/10.3892/ijmm.2018.3719 Avelin V, Sissonen S, Julkunen I, Österlund P (2022) Inactivation efficacy of H5N1 avian influenza virus by commonly used sample preparation reagents for safe laboratory practices. J Virol Methods 304:114527. https://doi.org/10.1016/j.jviromet.2022.114527 Sadowska-Bartosz I, Galiniak S, Bartosz G, Rachel M (2014) Oxidative modification of proteins in pediatric cystic fibrosis with bacterial infections. Oxid Med Cell Longev 2014:389629. https://doi.org/10.1155/2014/389629 Hanasand M, Omdal R, Norheim KB, Gøransson LG, Brede C, Jonsson G (2012) Improved detection of advanced oxidation protein products in plasma. Clin Chim Acta 413(9-10):901-906. https://doi.org/10.1016/j.cca.2012.01.038 Sedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Anal Biochem 25(1):192-205. https://doi.org/10.1016/0003-2697(68)90092-4 Smith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150(1):76-85. https://doi.org/10.1016/0003-2697(85)90442-7 Zhang Y, Tan F, Xu P, Qu S (2016) Recent Advance in the Relationship between Excitatory Amino Acid Transporters and Parkinson's Disease. Neural Plast 2016:8941327. https://doi.org/10.1155/2016/8941327 Kritis AA, Stamoula EG, Paniskaki KA, Vavilis TD (2015) Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Front Cell Neurosci 9:91. https://doi.org/10.3389/fncel.2015.00091 Basavarajappa D, Gupta V, Chitranshi N et al (2023) Anti-inflammatory Effects of Siponimod in a Mouse Model of Excitotoxicity-Induced Retinal Injury. Mol Neurobiol 60(12):7222-7237. https://doi.org/10.1007/s12035-023-03535-0 Oruc A, Oruc KY, Yanar K et al (2024) The Role of Glycogen Synthase Kinase-3β in the Zinc-Mediated Neuroprotective Effect of Metformin in Rats with Glutamate Neurotoxicity. Biol Trace Elem Res 202(1):233-245. https://doi.org/10.1007/s12011-023-03667-3 Williams TL, Nwokoye P, Kuc RE et al (2024) Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma. Front Neurosci 18:1379658. https://doi.org/10.3389/fnins.2024.1379658 Nopparat C, Chantadul V, Permpoonputtana K, Govitrapong P (2017) The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide. Mech Ageing Dev 164:49-60. https://doi.org/10.1016/j.mad.2017.04.001 Pickering M, Cumiskey D, O'Connor JJ (2005) Actions of TNF-alpha on glutamatergic synaptic transmission in the central nervous system. Exp Physiol 90(5):663-670. https://doi.org/10.1113/expphysiol.2005.030734 Jara JH, Singh BB, Floden AM, Combs CK (2007) Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. J Neurochem 100(5):1407-1420. https://doi.org/10.1111/j.1471-4159.2006.04330.x Yuan Y, Wang W, Zhang Y et al (2022) Apelin-13 Attenuates Lipopolysaccharide-Induced Inflammatory Responses and Acute Lung Injury by Regulating PFKFB3-Driven Glycolysis Induced by NOX4-Dependent ROS. J Inflamm Res 15:2121-2139. https://doi.org/10.2147/JIR.S348850 Lambeth JD (2007) Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med 43(3):332-347. https://doi.org/10.1016/j.freeradbiomed.2007.03.027 Jomova K, Raptova R, Alomar SY et al (2023) Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Arch Toxicol 97(10):2499-2574. https://doi.org/10.1007/s00204-023-03562-9 Fujii N, Kaji Y, Fujii N (2011) D-Amino acids in aged proteins: analysis and biological relevance. J Chromatogr B Analyt Technol Biomed Life Sci 879(29):3141-3147. https://doi.org/10.1016/j.jchromb.2011.05.051 Wu L, Pan Y (2019) Reactive oxygen species mediate TNF-α-induced inflammatory response in bone marrow mesenchymal cells. Iran J Basic Med Sci 22(11):1296-1301. https://doi.org/10.22038/ijbms.2019.37893.9006 Zhang P, Wang AP, Yang HP et al (2020) Apelin-13 attenuates high glucose-induced calcification of MOVAS cells by regulating MAPKs and PI3K/AKT pathways and ROS-mediated signals. Biomed Pharmacother 128:110271. https://doi.org/10.1016/j.biopha.2020.110271 Reddy VP, Aryal P, Darkwah EK (2022) Advanced Glycation End Products in Health and Disease. Microorganisms 10(9):1848. https://doi.org/10.3390/microorganisms10091848 Wen R, Huang R, Xu K, Cheng Y, Yi X (2023) Beneficial effects of Apelin-13 on metabolic diseases and exercise. Front Endocrinol (Lausanne) 14:1285788. https://doi.org/10.3389/fendo.2023.1285788 Witko-Sarsat V, Friedlander M, Capeillère-Blandin C et al (1996) Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 49(5):1304-1313. https://doi.org/10.1038/ki.1996.186 Kamińska K, Borzuta H, Buczma K, Cudnoch-Jędrzejewska A (2024) Neuroprotective effect of apelin-13 and other apelin forms-a review. Pharmacol Rep 76(3):439-451. https://doi.org/10.1007/s43440-024-00587-4 Halliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35(Pt 5):1147-1150. https://doi.org/10.1042/BST0351147 Schwarcz R (2016) Kynurenines and Glutamate: Multiple Links and Therapeutic Implications. Adv Pharmacol 76:13-37. https://doi.org/10.1016/bs.apha.2016.01.005 Pathak S, Nadar R, Kim S et al (2024) The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies. Int J Mol Sci 25(2):853. https://doi.org/10.3390/ijms25020853 Muneer A (2020) Kynurenine Pathway of Tryptophan Metabolism in Neuropsychiatric Disorders: Pathophysiologic and Therapeutic Considerations. Clin Psychopharmacol Neurosci 18(4):507-526. https://doi.org/10.9758/cpn.2020.18.4.507 Schwarcz R, Bruno JP, Muchowski PJ, Wu HQ (2012) Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci 13(7):465-477. https://doi.org/10.1038/nrn3257 Kwak SE, Cho SC, Bae JH et al (2019) Effects of exercise-induced apelin on muscle function and cognitive function in aged mice. Exp Gerontol 127:110710. https://doi.org/10.1016/j.exger.2019.110710 Lewerenz J, Hewett SJ, Huang Y et al (2013) The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal 18(5):522-555. https://doi.org/10.1089/ars.2011.4391 Shivasharan BD, Nagakannan P, Thippeswamy BS, Veerapur VP (2013) Protective Effect of Calendula officinalis L. Flowers Against Monosodium Glutamate Induced Oxidative Stress and Excitotoxic Brain Damage in Rats. Indian J Clin Biochem 28(3):292-298. https://doi.org/10.1007/s12291-012-0256-1 Hamza RZ, Al-Salmi FA, El-Shenawy NS (2019) Evaluation of the effects of the green nanoparticles zinc oxide on monosodium glutamate-induced toxicity in the brain of rats. PeerJ 7:e7460. https://doi.org/10.7717/peerj.7460 Topcu A, Saral S, Mercantepe T, Akyildiz K, Tumkaya L, Yilmaz A (2023) The effects of apelin-13 against cisplatin-induced nephrotoxicity in rats. Drug Chem Toxicol 46(1):77-87. https://doi.org/10.1080/01480545.2021.2011309 Shanaki-Bavarsad M, Almolda B, González B, Castellano B (2022) Astrocyte-targeted Overproduction of IL-10 Reduces Neurodegeneration after TBI. Exp Neurobiol 31(3):173-195. https://doi.org/10.5607/en21035 Chen D, Lee J, Gu X, Wei L, Yu SP (2015) Intranasal Delivery of Apelin-13 Is Neuroprotective and Promotes Angiogenesis After Ischemic Stroke in Mice. ASN Neuro 7(5):1759091415605114. https://doi.org/10.1177/1759091415605114 Chen H, Wan D, Wang L et al (2015) Apelin protects against acute renal injury by inhibiting TGF-β1. Biochim Biophys Acta 1852(7):1278-1287. https://doi.org/10.1016/j.bbadis.2015.02.013 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4736431","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":328899878,"identity":"083f4362-7871-4266-83c1-4c1ca1b657a5","order_by":0,"name":"Kadriye Yağmur Oruç","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYBACCSjN2A8iEwpI0TKzAaTFgBQtGw6AKGK0SM5IfvzqZts92c3nVyd+eGDAIM8vdgC/FmmJNDPr3LZi42033m6WADrMcObsBPxa5CQSzIxz2xISt904uwGkJcHgNkEt6d/AWjbPOLv5B1FapCVyjB+DtGzg791GnC2SPW/KmHPOJRjPuMG7zSLBQIKwXySOp2/+nFOWINvff3bzzR8VNvL80gS0MAgksEHiRgKsUgKfWijgP8D8AcogQvUoGAWjYBSMSAAA3+pIGzPz3RMAAAAASUVORK5CYII=","orcid":"","institution":"Istanbul University-Cerrahpaşa","correspondingAuthor":true,"prefix":"","firstName":"Kadriye","middleName":"Yağmur","lastName":"Oruç","suffix":""},{"id":328899880,"identity":"8fff172c-1ba4-469d-a62d-0cabd391022b","order_by":1,"name":"Aykut Oruç","email":"","orcid":"","institution":"Istanbul University-Cerrahpaşa","correspondingAuthor":false,"prefix":"","firstName":"Aykut","middleName":"","lastName":"Oruç","suffix":""},{"id":328899882,"identity":"7a35a66a-b78e-4861-974a-1c331cb8b200","order_by":2,"name":"Gökhan Ağtürk","email":"","orcid":"","institution":"Haliç University","correspondingAuthor":false,"prefix":"","firstName":"Gökhan","middleName":"","lastName":"Ağtürk","suffix":""},{"id":328899883,"identity":"f171c7c8-26a9-4f9a-9900-d1c60b105c62","order_by":3,"name":"Karolin Yanar","email":"","orcid":"","institution":"Istanbul University-Cerrahpaşa","correspondingAuthor":false,"prefix":"","firstName":"Karolin","middleName":"","lastName":"Yanar","suffix":""},{"id":328899884,"identity":"ddd65b89-513a-47d2-8364-7a9b3543daac","order_by":4,"name":"Hakkı Oktay Seymen","email":"","orcid":"","institution":"Istanbul University-Cerrahpaşa","correspondingAuthor":false,"prefix":"","firstName":"Hakkı","middleName":"Oktay","lastName":"Seymen","suffix":""}],"badges":[],"createdAt":"2024-07-13 21:08:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4736431/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4736431/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":61999814,"identity":"0ecf8f8c-e7ff-4213-b899-23b5b112c725","added_by":"auto","created_at":"2024-08-08 05:32:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":3536490,"visible":true,"origin":"","legend":"\u003cp\u003ePercentage cell viabilities from MTT test results at the 12th and 24th hours. \u003cstrong\u003eA\u003c/strong\u003e Cell viability values (%) for D-glutamic acid at the 12th and 24th hours. \u003cstrong\u003eB\u003c/strong\u003e Cell viability values (%) for Apelin-13 at the 12th and 24th hours. \u003cstrong\u003eC\u003c/strong\u003e Cell viability values (%) when D-glutamic acid and Apelin-13 are applied together at the 12th and 24th hours. Results are presented as mean ± SD. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/13e2d47cf8b4ebf9f74c0930.png"},{"id":62000310,"identity":"be619eb0-37ee-4ebe-acad-dba71c0f3c99","added_by":"auto","created_at":"2024-08-08 05:40:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":21689515,"visible":true,"origin":"","legend":"\u003cp\u003eImages from MTT tests at the 12th and 24th hours. The \u003cstrong\u003eCTL-12\u003c/strong\u003e and \u003cstrong\u003eCTL-24\u003c/strong\u003e groups are control groups, showing that the cells maintain their normal and healthy morphology. In the images for the \u003cstrong\u003eA-2μg/ml 12h\u003c/strong\u003e and \u003cstrong\u003eA-2μg/ml 24h\u003c/strong\u003e groups, there are no signs of stress or damage in the cells, similar to the control group. In the \u003cstrong\u003eD-10mM 12h\u003c/strong\u003e group, early apoptosis stages are indicated by cytoplasmic condensation and membrane irregularities. In the \u003cstrong\u003eD-10mM 24h\u003c/strong\u003e group, cell morphology is disrupted and apoptotic bodies are observed. The image for the \u003cstrong\u003eD-20mM 12h\u003c/strong\u003egroup shows severe disruption of cell morphology, rounding, increased cytoplasmic condensation, and formation of apoptotic bodies. In the \u003cstrong\u003eD-20mM 24h\u003c/strong\u003e group, increased apoptotic bodies, complete rounding of cells, severe apoptosis, and cell death are observed. In the image for the \u003cstrong\u003eD-10mM+A-2μg/ml 12h\u003c/strong\u003e group, cell morphology is relatively preserved, cytoplasmic condensation is reduced, membrane blebbing is less visible, and mild apoptosis is present. In the \u003cstrong\u003eD-10mM+A-2μg/ml\u003c/strong\u003e 24h group, apoptosis is still present, but there are fewer apoptotic bodies. In the \u003cstrong\u003eD-20mM+A-2μg/ml 12h\u003c/strong\u003egroup, the protective effect of Apelin is evident, with reduced cytoplasmic condensation and fewer apoptotic bodies. However, in the \u003cstrong\u003eD-20mM+A-2μg/ml 24h\u003c/strong\u003egroup, significant apoptosis is still present.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/c1ec7314ad250696227d1c9a.png"},{"id":61999815,"identity":"6fa47da2-bdd5-472a-904c-2e3d4ff0ff4b","added_by":"auto","created_at":"2024-08-08 05:32:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":5711887,"visible":true,"origin":"","legend":"\u003cp\u003ePro-inflammatory cytokine and ROS levels at the 12th and 24th hours. \u003cstrong\u003eA\u003c/strong\u003e IL-1β levels at the 12th hour (pg/mL). \u003cstrong\u003eB\u003c/strong\u003e IL-1β levels at the 24th hour (pg/mL). \u003cstrong\u003eC\u003c/strong\u003e TNF-α levels at the 12th hour (ng/L). \u003cstrong\u003eD\u003c/strong\u003e TNF-α levels at the 24th hour (ng/L). \u003cstrong\u003eE\u003c/strong\u003eROS levels at the 12th hour (IU/ml). \u003cstrong\u003eF\u003c/strong\u003e ROS levels at the 24th hour (IU/ml). Results are presented as mean ± SD. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001. IL-1β, Interleukin-1β; TNF-α, Tumor necrosis factor-alpha; ROS, Reactive oxygen species\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/ac0af5c253d442415bc891fa.png"},{"id":61999816,"identity":"2cb38011-7bc7-49fa-bfc4-3572081bf1f7","added_by":"auto","created_at":"2024-08-08 05:32:29","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5742111,"visible":true,"origin":"","legend":"\u003cp\u003eAnti-inflammatory cytokine levels at the 12th and 24th hours. \u003cstrong\u003eA \u003c/strong\u003eIL-10 levels at the 12th hour (pg/mL). \u003cstrong\u003eB\u003c/strong\u003eIL-10 levels at the 24th hour (pg/mL). \u003cstrong\u003eC\u003c/strong\u003e TGF-β1 levels at the 12th hour (pg/mL). \u003cstrong\u003eD\u003c/strong\u003e TGF-β1 levels at the 24th hour (pg/mL). \u003cstrong\u003eE\u003c/strong\u003e BDNF levels at the 12th hour (ng/mL). \u003cstrong\u003eF\u003c/strong\u003e BDNF levels at the 24th hour (ng/mL). Results are presented as mean ± SD.* p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001. IL-10, Interleukin-10; TGF-β1, Tumor growth factor-Beta1; BDNF, Brain-Derived Neurotrophic Factor\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/9ce4f20ba62efe949bb3a50d.png"},{"id":61999818,"identity":"8c50a3e9-3cf4-41b9-bdfa-b0e361171501","added_by":"auto","created_at":"2024-08-08 05:32:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":9139243,"visible":true,"origin":"","legend":"\u003cp\u003eOxidant-antioxidant biochemical parameter levels at the 12th and 24th hours. \u003cstrong\u003eA\u003c/strong\u003e AGE/Protein levels at the 12th hour (FU/mg protein). \u003cstrong\u003eB\u003c/strong\u003e AGE/Protein levels at the 24th hour (FU/mg protein). \u003cstrong\u003eC\u003c/strong\u003e AOPP/Protein levels at the 12th hour (µM/L Cl-T). \u003cstrong\u003eD\u003c/strong\u003eAOPP/Protein levels at the 24th hour (µM/L Cl-T). \u003cstrong\u003eE\u003c/strong\u003e KYN/Protein levels at the 12th hour (FU/mg protein). \u003cstrong\u003eF\u003c/strong\u003e KYN/Protein levels at the 24th hour (FU/mg protein). \u003cstrong\u003eG\u003c/strong\u003e DT/Protein levels at the 12th hour (FU/mg protein). \u003cstrong\u003eH\u003c/strong\u003eDT/Protein levels at the 24th hour (FU/mg protein). \u003cstrong\u003eI\u003c/strong\u003e T-SH/Protein levels at the 12th hour (nmol/mg protein). \u003cstrong\u003eJ \u003c/strong\u003eT-SH/Protein levels at the 24th hour (nmol/mg protein). Results are presented as mean ± SD.. * p\u0026lt;0.05, ** p\u0026lt;0.01, *** p\u0026lt;0.001. AGE, Advanced glycation end products; AOPP, Advanced oxidation protein products; KYN, Kynurenine; DT, Dityrosine; Tsh, Total-thiol\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/971413d57633af893db34c64.png"},{"id":62388682,"identity":"8bdd77a6-68fb-4e5c-a269-13cfd83dc85a","added_by":"auto","created_at":"2024-08-13 15:26:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":57912954,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4736431/v1/b4cf9ae6-efc7-45ad-88eb-dd20717d86e1.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Effect of Apelin-13 on D-Glutamic Acid-Induced Excitotoxicity in SH-SY5Y Cell Line: An In-Vitro Study","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eExcitotoxicity is defined by the excessive buildup of glutamate or other excitatory amino acids in the extracellular space, leading to the heightened activation of glutamate receptors, particularly the N-methyl-D-aspartate (NMDA) subtype, in the adult central nervous system (CNS) [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This excessive activation causes a sustained influx of calcium (Ca\u003csup\u003e2+\u003c/sup\u003e) through receptor channels, leading to neuronal cell death. Elevated levels of glutamate in the CNS increase intracellular Ca\u003csup\u003e2+\u003c/sup\u003e concentrations, triggering apoptotic stimuli within sensitive organelles like mitochondria and the endoplasmic reticulum (ER). This results in the production of toxic radicals, disruption of cellular energy production, and ultimately, cell death through acute necrosis and/or delayed apoptosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Additionally, glutamate exposure can activate extrasynaptic NMDA Receptors (NMDARs), halt cAMP response element-binding protein (CREB) activity, cause mitochondrial membrane potential loss, and lead to cell death [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eApelin, an endogenous neuropeptide widely distributed throughout various physiological systems, particularly prevalent within the nervous system, undergoes intricate post-translational modifications, resulting in the generation of diverse mature apelin active peptides, with apelin-13 exhibiting the most robust biological activity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Apelin is a natural ligand for the G protein-coupled apelin receptor (APJ) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The apelin/APJ system has been shown to possess neuroprotective properties, including anti-inflammatory, anti-oxidative stress, anti-apoptotic effects, autophagy regulation, and inhibition of excitotoxicity, suggesting its potential as a therapeutic target for neurological disorders [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGlutamate excitotoxicity, neuroinflammation, and oxidative stress collectively manifest as a pathogenic \u0026ldquo;triad\u0026rdquo; in various brain disorders, leading to cellular demise and perturbations in network dynamics [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Neuroinflammation is characterized by the activation of neuronal cells, leading to dysregulation of anti-inflammatory/proinflammatory cytokine ratios due to the excessive secretion of proinflammatory cytokines. These cytokines influence glutamate homeostasis by upregulating glutamate receptors, enhancing glutamatergic neurotransmission, and exacerbating excitotoxicity [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOxidative stress, resulting from an imbalance in the redox state of cells due to excessive production of reactive oxygen species (ROS), can lead to oxidative damage to cellular macromolecules and contribute to various neurodegenerative diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Imbalances in the oxidant/antioxidant relationship and excessive ROS production increase the levels of molecules such as advanced glycation end products (AGE), advanced oxidation protein products (AOPP), and kynurenine (KYN), dityrosine (DT), inducing DNA methylation and creating toxic effects on neurons [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. High ROS levels activate inflammatory pathways such as nuclear factor-kappa beta (NF-κB) and mitogen-activated protein kinase (MAPK), causing cellular damage and decreasing Brain-Derived Neurotrophic Factor (BDNF) expression in neurons [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Apelin-13 increases intracellular cyclic Adenosin Monophosphate (cAMP) levels, activating the protein kinase A (PKA) pathway, which suppresses inflammatory signaling pathways and increases BDNF synthesis [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Additionally, Apelin-13 binds to the APJ receptor, initiating MAPK activation through G-protein coupled receptor (GPCR) signaling pathways, triggering phospholipase C (PLC) activation [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. PLC stimulates the production of inositol triphosphate (IP3) and diacylglycerol (DAG), leading to intracellular Ca\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e release and protein kinase C (PKC) activation. These signals activate the Extracellular signal-Regulated Kinases 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 MAPK pathways, regulating BDNF gene expression [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Total thiol levels reduce oxidative stress by enhancing cellular antioxidant defense capacity [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. However, the increase in ROS levels due to mitochondrial dysfunction and the direct interaction of ROS with thiol groups (-SH) leads to the oxidation of free thiol groups [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The increase in disulfide bonds (R-S-S-R) in thiol groups causes protein structural degradation and amino acid side chain oxidation, leading to carbonylation, decreased -SH reactivity, and peroxidation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. As a result of this process, total thiol levels have been reported to decrease in neurodegenerative diseases [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterleukin-1β (IL-1β) and Tumor necrosis Factor-α (TNF-α) have been shown to play a role in glutamate-mediated excitotoxicity in several studies [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. These cytokines are secreted by Human Neuroblastoma cell line (SH-SY5Y cell line) in response to NMDA and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-induced excitotoxicity, triggering inflammatory responses that lead to cellular damage, neurodegeneration, apoptosis, and further inflammation [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Apelin-13, known for its anti-inflammatory effects on neurons, can reduce inflammation and neuronal damage by suppressing the production of IL-1β and TNF-α [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Apelin-13 binds to the APJ receptor, enhancing adenylate cyclase activity and cAMP production, which leads to PKA activation and suppression of inflammatory pathways such as NF-κB [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAnti-inflammatory cytokines play a crucial role in regulating inflammatory responses and protecting cells from the damaging effects of inflammation. Transforming growth factor β (TGF-β) is a prominent anti-inflammatory cytokine, existing in three isoforms: TGF-β1, TGF-β2, and TGF-β3 [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Among these, TGF-β1 is the most abundant subtype in mammals, with significant anti-inflammatory and neuroprotective effects [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. TGF-β1 has been shown to stimulate the regrowth of damaged neurons after axonal injury [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Although its role in neural plasticity and memory in humans is not well understood, TGF-β1\u0026rsquo;s contribution to learning and memory mechanisms has been demonstrated in rodents [\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Evidence suggests that TGF-β1 may have a protective role in neurodegenerative and neuroinflammatory diseases such as Alzheimer\u0026rsquo;s and multiple sclerosis [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInterleukin-10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF), serves as an immunomodulator in the anti-inflammatory process [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. IL-10 possesses numerous immunoregulatory effects crucial for resolving inflammation and can inhibit the production of various inflammatory cytokines such as TNF-α, IL-1β, Interleukin-6 (IL-6), and Interferon-gamma (IFN-γ) [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is produced by nearly all leukocytes, including T cell subsets, monocytes, macrophages, neutrophils, eosinophils, mast cells, dendritic cells (DCs), B cells, and natural killer (NK) cells, as well as by keratinocytes, epithelial cells, and Nestin\u003csup\u003e+\u003c/sup\u003e neuroblasts [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In the central nervous system, IL-10 receptors (IL-10R) are expressed by microglia, astrocytes, oligodendrocytes, and even neurons under both physiological and pathological conditions, initiating the Janus kinase (Jak)/tyrosine kinase (Tyk) signaling pathway in neurons [\u003cspan additionalcitationids=\"CR40\" citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBDNF is a neurotrophic factor critical for maintaining neuronal health and enhancing synaptic plasticity in the central nervous system. BDNF supports the growth, differentiation, and survival of neurons while also contributing to the strengthening of synaptic connections [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Environmental factors such as stress and toxicity can affect BDNF levels; some studies report that BDNF levels increase with stress, while others indicate a decrease [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A reduction in BDNF levels can negatively impact neuronal health and function, and is associated with neurodegenerative diseases [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. High doses of glutamate increase intracellular Ca\u003csup\u003e2+\u003c/sup\u003e levels through the overactivation of NMDA and AMPA receptors, leading to mitochondrial dysfunction and increased ROS production. This process exacerbates neuronal stress and toxicity, suppressing BDNF production and negatively affecting neuronal health [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study uses D-glutamic acid instead of the more commonly studied L-glutamic acid. D-amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are stereospecific enzymes that metabolize D-amino acids (D-AAs). DDO specifically acts on acidic D-AAs like D-aspartate, D-glutamate, and NMDA, converting them into imino acids while reducing flavin adenine dinucleotide (FAD) [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Subsequently, FAD is reoxidized in the presence of oxygen, producing hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e). High levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e cause oxidative stress and trigger events such as mitochondrial dysfunction, pro-inflammatory cytokine activation, and apoptotic and autophagic cell death [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Additionally, the accumulation of D-AAs has been associated with immune activation, and cellular exposure to D-AAs is suggested to induce inflammation and cell death through H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and NF-κB activation [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eAim\u003c/strong\u003e \u003cp\u003eThe aim of this in vitro study is to evaluate the protective effect of apelin-13 in a glutamic acid-induced excitotoxicity model in SH-SY5Y cells and to determine the underlying protective mechanisms. Specifically, the study aims to assess the changes in levels of BDNF, IL-10, IL-1β, TNF-α, TGF-β1, and ROS as well as KYN, DT, AGEs, AOPP, and T-SH following apelin-13 treatment. The findings of this study may shed light on the neuroprotective effects of apelin-13, elucidate its mechanisms of action, and identify potential novel targets for the treatment of neurodegenerative diseases.\u003c/p\u003e \u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Cell Culture\u003c/h2\u003e \u003cp\u003eThe study was conducted in the research laboratories of Istanbul University Cerrahpaşa Faculty of Medicine and Hali\u0026ccedil; University Faculty of Medicine. SH-SY5Y cells used in our study were obtained from the American Type Culture Collection (SH-SY5Y (ATCC CRL-2266) ATCC; Rockville, USA) as the 5th and 7th passages. All cell culture procedures were performed in a sterile laminar flow hood. After thawing the cells stored in cryotubes in a liquid nitrogen tank at -196\u0026deg;C, they were transferred to 25 cm\u003csup\u003e2\u003c/sup\u003e flasks. The cells were cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific) and 1% antibiotics (Penicillin/Streptomycin, Gibco, Thermo Fisher Scientific). The cells were incubated at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Following the incubation period, SH-SY5Y cells were checked under a ZEISS Primo Vert (Inverted Microscope Carl Zeiss Microscopy GmbH 07745 Jena, Germany) microscope, and when the cell density reached approximately 85\u0026ndash;90%, trypsinization was performed using Trypsin-EDTA (Gibco, Thermo Fisher Scientific).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Cell Treatments with D-Glutamic Acid and Apelin-13, Cell Viability Test\u003c/h2\u003e \u003cp\u003eThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay is a common method used to measure cellular metabolic activity and thus cell viability. This test is based on the ability of mitochondrial dehydrogenases in living cells to convert MTT into formazan crystals [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. 1 mg of D-Glutamic acid (Sigma, CAS:6893-26-1, Saint Louis, USA) was dissolved in 1 mL of dimethyl-sulphoxide (DMSO) at room temperature (25\u0026deg;C) and adjusted to pH 7.5. Four experimental groups were created: Control, D-Glutamic acid, Apelin-13, and D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13. Cells in the control group received no treatment. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide, Invitrogen) assay was performed on SH-SY5Y cells at 12th and 24th hours to determine cytotoxicity values in groups treated with D-Glutamic acid, Apelin-13 (Cayman Chemicals, Item 13523, Michigan, USA), and D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13. Cells were seeded in 96-well plates at a density of 1x105 cells for MTT assay. Samples were read at 540, 570, and 590 nanometer (nm) wavelengths using a spectrophotometer for MTT analysis. D-Glutamic acid concentrations of 10 millimolar (mM) and 20 mM, resulting in 80% cell viability, were selected for treatments [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Apelin-13 concentrations of 1, 2, and 5 micrograms per milliliter (\u0026micro;g/ml), resulting in 80% cell viability, were selected for treatments [\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. In the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 group, cells were first treated with D-Glutamic acid at concentrations of 10 mM and 20 mM, followed by treatment with Apelin-13 at concentrations of 1, 2, and 5 \u0026micro;g/ml for 10 mM D-Glutamic acid, and 1, 2, and 5 \u0026micro;g/ml for 20 mM D-Glutamic acid. Subsequently, all samples were collected to analyze their status at 12th and 24th hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Biochemical Antioxidant Status Analyses\u003c/h2\u003e \u003cp\u003eCells collected for biochemical analysis were lysed using the Radio-Immunoprecipitation Assay (RIPA) buffer protocol as described in the literature [\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.3.1. The levels of Dityrosine, Kynurenine, and Advanced Glycation End Products:\u003c/h2\u003e \u003cp\u003eThe levels of DT, KYN, and AGEs were measured spectrofluorometrically described by Sadowska and Bartosz et al. [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. The concentrations were expressed as fluorescence units (FU) per milligram of protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.3.2. Advanced Oxidation Protein Products:\u003c/h2\u003e \u003cp\u003eThe spectrophotometric method developed by Hanasand et al. was used to evaluate AOPP levels [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. The levels of AOPP were expressed as micromoles per liter of Chloramine-T equivalents.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.3.3. Total-Thiol:\u003c/h2\u003e \u003cp\u003eThe T-SH levels in cell lysates were measured using 5,5-dithiobis (2-nitrobenzoic acid) (DTNB) following the method reported by Sedlak and Lindsay [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. The concentrations were expressed as nmol/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.3.4. Total protein content:\u003c/h2\u003e \u003cp\u003eTotal protein content was measured by Bicinchoninic acid (BCA) assay described by Smith et al. [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Enzyme-Linked Immunosorbent Assay (ELISA) analyses:\u003c/h2\u003e \u003cp\u003eThe levels of BDNF, IL-10, IL-1β, TNF- α, TGF-β1, and ROS in the supernatants of SH-SY5Y cells subjected to various treatments were quantified using respective ELISA kits (Brain-Derived Neurotrophic Factor BDNF Cat. No. E1302Hu, interleukin-10 IL-10 Cat. No. E0102Hu, interleukin-1β IL-1β Cat. No. E0143Hu, tumor necrosis factor-alpha TNF-α Cat. No. E0082Hu, transforming growth factor-beta1 TGF-β1 Cat. No. E0134Hu; BT LAB, Shanghai, China. Human reactive oxygen species ROS Cat. No. EK710415; AFG Bioscience, Northbrook, USA), in accordance with the manufacturer's instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Imaging\u003c/h2\u003e \u003cp\u003eCulture plates were carefully placed on a ZEISS Primo Vert (Inverted Microscope Carl Zeiss Microscopy GmbH 07745 Jena, Germany) microscope stage. The area where the cells were located was focused with a 40x lens. Photographs of cells were taken using imaging software (Borox, SCMOS, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Statistical Analyses\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using GraphPad Prism Version 10.2.0 (GraphPad Software, Inc., San Diego, CA, USA). The Shapiro-Wilk Test was used to assess the normality of data distribution, and the Levene Test was used to assess homogeneity. Data were found to be normally distributed and homogenous. Therefore, group comparisons were made using One-way ANOVA test followed by post hoc Tukey test. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation for ELISA and manual biochemical analyses, and as minimum, maximum, median, and percentage for MTT test. Values with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. MTT Tests\u003c/h2\u003e \u003cp\u003eD-Glutamic acid treated groups showed a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in comparison with the D-CTL-12h group at 12 hours in the D-40mM-12h, D-60mM-12h, and D-80mM-12h groups, while no statistically significant difference was observed in the other groups. In comparison with the D-CTL-24h group at 24 hours, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the D-40mM-24h, D-60mM-24h, and D-80mM-24h groups, and a slight significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in the D-20mM-24h group, while no statistically significant difference was observed among the other groups. For the Apelin-13 treated groups, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the A-10\u0026micro;g/mL-12h group compared to the A-CTL-12h group at 12 hours, while no statistically significant difference was observed in the other groups. At 24 hours, a slight significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in the A-2\u0026micro;g/mL-24h group, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed in the A-5\u0026micro;g/mL-24h group, and a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the A-10\u0026micro;g/mL-24h group compared to the A-CTL-24h group. In the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups, significant differences were observed in the D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/mL-12h, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/mL-12h, D-40mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/mL-12h, D-40mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/mL-12h, and D-40mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/mL-12h groups compared to the CTL group at 12 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively), while no statistically significant difference was observed in the other groups. At 24 hours, significant statistical differences were observed in the D-40mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/mL-24h and D-40mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/mL-24h groups compared to the CTL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively), while no statistically significant difference was observed among the other groups. Relevant graphics and images are in \u003cem\u003eFigure.1\u003c/em\u003e and \u003cem\u003eFigure.2.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. ELISA Tests\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.2.1. ROS:\u003c/h2\u003e \u003cp\u003eAt 12 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in ROS levels was observed in the D-10mM group compared to the CTL group and between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 12 hours, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Additionally, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group compared to the D-10mM group, while a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml group compared to the D-10mM group. When compared to the D-20mM group, a statistically significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001) was observed in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups. At 24 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in ROS levels was observed in the D-10mM group compared to the CTL group and between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 24 hours, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), and a slight significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml group. Additionally, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group compared to the D-10mM group, and a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml group compared to the D-10mM group. When compared to the D-20mM group, all groups showed a statistically significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.3.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.2.2. IL-1β\u003c/h2\u003e \u003cp\u003eAt 12 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed in IL-1β levels when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 12 hours, no statistically significant difference was found when compared to the CTL group. However, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group compared to the D-10mM group, and a significant decrease was observed when comparing the D-20mM group to the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, respectively). At 24 hours, a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in IL-1β levels was observed when comparing the CTL group with the D-10mM group and between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 24 hours, statistically significant increases (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were found in the D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group. Significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the D-10mM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, respectively). When comparing the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups with the D-20mM group, significant decreases were observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively). Relevant graphics are in \u003cem\u003eFigure.3.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.2.3. TNF-α\u003c/h2\u003e \u003cp\u003eAt 12 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 12 hours, statistically significant increases were observed in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while no statistically significant difference was found in the other groups. However, no statistically significant difference was found when comparing the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups to the D-10mM group. When compared to the D-20mM group, a statistically significant decrease was observed only in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 24 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the CTL group with the D-10mM group, and a slight significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed when comparing the D-10mM group with the D-20mM group. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 24 hours, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml group and a slight increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group compared to the CTL group. In all other groups, a statistically significant increase was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When comparing the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups to the D-10mM group, a significant decrease was observed in all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When comparing the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups to the D-20mM group, a significant decrease was observed in all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.3.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e3.2.4. IL-10\u003c/h2\u003e \u003cp\u003eAt 12 hours, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the CTL group with the D-10mM group, and no statistically significant difference was observed between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 12 hours, no statistically significant difference was found when compared to the CTL group. However, when compared to the D-10mM group, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in all groups, and when comparing the D-20mM group with the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups, a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in all groups. At 24 hours, a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the CTL group with the D-10mM group and between the D-10mM and D-20mM groups. For the D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups at 24 hours, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while no significant difference was observed in the other groups. When comparing the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups to the D-10mM group, a significant increase was observed in all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When comparing the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups to the D-20mM group, a significant increase was observed in all groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.4.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.2.5. TGF-β1\u003c/h2\u003e \u003cp\u003eAt 12 hours, there was a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the CTL group when compared to the D-10 mM group, and there was no statistically significant difference in TGF-β1 levels between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13, only the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group showed a statistically significant slight increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the CTL group. However, when compared to the D-10 mM group, all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When comparing the D-20 mM group with its respective D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups (D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml), all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 24 hours, there was a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the CTL group when compared to the D-10 mM group, and there was no statistically significant difference in TGF-β1 levels between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13, only the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group showed a statistically significant slight increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the CTL group. When compared to the D-10 mM group, all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups (D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml), all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.4.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.2.6. BDNF\u003c/h2\u003e \u003cp\u003eAt 12 hours, there was a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in BDNF levels compared to both the D-CTL and D-10 mM groups, and no statistically significant difference was observed between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13, there was a significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups compared to the CTL group. Additionally, there was a statistically significant but less pronounced increase in the D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, when compared to the D-10 mM group, all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups (D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml), all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). At 24 hours, there was a significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) compared to the CTL group and no statistically significant difference between the D-10 mM and D-20mM groups. In the groups treated with D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13, there was a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in all groups compared to the CTL group. When compared to the D-10 mM group, all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, when comparing the D-20 mM group with its respective D-Glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups (D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml), all groups showed a statistically significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.4.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.3. Oxidative Stress Markers\u003c/h2\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1. AGE\u003c/h2\u003e \u003cp\u003eAt the 12th hour, a significant increase in AGE levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. It was noted that the increase in AGE levels could accompany D-glutamic acid-mediated excitotoxicity, and this increase could be concentration-dependent on D-glutamic acid. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively), while no statistically significant difference was observed in other groups. However, significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). This suggests that Apelin-13 may have a protective effect against the increase in AGE levels in 10mM and 20 mM D-glutamic acid-mediated excitotoxicity. At the 24th hour, a significant increase in AGE levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. It was observed that AGE levels increased both depending on D-glutamic acid concentration and time in excitotoxicity. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), a slightly significant increase in the D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a significant increase in the D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). However, when compared to the D-10 mM group, significant decreases were observed only in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and when compared to the D-20 mM group, significant decreases were observed in all groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.5.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2. AOPP\u003c/h2\u003e \u003cp\u003eAt the 12th hour, a significant increase in AOPP levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while statistically significant differences were not observed in other groups. However, significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). At the 24th hour, a significant statistical increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group, and a slightly significant increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed when comparing the D-10 mM group with the D-20mM group in AOPP levels. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), a slightly significant increase in the D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and a slightly significant increase in the D-20mM\u0026thinsp;+\u0026thinsp;A5-\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Significant decreases were observed only in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group when compared to the D-10 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and in all groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.5.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3. DT\u003c/h2\u003e \u003cp\u003eAt the 12th hour, a significant increase in DT levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all), while no statistically significant difference was observed in other groups. However, significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). At the 24th hour, a significant statistical increase (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group, while no significant difference was found when comparing the D-10 mM group with the D-20mM group in DT levels. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all), a slightly significant increase in the D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and significant differences were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 respectively). Significant decreases were observed in all groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.5.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e \u003ch2\u003e3.3.4. KYN\u003c/h2\u003e \u003cp\u003eAt the 12th hour, a significant increase in KYN levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all), while no statistically significant difference was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group. However, significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001), and in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-20 mM group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). At the 24th hour, a significant increase in KYN levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant differences were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 respectively). Significant decreases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups when compared to the D-10 mM group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 respectively). In comparison to the D-20 mM group, all groups showed a significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.5.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003e3.3.5. T-SH\u003c/h2\u003e \u003cp\u003eAt the 12th hour, there was a significant increase in T-SH antioxidant levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all), while no statistically significant difference was observed in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group. However, when compared to the D-10 mM group, there was a slightly significant decrease in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and a highly significant decrease in the D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). When compared to the D-20 mM group, all groups showed a statistically significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). At the 24th hour, there was a statistically significant increase in T-SH levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) when comparing the D-CTL group with the D-10 mM group and between the D-10 mM and D-20mM groups. When the D-glutamic acid\u0026thinsp;+\u0026thinsp;Apelin-13 treated groups were compared with the control, statistically significant increases were observed in the D-10mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, D-10mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/ml, D-20mM\u0026thinsp;+\u0026thinsp;A2\u0026micro;g/ml, and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/ml groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 for all). When compared to the D-10 mM group, all groups showed a statistically significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, when compared to the D-20 mM group, all groups exhibited a highly significant decrease (p\u0026thinsp;\u0026gt;\u0026thinsp;0.001). Relevant graphics are in \u003cem\u003eFigure.5.\u003c/em\u003e\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, the protective effect of Apelin-13 against D-glutamic acid-induced excitotoxicity in the SH-SY5Y cell line was investigated. The obtained results support that Apelin-13 has a protective effect against excitotoxicity; however, this protective effect is dose- and time-dependent.\u003c/p\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.1. MTT Tests\u003c/h2\u003e \u003cp\u003eAccording to the MTT test results, D-glutamic acid was observed to have a significant cytotoxic effect on SH-SY5Y cells. D-glutamic acid, applied at doses ranging from 10 mM to 80 mM, was examined for its effect on cell viability at both 12-hour and 24-hour durations. At the 12-hour treatment duration, a low dose (10 mM) of D-glutamic acid caused a significant decrease in cell viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while higher doses (20 mM, 40 mM, 60 mM, and 80 mM) resulted in highly significant reductions (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Similarly, Zhang and Xu [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e] reported that glutamate induces cell death through oxidative stress and inflammation by increasing intracellular calcium entry via NMDA receptors. At the 24-hour treatment duration, the cytotoxic effects of D-glutamic acid became more pronounced. Similarly, Lewerenz et al. noted that the long-term effects of glutamate lead to mitochondrial dysfunction and oxidative damage in neuronal cells, highlighting the significant role of chronic excitotoxicity in neurodegenerative diseases [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Based on the MTT test results, 10 mM and 20 mM doses of D-glutamic acid were chosen to induce acute and chronic excitotoxicity in this study, as these doses caused significant cytotoxic effects in SH-SY5Y cells without exceeding 50% cell loss. Previous studies have reported toxic effects of L-glutamic acid in SH-SY5Y cells at doses ranging from 8 mM to 80 mM, with Palanivel V et al. identifying 40 mM as the ideal dose [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. In another study conducted on rats, we demonstrated D-glutamic acid-mediated excitotoxicity and suggested it occurs through increased active GSK-3β levels and oxidative stress [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. However, no study has been conducted on SH-SY5Y cells. Therefore, this study is the first to demonstrate that D-glutamic acid induces excitotoxicity in SH-SY5Y cells and shows effects at lower doses compared to L-glutamic acid.\u003c/p\u003e \u003cp\u003eApelin-13 was applied at various doses (1 \u0026micro;g/mL, 2 \u0026micro;g/mL, 5 \u0026micro;g/mL, and 10 \u0026micro;g/mL), and its effects on cell viability were evaluated over both 12-hour and 24-hour durations. During the 12-hour treatment duration, a high dose (10 \u0026micro;g/mL) of Apelin-13 resulted in a highly significant decrease in cell viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), with no significant difference observed at other doses. Furthermore, during the 24-hour treatment duration, the toxic effects of high-dose Apelin-13 became more pronounced. In the A-5\u0026micro;g/mL-24h group, a slight decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was observed, while a highly significant decrease (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) was observed in the A-10\u0026micro;g/mL-24h group. This suggests that Apelin-13 may exhibit toxic effects at high doses over short durations. Other studies have highlighted that high doses of Apelin may cause toxic effects and that high serum Apelin levels indicate poor prognosis in glioblastoma multiforme [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. The doses of 2 \u0026micro;g/mL and 5 \u0026micro;g/mL of Apelin-13 were found to have minimal toxic effects on cell viability, indicating these doses are ideal for supporting cellular viability.\u003c/p\u003e \u003cp\u003eThe combination of D-glutamic acid and Apelin-13 was effective in preserving cell viability. During the 12-hour treatment duration, combinations of D-glutamic acid (10 mM and 20 mM) and Apelin-13 (1 \u0026micro;g/mL, 2 \u0026micro;g/mL, and 5 \u0026micro;g/mL) provided significant protection for cell viability. Studies have shown that Apelin reduces ER stress-mediated oxidative stress and neuroinflammation via AMPK inhibition, and inhibits cleaved caspase-1, IL-1β and TNF-α, MPO, and ROS production, and programmed cell death in neurons [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Additionally, other studies report that Apelin-13 increases BDNF levels, reduces neuronal damage through AKT and ERK1/2 pathway activation, and exhibits neuroprotective effects via increased BDNF expression through cAMP/PKA pathways [15\u003c/p\u003e \u003cp\u003eHowever, at the 24-hour treatment duration, certain doses of Apelin-13 did not mitigate the toxic effects of D-glutamic acid as expected. Specifically, significant decreases in cell viability (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were observed in the D-20mM\u0026thinsp;+\u0026thinsp;A1\u0026micro;g/mL-24h and D-20mM\u0026thinsp;+\u0026thinsp;A5\u0026micro;g/mL-24h groups. This indicates that the most effective protective dose of Apelin-13 against acute and chronic toxicity induced by 10 mM and 20 mM D-glutamic acid is 2 \u0026micro;g/mL.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.2. Proinflammatory Cytokines\u003c/h2\u003e \u003cp\u003eD-glutamic acid application at both 10 mM and 20 mM doses caused a significant increase in IL-1β levels at the 12th hour (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Similarly, Nopparat C et al. reported that glutamate triggers IL-1β production by increasing intracellular calcium loading through NMDA receptors [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. In the groups treated with Apelin-13, especially at the 2 \u0026micro;g/mL dose, a significant reduction in IL-1β levels was observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Luo H et al. similarly demonstrated that Apelin-13 provides anti-inflammatory effects in neuronal cells by suppressing the production of inflammatory cytokines [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The combination of Apelin-13 with 20 mM D-glutamic acid also significantly reduced IL-1β levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings indicate that Apelin-13 modulates IL-1β production, particularly at the 2 \u0026micro;g/mL dose, controlling inflammatory responses. At the 24-hour duration of D-glutamic acid administration, more pronounced increases in IL-1β levels were observed (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Leverenz J et al. similarly reported that prolonged exposure to glutamate could increase IL-1β levels, exacerbating cell damage [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In the groups combined with Apelin-13, significant reductions in IL-1β levels were observed at the 24-hour treatment duration. The 1 \u0026micro;g/mL and 2 \u0026micro;g/mL doses of Apelin-13 significantly reduced IL-1β levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eTNF-α levels also significantly increased following D-glutamic acid administration. At the 12th hour, the D-10 mM group showed a significant increase in TNF-α levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), with no significant difference observed between the D-10 mM and D-20 mM groups. TNF-α is a cytokine that promotes inflammatory responses and cell death, and it has been reported that glutamate enhances this process through NMDA receptors [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Additionally, Jara et al. indicated that TNF-α and glutamate work synergistically on NMDA receptor activation, with TNF-α enhancing calcium influx dependent on NMDA receptors, facilitating glutamate-mediated neurotoxicity through ERK activation [\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e]. In the groups treated with Apelin-13, significant reductions in TNF-α levels were observed. Combinations of 20 mM D-glutamic acid and Apelin-13 (1 \u0026micro;g/mL and 5 \u0026micro;g/mL) resulted in significant decreases in TNF-α levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The 2 \u0026micro;g/mL dose of Apelin-13 effectively reduced D-glutamic acid-induced inflammation.\u003c/p\u003e \u003cp\u003eDuring the 24-hour treatment duration, more pronounced increases in TNF-α levels were observed. Significant increases in TNF-α levels were noted between the D-10 mM and D-20 mM groups (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Literature indicates that prolonged application of glutamate can enhance inflammatory responses, leading to cell damage [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. In the groups combined with Apelin-13, significant reductions in TNF-α levels were observed during the 24-hour treatment duration. The 1 \u0026micro;g/mL and 2 \u0026micro;g/mL doses of Apelin-13 significantly reduced TNF-α levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). The suppressive effect of Apelin-13 on the production of inflammatory cytokines has also been demonstrated by Yuan et al., showing that Apelin-13 inhibits TNF-α through APJ receptors via the inhibition of Nicotinamide-Adenine Dinucleotide Phosphate Hydrogen (NADPH) oxidase (NOX-4), with its effect increasing over time [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.3. Oxidative Stress Markers\u003c/h2\u003e \u003cp\u003eAn imbalance in the oxidant/antioxidant relationship and excessive production of ROS can increase the levels of molecules such as AGE, AOPP, and DT, inducing DNA methylation and creating toxic effects on neurons [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e]. D-glutamic acid application resulted in significant oxidative stress in SH-SY5Y cells, leading to notable increases in ROS, AGE, AOPP, and DT levels. It has been found that D-amino acids accumulate in age-related diseases such as Alzheimer\u0026rsquo;s disease, chronic kidney disease, and cataracts, suggesting that D-amino acids may play a role in aging and neurodegeneration processes [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, D-glutamic acid application led to significant increases in ROS levels at both 12-hour and 24-hour treatment durations (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). ROS enhance inflammatory responses by causing oxidative damage to cellular structures and increase the production of IL-1β and TNF-α [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. The 1 \u0026micro;g/mL and 2 \u0026micro;g/mL doses of Apelin-13 significantly reduced ROS levels, mitigating cellular damage. Zhang et al. reported that Apelin-13 controls ROS production through the MAPKs and Phosphoinositide 3-Kinase (PI3K)/AKT pathways and reduces oxidative damage [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAGE levels showed significant increases following D-glutamic acid application (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). AGEs form through the non-enzymatic reaction of reduced sugars with lysine or arginine amino groups of proteins and through glycoxidation reactions under oxidative stress. Their interactions with the receptor for advanced glycation end-products (RAGE) trigger inflammatory events and are particularly effective in neurodegenerative diseases such as Alzheimer\u0026rsquo;s disease, traumatic brain injury (TBI), and amyotrophic lateral sclerosis (ALS) [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e]. Apelin-13 was effective in reducing AGE levels at certain doses. Similarly, Wen et al. demonstrated that Apelin-13 modulates cellular stress responses by inhibiting AGE production [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAOPP levels showed significant increases following D-glutamic acid application (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). AOPP is a marker indicating the oxidative modification of proteins and is considered an important indicator of cellular damage [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Apelin-13 reduced AOPP levels by mitigating oxidative protein modifications induced by D-glutamic acid. This suggests that Apelin-13 may alleviate the harmful effects of oxidative stress by supporting intracellular antioxidant defense mechanisms. Similarly, Kamińska et al. reported that Apelin-13 reduces AOPP levels, preserving cellular functions and preventing oxidative damage [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDT levels showed significant increases following D-glutamic acid application (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Tyrosine is one of the primary targets of protein oxidation. DT, a tyrosine dimer formed through reactive oxygen species, metal-catalyzed oxidation, ultraviolet irradiation, and peroxidases, is considered a good indicator of protein oxidation [\u003cspan citationid=\"CR80\" class=\"CitationRef\"\u003e80\u003c/span\u003e]. It was observed that Apelin-13, when combined with D-glutamic acid, reduced DT levels. This indicates that Apelin-13 contributes to the reduction of intracellular oxidative stress by preventing protein oxidation.\u003c/p\u003e \u003cp\u003eKYN levels showed significant increases following D-glutamic acid application (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). KYN is the primary catabolic pathway of the essential amino acid tryptophan and affects glutamatergic activity in various ways, including direct effects on ionotropic and metabotropic glutamate receptors or vesicular glutamate transport. Additionally, high doses of KYN contribute to neuronal damage by producing highly reactive free radicals through both direct and glutamatergic pathways [\u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. In this study, it was observed that Apelin-13 was also effective in reducing KYN levels. This suggests that Apelin-13 may reduce KYN production by modulating intracellular metabolic pathways and reducing oxidative stress responses. The effects observed in this study are consistent with Muneer A\u0026rsquo;s study examining the effects of KYN on inflammatory and oxidative stress responses [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. The findings are also in line with Schwarcz et al.\u0026rsquo;s report that KYN is associated with neuroinflammation and that Apelin-13 may provide neuroprotective effects by modulating this metabolite [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. Kwak et al. demonstrated that Apelin-13 reduces KYN production, preventing cellular damage and protecting neurons [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdding D-glutamic acid at 10 mM and 20 mM concentrations to SH-SY5Y cells led to significant decreases in T-SH levels in the D-10mM and D-20mM groups at both 12-hour and 24-hour time points compared to the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001 in both groups). In a study by Shivasharan et al., 2g/kg of monosodium glutamate (MSG) was administered intraperitoneally for 7 days to induce glutamate-related neurotoxicity in rats, and only the neurotoxicity group showed reduced T-SH levels [\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. Hamza et al. also reported that high-dose oral MSG (17.5 mg/kg/day) administration for 30 days reduced thiol levels in rats [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. These findings may explain the decrease in T-SH levels due to oxidative stress in SH-SY5Y cells treated with D-glutamic acid in this study. However, Apelin-13 treatment at doses of 1 \u0026micro;g/mL, 2 \u0026micro;g/mL, and 5 \u0026micro;g/mL resulted in significantly elevated T-SH levels, indicating its protective effect. Specifically, Apelin-13 at 1 \u0026micro;g/mL and 5 \u0026micro;g/mL doses showed significant increases in T-SH levels at both 12 and 24 hours, with 2 \u0026micro;g/mL being the most effective dose. This finding aligns with Topcu et al., who reported that Apelin-13 reduces oxidative stress and toxicity by increasing T-SH levels. In their study, Apelin-13 mitigated cisplatin-induced renal toxicity by boosting T-SH levels and reducing oxidative stress [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. These results suggest that Apelin-13, by boosting T-SH levels at various doses and time points, particularly at 2 \u0026micro;g/mL, may offer protective benefits against oxidative stress in neuronal cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eAnti-inflammatory Cytokines\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn this study, the application of D-glutamic acid resulted in significant decreases in IL-10 and TGF-β1 levels at both 12 and 24 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Previous studies have shown that glutamate suppresses anti-inflammatory responses in neurons by both activating NMDA receptors and disrupting glial cell function [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. However, the similar effect of D-glutamic acid on SH-SY5Y cells has not been demonstrated before, making this study the first to show such an effect.\u003c/p\u003e \u003cp\u003eApelin-13 plays a crucial role in modulating inflammatory responses and has been effective in reducing D-glutamic acid-induced inflammation and cell death at all doses, especially at 2 \u0026micro;g/mL, at both 12 and 24 hours [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. It has been shown that Apelin-13 increases adenylate cyclase activation and cAMP production by binding to APJ receptors, and through these pathways, it reduces monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1 (MIP-1) chemokines, thereby increasing IL-10 [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. Similarly, while it has been demonstrated that Apelin-13 increases TGF-β1 levels in renal tubular cells, there is insufficient research on its effects in neurons [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Thus, this study is the first to show the effects of Apelin-13 on TGF-β1 levels in SH-SY5Y cells.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e4.4. BDNF\u003c/h2\u003e \u003cp\u003eIn this study, D-glutamic acid caused a significant decrease in BDNF levels in SH-SY5Y cells. At doses of 10 mM and 20 mM, significant reductions in BDNF levels were observed at both 12-hour and 24-hour treatment durations. When co-administered with D-glutamic acid, Apelin-13 resulted in significant increases in BDNF levels. At 12 hours, low doses of Apelin-13 (1 \u0026micro;g/mL and 2 \u0026micro;g/mL) led to highly significant increases in BDNF levels compared to the D-10 mM group. In the 24-hour treatment duration, Apelin-13 at all doses, particularly at 5 \u0026micro;g/mL, caused more pronounced increases in BDNF levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). This finding indicates that Apelin-13 supports neuronal health in the short term and mitigates the effects of D-glutamic acid-induced cellular toxicity by increasing BDNF production. Similar studies in the field support our findings [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis study evaluated the excitotoxic effects of D-glutamic acid on SH-SY5Y cells and the protective effects of Apelin-13 against this toxicity. The study demonstrated that D-glutamic acid at doses of 10 mM and 20 mM caused significant cytotoxic effects on SH-SY5Y cells when applied for 12 and 24 hours. Particularly, at a dose of 20 mM with 24-hour application, cell viability dropped below 50%. This is the first study to show that D-glutamic acid induces excitotoxicity in SH-SY5Y cells, filling an important gap in the literature. Apelin-13, at specific doses (1\u0026ndash;10 \u0026micro;g/mL), alleviated D-glutamic acid-induced excitotoxicity, increased cell viability, and modulated inflammatory responses. Apelin-13 provided the most effective protection at a dose of 2 \u0026micro;g/mL, reducing levels of pro-inflammatory cytokines such as IL-1β and TNF-α while increasing levels of anti-inflammatory cytokines such as IL-10 and TGF-β1 to control inflammation. Additionally, Apelin-13 was observed to reduce levels of oxidative stress markers such as ROS, AGE, AOPP, and DT, and to support neuronal health and synaptic plasticity by increasing BDNF and T-SH levels. These findings suggest that Apelin-13 should be considered a protective agent against neurodegenerative processes and excitotoxicity and holds promising potential for future research. Future studies should more comprehensively investigate the effects of Apelin-13 on different cell lines and in vivo models. Additionally, the dose-response relationship and long-term effects of Apelin-13 should be thoroughly evaluated to explore its potential uses in the treatment of neurodegenerative diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis study was supported by\u0026nbsp;Istanbul University-Cerrahpaşa Scientific Research Projects Unit\u0026nbsp;(Project grant number:\u0026nbsp;TSA-2023-37359).\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u0026nbsp;\u003c/strong\u003eThe authors have no relevant financial or non-financial interests to disclose.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil;, G\u0026ouml;khan Ağt\u0026uuml;rk and Karolin Yanar. The first draft of the manuscript was written by Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil; and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: Kadriye Yağmur Oru\u0026ccedil;, Hakkı Oktay Seymen\u003c/p\u003e\n\u003cp\u003eMethodology: Kadriye Yağmur Oru\u0026ccedil;, G\u0026ouml;khan Ağt\u0026uuml;rk, Karolin Yanar\u003c/p\u003e\n\u003cp\u003eFormal analysis and investigation: Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil;, G\u0026ouml;khan Ağt\u0026uuml;rk, Karolin Yanar\u003c/p\u003e\n\u003cp\u003eWriting - original draft preparation: Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWriting - review and editing: Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil;, Hakkı Oktay Seymen\u003c/p\u003e\n\u003cp\u003eSupervision: Kadriye Yağmur Oru\u0026ccedil;, Aykut Oru\u0026ccedil;, Hakkı Oktay Seymen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe raw data supporting this study will be available by authors to any qualified researcher upon request.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics Approval:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is an in-vitro study. The Istanbul University-Cerrahpaşa Research Local Ethics Committee has confirmed that no ethical approval is required.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eNeves D, Salazar IL, Almeida RD, Silva RM (2023) Molecular mechanisms of ischemia and glutamate excitotoxicity. Life Sci 328:121814. https://doi.org/10.1016/j.lfs.2023.121814 \u003c/li\u003e\n\u003cli\u003eLai TW, Zhang S, Wang YT (2014) Excitotoxicity and stroke: identifying novel targets for neuroprotection. Prog Neurobiol 115:157-88. https://doi.org/10.1016/j.pneurobio.2013.11.006 \u003c/li\u003e\n\u003cli\u003eDutta R, Trapp BD (2011) Mechanisms of neuronal dysfunction and degeneration in multiple sclerosis. Prog Neurobiol 93(1):1-12. https://doi.org/10.1016/j.pneurobio.2010.09.005 \u003c/li\u003e\n\u003cli\u003eWang J, Wang F, Mai D, Qu S (2020) Molecular Mechanisms of Glutamate Toxicity in Parkinson\u0026apos;s Disease. Front Neurosci 14:585584. https://doi.org/10.3389/fnins.2020.585584 \u003c/li\u003e\n\u003cli\u003eLi A, Zhao Q, Chen L, Li Z (2023) Apelin/APJ system: an emerging therapeutic target for neurological diseases. Mol Biol Rep 50(2):1639-1653. https://doi.org/10.1007/s11033-022-08075-9 \u003c/li\u003e\n\u003cli\u003eZhang Y, Jiang W, Sun W et al (2023) Neuroprotective Roles of Apelin-13 in Neurological Diseases. Neurochem Res 48(6):1648-1662. https://doi.org/10.1007/s11064-023-03869-0 \u003c/li\u003e\n\u003cli\u003eZhou JX, Shuai NN, Wang B, Jin X, Kuang X, Tian SW (2021) Neuroprotective gain of Apelin/APJ system. Neuropeptides 87:102131. https://doi.org/10.1016/j.npep.2021.102131 \u003c/li\u003e\n\u003cli\u003eLewerenz J, Maher P (2015) Chronic Glutamate Toxicity in Neurodegenerative Diseases-What is the Evidence?. Front Neurosci 9:469. https://doi.org/10.3389/fnins.2015.00469 \u003c/li\u003e\n\u003cli\u003eAmbrogini P, Torquato P, Bartolini D, et al (2019) Excitotoxicity, neuroinflammation and oxidant stress as molecular bases of epileptogenesis and epilepsy-derived neurodegeneration: The role of vitamin E. Biochim Biophys Acta Mol Basis Dis 1865(6):1098-1112. https://doi.org/10.1016/j.bbadis.2019.01.026 \u003c/li\u003e\n\u003cli\u003eIovino L, Tremblay ME, Civiero L (2020) Glutamate-induced excitotoxicity in Parkinson\u0026apos;s disease: The role of glial cells. J Pharmacol Sci 144(3):151-164. https://doi.org/10.1016/j.jphs.2020.07.011 \u003c/li\u003e\n\u003cli\u003eKim GH, Kim JE, Rhie SJ, Yoon S (2015) The Role of Oxidative Stress in Neurodegenerative Diseases. Exp Neurobiol 24(4):325-340. https://doi.org/10.5607/en.2015.24.4.325 \u003c/li\u003e\n\u003cli\u003eLambeth JD (2007) Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med 43(3):332-347. https://doi.org/10.1016/j.freeradbiomed.2007.03.027 \u003c/li\u003e\n\u003cli\u003eOsredkar J, Gosar D, Maček J et al (2019) Urinary Markers of Oxidative Stress in Children with Autism Spectrum Disorder (ASD). Antioxidants (Basel) 8(6):187. https://doi.org/10.3390/antiox8060187 \u003c/li\u003e\n\u003cli\u003eHachem LD, Mothe AJ, Tator CH (2015) Effect of BDNF and Other Potential Survival Factors in Models of In Vitro Oxidative Stress on Adult Spinal Cord-Derived Neural Stem/Progenitor Cells. Biores Open Access 4(1):146-159. https://doi.org/10.1089/biores.2014.0058 \u003c/li\u003e\n\u003cli\u003eLuo H, Xiang Y, Qu X et al (2019) Apelin-13 Suppresses Neuroinflammation Against Cognitive Deficit in a Streptozotocin-Induced Rat Model of Alzheimer\u0026apos;s Disease Through Activation of BDNF-TrkB Signaling Pathway. Front Pharmacol 10:395. https://doi.org/10.3389/fphar.2019.00395 \u003c/li\u003e\n\u003cli\u003eSaral S, Top\u0026ccedil;u A, Alkanat M et al (2021) Apelin-13 activates the hippocampal BDNF/TrkB signaling pathway and suppresses neuroinflammation in male rats with cisplatin-induced cognitive dysfunction. Behav Brain Res 408:113290. https://doi.org/10.1016/j.bbr.2021.113290 \u003c/li\u003e\n\u003cli\u003eDai TT, Wang B, Xiao ZY, You Y, Tian SW (2018) Apelin-13 Upregulates BDNF Against Chronic Stress-induced Depression-like Phenotypes by Ameliorating HPA Axis and Hippocampal Glucocorticoid Receptor Dysfunctions. Neuroscience 390:151-159. https://doi.org/10.1016/j.neuroscience.2018.08.018 \u003c/li\u003e\n\u003cli\u003eKhaksari M, Aboutaleb N, Nasirinezhad F, Vakili A, Madjd Z (2012) Apelin-13 protects the brain against ischemic reperfusion injury and cerebral edema in a transient model of focal cerebral ischemia. J Mol Neurosci 48(1):201-208. https://doi.org/10.1007/s12031-012-9808-3 \u003c/li\u003e\n\u003cli\u003eBrancaccio M, Mennitti C, Cesaro A et al (2020) Dietary Thiols: A Potential Supporting Strategy against Oxidative Stress in Heart Failure and Muscular Damage during Sports Activity. Int J Environ Res Public Health 17(24):9424. https://doi.org/10.3390/ijerph17249424 \u003c/li\u003e\n\u003cli\u003ePeng JJ, Lin SH, Liu YT, Lin HC, Li TN, Yao CK (2019) A circuit-dependent ROS feedback loop mediates glutamate excitotoxicity to sculpt the Drosophilamotor system. Elife 8:e47372. https://doi.org/10.7554/eLife.47372 \u003c/li\u003e\n\u003cli\u003eCremers CM, Jakob U (2013) Oxidant sensing by reversible disulfide bond formation. J Biol Chem 288(37):26489-26496. https://doi.org/10.1074/jbc.R113.462929 \u003c/li\u003e\n\u003cli\u003eYe L, Huang Y, Zhao L et al (2013) IL-1\u0026beta; and TNF-\u0026alpha; induce neurotoxicity through glutamate production: a potential role for neuronal glutaminase. J Neurochem 125(6):897-908. https://doi.org/10.1111/jnc.12263 \u003c/li\u003e\n\u003cli\u003eLeal MC, Casabona JC, Puntel M, Pitossi FJ (2013) Interleukin-1\u0026beta; and tumor necrosis factor-\u0026alpha;: reliable targets for protective therapies in Parkinson\u0026apos;s Disease?. Front Cell Neurosci 7:53. https://doi.org/10.3389/fncel.2013.00053 \u003c/li\u003e\n\u003cli\u003eKania J, Barańska A, Guzdek A (2003) Cytokine action and oxidative stress response in differentiated neuroblastoma SH-SY5Y cells. Acta Biochim Pol 50(3):659-666. \u003c/li\u003e\n\u003cli\u003eBernardino L, Xapelli S, Silva AP et al (2005) Modulator effects of interleukin-1beta and tumor necrosis factor-alpha on AMPA-induced excitotoxicity in mouse organotypic hippocampal slice cultures. J Neurosci 25(29):6734-6744. https://doi.org/10.1523/JNEUROSCI.1510-05.2005 \u003c/li\u003e\n\u003cli\u003eXu W, Li T, Gao L et al (2019) Apelin-13/APJ system attenuates early brain injury via suppression of endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation and oxidative stress in a AMPK-dependent manner after subarachnoid hemorrhage in rats. J Neuroinflammation 16(1):247. https://doi.org/10.1186/s12974-019-1620-3 \u003c/li\u003e\n\u003cli\u003eZhang H, Chen S, Zeng M et al (2018) Apelin-13 Administration Protects Against LPS-Induced Acute Lung Injury by Inhibiting NF-\u0026kappa;B Pathway and NLRP3 Inflammasome Activation. Cell Physiol Biochem 49(5):1918-1932. https://doi.org/10.1159/000493653 \u003c/li\u003e\n\u003cli\u003eDobaczewski M, Chen W, Frangogiannis NG (2011) Transforming growth factor (TGF)-\u0026beta; signaling in cardiac remodeling. J Mol Cell Cardiol 51(4):600-606. https://doi.org/10.1016/j.yjmcc.2010.10.033 \u003c/li\u003e\n\u003cli\u003eHarder T, Guttek K, Philipsen L, Simeoni L, Schraven B, Reinhold D (2014) Selective targeting of transforming growth factor-beta1 into TCR/CD28 signalling plasma membrane domains silences T cell activation. Cell Commun Signal 12:74. https://doi.org/10.1186/s12964-014-0074-6\u003c/li\u003e\n\u003cli\u003eDiniz LP, Tortelli V, Matias I et al (2017) Astrocyte Transforming Growth Factor Beta 1 Protects Synapses against A\u0026beta; Oligomers in Alzheimer\u0026apos;s Disease Model. J Neurosci 37(28):6797-6809. https://doi.org/10.1523/JNEUROSCI.3351-16.2017 \u003c/li\u003e\n\u003cli\u003eShen WX, Chen JH, Lu JH, Peng YP, Qiu YH (2014) TGF-\u0026beta;1 protection against A\u0026beta;1-42-induced neuroinflammation and neurodegeneration in rats. Int J Mol Sci 15(12):22092-22108. https://doi.org/10.3390/ijms151222092\u003c/li\u003e\n\u003cli\u003eChen JH, Ke KF, Lu JH, Qiu YH, Peng YP (2015) Protection of TGF-\u0026beta;1 against neuroinflammation and neurodegeneration in A\u0026beta;1-42-induced Alzheimer\u0026apos;s disease model rats. PLoS One 10(2):e0116549. https://doi.org/10.1371/journal.pone.0116549 \u003c/li\u003e\n\u003cli\u003eRaschick M, Richter A, Fischer L et al (2023) Plasma concentrations of anti-inflammatory cytokine TGF-\u0026beta; are associated with hippocampal structure related to explicit memory performance in older adults. J Neural Transm (Vienna) 130(8):989-1002. https://doi.org/10.1007/s00702-023-02638-1\u003c/li\u003e\n\u003cli\u003eMart\u0026iacute;nez-Canabal A (2015) Potential neuroprotective role of transforming growth factor \u0026beta;1 (TGF\u0026beta;1) in the brain. Int J Neurosci 125(1):1-9. https://doi.org/10.3109/00207454.2014.903947 \u003c/li\u003e\n\u003cli\u003eCao BB, Zhang XX, Du CY, Liu Z, Qiu YH, Peng YP (2020) TGF-\u0026beta;1 Provides Neuroprotection via Inhibition of Microglial Activation in 3-Acetylpyridine-Induced Cerebellar Ataxia Model Rats. Front Neurosci 14:187. https://doi.org/10.3389/fnins.2020.00187 \u003c/li\u003e\n\u003cli\u003ePorro C, Cianciulli A, Panaro MA (2020) The Regulatory Role of IL-10 in Neurodegenerative Diseases. Biomolecules 10(7):1017. https://doi.org/10.3390/biom10071017 \u003c/li\u003e\n\u003cli\u003eBatista ML Jr, Lopes RD, Seelaender MC, Lopes AC (2010) Anti-inflammatory effect of physical training in heart failure: role of TNF-alpha and IL-10. Arq Bras Cardiol 95(2):279. \u003c/li\u003e\n\u003cli\u003ePereira L, Font-Nieves M, Van den Haute C, Baekelandt V, Planas AM, Pozas E (2015) IL-10 regulates adult neurogenesis by modulating ERK and STAT3 activity. Front Cell Neurosci 9:57. https://doi.org/10.3389/fncel.2015.00057 \u003c/li\u003e\n\u003cli\u003eMoore KW, de Waal Malefyt R, Coffman RL, O\u0026apos;Garra A (2001) Interleukin-10 and the interleukin-10 receptor. Annu Rev Immunol 19:683-765. https://doi.org/10.1146/annurev.immunol.19.1.683 \u003c/li\u003e\n\u003cli\u003eXin J, Wainwright DA, Mesnard NA, Serpe CJ, Sanders VM, Jones KJ (2011) IL-10 within the CNS is necessary for CD4+ T cells to mediate neuroprotection. Brain Behav Immun 25(5):820-829. https://doi.org/10.1016/j.bbi.2010.08.004 \u003c/li\u003e\n\u003cli\u003eCannella B, Raine CS (2004) Multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation. Ann Neurol 55(1):46-57. https://doi.org/10.1002/ana.10764 \u003c/li\u003e\n\u003cli\u003eHuang EJ, Reichardt LF (2001) Neurotrophins: roles in neuronal development and function. Annu Rev Neurosci 24:677-736. https://doi.org/10.1146/annurev.neuro.24.1.677 \u003c/li\u003e\n\u003cli\u003ePark H, Poo MM (2013) Neurotrophin regulation of neural circuit development and function. Nat Rev Neurosci 14(1):7-23. https://doi.org/10.1038/nrn3379 \u003c/li\u003e\n\u003cli\u003eDuman RS, Deyama S, Foga\u0026ccedil;a MV (2021) Role of BDNF in the pathophysiology and treatment of depression: Activity-dependent effects distinguish rapid-acting antidepressants. Eur J Neurosci 53(1):126-139. https://doi.org/10.1111/ejn.14630 \u003c/li\u003e\n\u003cli\u003eCorreia AS, Cardoso A, Vale N (2023) BDNF Unveiled: Exploring Its Role in Major Depression Disorder Serotonergic Imbalance and Associated Stress Conditions. Pharmaceutics 15(8):2081. https://doi.org/10.3390/pharmaceutics15082081 \u003c/li\u003e\n\u003cli\u003eMiao Z, Wang Y, Sun Z (2020) The Relationships Between Stress, Mental Disorders, and Epigenetic Regulation of BDNF. Int J Mol Sci 21(4):1375. https://doi.org/10.3390/ijms21041375 \u003c/li\u003e\n\u003cli\u003eMartin JL, Finsterwald C (2011) Cooperation between BDNF and glutamate in the regulation of synaptic transmission and neuronal development. Commun Integr Biol 4(1):14-16. https://doi.org/10.4161/cib.4.1.13761 \u003c/li\u003e\n\u003cli\u003eGulyaeva NV (2017) Interplay between Brain BDNF and Glutamatergic Systems: A Brief State of the Evidence and Association with the Pathogenesis of Depression. Biochemistry (Mosc) 82(3):301-307. https://doi.org/10.1134/S0006297917030087 \u003c/li\u003e\n\u003cli\u003eKatane M, Homma H (2010) D-aspartate oxidase: the sole catabolic enzyme acting on free D-aspartate in mammals. Chem Biodivers 7(6):1435-1449. https://doi.org/10.1002/cbdv.200900250 \u003c/li\u003e\n\u003cli\u003eSacchi S, Cappelletti P, Murtas G (2018) Biochemical Properties of Human D-amino Acid Oxidase Variants and Their Potential Significance in Pathologies. Front Mol Biosci 5:55. https://doi.org/10.3389/fmolb.2018.00055 \u003c/li\u003e\n\u003cli\u003eYap SH, Lee CS, Furusho A et al (2022) Plasma d-amino acids are associated with markers of immune activation and organ dysfunction in people with HIV. AIDS 36(7):911-921. https://doi.org/10.1097/QAD.0000000000003207 \u003c/li\u003e\n\u003cli\u003eMosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65(1-2):55-63. https://doi.org/10.1016/0022-1759(83)90303-4\u003c/li\u003e\n\u003cli\u003ePalanivel V, Gupta V, Mirshahvaladi SSO et al (2022) Neuroprotective Effects of Neuropeptide Y on Human Neuroblastoma SH-SY5Y Cells in Glutamate Excitotoxicity and ER Stress Conditions. Cells 11(22):3665. https://doi.org/10.3390/cells11223665 \u003c/li\u003e\n\u003cli\u003e\u0026Ccedil;etindağ \u0026Ccedil;iltaş A, G\u0026uuml;ndoğdu S, Yulak F (2022) Levetiracetam Protects Against Glutamate-Induced Excitotoxicity in SH-SY5Y Cell Line. Int J Nat Sci 6(2), 142-150. https://doi.org/10.47947/ijnls.1187054 \u003c/li\u003e\n\u003cli\u003eSamandari-Bahraseman MR, Elyasi L (2021) Apelin-13 protects human neuroblastoma SH-SY5Y cells against amyloid-beta induced neurotoxicity: Involvement of anti oxidant and anti apoptotic properties. J Basic Clin Physiol Pharmacol 33(5):599-605. https://doi.org/10.1515/jbcpp-2020-0294 \u003c/li\u003e\n\u003cli\u003eJiang Y, Liu H, Ji B et al (2018) Apelin‑13 attenuates ER stress‑associated apoptosis induced by MPP+ in SH‑SY5Y cells. Int J Mol Med 42(3):1732-1740. https://doi.org/10.3892/ijmm.2018.3719 \u003c/li\u003e\n\u003cli\u003eAvelin V, Sissonen S, Julkunen I, \u0026Ouml;sterlund P (2022) Inactivation efficacy of H5N1 avian influenza virus by commonly used sample preparation reagents for safe laboratory practices. J Virol Methods 304:114527. https://doi.org/10.1016/j.jviromet.2022.114527 \u003c/li\u003e\n\u003cli\u003eSadowska-Bartosz I, Galiniak S, Bartosz G, Rachel M (2014) Oxidative modification of proteins in pediatric cystic fibrosis with bacterial infections. Oxid Med Cell Longev 2014:389629. https://doi.org/10.1155/2014/389629 \u003c/li\u003e\n\u003cli\u003eHanasand M, Omdal R, Norheim KB, G\u0026oslash;ransson LG, Brede C, Jonsson G (2012) Improved detection of advanced oxidation protein products in plasma. Clin Chim Acta 413(9-10):901-906. https://doi.org/10.1016/j.cca.2012.01.038 \u003c/li\u003e\n\u003cli\u003eSedlak J, Lindsay RH (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman\u0026apos;s reagent. Anal Biochem 25(1):192-205. https://doi.org/10.1016/0003-2697(68)90092-4 \u003c/li\u003e\n\u003cli\u003eSmith PK, Krohn RI, Hermanson GT et al (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 1985;150(1):76-85. https://doi.org/10.1016/0003-2697(85)90442-7 \u003c/li\u003e\n\u003cli\u003eZhang Y, Tan F, Xu P, Qu S (2016) Recent Advance in the Relationship between Excitatory Amino Acid Transporters and Parkinson\u0026apos;s Disease. Neural Plast 2016:8941327. https://doi.org/10.1155/2016/8941327 \u003c/li\u003e\n\u003cli\u003eKritis AA, Stamoula EG, Paniskaki KA, Vavilis TD (2015) Researching glutamate - induced cytotoxicity in different cell lines: a comparative/collective analysis/study. Front Cell Neurosci 9:91. https://doi.org/10.3389/fncel.2015.00091 \u003c/li\u003e\n\u003cli\u003eBasavarajappa D, Gupta V, Chitranshi N et al (2023) Anti-inflammatory Effects of Siponimod in a Mouse Model of Excitotoxicity-Induced Retinal Injury. Mol Neurobiol 60(12):7222-7237. https://doi.org/10.1007/s12035-023-03535-0 \u003c/li\u003e\n\u003cli\u003eOruc A, Oruc KY, Yanar K et al (2024) The Role of Glycogen Synthase Kinase-3\u0026beta; in the Zinc-Mediated Neuroprotective Effect of Metformin in Rats with Glutamate Neurotoxicity. Biol Trace Elem Res 202(1):233-245. https://doi.org/10.1007/s12011-023-03667-3 \u003c/li\u003e\n\u003cli\u003eWilliams TL, Nwokoye P, Kuc RE et al (2024) Expression of the apelin receptor, a novel potential therapeutic target, and its endogenous ligands in diverse stem cell populations in human glioblastoma. Front Neurosci 18:1379658. https://doi.org/10.3389/fnins.2024.1379658 \u003c/li\u003e\n\u003cli\u003eNopparat C, Chantadul V, Permpoonputtana K, Govitrapong P (2017) The anti-inflammatory effect of melatonin in SH-SY5Y neuroblastoma cells exposed to sublethal dose of hydrogen peroxide. Mech Ageing Dev 164:49-60. https://doi.org/10.1016/j.mad.2017.04.001 \u003c/li\u003e\n\u003cli\u003ePickering M, Cumiskey D, O\u0026apos;Connor JJ (2005) Actions of TNF-alpha on glutamatergic synaptic transmission in the central nervous system. Exp Physiol 90(5):663-670. https://doi.org/10.1113/expphysiol.2005.030734 \u003c/li\u003e\n\u003cli\u003eJara JH, Singh BB, Floden AM, Combs CK (2007) Tumor necrosis factor alpha stimulates NMDA receptor activity in mouse cortical neurons resulting in ERK-dependent death. J Neurochem 100(5):1407-1420. https://doi.org/10.1111/j.1471-4159.2006.04330.x \u003c/li\u003e\n\u003cli\u003eYuan Y, Wang W, Zhang Y et al (2022) Apelin-13 Attenuates Lipopolysaccharide-Induced Inflammatory Responses and Acute Lung Injury by Regulating PFKFB3-Driven Glycolysis Induced by NOX4-Dependent ROS. J Inflamm Res 15:2121-2139. https://doi.org/10.2147/JIR.S348850 \u003c/li\u003e\n\u003cli\u003eLambeth JD (2007) Nox enzymes, ROS, and chronic disease: an example of antagonistic pleiotropy. Free Radic Biol Med 43(3):332-347. https://doi.org/10.1016/j.freeradbiomed.2007.03.027 \u003c/li\u003e\n\u003cli\u003eJomova K, Raptova R, Alomar SY et al (2023) Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Arch Toxicol 97(10):2499-2574. https://doi.org/10.1007/s00204-023-03562-9 \u003c/li\u003e\n\u003cli\u003eFujii N, Kaji Y, Fujii N (2011) D-Amino acids in aged proteins: analysis and biological relevance. J Chromatogr B Analyt Technol Biomed Life Sci 879(29):3141-3147. https://doi.org/10.1016/j.jchromb.2011.05.051 \u003c/li\u003e\n\u003cli\u003eWu L, Pan Y (2019) Reactive oxygen species mediate TNF-\u0026alpha;-induced inflammatory response in bone marrow mesenchymal cells. Iran J Basic Med Sci 22(11):1296-1301. https://doi.org/10.22038/ijbms.2019.37893.9006 \u003c/li\u003e\n\u003cli\u003eZhang P, Wang AP, Yang HP et al (2020) Apelin-13 attenuates high glucose-induced calcification of MOVAS cells by regulating MAPKs and PI3K/AKT pathways and ROS-mediated signals. Biomed Pharmacother 128:110271. https://doi.org/10.1016/j.biopha.2020.110271 \u003c/li\u003e\n\u003cli\u003eReddy VP, Aryal P, Darkwah EK (2022) Advanced Glycation End Products in Health and Disease. Microorganisms 10(9):1848. https://doi.org/10.3390/microorganisms10091848 \u003c/li\u003e\n\u003cli\u003eWen R, Huang R, Xu K, Cheng Y, Yi X (2023) Beneficial effects of Apelin-13 on metabolic diseases and exercise. Front Endocrinol (Lausanne) 14:1285788. https://doi.org/10.3389/fendo.2023.1285788 \u003c/li\u003e\n\u003cli\u003eWitko-Sarsat V, Friedlander M, Capeill\u0026egrave;re-Blandin C et al (1996) Advanced oxidation protein products as a novel marker of oxidative stress in uremia. Kidney Int 49(5):1304-1313. https://doi.org/10.1038/ki.1996.186 \u003c/li\u003e\n\u003cli\u003eKamińska K, Borzuta H, Buczma K, Cudnoch-Jędrzejewska A (2024) Neuroprotective effect of apelin-13 and other apelin forms-a review. Pharmacol Rep 76(3):439-451. https://doi.org/10.1007/s43440-024-00587-4 \u003c/li\u003e\n\u003cli\u003eHalliwell B (2007) Biochemistry of oxidative stress. Biochem Soc Trans 35(Pt 5):1147-1150. https://doi.org/10.1042/BST0351147 \u003c/li\u003e\n\u003cli\u003eSchwarcz R (2016) Kynurenines and Glutamate: Multiple Links and Therapeutic Implications. Adv Pharmacol 76:13-37. https://doi.org/10.1016/bs.apha.2016.01.005 \u003c/li\u003e\n\u003cli\u003ePathak S, Nadar R, Kim S et al (2024) The Influence of Kynurenine Metabolites on Neurodegenerative Pathologies. Int J Mol Sci 25(2):853. https://doi.org/10.3390/ijms25020853 \u003c/li\u003e\n\u003cli\u003eMuneer A (2020) Kynurenine Pathway of Tryptophan Metabolism in Neuropsychiatric Disorders: Pathophysiologic and Therapeutic Considerations. Clin Psychopharmacol Neurosci 18(4):507-526. https://doi.org/10.9758/cpn.2020.18.4.507 \u003c/li\u003e\n\u003cli\u003eSchwarcz R, Bruno JP, Muchowski PJ, Wu HQ (2012) Kynurenines in the mammalian brain: when physiology meets pathology. Nat Rev Neurosci 13(7):465-477. https://doi.org/10.1038/nrn3257 \u003c/li\u003e\n\u003cli\u003eKwak SE, Cho SC, Bae JH et al (2019) Effects of exercise-induced apelin on muscle function and cognitive function in aged mice. Exp Gerontol 127:110710. https://doi.org/10.1016/j.exger.2019.110710 \u003c/li\u003e\n\u003cli\u003eLewerenz J, Hewett SJ, Huang Y et al (2013) The cystine/glutamate antiporter system x(c)(-) in health and disease: from molecular mechanisms to novel therapeutic opportunities. Antioxid Redox Signal 18(5):522-555. https://doi.org/10.1089/ars.2011.4391 \u003c/li\u003e\n\u003cli\u003eShivasharan BD, Nagakannan P, Thippeswamy BS, Veerapur VP (2013) Protective Effect of Calendula officinalis L. Flowers Against Monosodium Glutamate Induced Oxidative Stress and Excitotoxic Brain Damage in Rats. Indian J Clin Biochem 28(3):292-298. https://doi.org/10.1007/s12291-012-0256-1 \u003c/li\u003e\n\u003cli\u003eHamza RZ, Al-Salmi FA, El-Shenawy NS (2019) Evaluation of the effects of the green nanoparticles zinc oxide on monosodium glutamate-induced toxicity in the brain of rats. PeerJ 7:e7460. https://doi.org/10.7717/peerj.7460 \u003c/li\u003e\n\u003cli\u003eTopcu A, Saral S, Mercantepe T, Akyildiz K, Tumkaya L, Yilmaz A (2023) The effects of apelin-13 against cisplatin-induced nephrotoxicity in rats. Drug Chem Toxicol 46(1):77-87. https://doi.org/10.1080/01480545.2021.2011309\u003c/li\u003e\n\u003cli\u003eShanaki-Bavarsad M, Almolda B, Gonz\u0026aacute;lez B, Castellano B (2022) Astrocyte-targeted Overproduction of IL-10 Reduces Neurodegeneration after TBI. Exp Neurobiol 31(3):173-195. https://doi.org/10.5607/en21035 \u003c/li\u003e\n\u003cli\u003eChen D, Lee J, Gu X, Wei L, Yu SP (2015) Intranasal Delivery of Apelin-13 Is Neuroprotective and Promotes Angiogenesis After Ischemic Stroke in Mice. ASN Neuro 7(5):1759091415605114. https://doi.org/10.1177/1759091415605114 \u003c/li\u003e\n\u003cli\u003eChen H, Wan D, Wang L et al (2015) Apelin protects against acute renal injury by inhibiting TGF-\u0026beta;1. Biochim Biophys Acta 1852(7):1278-1287. https://doi.org/10.1016/j.bbadis.2015.02.013\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"apelin-13, excitotoxicity, D-glutamic acid, neuroinflammation, SH-SY5Y cell line","lastPublishedDoi":"10.21203/rs.3.rs-4736431/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4736431/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExcitotoxicity, resulting from excessive accumulation of glutamate in the extracellular space, leads to neuronal cell death. This study investigates the protective effects of Apelin-13 on D-glutamic acid-induced excitotoxicity in SH-SY5Y human neuroblastoma cells, an in vitro model for neurodegenerative diseases. Unlike the commonly studied L-glutamic acid, this research focuses on D-glutamic acid to understand its specific impacts. SH-SY5Y cells were treated with varying concentrations of D-glutamic acid and Apelin-13, followed by analyses at 12 and 24 hours to evaluate cell viability, oxidative stress markers, and inflammatory cytokine levels. Cell viability assays revealed significant cytotoxic effects of D-glutamic acid at doses of 10 mM and 20 mM, reducing viability by over 50%. However, Apelin-13 treatment mitigated these effects, especially at 2 \u0026micro;g/mL, enhancing cell viability and reducing inflammatory cytokine levels (IL-1β and TNF-α). Apelin-13 also increased anti-inflammatory cytokine levels (IL-10 and TGF-β1) and brain-derived neurotrophic factor (BDNF), indicating its neuroprotective role. Oxidative stress markers, including ROS, AGE, AOPP, DT, and T-SH, were significantly elevated by D-glutamic acid but effectively reduced by Apelin-13. The neuroprotective mechanisms of Apelin-13 involve modulation of cAMP/PKA and MAPK signaling pathways, enhancing BDNF synthesis and suppressing oxidative stress and inflammatory responses. This study is the first to demonstrate the effects of D-glutamic acid on SH-SY5Y cells. It highlights Apelin-13\u0026rsquo;s potential as a therapeutic agent against excitotoxicity-induced neuronal damage, emphasizing its ability to modulate key molecular pathways involved in inflammation and oxidative stress. Further in vivo studies are warranted to explore the long-term neuroprotective effects of Apelin-13 in treating neurodegenerative diseases.\u003c/p\u003e","manuscriptTitle":"Protective Effect of Apelin-13 on D-Glutamic Acid-Induced Excitotoxicity in SH-SY5Y Cell Line: An In-Vitro Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-08-08 05:32:24","doi":"10.21203/rs.3.rs-4736431/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41457146-a17e-4cae-a916-731d25bbd94b","owner":[],"postedDate":"August 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-16T12:11:15+00:00","versionOfRecord":[],"versionCreatedAt":"2024-08-08 05:32:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4736431","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4736431","identity":"rs-4736431","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}