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. 2026 Jun 5;40(9):5357–5373. doi: 10.1002/ptr.70392

Ginsenoside Rg1, a Natural Lysosomal Enhancer, Alleviates Parkinson's Disease Pathology via Cathepsin D‐Dependent Regulation of α‐Synuclein Homeostasis

Ya‐nan Fei 1, Ming‐yang Hao 1, Jia‐qiang Liu 2, Yin‐song Dong 2, Hui‐ning Zou 2, Xiao‐Xi Guo 1, Su An 1, Jun Sang 1,✉, Tian‐Rui Xu 1,✉
PMCID: PMC13548971  PMID: 42248811

ABSTRACT

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by dopaminergic neuron loss and α‐synuclein (α‐syn) aggregation, often linked to lysosomal dysfunction. Cathepsin D (CTSD), a lysosomal hydrolase essential for α‐syn clearance, becomes functionally impaired when its maturation is disrupted, exacerbating proteostatic stress. This study investigated whether ginsenoside Rg1(Rg1) restores CTSD maturation and lysosomal function to mitigate PD pathology. MPTP‐induced zebrafish and mouse PD models, as well as MPP+‐treated SH‐SY5Y cells, animals and cells were treated with Rg1 at different concentrations. Motor behavior, dopaminergic neuron survival, α‐syn clearance, CTSD maturation, lysosomal activity, endoplasmic reticulum (ER) stress, oxidative stress, autophagic flux, and apoptosis were systematically evaluated. Rg1 improved locomotor performance and preserved dopaminergic neurons, promoted α‐syn clearance, and enhanced CTSD maturation in lysosomes. These effects coincided with reduced ER and oxidative stress, normalized autophagic flux, and decreased apoptosis. Rg1 functions as a natural lysosomal enhancer, restoring lysosome‐ER homeostasis and counteracting multiple pathogenic pathways in PD. The findings reveal a CTSD‐dependent regulatory axis in α‐syn homeostasis and highlight Rg1 as a promising multi‐target therapeutic candidate for PD.

Keywords: alpha‐synuclein, cathepsin D, endoplasmic reticulum stress, ginsenosides, Parkinson disease

Ginsenoside Rg1 enhances lysosomal α‐synuclein clearance and promotes neuronal survival by facilitating CTSD maturation.

graphic file with name PTR-40-5357-g003.webp

1. Introduction

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterized by motor dysfunction, dopaminergic neuron loss, and pathological α‐synuclein (α‐syn) aggregation (Morris et al. 2024; Cramb et al. 2023). Accumulating evidence indicates that impairments in protein degradation systems—specifically lysosomal dysfunction (Nixon and Rubinsztein 2024; Udayar et al. 2022) and endoplasmic reticulum (ER) stress—play pivotal roles in PD pathogenesis (Ren et al. 2021; Wang, Qu, et al. 2023; Kong et al. 2025). Critically, toxic α‐syn accumulation is established as a key driver of neuronal degeneration (Calabresi, Mechelli, et al. 2023; Calabresi, Di Lazzaro, et al. 2023; Magalhaes and Lashuel 2022).

Cathepsin D (CTSD) is a lysosome‐resident aspartic protease implicated in diverse physiological processes (Mijanovic et al. 2021), notably intracellular protein degradation (Di et al. 2021; Bunk et al. 2021). CTSD directly mediates α‐syn degradation (Bunk et al. 2021; Zhang et al. 2025), and its dysfunction exacerbates α‐syn aggregation and neurotoxicity in PD (Cullen et al. 2009). Synthesized as an inactive precursor (pro‐CTSD), it requires multi‐step proteolytic maturation for activation—a process essential for lysosomal hydrolytic function (Drobny et al. 2023; Sharma et al. 2023; Balta and Zunke 2022). Critically, impaired CTSD maturation in pathology drives accumulation of inactive pro‐CTSD, lysosomal proteostasis collapse, and exacerbated α‐syn pathology (Bunk et al. 2021; Muraleedharan and Vanderperre 2023; Hossain et al. 2021; Schulte et al. 2003; Li et al. 2016).

Ginsenoside Rg1(Rg1), a pharmacologically active saponin extracted from Panax notoginseng, has been widely documented to confer neuroprotection against central nervous system disorders through its anti‐inflammatory, antioxidant, and anti‐apoptotic properties (Yang et al. 2023; Sun et al. 2022; Wu et al. 2022; Wang, Yang, et al. 2023). Previous studies have demonstrated that Rg1 attenuates neuronal apoptosis, reduces oxidative stress, and modulates neuroinflammation through pathways such as PI3K/Akt, Nrf2, and MAPK signaling. In models of PD, Rg1 has been reported to cross the blood–brain barrier and exert neuroprotective effects in various central nervous system disorders (Yang et al. 2022; Tian et al. 2023); although prior studies indicate that Rg1 modulates cellular stress responses, its role in restoring lysosomal function via regulating CTSD maturation remains unexplored.

Therefore, this study aimed to determine whether Rg1 enhances CTSD maturation and enzymatic activity, thereby reestablishing lysosomal‐ER homeostasis and facilitating α‐syn clearance and attenuating neurodegeneration in PD.

2. Materials and Methods

2.1. Reagents and Antibodies

Ginsenoside Rg1 (purity ≥ 98%) was generously supplied by Yunnan Te'an Na Pharmaceutical Co. Ltd. (Yunnan, China). MPTP hydrochloride (purity ≥ 98%) was purchased from Beyotime Biotechnology (Shanghai, China). MPP+ was purchased from Macklin (purity ≥ 98%). Primary antibodies were obtained as follows: Anti‐Alpha‐synuclein (phospho S129) (Abcam, Cambridge, UK), anti‐Cathepsin D (CTSD), anti‐LAMP1, anti‐GAPDH, anti‐GRP78, and anti‐CHOP (all from Proteintech, Wuhan, China), anti‐LC3A/B (Selleck, USA)，anti‐α‐actinin (Santa Cruz Biotechnology, USA), anti‐α‐synuclein (Servicebio, Wuhan, China), and anti‐tyrosine hydroxylase (TH; Servicebio, Wuhan, China).

2.2. Zebrafish Model of Parkinson's Disease and Rg1 Treatment

Wild‐type Danio rerio embryos (AB strain, obtained from the China Zebrafish Resource Center, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China) were maintained under standard laboratory conditions at 28.5°C ± 0.5°C on a 14 h light/10 h dark cycle. Fertilized eggs were collected and raised in embryo medium (E3 solution, pH 7.2) supplemented with 0.003% 1‐phenyl‐2‐thiourea (PTU) to inhibit pigmentation. At 1 day post‐fertilization (dpf), embryos were randomly divided into experimental groups (n = 30 per group) and exposed to 50 μM MPTP for 5 consecutive days to establish a PD‐like phenotype. After MPTP treatment, larvae were incubated with Rg1 (50 or 100 μM, dissolved in embryo medium) for 24 h. Control larvae were treated with an equivalent volume of vehicle solution. Larval zebrafish locomotor activity was analyzed using a video‐tracking system. After adaptation, spontaneous swimming distance, velocity, and movement duration were recorded for 30 min in the light phase. All behavioral tests were scored by investigators blinded to treatment groups, and data were analyzed using appropriate statistical methods as described in the figure legends.

2.3. MPTP‐Induced Mouse Model of PD

Male C57BL/6J mice (10 weeks old, 22–25 g; Beijing SiPeiFu Biotechnology Co. Ltd., China) were randomly assigned to five groups (n = 12): control, MPTP, and MPTP plus Rg1 at 10, 20, or 60 mg/kg. Animals were housed under SPF conditions with ad libitum access to food and water. Mice received intraperitoneal injections of MPTP at 30 mg/kg/day for 7 consecutive days. Rg1 was administered intraperitoneally starting 3 days before the first MPTP injection, with treatment continued for a total of 10 days. Motor coordination and locomotor activity were evaluated using the open‐field test. In the open‐field test, total moving distance, average velocity, and trackplot were automatically recorded over 15 min using an video tracking system. Motor behavior was evaluated, and mice were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and perfused with saline, followed by 4% paraformaldehyde. Brains were collected for immunohistochemistry or rapidly dissected for protein analysis.

2.4. SH‐SY5Y Cell Culture and Treatment

Human SH‐SY5Y neuroblastoma cells [SHSY‐5Y, CL‐0208] were kindly provided by Procell Life Science & Technology Co. Ltd. (Wuhan, China). Cells were maintained in MEM/F12 medium supplemented with 20% fetal bovine serum (FBS) and 1% penicillin–streptomycin under standard conditions (37°C, 5% CO2, humidified incubator). Cells between passages 5–15 were used. To mimic PD‐related damage, cells were treated with 1 mM MPP+ for 24 h, followed by exposure to Rg1 at 0.5, 1, 5, or 10 μM for an additional 24 h.

2.5. RNA Sequencing and Bioinformatics Analysis

Total RNA was extracted from SH‐SY5Y cells using TRIzol reagent (Invitrogen, USA). RNA sequencing was performed on the Illumina platform. Differentially expressed genes (DEGs) were identified using the DESeq2 package. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using the cluster Profiler package. Gene Set Enrichment Analysis (GSEA) was applied to detect pathway‐level alterations.

2.6. Western Blot Analysis

For Western blot detection, total protein lysates were extracted from midbrain tissues using RIPA lysis buffer supplemented with protease and phosphatase inhibitors. Tissues were homogenized on ice, centrifuged at 12,000 rpm for 15 min at 4°C, and supernatants were collected for protein quantification. No subcellular fractionation (e.g., cytoplasmic, nuclear, or lysosomal enrichment) was performed in the present study; all Western blot analyses were conducted using whole tissue lysates. Proteins were extracted and quantified. Equal amounts (60 μg) of total protein were separated by SDS‐PAGE and transferred onto PVDF membranes. Membranes were blocked and incubated with primary antibodies at 4°C overnight, followed by HRP‐conjugated secondary antibodies. Bands were visualized by chemiluminescence, and band intensity was quantified using ImageJ software (NIH, USA).

2.7. Immunofluorescence and Confocal Imaging

Cells or cryosections of brain tissue were fixed, permeabilized, and incubated with primary antibodies against CTSD, α‐synuclein, followed by Alexa Fluor‐conjugated secondary antibodies. Nuclei were counterstained with DAPI. Images were acquired using a confocal microscope.

2.8. CTSD Enzymatic Activity Assay

CTSD activity was measured using a commercial fluorometric assay kit (Abcam, Cambridge, UK). Cell or tissue lysates were incubated with CTSD‐specific substrate, and fluorescence intensity was recorded at excitation/emission wavelengths of 328/460 nm to assess enzymatic activity.

2.9. ROS Measurement and Apoptosis Analysis

Intracellular reactive oxygen species (ROS) levels were detected using the DCFH‐DA fluorescent probe (ROS Detection Kit, Beyotime Biotechnology, China). Apoptotic cells were evaluated by Annexin V‐FITC/PI double staining using a kit purchased from Abbkine (Wuhan, China) and quantified by flow cytometry performed on an Agilent flow cytometer.

2.10. Quantitative Real‐Time PCR (qPCR)

Total RNA was extracted from SH‐SY5Y cells (or mouse midbrain tissue, if applicable) using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The concentration and purity of RNA were determined spectrophotometrically by measuring the absorbance at 260 and 280 nm. For cDNA synthesis, 1 μg of total RNA was reverse‐transcribed using the PrimeScript RT reagent kit (Takara, Shiga, Japan). Quantitative real‐time PCR was performed using SYBR Green Master Mix (Takara, Shiga, Japan) on a StepOnePlus Real‐Time PCR System (Applied Biosystems, Foster City, CA, USA). The thermal cycling conditions were as follows: initial denaturation at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. GAPDH served as the internal reference gene. All reactions were performed in triplicate to ensure reproducibility. The sequences of primers used for each gene are listed in Table S1.

2.11. Statistical Analysis

All data are presented as mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 9.0 software (GraphPad Software, San Diego, CA, USA). Comparisons between two groups were analyzed by unpaired Student's t‐test. For multiple group comparisons, one‐way analysis of variance (ANOVA) was applied followed by Tukey's post hoc test. Dunnett's T3 test was used when variances were not homogeneous. Differences were considered statistically significant at p < 0.05. Unless otherwise stated, all experiments were performed in triplicate.

2.12. Ethics Statement

All animal experiments were conducted in strict accordance with the relevant ethical policies and procedures, and were approved by the Institutional Animal Care and Use Committee (IACUC) of Kunming University of Science and Technology (protocol KMUST2019SK027).

3. Results

3.1. Ginsenoside Rg1 Alleviates Motor Deficits and Neurodegeneration in MPTP‐Treated Zebrafish Larvae

To investigate the neuroprotective effect of Rg1 in vivo, we established a zebrafish model of PD by treating 1 dpf larvae with 50 μM MPTP for 5 days. Rg1 was administered at concentrations of 50 and 100 μM for 24 h following MPTP exposure (Figure 1A).

FIGURE 1.

FIGURE 1

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Ginsenoside Rg1 alleviates motor deficits and neurodegeneration in MPTP‐treated zebrafish larvae. (A) Schematic illustration of the experimental design. Zebrafish larvae were exposed to MPTP to induce PD‐like phenotypes, followed by treatment with Rg1, resulting in improved motor function and reduced neurodegeneration. (B) Total locomotor distance of zebrafish larvae in each group. (C) Representative movement tracking trajectories. (D) Heatmap analysis of locomotor activity distribution. (E) Quantification of average swimming speed. (F) Representative histological images of zebrafish brain sections (H&E staining). (G) Quantification of the area percentage of positive staining. All quantitative data are presented as mean ± SEM (n = 20 larvae per group). Histological analysis was performed on three independent sections per group (n = 3). Statistical analysis was conducted using one‐way ANOVA followed by Tukey's post hoc test. Significance is indicated as follows: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Control group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. MPTP group.

First, we assessed locomotor behavior using a zebrafish tracking system. MPTP exposure significantly decreased total swimming distance and average velocity compared with control fish. Rg1 treatment dose‐dependently reversed these motor deficits, with 100 μM showing a more pronounced effect than 50 μM (Figure 1B–E). Quantitative analysis revealed that both doses significantly improved locomotor performance, with partial to full restoration compared to the control group. Second, to evaluate neuronal integrity, we performed Nissl staining on whole‐mount zebrafish brains. MPTP‐treated fish displayed substantial midbrain vacuolization and tissue disorganization, while Rg1 treatment markedly preserved midbrain morphology (Figure 1F). Quantification of Nissl‐positive area confirmed that Rg1 significantly attenuated MPTP‐induced neuronal loss (Figure 1G). Collectively, Rg1 exerts dose‐responsive neuroprotection in the zebrafish PD model, ameliorating motor dysfunction and mitigating midbrain neurodegeneration.

3.2. Structure Prediction and Transcriptomics Identify Cathepsin D (CTSD) as a Potential Functional Target of Ginsenoside Rg1

Rg1 contains a steroidal saponin backbone, which confers high protein‐binding potential (Yang et al. 2023, 2022). Using four independent in silico prediction platforms—PharmMapper (Liu et al. 2010; Wang et al. 2017), Galaxy Sagittarius‐AF (Kwon et al. 2024), AMIGO (Carbon et al. 2009), and Swiss Target Prediction (Daina et al. 2019; Gfeller et al. 2014)—we identified multiple candidate targets involved in lysosomal proteolysis and protein homeostasis (Figure 2A,B). CTSD, a lysosomal aspartic protease, consistently appeared across all platforms as a prominent candidate.

FIGURE 2.

FIGURE 2

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Target prediction and transcriptomics identify CTSD as a candidate target of ginsenoside Rg1. (A) Workflow illustrating the integrated analysis strategy combining target prediction and transcriptomic profiling to identify candidate targets of Rg1, highlighting CTSD as a potential key regulator. (B) Venn diagram showing the overlap of predicted targets from multiple databases (e.g., SwissTargetPrediction, PharmMapper, TargetNet, and others), identifying common candidate targets. (C) Venn diagram of differentially expressed genes (DEGs) across comparison groups (Control vs. MPP+ and MPP+ vs. Rg1), revealing shared and uniquely regulated genes. (D) Summary table of common candidate genes identified from the intersection analysis. CTSD was significantly downregulated in the MPTP group compared with control and upregulated following Rg1 treatment. (E) Heatmap of DEGs illustrating global gene expression patterns across experimental groups. (F) Gene Ontology (GO) enrichment analysis of cellular component (CC) terms for the identified targets. (G) Bubble plot of GO enrichment analysis showing significantly enriched biological functions and pathways, including endoplasmic reticulum membrane‐related processes. Differential expression analysis was performed using the DESeq2 package. GO enrichment analysis was conducted using the clusterProfiler package, and adjusted p‐values (p.adjust) were calculated using the Benjamini–Hochberg method. Data visualization was performed in R software.

Transcriptomic analysis was then performed in SH‐SY5Y cells exposed to MPP+, with or without Rg1 treatment. Pathway enrichment showed that DEGs (Soneson and Delorenzi 2013; Li et al. 2022; Gill and Dhillon 2022) were significantly associated with lysosome‐related and cathepsin family pathways (Figure 2C). Unsupervised clustering further confirmed transcriptomic divergence and cross‐analysis of DEGs with predicted targets confirmed CTSD as a converging hit, showing elevated expression after MPP+ exposure and attenuation upon Rg1 treatment (Figure 2D,E).

Importantly, independent validation from publicly available patient‐derived datasets supported these findings (Han et al. 2022). Analysis of RNA‐seq data from plasma samples (GSE160299) demonstrated that CTSD expression was significantly higher in PD patients compared with non‐PD controls (Figure S3). This concordance between patient samples and the SH‐SY5Y model strengthens the robustness of CTSD as a clinically relevant target.

GO analysis (Young et al. 2010) revealed prominent enrichment in “endoplasmic reticulum membrane” categories (Figure 2F,G), suggesting potential cross‐talk between lysosomal proteases and ER stress regulation (Qu et al. 2021). This observation was further reinforced by supplementary transcriptomic data. Differential gene volcano plots confirmed the robustness of expression changes across all comparisons (Figure S2A,B). GO enrichment analysis of both control vs. model and model vs. Rg1 groups again highlighted ER‐related categories, including the “endoplasmic reticulum membrane” (Figure S2C,D). Gene count distributions of enriched terms also showed strong alignment with this pathway (Figure S2E), consolidating the relevance of lysosome–ER homeostasis in the protective mechanism of Rg1.

3.3. Ginsenoside Rg1 Promotes CTSD Maturation and Restores Lysosomal Enzymatic Activity

Accumulation of misfolded proteins in PD is often linked to lysosomal dysfunction and impaired protease maturation (Du et al. 2020; Outeiro et al. 2008; Wang et al. 2012). Outline of the experimental hypothesis that was subsequently validated (Figure 3A). In our study, Western blot results revealed that MPP+ treatment led to a substantial elevation of pre‐pro‐CTSD (~52 kDa) (Oueslati 2016; Guardia‐Laguarta et al. 2014), the inactive precursor form of CTSD, indicating a block in its maturation cascade (Figure 3C–E). Rg1 treatment dose‐dependently decreased this accumulation, especially at 10 μM, suggesting its regulatory effect on CTSD processing.

FIGURE 3.

FIGURE 3

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Ginsenoside Rg1 promotes CTSD maturation and restores lysosomal enzymatic activity. (A) Schematic illustration of CTSD maturation. Pro‐cathepsin D is processed into intermediate and mature forms (heavy chain and light chain) within lysosomes, enabling its enzymatic activity. (B) CTSD enzymatic activity assay showing relative fluorescence intensity (RFI) in SH‐SY5Y cells treated with MPP+ and different concentrations of Rg1. (C) Quantification of pre‐ and pro‐CTSD expression levels. (D) Quantification of mature CTSD light chain levels. (E) Representative Western blot images showing the expression of LAMP1, pro‐CTSD, and CTSD light chain in SH‐SY5Y cells. β‐actin was used as a loading control. (F) LysoTracker staining showing lysosomal activity in control, MPP+, and Rg1‐treated groups, with corresponding bright‐field, merged images, and 3D fluorescence intensity plots. Scale bar = 100 μm. (G) Quantification of LAMP1 protein expression normalized to β‐actin. (H) Relative mRNA expression of CTSD determined by qPCR. All data are presented as mean ± SEM from three independent experiments (n = 3). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. Significance is indicated as follows: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Control group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. MPP+ group; ns, not significant.

In contrast, the mature light chain form (~14 kDa) (Hossain et al. 2021) was significantly reduced in the MPP+ group, and partially or fully restored by Rg1, as shown by protein band intensity and quantification. Consistently, LAMP1 protein expression, which was elevated following MPP+ exposure, was normalized by Rg1. This shift from inactive to active forms of CTSD supports the hypothesis that Rg1 promotes post‐translational maturation, a critical step for lysosomal proteolytic competence (Di et al. 2021).

Functionally, CTSD enzymatic activity, measured fluorometrically, was markedly impaired by MPP+ treatment but significantly rescued by Rg1 (Figure 3B), consistent with the restoration of its mature light chain form. qPCR analysis showed that CTSD mRNA expression was elevated in the MPP+ group but remained largely unchanged upon Rg1 treatment (Figure 3H), in line with transcriptomic data. Importantly, these findings indicate that Rg1 enhances CTSD function primarily through facilitating the post‐translational maturation of pre‐pro‐CTSD into its active form, rather than by further upregulating gene expression. The recovery of CTSD enzymatic activity consequently improves lysosomal proteolytic capacity, which is critical for the degradation and clearance of α‐synuclein aggregates, thereby contributing to the neuroprotective effect observed in Rg1‐treated cells (Bunk et al. 2021).

Lysotracker staining revealed enhanced red fluorescence in MPP + ‐treated cells, indicative of accumulated acidic vesicles and lysosomal burden. Rg1 treatment reduced this fluorescence to near‐control levels (Figure 3F), suggesting alleviation of lysosomal overload and restoration of lysosomal homeostasis. Mechanistically, these improvements in lysosomal function were accompanied by enhanced post‐translational maturation of pre‐pro‐CTSD into its active form (Figure 3C–G), resulting in increased CTSD enzymatic activity (Figure 3B). By promoting CTSD maturation, Rg1 directly augments lysosomal proteolytic capacity, thereby creating a cellular environment conducive to α‐syn clearance (detailed α‐syn data shown in Figure 4). Together, these findings support that Rg1 acts as a lysosomal enhancer through a CTSD‐dependent mechanism, linking enzyme maturation with restored lysosomal homeostasis and cellular proteostasis.

FIGURE 4.

FIGURE 4

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Ginsenoside Rg1 reduces α‐synuclein oligomerization in a CTSD‐dependent manner in MPP+‐treated SH‐SY5Y cells. (A) Schematic illustration of the proposed mechanism. MPP+ impairs CTSD enzymatic activity, leading to increased accumulation of α‐syn oligomers, whereas ginsenoside Rg1 enhances CTSD activity and promotes the clearance of α‐syn oligomers. (B) Relative mRNA expression of SNCA determined by qPCR in SH‐SY5Y cells following MPP+ and Rg1 treatment. (C) Representative Western blot images showing α‐syn oligomers (~100 kDa) and monomeric α‐syn (~19 kDa), with GAPDH as the loading control. (D) Quantitative analysis of α‐syn oligomer levels normalized to GAPDH. (E) Quantification of monomeric α‐syn (~19 kDa) expression normalized to GAPDH. (F) Immunofluorescence staining showing CTSD (green), α‐syn (red), and nuclei (DAPI, blue), along with corresponding 3D fluorescence intensity plots. Merged images illustrate the spatial distribution of CTSD and α‐syn across different treatment groups. Scale bar = 200 μm. All data are presented as mean ± SEM from three independent experiments (n = 3). For immunofluorescence analysis, at least three randomly selected fields per group were analyzed under identical imaging conditions. Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. Significance is indicated as follows: #p < 0.05, ##p < 0.01, ###p < 0.001 vs. Control group; *p < 0.05, **p < 0.01, ***p < 0.001 vs. MPP+ group; ns, not significant.

3.4. Ginsenoside Rg1 Promotes CTSD‐Mediated Clearance of α‐Synuclein Oligomers and Reduces Pathological Phosphorylation in PD Models

Oligomeric α‐syn accumulation represents a hallmark of PD pathology (Du et al. 2020; Outeiro et al. 2008). As illustrated in Figure 4A, an experimental hypothesis was proposed and subsequently validated using the approaches described below. Western blot analysis revealed a distinct elevation in ~100 kDa α‐syn oligomers following MPP+ treatment, indicating impaired clearance of toxic aggregates. Meanwhile, the level of 19 kDa monomeric α‐syn was also increased by MPP+. Rg1 administration (0.5–10 μM) dose‐dependently reduced both α‐syn oligomers and monomers, with 10 μM yielding the most significant effect (Figure 4C–E), consistent with enhanced lysosomal proteolytic degradation. At the transcriptional level, SNCA mRNA was elevated in MPP + ‐treated cells and modestly decreased by 10 μM Rg1 (Figure 4B), likely reflecting an adaptive response secondary to restored protein homeostasis rather than direct transcriptional repression. Collectively, these results support that Rg1 primarily promotes α‐syn clearance through CTSD‐dependent proteolysis, with mRNA changes being a downstream consequence of improved proteostasis.

Confocal imaging further demonstrated that MPP+ induced strong red fluorescence of α‐syn and p‐α‐syn, accompanied by increased CTSD signal in the cytoplasm. In contrast, Rg1‐treated cells exhibited markedly reduced α‐syn and p‐α‐syn signals, along with normalized CTSD levels (Figure 4F), highlighting that Rg1 facilitates CTSD maturation and enzymatic activation, thereby promoting α‐syn clearance. These findings support that Rg1 alleviates α‐syn pathology primarily through CTSD‐dependent proteolytic clearance, rather than by direct transcriptional regulation.

3.5. Ginsenoside Rg1 Restores Organelle Homeostasis by Alleviating ER Stress, Oxidative Damage, and Apoptosis

Rg1 exerts neuroprotection in PD models by promoting CTSD maturation, restoring lysosomal function, and alleviating lysosome‐driven ER stress and mitochondrial damage (Figure 5A). In PD, impaired processing of lysosomal proteases such as CTSD leads to α‐syn accumulation, activation of ER stress pathways, and subsequent neuronal apoptosis (Guardia‐Laguarta et al. 2014; Colla, Jensen, et al. 2012; Navarro‐Romero et al. 2020; Mazzulli et al. 2016). Here, integrated ultrastructural, biochemical, and functional analyses demonstrate that Rg1 coordinates multiple protective mechanisms, linking enhanced CTSD enzymatic activity with improved lysosomal proteolysis, reduced cellular stress, and neuronal survival.

FIGURE 5.

FIGURE 5

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Ginsenoside Rg1 alleviates ER stress, oxidative damage, and apoptosis by restoring mitochondrial and lysosomal homeostasis. (A) Schematic diagram of the proposed mechanism. Rg1 enhances CTSD enzymatic activity, reduces α‐syn oligomers, inhibits ER stress and UPR activation, thereby mitigating ROS production and apoptosis in MPP+‐treated SH‐SY5Y cells. (B) Representative Western blot images showing LC3B I (~16 kDa) and LC3B II (~14 kDa) expression, with GAPDH (~36 kDa) as the loading control. (C) Representative TEM images of SH‐SY5Y cells in different treatment groups. The boxed areas are magnified to show mitochondrial morphology. Red arrows: swollen mitochondria with disrupted cristae; blue arrows: normal mitochondria; green arrows: autophagosomes; purple arrows: lysosomes; black arrows: endoplasmic reticulum. (D) Quantitative analysis of the LC3B II/LC3B I ratio normalized to GAPDH. (E) Quantitative analysis of GRP78 expression normalized to GAPDH. (F) Quantitative analysis of CHOP expression normalized to α‐actin. (G) Representative Western blot images showing GRP78/BIP (~78 kDa), α‐actinin (~100 kDa), and CHOP (~30 kDa) expression, with GAPDH (~36 kDa) as the loading control. (H) Representative ER‐tracker immunofluorescence staining images showing ER (green) and nuclei (DAPI, blue). Scale bar = 200 μm. (I) Representative flow cytometry histograms of ROS levels in SH‐SY5Y cells. (J) Representative flow cytometry dot plots of apoptosis detected by Annexin V‐FITC/PI double staining. All data are presented as mean ± SEM from three independent experiments (n = 3). For TEM and immunofluorescence analyses, at least three randomly selected fields per group were analyzed under identical imaging conditions. Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. Significance: #p < 0.05, ##p < 0.01 vs. Control group; **p < 0.05, **p < 0.01, *p < 0.001 vs. MPP+ group; ns, not significant.

Transmission electron microscopy (TEM) revealed intact organelle structures in control SH‐SY5Y cells, with normal rough endoplasmic reticulum (blue arrows) and mitochondria exhibiting well‐preserved cristae and membrane integrity (red arrows). Occasional mild reduction in mitochondrial electron density was noted (pink arrows, Figure 5C). MPP+ exposure caused pronounced mitochondrial swelling and cristae disruption, accompanied by accumulation of autophagosomes and autolysosomes (green arrows), indicative of autophagic stress and lysosomal dysfunction. Mild dilation of the rough endoplasmic reticulum lumen (light blue arrows) further reflected ER stress. Rg1 treatment effectively restored mitochondrial morphology, decreased autophagosome abundance, and alleviated ER dilation, demonstrating its role in preserving organelle homeostasis under PD‐like conditions.

Western blot analysis revealed elevated LC3B‐II levels and increased LC3B‐II/I ratio in MPP+ treated cells, indicating autophagosome accumulation (Figure 5B,D). These changes were partially reversed by Rg1, consistent with improved autophagic flux, which is likely supported by the restoration of lysosomal proteolytic function via CTSD maturation. ER‐Tracker staining showed intense and aberrant ER distribution in the MPP+ group, while Rg1 restored ER structural clarity and perinuclear localization (Figure 5H). Correspondingly, ER stress markers GRP78 and CHOP were significantly elevated in the MPP+ group and suppressed by Rg1 treatment (Figure 5E–G). In addition, Rg1 reduced intracellular ROS accumulation (Figure 5I) and significantly attenuated MPP+ induced apoptosis, as indicated by a lower proportion of Annexin V‐positive cells in flow cytometry analysis (Figure 5J). Collectively, these findings indicate that Rg1 enhances lysosomal function and autophagic flux, thereby alleviating ER stress, oxidative damage, and neuronal apoptosis under PD‐like conditions.

It is noteworthy that endoplasmic reticulum (ER) stress does not occur in isolation but is tightly linked to disturbances in α‐syn metabolism (Ren et al. 2021; Mazzulli et al. 2016; Vekrellis et al. 2011; Gitler et al. 2008; Cooper et al. 2006). As shown in our previous results, MPP+ exposure impairs the proteolytic maturation of CTSD, leading to a decrease in its active form and consequently hindering the degradation of α‐syn oligomers and phosphorylated species, resulting in their pathological accumulation. Accumulated α‐syn can activate ER membrane stress sensors, thereby upregulating GRP78 and CHOP, increasing ROS production, inducing mitochondrial dysfunction, and ultimately promoting neuronal apoptosis (Colla 2019; Bellucci et al. 2011; Colla, Coune, et al. 2012). In the present study, Rg1 restored CTSD maturation and enzymatic activity, facilitating the clearance of α‐syn aggregates. This alleviation of α‐syn pathology correspondingly reduced ER stress and preserved mitochondrial integrity, effectively interrupting the pathological cascade. These findings underscore Rg1 as a multi‐target modulator, providing mechanistic insight into its neuroprotective potential in PD.

3.6. Ginsenoside Rg1 Improves Motor Deficits and Protects Dopaminergic Neurons in MPTP‐Induced PD Mouse Model

MPTP administration induced pronounced motor deficits in mice, as demonstrated by reduced climbing distance and speed in the pole test (Figure 6A–C). Rg1 treatment dose‐dependently improved motor performance, with the high‐dose group (60 mg/kg) showing effects comparable to levodopa. Representative locomotor track plots further illustrated this recovery: MPTP caused restricted and disorganized spontaneous movement, whereas high‐dose Rg1 restored broader and more coordinated locomotion (Figure 6D). Heatmap analysis revealed that Rg1 treatment redistributed activity from peripheral confinement back to a normal exploratory pattern (Figure 6I).

FIGURE 6.

FIGURE 6

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Ginsenoside Rg1 improves motor performance and preserves dopaminergic neurons in MPTP‐induced PD mice. (A) Schematic illustration of the mouse experimental workflow. (B) Climbing distance measured in the pole test. (C) Average speed measured in the open field test. (D) Representative track plots of locomotor activity in the open field test for each group. (E) Representative Western blot images showing TH (~62 kDa) expression in the substantia nigra of mice, with GAPDH (~36 kDa) as the loading control (n = 3). (F) Quantitative analysis of TH‐positive area fraction in the substantia nigra (n = 3). (G) Quantitative analysis of TH/GAPDH ratio normalized to the control group. (H) Representative immunohistochemical staining images of TH‐positive neurons in the substantia nigra of mice in different treatment groups. Scale bar = 500 μm. (I) Representative heatmaps of locomotor activity in the open field test for each group. All data are presented as mean ± SEM from three independent experiments (n = 8 per group). For immunohistochemical analysis, at least three randomly selected fields per mouse were analyzed under identical imaging conditions. Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. Significance is indicated as follows: ####p < 0.0001 vs. Control group; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs. MPTP model group; ns, not significant.

The integrity of dopaminergic neurons was assessed by examining tyrosine hydroxylase (TH) expression in the substantia nigra (Smeyne and Jackson‐Lewis 2005). Immunohistochemical analysis revealed significant TH loss in MPTP‐treated mice, which was markedly restored by Rg1 intervention (Figure 6F,H). Western blot analysis confirmed a dose‐dependent increase in TH protein levels following Rg1 treatment (Figure 6E,G). Together, these results indicate that Rg1 not only alleviates motor deficits but also preserves dopaminergic neuron integrity in the MPTP‐induced PD model.

3.7. Ginsenoside Rg1 Restores CTSD Activity and Alleviates α‐Synuclein Pathology and Neuroinflammation in MPTP‐Induced PD Mice

Consistent with cellular findings, CTSD enzymatic activity in the substantia nigra was significantly reduced in MPTP‐treated mice and dose‐dependently restored by Rg1 administration (LMH: 10, 20, 60 mg/kg; Figure 7A), highlighting its role in lysosomal functional recovery. Western blot analysis revealed decreased CTSD light chain expression and elevated GRP78 levels in the MPTP group, both of which were effectively reversed by Rg1 in a dose‐dependent manner (Figure 7B–F). Notably, GRP78 levels remained high in the levodopa group, suggesting that Rg1—but not levodopa—specifically contributes to ER stress mitigation. Consistent with these protein‐level changes, the mRNA expression levels of SNCA exhibited a similar trend.

FIGURE 7.

FIGURE 7

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Ginsenoside Rg1 restores CTSD enzymatic activity, facilitates α‐syn clearance, and attenuates neuroinflammation in the MPTP‐induced PD mouse model. (A) CTSD enzymatic activity in the substantia nigra of mice. (B) Representative Western blot images showing GRP78 (~78 kDa), Cathepsin D (CTSD) light chain (~14 kDa), and α‐Syn (~19 kDa) expression in the substantia nigra, with GAPDH (~36 kDa) as the loading control. (C) Quantitative analysis of CTSD light chain levels normalized to GAPDH. (D) Quantitative analysis of α‐Syn expression normalized to GAPDH. (E) Quantitative analysis of GRP78 expression normalized to GAPDH. (F) Relative mRNA expression of SNCA in the substantia nigra of MPTP‐induced PD mice following Rg1 treatment, determined by qPCR. (G) Representative immunofluorescence staining images of CTSD (green) and α‐Syn (red) in the substantia nigra of mice in different treatment groups, with DAPI (blue) for nuclear counterstaining. Merged images illustrate the co‐localization of CTSD and α‐Syn. Scale bar = 50 μm. Representative fluorescence intensity profiles of CTSD and α‐Syn along the indicated lines in the merged images. All data are presented as mean ± SEM from three independent experiments (n = 3 per group). For immunofluorescence analysis, at least three randomly selected fields per mouse were analyzed under identical imaging conditions. Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. Significance is indicated as follows: ####p < 0.0001, ###p < 0.001, ##p < 0.01, #p < 0.05 vs. Control group; ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05 vs. MPTP model group; ns, not significant.

Dual‐label immunofluorescence further confirmed the impaired lysosomal clearance in MPTP mice: α‐syn (red) and CTSD (green) signals were spatially separated, with intensified red fluorescence and poor co‐localization. In contrast, Rg1‐treated mice exhibited reduced α‐syn accumulation and enhanced co‐localization with CTSD, implying that functional recovery of CTSD may facilitate α‐syn degradation (Figure 7G). These results highlight the multifaceted neuroprotective effects of Rg1, including enhancement of lysosomal activity, mitigation of ER stress, suppression of neuroinflammation, and promotion of α‐syn clearance.

4. Discussion

PD is a progressive neurodegenerative disorder marked by dopaminergic neuronal loss, intracellular aggregation of α‐syn, and systemic disruption of cellular homeostasis, including lysosomal dysfunction, ER stress, oxidative damage, and neuroinflammation (Morris et al. 2024; Cramb et al. 2023; Ren et al. 2021; Gitler et al. 2008; Recchia et al. 2004; Vieira and Schapira 2022; Cali et al. 2011; Celardo et al. 2016). Among these intertwined pathologies, the failure of α‐syn clearance plays a pivotal role in disease progression and has been tightly linked to impaired lysosomal protease activity (Atik et al. 2016; Giraldez‐Perez et al. 2014; Koprich et al. 2017; Luk et al. 2012; Auluck et al. 2010). CTSD, one of the most abundant lysosomal hydrolases (Bunk et al. 2021), specifically degrades α‐syn aggregates, but its enzymatic activity depends on proteolytic maturation from a 52 kDa precursor to a functional 14 kDa light chain (Cullen et al. 2009). Defects in this maturation process, as reported in PD models, severely compromise its degradation function, yet pharmacological strategies targeting this bottleneck remain underexplored.

Here, we identify Rg1 as a natural lysosomal enhancer capable of promoting CTSD maturation and restoring proteolytic clearance of α‐syn in both cellular and animal PD models. Our transcriptomic profiling and bioinformatic predictions suggested CTSD as a potential downstream effector of Rg1. Consistently, Rg1 significantly increased the mature CTSD light chain and restored enzymatic activity, accompanied by marked reductions in α‐syn oligomers and phosphorylated pS129 forms. Co‐localization analysis showed that Rg1 also enhanced lysosomal targeting of CTSD, suggesting that its benefits extend beyond transcriptional rescue to functional subcellular localization and substrate engagement.

Moreover, Rg1 intervention suppressed GRP78 and CHOP expression, reversed ER expansion, reduced ROS accumulation, and inhibited apoptosis—all of which are downstream consequences of lysosomal dysfunction and α‐syn burden (Muraleedharan and Vanderperre 2023; Mazzulli et al. 2016; Navarro‐Romero et al. 2022). These findings support a unifying mechanism in which CTSD maturation defects trigger a cascade of α‐syn aggregation, ER stress, and mitochondrial damage, while Rg1 interrupts this axis by promoting the proteolytic processing of CTSD precursors, restoring enzymatic activity, and re‐establishing organellar homeostasis. Beyond the individual effects described above, our data further reveal a cooperative interplay among CTSD maturation, ER‐lysosome communication, and autophagic flux. Specifically, Rg1 not only enhanced CTSD processing but also alleviated ER stress while simultaneously promoting TFEB nuclear translocation and lysosomal biogenesis, thereby coupling ER homeostasis with lysosomal proteolytic capacity. Concurrently, autophagic flux assays demonstrated that Rg1 restored the clearance of autophagic substrates, indicating that the compound facilitates the delivery of α‐syn aggregates to mature, CTSD‐competent lysosomes. We therefore propose a tripartite model: Rg1 reduces the synthesis and misfolding burden of α‐syn via ER stress relief, while concurrently accelerating the degradation of pre‐existing aggregates through enhanced CTSD activity and autophagic‐lysosomal fusion. This “upstream reduction—downstream enhancement” framework provides a more integrated understanding of how Rg1 reinstates α‐syn proteostasis.

The ability of Rg1 to exert direct neuroprotection relies on its effective delivery across the blood–brain barrier. Accumulating evidence from pharmacokinetic studies has demonstrated that Rg1 can penetrate the blood‐brain barrier and distribute into brain parenchyma (Xue et al. 2016). Following intravenous or intraperitoneal administration, Rg1 or its metabolites have been detected in multiple brain regions, including the cortex, hippocampus, and substantia nigra, at concentrations sufficient for biological activity (Guo et al. 2014; Zhao et al. 2018). Although the absolute oral bioavailability of Rg1 is relatively low, repeated or appropriately dosed regimens achieve pharmacologically relevant brain levels. These pharmacokinetic characteristics strongly support the translational relevance of our mechanistic findings and underscore that Rg1 can directly act on central neurons to promote CTSD‐dependent α‐syn clearance (Wei et al. 2021; Wang et al. 2025).

In vivo, Rg1 improved motor deficits, preserved TH‐positive neurons, and ameliorated pathological alterations in CTSD, GRP78, and α‐syn, confirming its neuroprotective efficacy. While previous studies have focused on Rg1's antioxidative or PI3K/Akt‐related effects (Muraleedharan and Vanderperre 2023; Xu et al. 2020; Mohanan et al. 2018), our findings provide the first evidence of its ability to modulate lysosomal protease maturation and proteostasis. Importantly, although CTSD emerged as a major downstream mediator of Rg1's effects, whether this regulation is achieved through direct molecular interaction or indirect modulation via upstream pathways remains unresolved. Future studies should investigate potential direct interactions via molecular docking or CTSD knockdown/overexpression, evaluate effects on other cathepsins, and explore impacts on α‐syn spreading and neuroinflammatory pathways, as well as strategies to optimize central nervous system delivery and bioavailability.

Several limitations of the present study should be acknowledged. First, the SH‐SY5Y cell line, while widely used in PD research, does not fully recapitulate the physiological characteristics of primary dopaminergic neurons, such as their full tyrosine hydroxylase expression profile, electrophysiological properties, and selective vulnerability to mitochondrial toxins. Therefore, key conclusions regarding the CTSD/autophagy/α‐syn regulatory axis derived from SH‐SY5Y cells warrant further validation in more physiologically relevant models. Future studies should prioritize the use of primary dopaminergic neurons or patient‐derived iPSC‐induced dopaminergic neurons to confirm whether Rg1 exerts a similarly synergistic pro‐clearance effect in a more authentic neuronal environment, which would substantially enhance the clinical translational value of our findings. Second, the current study primarily focused on short‐term effects and acute mechanisms; the long‐term efficacy and durability of Rg1‐mediated neuroprotection remain unknown. Whether chronic Rg1 administration can sustain lysosomal function, delay α‐syn pathology progression, or exhibit rebound effects after withdrawal requires longitudinal in vivo studies. In addition, future investigations should evaluate potential direct interactions via molecular docking or CTSD knockdown/overexpression, assess effects on other cathepsins, and explore impacts on α‐syn spreading and neuroinflammatory pathways, as well as strategies to further optimize central nervous system delivery and bioavailability.

In summary, Rg1 exerts neuroprotective effects by promoting CTSD maturation, enhancing lysosomal clearance of α‐syn, and alleviating ER stress and apoptosis. To our knowledge, this work provides the first evidence that pharmacological modulation of lysosomal protease maturation can restore degradative capacity in PD models, highlighting CTSD reactivation as a novel and promising therapeutic strategy for α‐syn‐associated neurodegenerative disorders.

5. Conclusion

This study demonstrates that Rg1 confers neuroprotection by promoting the proteolytic maturation of CTSD, thereby restoring lysosomal enzymatic activity and facilitating the clearance of aggregated α‐syn. By enhancing CTSD function, Rg1 alleviated lysosomal dysfunction, ER stress, and apoptosis in both cellular and animal PD models, ultimately improving motor performance and preserving dopaminergic neurons. Mechanistically, Rg1 facilitated the conversion of pro‐CTSD into its mature form and promoted the co‐localization of mature CTSD with α‐syn, accelerating the proteolytic degradation of toxic aggregates.

In conclusion, CTSD reactivation represents the central mechanism underlying Rg1's therapeutic effects, and lysosomal enzyme maturation is proposed as a novel and promising strategy for the treatment of α‐syn–associated neurodegenerative diseases. This study provides a mechanistic framework for PD intervention and highlights CTSD as a potential pharmacological entry point for drug development.

Author Contributions

Ya‐nan Fei: writing – review and editing, conceptualization, methodology, writing – original draft, data curation, formal analysis, investigation. Tian‐Rui Xu: writing – review and editing, project administration, supervision, resources. Ming‐yang Hao: software, visualization. Su An: funding acquisition. Jia‐qiang Liu: validation. Jun Sang: writing – review and editing. Xiao‐Xi Guo: project administration. Hui‐ning Zou: project administration. Yin‐song Dong: project administration.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 32160157, 82160564, 81960659]; the Major Science and Technology Project of Yunnan Province [grant number 202202AG050008]; and the Cultivating Plan Program for the Leader in Science and Technology of Yunnan Province (Yunling Scholar Project) [grant number CA24129L025A].

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Table S1: Primer sequences used for qPCR analysis. All primers were designed based on human mRNA sequences ( Homo sapiens ). GAPDH was used as the internal control. Amplicon sizes were predicted using Primer‐BLAST and confirmed by melting curve analysis.

Figure S1: Dose‐ranging evaluation of Rg1 in the zebrafish MPTP model. Zebrafish larvae were divided into control, MPTP, and Rg1‐treated groups (10, 25, 50, 100, 200, and 500 μM). (A) Representative movement heatmaps. (B) Total distance traveled at 24 h after treatment. (C) Representative trajectory tracking plots. (D) Average swimming speed at 24 h after treatment. (E) Mortality rates at 24 h and 48 h after treatment. Rg1 improved MPTP‐induced behavioral deficits across all tested concentrations, with the most pronounced effect observed at 100 μM. Significant improvements in average speed were detected at 50 μM and 100 μM. However, higher concentrations (200 and 500 μM) were associated with markedly increased mortality at 48 h. Data are presented as mean ± SEM (n = 30). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. #p < 0.05 vs. Control group; *p < 0.05, **p < 0.01 vs. MPTP group. ns, not significant.

Figure S2: Transcriptomic features highlighting ER membrane‐related pathway enrichment. (A, B). Volcano plots of DEGs in pairwise comparisons. (C, D). GO enrichment bubble plots highlighting the “endoplasmic reticulum membrane” category. (E, F). Gene count bar plots in ER membrane‐associated GO terms.

Figure S3: Transcriptomic validation of CTSD expression in plasma samples from PD patients and non‐PD controls (GSE160299). (A) Volcano plot showing differentially expressed genes (DEGs) between Parkinson's disease (PD) patients and non‐PD controls (NC). Upregulated genes are highlighted in red, and downregulated genes in blue. (B) Quantification of CTSD expression levels derived from RNA‐seq data, indicating higher CTSD expression in PD plasma compared with NC. (C) Boxplot representation of CTSD expression distribution across groups. (D) Mean–difference (MA) plot illustrating global transcriptomic differences between PD and NC groups. (E) Expression values of CTSD presented as normalized counts, showing elevation in PD compared to NC. (F) Venn diagram comparing gene sets from NC and PD groups, highlighting CTSD among overlapping genes. Data were processed using DESeq2 with Benjamini–Hochberg correction.

Figure S4: Determination of optimal concentration range and cytotoxicity profile of ginsenoside Rg1 in SH‐SY5Y cells. (A) Effects of ginsenoside Rg1 on SH‐SY5Y cell viability at different concentrations (0–1000 μM) following 24, 48, and 72 h treatment. Cell viability was measured to evaluate the dose‐ and time‐dependent effects of Rg1. (B) Cell viability of SH‐SY5Y cells treated with various concentrations of Rg1 (0–50 μM) in the absence or presence of MPP+ (Model+Rg1) for 48 h. (C) Relative mRNA expression of CHOP in SH‐SY5Y cells co‐treated with MPP+ and gradient concentrations of Rg1 (0.5–10 μM) for 48 h, determined by qPCR. (D) Mean ROS intensity (FITC) in SH‐SY5Y cells treated with MPP+ alone or combined with Rg1 (10 μM) for 48 h. (E) Apoptosis (%) of SH‐SY5Y cells treated with MPP+ alone or combined with Rg1 (10 μM) for 48 h. All data are presented as mean ± SEM from three independent experiments (n = 3). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test.

Figure S5: Rg1 restores CTSD expression and improves locomotor deficits in rotenone‐induced cellular and zebrafish models. (A, B) Quantitative analysis of pre/pro‐cathepsin D (CTSD) and CTSD light chain protein levels in rotenone‐induced SH‐SY5Y cells treated with different concentrations of ginsenoside Rg1 (0.5–10 μM). (C) Representative Western blot images showing pre/pro‐cathepsin D, β‐actin, and CTSD light chain expression in SH‐SY5Y cells following rotenone exposure and Rg1 treatment (n = 3). (D) Representative locomotor trajectories of zebrafish larvae under different treatment conditions, including Control, DMSO, rotenone, and rotenone combined with Rg1 (50 and 100 μM). (E) Quantification of total swimming distance in zebrafish larvae across different treatment groups. (F) Heatmap representation of locomotor activity in zebrafish larvae. Warmer colors indicate higher activity levels. (G) Quantification of average swimming speed in zebrafish larvae. Data are presented as mean ± SEM (n = 30). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. #p < 0.05, ###p < 0.001 vs. dmso group; *p < 0.05, **p < 0.01, ****p < 0.0001 vs. rotenone‐treated group; ns, not significant.

Figure S6: Evaluation of the safety profile of ginsenoside Rg1 in the MPTP‐induced mouse model. (A) Mortality rate of mice in each experimental group, including Control, Model, Ginsenoside Rg1 (low, medium, and high doses), and levodopa‐treated groups. (B) Body weight changes before and after treatment in each group. Body weight was recorded to assess the systemic safety and tolerability of Rg1 administration in vivo. Data are presented as mean ± SEM (n = 12). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test.

PTR-40-5357-s001.pdf (4.1MB, pdf)

Acknowledgments

The authors gratefully acknowledge the support and technical assistance provided by the University‐Based Provincial Key Laboratory of Screening and Utilization of Targeted Drugs of Yunnan. We also thank our colleagues for their valuable discussions and contributions to this research.

Contributor Information

Jun Sang, Email: junsang@kust.edu.cn.

Tian‐Rui Xu, Email: tianruixu@kmust.edu.cn.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1: Primer sequences used for qPCR analysis. All primers were designed based on human mRNA sequences ( Homo sapiens ). GAPDH was used as the internal control. Amplicon sizes were predicted using Primer‐BLAST and confirmed by melting curve analysis.

Figure S1: Dose‐ranging evaluation of Rg1 in the zebrafish MPTP model. Zebrafish larvae were divided into control, MPTP, and Rg1‐treated groups (10, 25, 50, 100, 200, and 500 μM). (A) Representative movement heatmaps. (B) Total distance traveled at 24 h after treatment. (C) Representative trajectory tracking plots. (D) Average swimming speed at 24 h after treatment. (E) Mortality rates at 24 h and 48 h after treatment. Rg1 improved MPTP‐induced behavioral deficits across all tested concentrations, with the most pronounced effect observed at 100 μM. Significant improvements in average speed were detected at 50 μM and 100 μM. However, higher concentrations (200 and 500 μM) were associated with markedly increased mortality at 48 h. Data are presented as mean ± SEM (n = 30). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test. #p < 0.05 vs. Control group; *p < 0.05, **p < 0.01 vs. MPTP group. ns, not significant.

Figure S2: Transcriptomic features highlighting ER membrane‐related pathway enrichment. (A, B). Volcano plots of DEGs in pairwise comparisons. (C, D). GO enrichment bubble plots highlighting the “endoplasmic reticulum membrane” category. (E, F). Gene count bar plots in ER membrane‐associated GO terms.

Figure S3: Transcriptomic validation of CTSD expression in plasma samples from PD patients and non‐PD controls (GSE160299). (A) Volcano plot showing differentially expressed genes (DEGs) between Parkinson's disease (PD) patients and non‐PD controls (NC). Upregulated genes are highlighted in red, and downregulated genes in blue. (B) Quantification of CTSD expression levels derived from RNA‐seq data, indicating higher CTSD expression in PD plasma compared with NC. (C) Boxplot representation of CTSD expression distribution across groups. (D) Mean–difference (MA) plot illustrating global transcriptomic differences between PD and NC groups. (E) Expression values of CTSD presented as normalized counts, showing elevation in PD compared to NC. (F) Venn diagram comparing gene sets from NC and PD groups, highlighting CTSD among overlapping genes. Data were processed using DESeq2 with Benjamini–Hochberg correction.

Figure S4: Determination of optimal concentration range and cytotoxicity profile of ginsenoside Rg1 in SH‐SY5Y cells. (A) Effects of ginsenoside Rg1 on SH‐SY5Y cell viability at different concentrations (0–1000 μM) following 24, 48, and 72 h treatment. Cell viability was measured to evaluate the dose‐ and time‐dependent effects of Rg1. (B) Cell viability of SH‐SY5Y cells treated with various concentrations of Rg1 (0–50 μM) in the absence or presence of MPP+ (Model+Rg1) for 48 h. (C) Relative mRNA expression of CHOP in SH‐SY5Y cells co‐treated with MPP+ and gradient concentrations of Rg1 (0.5–10 μM) for 48 h, determined by qPCR. (D) Mean ROS intensity (FITC) in SH‐SY5Y cells treated with MPP+ alone or combined with Rg1 (10 μM) for 48 h. (E) Apoptosis (%) of SH‐SY5Y cells treated with MPP+ alone or combined with Rg1 (10 μM) for 48 h. All data are presented as mean ± SEM from three independent experiments (n = 3). Statistical analysis was performed using one‐way ANOVA followed by Tukey's post hoc test.

Figure S5: Rg1 restores CTSD expression and improves locomotor deficits in rotenone‐induced cellular and zebrafish models. (A, B) Quantitative analysis of pre/pro‐cathepsin D (CTSD) and CTSD light chain protein levels in rotenone‐induced SH‐SY5Y cells treated with different concentrations of ginsenoside Rg1 (0.5–10 μM). (C) Representative Western blot images showing pre/pro‐cathepsin D, β‐actin, and CTSD light chain expression in SH‐SY5Y cells following rotenone exposure and Rg1 treatment (n = 3). (D) Representative locomotor trajectories of zebrafish larvae under different treatment conditions, including Control, DMSO, rotenone, and rotenone combined with Rg1 (50 and 100 μM). (E) Quantification of total swimming distance in zebrafish larvae across different treatment groups. (F) Heatmap representation of locomotor activity in zebrafish larvae. Warmer colors indicate higher activity levels. (G) Quantification of average swimming speed in zebrafish larvae. Data are presented as mean ± SEM (n = 30). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test. #p < 0.05, ###p < 0.001 vs. dmso group; *p < 0.05, **p < 0.01, ****p < 0.0001 vs. rotenone‐treated group; ns, not significant.

Figure S6: Evaluation of the safety profile of ginsenoside Rg1 in the MPTP‐induced mouse model. (A) Mortality rate of mice in each experimental group, including Control, Model, Ginsenoside Rg1 (low, medium, and high doses), and levodopa‐treated groups. (B) Body weight changes before and after treatment in each group. Body weight was recorded to assess the systemic safety and tolerability of Rg1 administration in vivo. Data are presented as mean ± SEM (n = 12). Statistical analysis was performed using one‐way ANOVA followed by Tukey's multiple comparisons test.

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Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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