Ginsenoside Rh2 Protects against Glutamate-Induced Neurotoxicity in PC12 Cells via Activation of the VEGF-Mediated PI3K/Akt/mTOR Signaling Pathway

Ginsenoside Rh2 Protects against Glutamate-Induced Neurotoxicity in PC12 Cells via Activation of the VEGF-Mediated PI3K/Akt/mTOR Signaling Pathway | 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 Ginsenoside Rh2 Protects against Glutamate-Induced Neurotoxicity in PC12 Cells via Activation of the VEGF-Mediated PI3K/Akt/mTOR Signaling Pathway Chun-Yue Zhang, Chang Liu, Liang-Jing Liu, Jian-Ming Yang, Li-Xia Shen, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8880846/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 7 You are reading this latest preprint version Abstract Ginsenoside Rh2 (GRh2), a primary active constituent of red ginseng, demonstrates significant neuroprotection against glutamate-induced excitotoxic damage in differentiated PC12 cells, a model of depression. This study found that GRh2 concentration-dependently reversed the loss of cell viability caused by glutamate. It effectively mitigated key pathological events, including intracellular calcium overload, reactive oxygen species accumulation, and the collapse of mitochondrial membrane potential. Furthermore, GRh2 enhanced synaptic plasticity, as evidenced by improved neurite morphology and increased levels of the synaptic markers neurogranin and neuromodulin. Mechanistic investigations revealed that GRh2 upregulated vascular endothelial growth factor (VEGF) expression and subsequently activated the PI3K/Akt/mTOR signaling pathway. This activation led to increased expression of synaptic proteins (PSD-95 and synaptophysin), an elevated Bcl-2/Bax ratio. Critically, the specific VEGF inhibitor SU11248 and PI3K inhibitor LY294002 abolished all the protective effects of GRh2, confirming the indispensable role of the VEGF/PI3K/Akt/mTOR axis. These results indicate that GRh2 alleviates glutamate-induced neurotoxicity by activating the VEGF-mediated PI3K/Akt/mTOR pathway, thereby improving mitochondrial function, inhibiting oxidative stress and apoptosis, and promoting synaptic integrity. This work provides novel molecular insights into the antidepressant potential of GRh2. ginsenoside Rh2 depression glutamate excitotoxicity VEGF PI3K/Akt/mTOR pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Introduction Major depressive disorder is one of the most common mental illnesses worldwide, affecting over 300 million people and posing a substantial societal burden[ 1 – 3 ]. Current first-line antidepressant therapies are often hampered by a slow onset of action, unfavorable side effects, and prolonged treatment duration. Consequently, developing novel antidepressants with rapid efficacy and improved tolerability remains an urgent priority in neuropsychopharmacology. Accumulating evidence implicates dysfunction of the central glutamatergic system as a key pathological mechanism in depression, involving aberrant glutamate release, reuptake, and receptor expression[ 4 – 6 ]. Under conditions such as chronic stress, elevated extracellular glutamate levels lead to overactivation of N-methyl-D-aspartate (NMDA) receptors, triggering intracellular calcium overload. Sustained calcium influx impairs mitochondrial function, exacerbates oxidative stress, and ultimately induces neuronal apoptosis or necrosis[ 7 , 8 ]. Therefore, targeting the glutamatergic system and its downstream neurotoxic pathways presents a promising new avenue for antidepressant development. Vascular Endothelial Growth Factor (VEGF), renowned for its angiogenic properties, has recently been recognized for its crucial neurotrophic and neuroprotective roles. Notably, some fast-acting antidepressants, such as ketamine, rapidly upregulate brain VEGF expression following NMDA receptor blockade, subsequently ameliorating depressive-like behaviors[ 9 , 10 ]. The neurotrophic effects of VEGF and its promotion of synaptic plasticity largely depend on the activation of the Phosphoinositide 3-kinase/Protein Kinase B (PI3K/Akt) signaling pathway[ 11 , 12 ]. This pathway further regulates its key downstream effector, the mammalian target of rapamycin (mTOR), which ultimately improves depressive symptoms by promoting synaptic protein synthesis and inhibiting apoptosis, among other mechanisms[ 13 – 16 ]. This positions the VEGF-PI3K/Akt/mTOR signaling axis as a critical bridge linking glutamate receptor modulation to neuroprotection and plasticity. Ginsenoside Rh2 (GRh2) is a rare diol-type saponin monomer extracted from red ginseng. As a primary active constituent of Panax ginseng, GRh2 exhibits broad therapeutic potential, including anti-tumor, immunomodulatory, anti-inflammatory, and neuroprotective activities[ 17 – 20 ]. Recent studies suggest that GRh2 may exert antidepressant effects by inhibiting oxidative stress and neuroinflammation[ 21 , 22 ]. However, whether GRh2 can antagonize glutamate-induced neuronal damage, and specifically, whether its protective effects involve the regulation of the VEGF/PI3K/Akt/mTOR pathway, remains unclear. This study utilized the PC12 cell line, which exhibits typical neuronal characteristics, to establish a glutamate-induced neuronal injury model. Our aim was to thoroughly investigate the neuroprotective effects of GRh2 and to elucidate its potential mechanism of action via the VEGF/PI3K/Akt/mTOR pathway. This research is expected to provide novel molecular insights into the neuroprotective role of GRh2 and open new avenues for antidepressant drug discovery. Materials and Methods Materials PC12 cells were purchased from the Kunming Cell Bank, Chinese Academy of Sciences. S-type Ginsenoside Rh2 (Batch No.: YZ-111748) was obtained from the National Institutes for Food and Drug Control (China). L-Glutamic acid solution (Batch No.: CB1321) was from Procell (Beijing). SU11248 (Batch No.: A157426) and LY294002 (Batch No.: A133122) were purchased from Shanghai Bide Pharmatech. Reactive Oxygen Species Assay Kit (Batch No.: S0033S), Fluo-4 AM Calcium Assay Kit (Batch No.: S1061S), JC-1 Mitochondrial Membrane Potential Assay Kit (Batch No.: C2006), and VEGF Rabbit Monoclonal Antibody (Batch No.: AF1309) were sourced from Beyotime Biotechnology (Shanghai). Antibodies against PI3K p85α (CY5355), p-PI3K p85α (Y607) (CY6427), Akt (CY5561), p-Akt (S473) (CY6569), mTOR (CY5306), p-mTOR (S2448) (CY6571), Synaptophysin (SYN, CY5245), PSD-95 (DLG4, CY5407), Bcl-2 (CY6717), Bax (CY5059), and Goat Anti-Rabbit IgG HRP (F300405) were from Abways Technology. Caspase-3 (CY5384) and β-actin (AC028) antibodies were from ABclonal. GAP43 Mouse mAb (bsm-33192M) was from Bioss (Beijing). Neurogranin (Ng) Polyclonal antibody (10440-1-AP) was from Proteintech. Instruments Cell culture was performed in a Thermo Scientific CO 2 incubator. Absorbance for cell viability was measured using a BioTek Cytation5 microplate reader. Imaging was conducted with an OLYMPUS FV3000 confocal laser scanning microscope. Centrifugation used a Sigma high-speed refrigerated centrifuge. Protein bands were visualized with a GE Amersham Imager 600. Routine cell observation used a Nikon inverted microscope. Cell Culture Rat pheochromocytoma (PC12) cells (highly differentiated) were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C in a humidified 5% CO 2 atmosphere. The medium was replaced every two days. Upon reaching 70–80% confluence, cells were passaged using 0.25% trypsin. All experiments were performed using cells in the logarithmic growth phase. Cell Viability Assay (CCK-8) Log-phase PC12 cells were seeded into 96-well plates at a density of 8×10³ cells/well in 100 µL complete medium and cultured for 24 h for attachment. Glu Modeling Concentration Screening: Cells were treated with 6.5, 7.5, 8.5, 9.5, or 10.5 mmol/L Glu for 24 h, alongside normal control and blank (medium-only) groups. GRh2 Treatment Concentration Screening: Cells were treated with 1, 4, 8, 12, 16, or 20 µmol/L GRh2. Inhibitor Validation: The following groups were established: (i) Normal control; (ii) Model (8.5 mmol/L Glu); (iii) GRh2 protection (8.5 mmol/L Glu + 12 µmol/L GRh2); (iv) Pathway blockade 1 (8.5 mmol/L Glu + 12 µmol/L GRh2 + 0.4 µmol/L SU11248); (v) Pathway blockade 2 (8.5 mmol/L Glu + 12 µmol/L GRh2 + 2.5 µmol/L LY294002).After treatments, 10 µL of CCK-8 solution was added to each well, followed by incubation for 2–4 h. Absorbance was measured at 450 nm. Cell survival rate was calculated as: Cell Viability = [(As-Ab)/ (Ac-Ab)]×100%. As is the absorbance of the experimental well (containing cells, culture medium, CCK-8, and different concentrations of the drug); Ac is the absorbance of the control well (containing cells, culture medium, and CCK-8); Ab is the absorbance of the blank well (containing culture medium and CCK-8). Intracellular Ca²⁺ Measurement (Fluo-4 AM) Cells were seeded into confocal dishes (1×10⁵ cells/dish) and treated as per the inhibitor validation groups after 24 h. Post-treatment, cells were processed according to the Fluo-4 AM kit instructions. Fluorescence images were captured using a confocal microscope and analyzed with ImageJ software. Intracellular ROS Measurement (DCFH-DA) ROS levels were detected using the DCFH-DA kit per the manufacturer's protocol. Fluorescence was observed and quantified via confocal microscopy and ImageJ. Mitochondrial Membrane Potential (ΔΨm) Assay (JC-1) The JC-1 assay was performed as per the kit instructions. Cells were immediately observed under a confocal microscope. The ratio of JC-1 aggregates (red fluorescence) to monomers (green fluorescence) was calculated using ImageJ to assess ΔΨm. Immunofluorescence Staining for Ng and Nm Post-treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. Cells were incubated with primary antibodies (Rabbit anti-Ng, 1:500; Mouse anti-Nm, 1:500) overnight at 4°C, followed by appropriate fluorescently-labeled secondary antibodies (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI-containing mounting medium. Images were acquired using a confocal microscope. Western Blot Analysis Total protein was extracted using RIPA lysis buffer and quantified via the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4°C with specific primary antibodies: p-PI3K (1:1000), PI3K (1:1000), p-Akt (1:2000), Akt (1:2000), p-mTOR (1:1000), mTOR (1:1000), VEGF (1:1000), PSD-95 (1:1000), SYN (1:1000), Bax (1:1000), Bcl-2 (1:1000), Caspase-3 (1:1000), β-actin (1:5000). Membranes were then incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using an ECL kit, and band density was quantified with ImageJ software, normalized to β-actin. Statistical Analysis All experiments were independently repeated at least three times. Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.5.0. Data passed normality and homogeneity of variance tests. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by Dunnett's t-test for post-hoc comparisons against specific control groups. A P -value < 0.05 was considered statistically significant. Results Establishment of Glu-induced PC12 Cell Injury Model and Determination of GRh2 Protective Concentration To establish an in vitro depression model, the effect of different Glu concentrations on PC12 cell viability was assessed. As shown in Fig. 1 , treatment with 6.5 to 10.5 mmol/L Glu for 24 h significantly inhibited cell viability compared to the control group ( P < 0.01). Since 8.5 mmol/L Glu reduced cell viability to (55.87 ± 2.42)%, this concentration was selected for subsequent modeling. The protective effect of GRh2 was then evaluated. As shown in Fig. 2 , compared to the model group (viability reduced to (51.51 ± 3.06)%), 1–20 µmol/L GRh2 concentration-dependently reversed the Glu-induced decrease in cell viability ( P < 0.01). Based on cell viability and preliminary results, 12 µmol/L GRh2 was chosen as the optimal concentration for subsequent experiments. GRh2 Antagonizes Glu-induced Cytotoxicity via the VEGF/PI3K/Akt Pathway To investigate whether GRh2's protection depends on the VEGF/PI3K/Akt pathway, pathway inhibitors were employed. CCK-8 results (Fig. 3 ) showed that co-treatment with the VEGF inhibitor SU11248 (0.4 µmol/L) or the PI3K inhibitor LY294002 (2.5 µmol/L) significantly attenuated the protective effect of GRh2 on cell viability ( P < 0.01). This preliminarily confirms that GRh2's protective effect relies on the VEGF-mediated PI3K/Akt signaling pathway. GRh2 Alleviates Glu-induced Oxidative Stress and Calcium Overload The role of GRh2 in mitigating key excitotoxicity events was further explored. Intracellular Ca²⁺ Fluo-4 AM staining (Fig. 4 ) revealed a significant increase in Ca²⁺ fluorescence intensity in the Glu model group ( P < 0.01), which was effectively reduced by GRh2 treatment ( P < 0.01). Both SU11248 and LY294002 reversed GRh2's effect ( P < 0.01), with LY294002 showing a stronger inhibitory effect ( P < 0.05), suggesting the PI3K/Akt pathway plays a central role downstream of VEGF in regulating calcium homeostasis. ROS Levels: DCFH-DA probe results (Fig. 5 ) were consistent with Ca²⁺ findings. Glu significantly increased intracellular ROS levels ( P < 0.01), while GRh2 significantly scavenged ROS ( P < 0.01). This effect was also blocked by both inhibitors ( P < 0.01). Mitochondrial Membrane Potential: JC-1 staining (Fig. 6 ) indicated that Glu significantly decreased ΔΨm (P < 0.01), whereas GRh2 effectively restored it ( P < 0.01). SU11248 and LY294002 inhibited GRh2's protective effect on mitochondria ( P < 0.01). GRh2 Improves Synaptic Plasticity after Glu Injury Immunofluorescence observation and quantification (Fig. 7 ) showed that Glu injury caused shortened neurites, loose connections, and significantly reduced levels of Neurogranin (Ng) and Neuromodulin (Nm) ( P < 0.01). GRh2 treatment significantly reversed these morphological and molecular abnormalities ( P < 0.01). The SU11248 and LY294002 treatment groups exhibited phenotypes similar to the Glu injury group, with Ng and Nm levels significantly lower than the GRh2 protection group ( P < 0.01, Fig. 7 B). This indicates that GRh2 improves synaptic plasticity via the VEGF/PI3K/Akt pathway. GRh2 Activates the VEGF/PI3K/Akt/mTOR Pathway and Regulates Downstream Targets Western blot analysis was conducted to confirm the mechanism at the molecular level. Pathway Activation: As shown in Fig. 8 , compared to the control group, protein levels of VEGF, p-PI3K, p-Akt, and p-mTOR were significantly downregulated in the Glu model group ( P < 0.01). GRh2 treatment significantly increased the phosphorylation levels or expression of these proteins ( P < 0.05). The activation of this pathway was effectively inhibited by SU11248 or LY294002. Synaptic-related Proteins: Detection of key synaptic plasticity proteins (Fig. 9 ) revealed that GRh2 significantly reversed the Glu-induced downregulation of PSD-95 and SYN expression ( P < 0.05), effects that were blocked by pathway inhibitors. Apoptosis-related Proteins: As shown in Fig. 10 , GRh2 treatment upregulated the anti-apoptotic protein Bcl-2 ( P < 0.01) and downregulated the pro-apoptotic proteins Bax and Caspase-3 ( P < 0.05), shifting the Bcl-2/Bax ratio towards anti-apoptosis. Similarly, pathway inhibitors abolished the anti-apoptotic effects of GRh2. Discussion This study demonstrates that Ginsenoside Rh2 significantly alleviates glutamate-induced damage in PC12 cells. The protective mechanism is closely associated with the activation of the VEGF/PI3K/Akt/mTOR signaling pathway, leading to improved mitochondrial function, inhibition of oxidative stress and apoptosis, and enhanced synaptic plasticity. Glutamate excitotoxicity is a core pathological mechanism in depression and other neuropsychiatric disorders. NMDA receptor overactivation induces calcium overload, mitochondrial dysfunction, excessive ROS production[ 23 – 25 ], and ultimately neuronal apoptosis via Bcl-2/Bax imbalance and Caspase-3 activation[ 26 – 29 ]. Our results show that GRh2 effectively restores ΔΨm, reduces ROS levels, and modulates the Bcl-2/Bax ratio and Caspase-3 expression, confirming its neuroprotection via mitigating oxidative stress and mitochondria-dependent apoptosis(Fig. 11 ). VEGF, a key neurotrophic factor, plays an increasingly recognized role in depression pathophysiology. Studies show significantly reduced VEGF levels in the hippocampus of depressive models and in depressed patients[ 30 , 31 ], while fast-acting antidepressants like ketamine exert effects by upregulating VEGF[ 9 , 32 ]. Consistent with these findings, our results indicate that GRh2 treatment significantly elevates VEGF expression in PC12 cells, identifying VEGF as a critical upstream mediator of its antidepressant action. VEGF's neuroprotective effects largely depend on downstream PI3K/Akt pathway activation [ 11 , 33 ]. This study proves that GRh2, while increasing VEGF expression, also significantly enhances the phosphorylation of PI3K, Akt, and the pivotal hub molecule mTOR. This aligns with reports that various antidepressants activate this pathway in cellular and animal models[ 34 – 36 ]. To establish the causal role of the VEGF/PI3K/Akt/mTOR axis, we employed the inhibitors SU11248 and LY294002. Results demonstrated that both inhibitors effectively reversed GRh2's protection of cell viability, its suppression of oxidative stress, its restoration of ΔΨm, and concurrently blocked pathway activation. This key evidence strongly affirms that GRh2's effects are initiated by VEGF and transmitted through the PI3K/Akt/mTOR pathway. The activated PI3K/Akt/mTOR pathway synergistically improves neuronal health through multiple downstream mechanisms. Firstly, it enhances synaptic plasticity by promoting the synthesis of proteins like PSD-95 and SYN via mTOR[ 35 , 37 ]. Secondly, it inhibits apoptosis by regulating the Bcl-2 family protein balance and suppressing Caspase-3 activity[ 38 , 39 ] (Fig. 12 ). Our study observed that GRh2 treatment upregulated PSD-95, SYN, and Bcl-2, while downregulating Bax and Caspase-3. Furthermore, fluorescence tracing of Ng and Nm visually confirmed that GRh2 reverses Glu-induced synaptic atrophy and sparse connectivity, an effect also blocked by SU11248 and LY294002. These results collectively illustrate that GRh2, via the core VEGF/PI3K/Akt/mTOR pathway, simultaneously improves synaptic plasticity and cell survival. Conclusions In summary, this study systematically elucidates the effects and mechanisms of GRh2 in alleviating glutamate excitotoxicity in a cellular model. We propose and validate for the first time that GRh2 upregulates VEGF, subsequently activating the PI3K/Akt/mTOR signaling pathway, thereby inhibiting oxidative stress and apoptosis while promoting synaptic reconstruction. These findings provide novel molecular insights into the antidepressant potential of GRh2. A limitation of this study is that its conclusions are based solely on a cell model. Future research should validate GRh2's efficacy and mechanism in animal models of depression and explore the upstream signaling events regulating VEGF expression. These investigations will provide a more robust theoretical foundation for developing GRh2 as a novel, multi-target antidepressant candidate drug. Declarations Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the Natural Science Foundation of Hebei Province (Grant No. H2025405005); the Natural Science Research Program of Hebei Provincial Department of Education for Basic Scientific Research Operating Funds of Provincial Universities (Grant No. JYT2024020); the Scientific Research Project of Hebei Provincial Health Department (Grant No. 20160031); and the Hebei North University Institutional Scientific Research Project (Grant No. XJ2023034). Author Contribution Chun-Yue Zhang: Conceptualization, Methodology, Investigation, Formal analysis, Writing-Original Draft. Chang Liu: Methodology, Data curation, Validation, Visualization, Writing-Original Draft. Liang-Jing Liu: Investigation, Resources, Project administration. 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Front Cell Neurosci 19:1645437 Additional Declarations No competing interests reported. Supplementary Files supplementarymaterials.rar Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 03 Mar, 2026 Reviews received at journal 26 Feb, 2026 Reviewers agreed at journal 24 Feb, 2026 Reviewers invited by journal 23 Feb, 2026 Editor assigned by journal 17 Feb, 2026 Submission checks completed at journal 16 Feb, 2026 First submitted to journal 14 Feb, 2026 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. 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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-8880846","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":596493711,"identity":"f10343ae-eadf-4292-8471-626d80182fbc","order_by":0,"name":"Chun-Yue Zhang","email":"","orcid":"","institution":"Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Chun-Yue","middleName":"","lastName":"Zhang","suffix":""},{"id":596493714,"identity":"f0ab73c4-4700-44c8-ab8b-36958a9fabd4","order_by":1,"name":"Chang Liu","email":"","orcid":"","institution":"Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Chang","middleName":"","lastName":"Liu","suffix":""},{"id":596493721,"identity":"e3cf760c-a9b9-4295-bd39-748a008febe6","order_by":2,"name":"Liang-Jing Liu","email":"","orcid":"","institution":"Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Liang-Jing","middleName":"","lastName":"Liu","suffix":""},{"id":596493722,"identity":"5b27e10d-c54a-401a-af8c-aaece6c69317","order_by":3,"name":"Jian-Ming Yang","email":"","orcid":"","institution":"Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Jian-Ming","middleName":"","lastName":"Yang","suffix":""},{"id":596493727,"identity":"f34133c9-8bba-4667-9826-3416a6cd3b1b","order_by":4,"name":"Li-Xia Shen","email":"","orcid":"","institution":"Hebei North University","correspondingAuthor":false,"prefix":"","firstName":"Li-Xia","middleName":"","lastName":"Shen","suffix":""},{"id":596493729,"identity":"6b11bf49-b5df-4fd2-a33d-6d4958923487","order_by":5,"name":"Zhi-Gang Wu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA+0lEQVRIiWNgGAWjYHACNoYEEMXMfPDBhwoJOX7itbCzJRvOOGNhLNlAjBYw4Ocxk+Ztq0jcQEiLwY30Zw8e/LLLk3fmMTbgnSfBuIGB+eGjG3i1JKQbJPYlFxseZit8ILlNgtmcgc3YOAePFrMbCcckEnuYEzc2M282MNwmwWbZwMMmjV9LYhtQSz1QC4OZROIcCR6DAwS1JLNJJPw4nDifmcVM4mCDhARBLfZnnrFJJDYcT9zADAzkhmMSBpLNBPwi2Z7+TPLHn+rE+f2HDz7+U1NX38/e/PAxPi1gwNgGDLoDMB4zIeVg8IeBQb6BKJWjYBSMglEwEgEArB5OkOycZ8YAAAAASUVORK5CYII=","orcid":"","institution":"Hebei North University","correspondingAuthor":true,"prefix":"","firstName":"Zhi-Gang","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2026-02-14 14:53:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8880846/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8880846/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":103472814,"identity":"c016750f-7df6-452e-bfd0-354937f2e77e","added_by":"auto","created_at":"2026-02-26 06:15:41","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25664,"visible":true,"origin":"","legend":"\u003cp\u003eGlutamate induces concentration-dependent cytotoxicity in PC12 cells. Cell viability was assessed by CCK-8 assay after 24 h treatment with the indicated concentrations of glutamate (Glu). Data are presented as mean ± SD (n=5). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. control group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/106dc20202bb2fb34074f9d7.png"},{"id":103507841,"identity":"cdc06870-eafc-47d1-b7fa-19e931e23745","added_by":"auto","created_at":"2026-02-26 13:45:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":31120,"visible":true,"origin":"","legend":"\u003cp\u003eGinsenoside Rh2 (GRh2) protects PC12 cells from glutamate-induced cytotoxicity.Cell viability was measured by CCK-8 assay after co-treatment with 8.5 mmol/L Glu and the indicated concentrations of GRh2 for 24 h. Data are presented as mean ± SD (n=5). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu-only group.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/c9507a09738931772bae5d23.png"},{"id":103472820,"identity":"0784b58c-3994-4a0b-b31d-bd44e0d1cf12","added_by":"auto","created_at":"2026-02-26 06:15:41","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":29513,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of VEGF or PI3K blocks the protective effect of GRh2 on cell viability.Cell viability was measured after treatment with 8.5 mmol/L Glu, 12 μmol/L GRh2, and the VEGF inhibitor SU11248 (0.4 μmol/L) or the PI3K inhibitor LY294002 (2.5 μmol/L) for 24 h. Data are presented as mean ± SD (n=5). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu + GRh2 group.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/c5225bf167b96673a2bb72bc.png"},{"id":103507853,"identity":"689fc62e-1be5-4eee-9b77-84a7bd6655d6","added_by":"auto","created_at":"2026-02-26 13:46:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":224816,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 attenuates glutamate-induced intracellular calcium overload. Intracellular Ca²⁺ levels were detected by Fluo-4 AM staining. (A) Representative fluorescence images. (B) Quantitative analysis of fluorescence intensity. Data are presented as mean ± SD (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. Glu + GRh2 group. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/2740d4c3535909d654ebe221.png"},{"id":103507192,"identity":"b03f3ae5-1b3e-4131-88c6-9a626195e085","added_by":"auto","created_at":"2026-02-26 13:40:42","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":261963,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 reduces glutamate-induced reactive oxygen species (ROS) generation.Intracellular ROS levels were detected by DCFH-DA staining. (A) Representative fluorescence images. (B) Quantitative analysis of fluorescence intensity. Data are presented as mean ± SD (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt; \u003c/em\u003e0.01 vs. control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05 vs. Glu + GRh2 group. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/dd6b81c16420882bc1cff10d.png"},{"id":103472819,"identity":"76e0df17-0b2e-44e9-b71a-0e157933d6ad","added_by":"auto","created_at":"2026-02-26 06:15:41","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":682853,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 restores glutamate-induced loss of mitochondrial membrane potential (ΔΨm). ΔΨm was assessed by JC-1 staining (red/green fluorescence ratio). (A) Representative fluorescence images (red: aggregates, green: monomers). (B) Quantitative analysis of the red/green fluorescence ratio. Data are presented as mean ± SD (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 vs. control group;\u003csup\u003e ##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Glu group; \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Glu + GRh2 group. Scale bar: 50 μm.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/16b078f259b04fe7bbf51c15.png"},{"id":103508008,"identity":"66d14f4c-ce55-4c64-84f8-8c27f0dc7e8a","added_by":"auto","created_at":"2026-02-26 13:46:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":433702,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 enhances the expression of synaptic plasticity markers neuromodulin (Nm) and neurogranin (Ng). (A) Representative immunofluorescence images showing Nm (green) and Ng (red) expression. Nuclei were counterstained with DAPI (blue). Quantitative analysis of fluorescence intensity for (B) Nm and (C) Ng. Data are presented as mean ± SD (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.01 vs. control group; \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u003cem\u003e\u0026lt;\u003c/em\u003e 0.01 vs. Glu + GRh2 group. Scale bar: 20 μm.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/2536490c126dfacf32a8ebb1.png"},{"id":103472825,"identity":"24a89c57-b948-43ce-a5f2-2153d8f10e20","added_by":"auto","created_at":"2026-02-26 06:15:41","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":283805,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 activates the VEGF/PI3K/Akt/mTOR signaling pathway.(A) Representative western blots of VEGF, PI3K, p-PI3K, Akt, p-Akt, mTOR, and p-mTOR. Quantitative analysis of (B) VEGF protein level, and the phosphorylation ratios of (C) p-PI3K/PI3K, (D) p-Akt/Akt, and (E) p-mTOR/mTOR. Data are presented as mean ± SD (n=3). \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu + GRh2 group.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/c07c40eea39d0dba1bc0ce51.png"},{"id":104397564,"identity":"c01b75f7-00bb-4bb8-a4d4-90286f52b61d","added_by":"auto","created_at":"2026-03-11 11:51:56","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":197458,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 upregulates synaptic protein expression.(A) Representative western blots of PSD-95 and synaptophysin (SYN). Quantitative analysis of (B) PSD-95 and (C) SYN protein levels. Data are presented as mean ± SD (n=3). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu group; \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.05, \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP \u0026lt;\u003c/em\u003e 0.01 vs. Glu + GRh2 group.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/cef207c31767f34ab4099542.png"},{"id":103507277,"identity":"bcafa939-18b0-4cfc-be59-5c1831a019e3","added_by":"auto","created_at":"2026-02-26 13:40:52","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":200354,"visible":true,"origin":"","legend":"\u003cp\u003eGRh2 exerts anti-apoptotic effects.(A) Representative western blots of Bcl-2, Bax, and Caspase-3. Quantitative analysis of (B) Bcl-2, (C) Bax, and (D) Caspase-3 protein levels. Data are presented as mean ± SD (n=3). \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. control group; \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Glu group; \u003csup\u003e▲\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e▲▲\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 vs. Glu + GRh2 group.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/2c15e202524c79e66df279fa.png"},{"id":103472823,"identity":"d4638f2b-3326-4273-833e-cabee0d9b673","added_by":"auto","created_at":"2026-02-26 06:15:41","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":307858,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of glutamate-induced excitotoxic injury. Excessive glutamate activates NMDA receptors, leading to calcium influx and intracellular Ca²⁺ overload. This disrupts mitochondrial function, increasing ROS production, reducing ATP, and diminishing the mitochondrial membrane potential. These changes disrupt Bcl-2/Bax balance, upregulate Caspase-3, and ultimately trigger neuronal apoptosis.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/16049f16dc66e07b8385b820.png"},{"id":103472827,"identity":"425d84ac-6272-425b-baf5-c7e67cd0275a","added_by":"auto","created_at":"2026-02-26 06:15:43","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":164823,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic diagram of the proposed neuroprotective mechanism of GRh2 via the VEGF/PI3K/Akt/mTOR pathway.GRh2 upregulates VEGF, which binds to its receptor and triggers autophosphorylation. This recruits and activates PI3K, which catalyzes PIP2 to PIP3. PIP3 then activates Akt, which subsequently phosphorylates and activates mTOR. The activated mTOR pathway promotes cell survival, neurogenesis, and synaptic plasticity, counteracting glutamate-induced damage.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/a09f72510669803f262060ac.png"},{"id":104410052,"identity":"0ef4e02b-6107-4d5a-a8fd-ce2641cd21ef","added_by":"auto","created_at":"2026-03-11 12:49:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3470102,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/461c4adb-325f-4988-b646-ab0753fca5cf.pdf"},{"id":103472826,"identity":"dd9743a0-d231-4ae1-816e-dbbae524bca8","added_by":"auto","created_at":"2026-02-26 06:15:42","extension":"rar","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":19919419,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterials.rar","url":"https://assets-eu.researchsquare.com/files/rs-8880846/v1/14168a660fef517e2df2372f.rar"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ginsenoside Rh2 Protects against Glutamate-Induced Neurotoxicity in PC12 Cells via Activation of the VEGF-Mediated PI3K/Akt/mTOR Signaling Pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eMajor depressive disorder is one of the most common mental illnesses worldwide, affecting over 300\u0026nbsp;million people and posing a substantial societal burden[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Current first-line antidepressant therapies are often hampered by a slow onset of action, unfavorable side effects, and prolonged treatment duration. Consequently, developing novel antidepressants with rapid efficacy and improved tolerability remains an urgent priority in neuropsychopharmacology.\u003c/p\u003e \u003cp\u003eAccumulating evidence implicates dysfunction of the central glutamatergic system as a key pathological mechanism in depression, involving aberrant glutamate release, reuptake, and receptor expression[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Under conditions such as chronic stress, elevated extracellular glutamate levels lead to overactivation of N-methyl-D-aspartate (NMDA) receptors, triggering intracellular calcium overload. Sustained calcium influx impairs mitochondrial function, exacerbates oxidative stress, and ultimately induces neuronal apoptosis or necrosis[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, targeting the glutamatergic system and its downstream neurotoxic pathways presents a promising new avenue for antidepressant development.\u003c/p\u003e \u003cp\u003eVascular Endothelial Growth Factor (VEGF), renowned for its angiogenic properties, has recently been recognized for its crucial neurotrophic and neuroprotective roles. Notably, some fast-acting antidepressants, such as ketamine, rapidly upregulate brain VEGF expression following NMDA receptor blockade, subsequently ameliorating depressive-like behaviors[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. The neurotrophic effects of VEGF and its promotion of synaptic plasticity largely depend on the activation of the Phosphoinositide 3-kinase/Protein Kinase B (PI3K/Akt) signaling pathway[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. This pathway further regulates its key downstream effector, the mammalian target of rapamycin (mTOR), which ultimately improves depressive symptoms by promoting synaptic protein synthesis and inhibiting apoptosis, among other mechanisms[\u003cspan additionalcitationids=\"CR14 CR15\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. This positions the VEGF-PI3K/Akt/mTOR signaling axis as a critical bridge linking glutamate receptor modulation to neuroprotection and plasticity.\u003c/p\u003e \u003cp\u003eGinsenoside Rh2 (GRh2) is a rare diol-type saponin monomer extracted from red ginseng. As a primary active constituent of Panax ginseng, GRh2 exhibits broad therapeutic potential, including anti-tumor, immunomodulatory, anti-inflammatory, and neuroprotective activities[\u003cspan additionalcitationids=\"CR18 CR19\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recent studies suggest that GRh2 may exert antidepressant effects by inhibiting oxidative stress and neuroinflammation[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. However, whether GRh2 can antagonize glutamate-induced neuronal damage, and specifically, whether its protective effects involve the regulation of the VEGF/PI3K/Akt/mTOR pathway, remains unclear.\u003c/p\u003e \u003cp\u003eThis study utilized the PC12 cell line, which exhibits typical neuronal characteristics, to establish a glutamate-induced neuronal injury model. Our aim was to thoroughly investigate the neuroprotective effects of GRh2 and to elucidate its potential mechanism of action via the VEGF/PI3K/Akt/mTOR pathway. This research is expected to provide novel molecular insights into the neuroprotective role of GRh2 and open new avenues for antidepressant drug discovery.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003ePC12 cells were purchased from the Kunming Cell Bank, Chinese Academy of Sciences. S-type Ginsenoside Rh2 (Batch No.: YZ-111748) was obtained from the National Institutes for Food and Drug Control (China). L-Glutamic acid solution (Batch No.: CB1321) was from Procell (Beijing). SU11248 (Batch No.: A157426) and LY294002 (Batch No.: A133122) were purchased from Shanghai Bide Pharmatech. Reactive Oxygen Species Assay Kit (Batch No.: S0033S), Fluo-4 AM Calcium Assay Kit (Batch No.: S1061S), JC-1 Mitochondrial Membrane Potential Assay Kit (Batch No.: C2006), and VEGF Rabbit Monoclonal Antibody (Batch No.: AF1309) were sourced from Beyotime Biotechnology (Shanghai). Antibodies against PI3K p85α (CY5355), p-PI3K p85α (Y607) (CY6427), Akt (CY5561), p-Akt (S473) (CY6569), mTOR (CY5306), p-mTOR (S2448) (CY6571), Synaptophysin (SYN, CY5245), PSD-95 (DLG4, CY5407), Bcl-2 (CY6717), Bax (CY5059), and Goat Anti-Rabbit IgG HRP (F300405) were from Abways Technology. Caspase-3 (CY5384) and β-actin (AC028) antibodies were from ABclonal. GAP43 Mouse mAb (bsm-33192M) was from Bioss (Beijing). Neurogranin (Ng) Polyclonal antibody (10440-1-AP) was from Proteintech.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInstruments\u003c/h3\u003e\n\u003cp\u003eCell culture was performed in a Thermo Scientific CO\u003csub\u003e2\u003c/sub\u003e incubator. Absorbance for cell viability was measured using a BioTek Cytation5 microplate reader. Imaging was conducted with an OLYMPUS FV3000 confocal laser scanning microscope. Centrifugation used a Sigma high-speed refrigerated centrifuge. Protein bands were visualized with a GE Amersham Imager 600. Routine cell observation used a Nikon inverted microscope.\u003c/p\u003e\n\u003ch3\u003eCell Culture\u003c/h3\u003e\n\u003cp\u003eRat pheochromocytoma (PC12) cells (highly differentiated) were cultured in RPMI-1640 complete medium supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin at 37\u0026deg;C in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere. The medium was replaced every two days. Upon reaching 70\u0026ndash;80% confluence, cells were passaged using 0.25% trypsin. All experiments were performed using cells in the logarithmic growth phase.\u003c/p\u003e\n\u003ch3\u003eCell Viability Assay (CCK-8)\u003c/h3\u003e\n\u003cp\u003eLog-phase PC12 cells were seeded into 96-well plates at a density of 8\u0026times;10\u0026sup3; cells/well in 100 \u0026micro;L complete medium and cultured for 24 h for attachment.\u003c/p\u003e \u003cp\u003eGlu Modeling Concentration Screening: Cells were treated with 6.5, 7.5, 8.5, 9.5, or 10.5 mmol/L Glu for 24 h, alongside normal control and blank (medium-only) groups.\u003c/p\u003e \u003cp\u003eGRh2 Treatment Concentration Screening: Cells were treated with 1, 4, 8, 12, 16, or 20 \u0026micro;mol/L GRh2.\u003c/p\u003e \u003cp\u003eInhibitor Validation: The following groups were established: (i) Normal control; (ii) Model (8.5 mmol/L Glu); (iii) GRh2 protection (8.5 mmol/L Glu\u0026thinsp;+\u0026thinsp;12 \u0026micro;mol/L GRh2); (iv) Pathway blockade 1 (8.5 mmol/L Glu\u0026thinsp;+\u0026thinsp;12 \u0026micro;mol/L GRh2\u0026thinsp;+\u0026thinsp;0.4 \u0026micro;mol/L SU11248); (v) Pathway blockade 2 (8.5 mmol/L Glu\u0026thinsp;+\u0026thinsp;12 \u0026micro;mol/L GRh2\u0026thinsp;+\u0026thinsp;2.5 \u0026micro;mol/L LY294002).After treatments, 10 \u0026micro;L of CCK-8 solution was added to each well, followed by incubation for 2\u0026ndash;4 h. Absorbance was measured at 450 nm. Cell survival rate was calculated as: Cell Viability = [(As-Ab)/ (Ac-Ab)]\u0026times;100%. As is the absorbance of the experimental well (containing cells, culture medium, CCK-8, and different concentrations of the drug); Ac is the absorbance of the control well (containing cells, culture medium, and CCK-8); Ab is the absorbance of the blank well (containing culture medium and CCK-8).\u003c/p\u003e\n\u003ch3\u003eIntracellular Ca²⁺ Measurement (Fluo-4 AM)\u003c/h3\u003e\n\u003cp\u003eCells were seeded into confocal dishes (1\u0026times;10⁵ cells/dish) and treated as per the inhibitor validation groups after 24 h. Post-treatment, cells were processed according to the Fluo-4 AM kit instructions. Fluorescence images were captured using a confocal microscope and analyzed with ImageJ software.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ROS Measurement (DCFH-DA)\u003c/h2\u003e \u003cp\u003eROS levels were detected using the DCFH-DA kit per the manufacturer's protocol. Fluorescence was observed and quantified via confocal microscopy and ImageJ.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMitochondrial Membrane Potential (ΔΨm) Assay (JC-1)\u003c/h3\u003e\n\u003cp\u003eThe JC-1 assay was performed as per the kit instructions. Cells were immediately observed under a confocal microscope. The ratio of JC-1 aggregates (red fluorescence) to monomers (green fluorescence) was calculated using ImageJ to assess ΔΨm.\u003c/p\u003e\n\u003ch3\u003eImmunofluorescence Staining for Ng and Nm\u003c/h3\u003e\n\u003cp\u003ePost-treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA. Cells were incubated with primary antibodies (Rabbit anti-Ng, 1:500; Mouse anti-Nm, 1:500) overnight at 4\u0026deg;C, followed by appropriate fluorescently-labeled secondary antibodies (e.g., Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 594-conjugated goat anti-mouse IgG) for 1 h at room temperature in the dark. Nuclei were counterstained with DAPI-containing mounting medium. Images were acquired using a confocal microscope.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eTotal protein was extracted using RIPA lysis buffer and quantified via the BCA method. Equal amounts of protein were separated by SDS-PAGE and transferred to PVDF membranes. After blocking with 5% non-fat milk, membranes were incubated overnight at 4\u0026deg;C with specific primary antibodies: p-PI3K (1:1000), PI3K (1:1000), p-Akt (1:2000), Akt (1:2000), p-mTOR (1:1000), mTOR (1:1000), VEGF (1:1000), PSD-95 (1:1000), SYN (1:1000), Bax (1:1000), Bcl-2 (1:1000), Caspase-3 (1:1000), β-actin (1:5000). Membranes were then incubated with HRP-conjugated secondary antibodies. Protein bands were visualized using an ECL kit, and band density was quantified with ImageJ software, normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll experiments were independently repeated at least three times. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using GraphPad Prism 9.5.0. Data passed normality and homogeneity of variance tests. Comparisons among multiple groups were analyzed by one-way ANOVA, followed by \u003cem\u003eDunnett's t-test\u003c/em\u003e for post-hoc comparisons against specific control groups. A \u003cem\u003eP\u003c/em\u003e-value\u0026thinsp;\u003cem\u003e\u0026lt;\u003c/em\u003e\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of Glu-induced PC12 Cell Injury Model and Determination of GRh2 Protective Concentration\u003c/h2\u003e \u003cp\u003eTo establish an in vitro depression model, the effect of different Glu concentrations on PC12 cell viability was assessed. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, treatment with 6.5 to 10.5 mmol/L Glu for 24 h significantly inhibited cell viability compared to the control group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). Since 8.5 mmol/L Glu reduced cell viability to (55.87\u0026thinsp;\u0026plusmn;\u0026thinsp;2.42)%, this concentration was selected for subsequent modeling.\u003c/p\u003e \u003cp\u003eThe protective effect of GRh2 was then evaluated. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, compared to the model group (viability reduced to (51.51\u0026thinsp;\u0026plusmn;\u0026thinsp;3.06)%), 1\u0026ndash;20 \u0026micro;mol/L GRh2 concentration-dependently reversed the Glu-induced decrease in cell viability (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). Based on cell viability and preliminary results, 12 \u0026micro;mol/L GRh2 was chosen as the optimal concentration for subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGRh2 Antagonizes Glu-induced Cytotoxicity via the VEGF/PI3K/Akt Pathway\u003c/h2\u003e \u003cp\u003eTo investigate whether GRh2's protection depends on the VEGF/PI3K/Akt pathway, pathway inhibitors were employed. CCK-8 results (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) showed that co-treatment with the VEGF inhibitor SU11248 (0.4 \u0026micro;mol/L) or the PI3K inhibitor LY294002 (2.5 \u0026micro;mol/L) significantly attenuated the protective effect of GRh2 on cell viability (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). This preliminarily confirms that GRh2's protective effect relies on the VEGF-mediated PI3K/Akt signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eGRh2 Alleviates Glu-induced Oxidative Stress and Calcium Overload\u003c/h2\u003e \u003cp\u003eThe role of GRh2 in mitigating key excitotoxicity events was further explored.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eIntracellular Ca\u0026sup2;⁺\u003c/strong\u003e \u003cp\u003eFluo-4 AM staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) revealed a significant increase in Ca\u0026sup2;⁺ fluorescence intensity in the Glu model group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01), which was effectively reduced by GRh2 treatment (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). Both SU11248 and LY294002 reversed GRh2's effect (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01), with LY294002 showing a stronger inhibitory effect (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05), suggesting the PI3K/Akt pathway plays a central role downstream of VEGF in regulating calcium homeostasis.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eROS Levels: DCFH-DA probe results (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) were consistent with Ca\u0026sup2;⁺ findings. Glu significantly increased intracellular ROS levels (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01), while GRh2 significantly scavenged ROS (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). This effect was also blocked by both inhibitors (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eMitochondrial Membrane Potential: JC-1 staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e) indicated that Glu significantly decreased ΔΨm (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas GRh2 effectively restored it (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). SU11248 and LY294002 inhibited GRh2's protective effect on mitochondria (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eGRh2 Improves Synaptic Plasticity after Glu Injury\u003c/h2\u003e \u003cp\u003eImmunofluorescence observation and quantification (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e) showed that Glu injury caused shortened neurites, loose connections, and significantly reduced levels of Neurogranin (Ng) and Neuromodulin (Nm) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). GRh2 treatment significantly reversed these morphological and molecular abnormalities (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The SU11248 and LY294002 treatment groups exhibited phenotypes similar to the Glu injury group, with Ng and Nm levels significantly lower than the GRh2 protection group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). This indicates that GRh2 improves synaptic plasticity via the VEGF/PI3K/Akt pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eGRh2 Activates the VEGF/PI3K/Akt/mTOR Pathway and Regulates Downstream Targets\u003c/h2\u003e \u003cp\u003eWestern blot analysis was conducted to confirm the mechanism at the molecular level.\u003c/p\u003e \u003cp\u003ePathway Activation: As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e, compared to the control group, protein levels of VEGF, p-PI3K, p-Akt, and p-mTOR were significantly downregulated in the Glu model group (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). GRh2 treatment significantly increased the phosphorylation levels or expression of these proteins (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05). The activation of this pathway was effectively inhibited by SU11248 or LY294002.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSynaptic-related Proteins: Detection of key synaptic plasticity proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) revealed that GRh2 significantly reversed the Glu-induced downregulation of PSD-95 and SYN expression (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05), effects that were blocked by pathway inhibitors.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eApoptosis-related Proteins: As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, GRh2 treatment upregulated the anti-apoptotic protein Bcl-2 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01) and downregulated the pro-apoptotic proteins Bax and Caspase-3 (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05), shifting the Bcl-2/Bax ratio towards anti-apoptosis. Similarly, pathway inhibitors abolished the anti-apoptotic effects of GRh2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study demonstrates that Ginsenoside Rh2 significantly alleviates glutamate-induced damage in PC12 cells. The protective mechanism is closely associated with the activation of the VEGF/PI3K/Akt/mTOR signaling pathway, leading to improved mitochondrial function, inhibition of oxidative stress and apoptosis, and enhanced synaptic plasticity.\u003c/p\u003e \u003cp\u003eGlutamate excitotoxicity is a core pathological mechanism in depression and other neuropsychiatric disorders. NMDA receptor overactivation induces calcium overload, mitochondrial dysfunction, excessive ROS production[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and ultimately neuronal apoptosis via Bcl-2/Bax imbalance and Caspase-3 activation[\u003cspan additionalcitationids=\"CR27 CR28\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Our results show that GRh2 effectively restores ΔΨm, reduces ROS levels, and modulates the Bcl-2/Bax ratio and Caspase-3 expression, confirming its neuroprotection via mitigating oxidative stress and mitochondria-dependent apoptosis(Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eVEGF, a key neurotrophic factor, plays an increasingly recognized role in depression pathophysiology. Studies show significantly reduced VEGF levels in the hippocampus of depressive models and in depressed patients[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], while fast-acting antidepressants like ketamine exert effects by upregulating VEGF[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistent with these findings, our results indicate that GRh2 treatment significantly elevates VEGF expression in PC12 cells, identifying VEGF as a critical upstream mediator of its antidepressant action.\u003c/p\u003e \u003cp\u003eVEGF's neuroprotective effects largely depend on downstream PI3K/Akt pathway activation [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This study proves that GRh2, while increasing VEGF expression, also significantly enhances the phosphorylation of PI3K, Akt, and the pivotal hub molecule mTOR. This aligns with reports that various antidepressants activate this pathway in cellular and animal models[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. To establish the causal role of the VEGF/PI3K/Akt/mTOR axis, we employed the inhibitors SU11248 and LY294002. Results demonstrated that both inhibitors effectively reversed GRh2's protection of cell viability, its suppression of oxidative stress, its restoration of ΔΨm, and concurrently blocked pathway activation. This key evidence strongly affirms that GRh2's effects are initiated by VEGF and transmitted through the PI3K/Akt/mTOR pathway.\u003c/p\u003e \u003cp\u003eThe activated PI3K/Akt/mTOR pathway synergistically improves neuronal health through multiple downstream mechanisms. Firstly, it enhances synaptic plasticity by promoting the synthesis of proteins like PSD-95 and SYN via mTOR[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Secondly, it inhibits apoptosis by regulating the Bcl-2 family protein balance and suppressing Caspase-3 activity[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e). Our study observed that GRh2 treatment upregulated PSD-95, SYN, and Bcl-2, while downregulating Bax and Caspase-3. Furthermore, fluorescence tracing of Ng and Nm visually confirmed that GRh2 reverses Glu-induced synaptic atrophy and sparse connectivity, an effect also blocked by SU11248 and LY294002. These results collectively illustrate that GRh2, via the core VEGF/PI3K/Akt/mTOR pathway, simultaneously improves synaptic plasticity and cell survival.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn summary, this study systematically elucidates the effects and mechanisms of GRh2 in alleviating glutamate excitotoxicity in a cellular model. We propose and validate for the first time that GRh2 upregulates VEGF, subsequently activating the PI3K/Akt/mTOR signaling pathway, thereby inhibiting oxidative stress and apoptosis while promoting synaptic reconstruction. These findings provide novel molecular insights into the antidepressant potential of GRh2.\u003c/p\u003e \u003cp\u003eA limitation of this study is that its conclusions are based solely on a cell model. Future research should validate GRh2's efficacy and mechanism in animal models of depression and explore the upstream signaling events regulating VEGF expression. These investigations will provide a more robust theoretical foundation for developing GRh2 as a novel, multi-target antidepressant candidate drug.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eConflict of Interest\u003c/strong\u003e \u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis work was supported by the Natural Science Foundation of Hebei Province (Grant No. H2025405005); the Natural Science Research Program of Hebei Provincial Department of Education for Basic Scientific Research Operating Funds of Provincial Universities (Grant No. JYT2024020); the Scientific Research Project of Hebei Provincial Health Department (Grant No. 20160031); and the Hebei North University Institutional Scientific Research Project (Grant No. XJ2023034).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eChun-Yue Zhang: Conceptualization, Methodology, Investigation, Formal analysis, Writing-Original Draft. Chang Liu: Methodology, Data curation, Validation, Visualization, Writing-Original Draft. Liang-Jing Liu: Investigation, Resources, Project administration. Jian-Ming Yang: Software, Validation, Formal analysis. Li-Xia Shen: Funding acquisition. Zhi-Gang Wu: Supervision, Funding acquisition, Writing-Review \u0026amp; Editing.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article and its supplementary materials.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGaebel W, Stricker J, Kerst A (2020) Changes from ICD-10 to ICD-11 and future directionsin psychiatric classification. Dialog Clin Neurosci 22:7\u0026ndash;15\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSowa-Kućma M, Stachowicz K (2025) Special issue: molecular research on depression. 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Front Cell Neurosci 19:1645437\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ginsenoside Rh2, depression, glutamate excitotoxicity, VEGF, PI3K/Akt/mTOR pathway","lastPublishedDoi":"10.21203/rs.3.rs-8880846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8880846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGinsenoside Rh2 (GRh2), a primary active constituent of red ginseng, demonstrates significant neuroprotection against glutamate-induced excitotoxic damage in differentiated PC12 cells, a model of depression. This study found that GRh2 concentration-dependently reversed the loss of cell viability caused by glutamate. It effectively mitigated key pathological events, including intracellular calcium overload, reactive oxygen species accumulation, and the collapse of mitochondrial membrane potential. Furthermore, GRh2 enhanced synaptic plasticity, as evidenced by improved neurite morphology and increased levels of the synaptic markers neurogranin and neuromodulin. Mechanistic investigations revealed that GRh2 upregulated vascular endothelial growth factor (VEGF) expression and subsequently activated the PI3K/Akt/mTOR signaling pathway. This activation led to increased expression of synaptic proteins (PSD-95 and synaptophysin), an elevated Bcl-2/Bax ratio. Critically, the specific VEGF inhibitor SU11248 and PI3K inhibitor LY294002 abolished all the protective effects of GRh2, confirming the indispensable role of the VEGF/PI3K/Akt/mTOR axis. These results indicate that GRh2 alleviates glutamate-induced neurotoxicity by activating the VEGF-mediated PI3K/Akt/mTOR pathway, thereby improving mitochondrial function, inhibiting oxidative stress and apoptosis, and promoting synaptic integrity. This work provides novel molecular insights into the antidepressant potential of GRh2.\u003c/p\u003e","manuscriptTitle":"Ginsenoside Rh2 Protects against Glutamate-Induced Neurotoxicity in PC12 Cells via Activation of the VEGF-Mediated PI3K/Akt/mTOR Signaling Pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-26 06:15:36","doi":"10.21203/rs.3.rs-8880846/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-03T20:01:09+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-02-26T19:02:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"248280126534938894368146647686191961979","date":"2026-02-24T16:02:17+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-23T22:52:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-17T18:38:41+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-16T07:04:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"Neurochemical Research","date":"2026-02-14T14:50:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"neurochemical-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"nere","sideBox":"Learn more about [Neurochemical Research](https://www.springer.com/journal/11064)","snPcode":"11064","submissionUrl":"https://submission.nature.com/new-submission/11064/3","title":"Neurochemical Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9441de59-13bf-4457-a4a0-dcc9e6e71517","owner":[],"postedDate":"February 26th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-20T17:39:28+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-26 06:15:36","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8880846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8880846","identity":"rs-8880846","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}