The CRSJ formulation consisted of Cistanche deserticola (6 g), processed Polygonatum (12 g), Salvia miltiorrhiza (15 g), Paeonia lactiflora (12 g), and Moutan cortex (10 g) (Supplementary Table 2). All herbal materials were supplied by the Third Affiliated Hospital of Fujian University of Traditional Chinese Medicine. Each component was decocted twice in water for 1–2 h, and the combined filtrates were concentrated at 50–85°C, dried, and homogenized to obtain the crude extract. Extracts were mixed according to the prescribed ratios and processed into 12–40 mesh granules by dry granulation. Based on body surface area conversion (factor = 9.1), the mouse-equivalent dose for a 20 g animal was calculated as 3.71 g/kg. Three CRSJ dosage groups were therefore established: low (1.68 g/kg), medium (3.71 g/kg), and high (7.42 g/kg), corresponding to 0.5 × , 1 × , and 2 × the adult equivalent dose.

A precisely weighed 0.2 g aliquot of CRSJ powder was extracted with 10 mL of 80% methanol containing grinding beads. After grinding for 5 min and vortexing for 10 min, the mixture was centrifuged at 13,000 rpm for 10 min, and the supernatant was collected for analysis. Mass spectrometry was performed on a Q Exactive instrument (Thermo Fisher Scientific, Shanghai, China) using electrospray ionization (ESI) in both positive and negative switching modes. Chromatographic separation employed an UltiMate 3000 RS HPLC system (Thermo Fisher Scientific, Shanghai, China) with an AQ-C18 column (150 × 2.1 mm, 1.8 μm; Welch) at a flow rate of 0.7 mL/min, using water with 0.1% formic acid as the mobile phase. The gradient elution program is provided in Supplementary Table 5. High-resolution spectra were preprocessed with Compound Discoverer 3.3 (Thermo Fisher Scientific) and searched against the mzCloud database. Compounds scoring above the threshold were initially selected and subsequently confirmed by MS² and MS² spectral matching.

Two-dimensional ligand structures were retrieved from PubChem, and their three-dimensional conformations were generated in ChemOffice and saved as mol2 files. High-resolution crystal structures of target proteins were obtained from the RCSB PDB. All protein structures were prepared in PyMOL by removing water molecules and other heteroatoms. Docking was performed using AutoDock Vina (version 1.5.6). Protein and ligand structures were processed by adding hydrogens, assigning charges, and defining rotatable bonds. Grid box coordinates were set according to the predicted binding pocket. The best binding pose was selected based on docking affinity scores. Protein–ligand interactions were visualized using Discovery Studio 2019. Binding affinity < −5.0 kcal/mol was considered indicative of good binding, whereas < −7.0 kcal/mol indicated strong binding. Lower binding energy reflects higher affinity and greater conformational stability.

Molecular dynamics (MD) simulations were performed using GROMACS 2022 to evaluate the stability of protein–ligand complexes. The CHARMM36 force field was applied to proteins, and ligand parameters were generated using CGenFF. Complexes were solvated in a TIP3P water box with a 1.2 nm margin and neutralized with counterions. Long-range electrostatics were treated with the particle-mesh Ewald (PME) method, and the Verlet cutoff scheme was used. After energy minimization, NVT and NPT equilibration were performed for 200 ps each with position restraints on protein heavy atoms. Production MD simulations were run for 100 ns at 310 K and 1 bar. RMSD and RMSF analyses were performed to assess structural stability and residue flexibility (Li X. et al., 2025).

Male C57BL/6J mice (8 weeks, 20 ± 2 g) were obtained from the Laboratory Animal Center of Fujian University of Traditional Chinese Medicine and housed in SPF conditions [license: SYXK (Min) 2023-0004]. All procedures were approved by the Institutional Animal Care and Use Committee (FJTCM IACUC 0024037). After 1-week acclimation, 88 mice received intraperitoneal injections of MPTP (30 mg/kg/day) for 7 days to induce PD-like symptoms, while 12 mice served as untreated controls. Based on behavioral scoring (Supplementary Table 3), 60 successfully modeled mice were randomized into five groups: model, CRSJ-L (low-dose Congrong Shujing Granules, 1.68 g/kg), CRSJ-M (medium-dose Congrong Shujing Granules, 3.71 g/kg), and CRSJ-H (high-dose Congrong Shujing Granules, 7.42 g/kg), and Madopar (0.05 g/kg). Treatments were administered once daily by oral gavage for 14 days. Body weight was recorded weekly, and doses were adjusted accordingly. At the study endpoint, mice were anesthetized with pentobarbital sodium (150 mg/kg), and striatum and spleen were collected for analysis.

Mice were habituated to the testing room for 1 h before each assessment. Behavioral tests—including the wire hang test, pole test, and open-field test—were conducted on day 9 after MPTP induction and on days 7 and 14 of treatment. Detailed scoring criteria are provided in Supplementary Tables 4, 5. In the pole test, mice were placed head-up at the top of a gauze-wrapped wooden pole (50 × 1 cm) and the time to descend was recorded. In the open-field test, mice were placed in the center of a 40 × 40 × 25 cm arena divided into 16 squares, and locomotor activity was monitored for 5 min using the TOP SCAN Super Maze 3.0 system. The apparatus was cleaned with 75% ethanol between trials.

Brain tissues were collected from treated mice and fixed in 4% paraformaldehyde, followed by dehydration, clearing, paraffin embedding, and sectioning into 5 μm slices. After deparaffinization and rehydration, antigen retrieval was performed in citrate buffer (pH 6.0) at 95–100°C for 15 min. Sections were then blocked with 5% goat serum for 30 min at room temperature. Subsequently, they were incubated overnight at 4°C with an anti–tyrosine hydroxylase primary antibody (Proteintech, 25859-1-AP). The next day, sections were incubated for 1 h with an HRP-conjugated goat anti-mouse/rabbit IgG polymer secondary antibody (Boster, SA1020). DAB chromogenic development was carried out using a commercial kit (Boster, AR1022) for 1–3 min until optimal staining was achieved, followed by dehydration and mounting. Images were acquired under a light microscope at 100 × magnification. For quantification, 3–5 sections per mouse were analyzed, and three randomly selected fields per section were imaged. TH-positive neurons in the substantia nigra were quantified using ImageJ to obtain the mean neuronal count for each animal.

Fresh spleens were gently dissociated through a 70-μm cell strainer to obtain single-cell suspensions, followed by red blood cell lysis. Cells were stimulated with PMA (Elabscience, No. E-CK-A091) for 5 h at 37°C in the presence of a protein transport inhibitor. After stimulation, cells were fixed and permeabilized using the FoxP3/Transcription Factor Staining Buffer Kit (LianKe, No. IC001). For Treg staining, cells were incubated with FITC-conjugated anti-CD4 (Elabscience, No. E-AB-F1353C), APC-conjugated anti-CD25 (Elabscience, No. E-AB-F1102E), and PE-conjugated anti-Foxp3 (Elabscience, No. E-AB-F1238D). For Th17 staining, cells were labeled with FITC-anti-CD4 and APC-anti-IL-17A antibodies (Elabscience, No. E-AB-F1199E). After washing and filtration to remove bubbles and aggregates, samples were analyzed on an LSRFortessa™ flow cytometer (BD Biosciences, New Jersey, United States). Based on established gating strategies, CD4⁺CD25⁺Foxp3⁺ cells were defined as Tregs, and CD4⁺IL-17A⁺ cells were identified as Th17 cells.

Paraffin-embedded brain tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval. Sections were permeabilized with 1% Triton X-100 (Servicebio, No. G1204), washed with 0.1% PBS-T, and blocked with 10% goat serum at 37°C for 30 min. Primary antibodies against Foxp3 (Servicebio, No. GB112325-50), CX3CL1 (Proteintech, No. 60339), RORγt (Affinity, No. DF3196), IBA1 (Servicebio, No. GB12105), CD206 (Servicebio, No. GB113497), CD86 (Servicebio, No. GB150054), and IL-17 (Servicebio, No. GB11110) were applied and incubated overnight at 4°C. The next day, fluorescently labeled secondary antibodies (Servicebio, No. GB22303, GB27301, or GB28301) were added and incubated for 1 h at room temperature in the dark. Slides were mounted using DAPI-containing mounting medium (Servicebio, No. G1401). Fluorescence images were captured under identical exposure settings using a fluorescence microscope, and positive signals were quantified using Fiji software.

Brain tissue samples were lysed using a protein extraction buffer (Servicebio, No. G2002), and protein concentrations were determined using the BCA assay. Equal amounts of protein were separated by SDS-PAGE and transferred onto PVDF membranes (Servicebio, No. G6045). Membranes were blocked with 5% non-fat milk at room temperature for 1.5 h, followed by overnight incubation at 4°C with primary antibodies against RORγt (Affinity, No. DF3196), CX3CR1 (Proteintech, No.60339), α-synuclein (Servicebio, No. GB11773), Foxp3 (Servicebio, No. GB112325), IL-17A (No. GB11110), Arg1 (Servicebio, No. GB11285), iNOS (Servicebio, No. GB153965), TGF-β (Servicebio, No. GB111876), smad3 (Servicebio, No. GB150085), and β-actin (Servicebio, No. GB15003). Membranes were then incubated with appropriate secondary antibodies for 1 h (Servicebio, No. GB23303, GB23301). Protein bands were visualized using a Bio-Rad chemiluminescence imaging system, and relative expression levels were quantified using AIWBwell™ analysis software.

Serum samples were fully thawed at room temperature and diluted 1:4 before loading into 96-well plates for multiplex cytokine analysis using the Luminex^® 200 system (Luminex Corporation, Texas, United States). A 31-plex cytokine/chemokine panel (Bio-Rad, Bio-Plex Pro™ Mouse Chemokine Panel 31-Plex, No. 12009159) was used according to the manufacturer’s instructions. Samples and standards were incubated with magnetic beads for 1.5 h at room temperature in the dark with gentle shaking, followed by a series of wash steps and incubation with detection antibodies and streptavidin–PE. All serum samples were measured in duplicate. Quantification was based on bead classification and fluorescence intensity, and data acquisition and analysis were performed using Bio-Plex Manager™ software.

Two mice were randomly selected from each group for collection of bilateral striatum tissues. For RNA-seq, tissues from each mouse were processed independently, with each sample treated as one biological replicate. Total RNA was extracted using the MJzol Animal RNA Isolation Kit (Majorbio) and further purified with the RNAClean XP Kit (Beckman Coulter) and RNase-Free DNase Set (QIAGEN). rRNA-depleted, strand-specific libraries were constructed from each sample.

Sequencing was performed on the Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads, yielding approximately 30 million reads per sample. Raw reads were subjected to quality control and trimming using TrimGalore (v0.6.x) and seqtk (v1.0). Clean reads were aligned to the mouse reference genome (GRCm39) using HISAT2 (v2.2.x), and alignment files were processed with SAMtools (v1.9) and Sambamba (v0.6.4). Transcript quantification was performed using StringTie (v1.3.3b) to obtain gene-level expression matrices. Differentially expressed genes (DEGs) were identified using edgeR (v3.2.0) with thresholds of Q-value ≤ 0.05 and | log2 fold change| ≥ 1. To investigate Th17/Treg-related immunoregulatory programs, single-sample gene set enrichment–like analyses were performed using curated Th17/Treg, IL-6–STAT3, and TGFβ–SMAD pathway signatures. Pathway scores and immune marker expression patterns were visualized using heatmaps. Given the limited sample size, pathway-level trends and coordinated transcriptional patterns were emphasized.

The antibodies used in the IHC, IF, FCM, and WB are listed in Supplementary Tables 6–8.

All data are presented as mean ± standard deviation (mean ± SD). Statistical analyses were conducted using IBM SPSS Statistics 26.0, and graphical representations were generated with GraphPad Prism 9.2. Data normality and homogeneity of variance were assessed using the Shapiro–Wilk test, respectively. For data meeting the assumptions of normal distribution and homogeneity of variance, one-way analysis of variance followed by Tukey’s post-hoc test was used for multiple group comparisons. When the assumption of homogeneity of variance was violated, the Games–Howell post-hoc test was applied. Non-normally distributed data were analyzed using non-parametric tests. The significance level was set at α, = 0.05, with p < 0.05 considered statistically significant.

Liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis of the CRSJ aqueous extract identified 44 representative compounds derived from its constituent herbs (Figure 1 and Table 1). Based on prior pharmacological evidence, these compounds were functionally associated with immunomodulatory and neuroprotective activities through partially overlapping pathways. Specifically, paeoniflorin and its analogs (albiflorin and oxypaeoniflorin), together with verbascoside, genistein, and paeonol, have been linked to regulation of the Th17/Treg axis via suppression of RORγt/IL-17 signaling and enhancement of Foxp3- and IL-10–related pathways. Salvianolic acid A and B, rosmarinic acid, danshensu, tanshinone IIA, and cryptotanshinone have been associated with attenuation of microglial activation, oxidative stress, and α-synuclein aggregation (Table 1). Additional phenolic compounds, including tyrosol, caffeic acid, ferulic acid, gallic acid, ellagic acid, and isomangiferin, are implicated in mitochondrial and redox homeostasis (Table 1). Oligosaccharides such as stachyose, raffinose, and nystose have been reported to contribute to peripheral immune regulation and Treg induction (Table 1). Collectively, the identified compounds span multiple chemical classes and suggest a multi-component profile consistent with coordinated regulation of immune balance, neuroinflammation, and oxidative stress.

CRSJ-medicated serum contains multiple bioactive constituents, including echinacoside, paeoniflorin, salvianolic acid B, acteoside, and tanshinone IIA, consistent with a cooperative anti-inflammatory profile. Among these, paeoniflorin (PF) has been extensively implicated in Th17/Treg regulation across inflammatory and autoimmune models, primarily through suppression of Th17-associated signaling and enhancement of Treg-related pathways (Figures 2A). Based on this established immunomodulatory relevance, PF was prioritized for molecular docking and molecular dynamics analyses with Foxp3, RORγt, and α-synuclein. Paeoniflorin exhibited favorable binding affinities toward all three targets, with relatively stronger interactions observed for RORγt (Figures 2B and Supplementary Table 9). Stable protein–ligand associations were supported by hydrogen bonding and hydrophobic interactions, and molecular dynamics simulations confirmed conformational stability of the complexes, particularly for the RORγt–paeoniflorin interaction (Figures 2C–F). Rather than assuming uniform bioactivity across all detected constituents, we integrated serum exposure, prior pharmacological evidence, and pathway relevance to prioritize functionally plausible components. Within this framework, paeoniflorin emerges as a key immunoregulatory node linked to Th17/Treg modulation, while other constituents likely provide complementary anti-inflammatory, antioxidant, and neuroprotective support. This systems-level chemical architecture aligns with the multi-target pharmacology of traditional formulations and offers a mechanistic basis for the immuno-neuroprotective effects of CRSJ.

To assess CRSJ’s therapeutic efficacy, we established an MPTP-induced PD mouse model and evaluated motor performance (Figure 3A). Baseline body weight and motor performance were comparable across all groups prior to MPTP administration (Figure 3B). Following MPTP induction, PD model mice exhibited significant impairments in body weight gain, neuromuscular strength, and motor coordination, as reflected by altered wire hang and pole test performance, confirming successful model establishment (Figures 3B,C). CRSJ treatment dose-dependently improved body weight and motor function after 1 and 2 weeks of intervention, with the mid- and high-dose groups showing the most pronounced recovery, comparable to Madopar. Although motor performance did not fully return to baseline, all CRSJ-treated groups performed significantly better than the untreated model group, indicating that CRSJ effectively ameliorates MPTP-induced motor dysfunction. Overall, these findings indicate that CRSJ effectively alleviates MPTP-induced motor dysfunction and enhances neuromuscular performance in PD mice.

To assess the neuroprotective effects of CRSJ, we examined dopaminergic neuron survival in the substantia nigra and α-syn protein expression in the striatum of MPTP-induced PD mice. TH immunohistochemistry revealed a marked reduction of TH-positive neurons in the model group (P < 0.05), confirming dopaminergic degeneration (Figures 4A,C). CRSJ treatment significantly preserved TH-positive cells in a dose-dependent manner, with mid- and high-dose groups showing the strongest protection, comparable to the Madopar control (Figure 4C). Western blot analysis further demonstrated elevated α-syn expression in the striatum of model mice relative to controls (P < 0.05), consistent with pathological aggregation. CRSJ intervention significantly reduced α-syn levels, particularly in the mid- and high-dose groups, whereas the low-dose group showed a non-significant downward trend (Figure 4B). Together, these findings indicate that CRSJ suppresses abnormal α-syn accumulation and preserves dopaminergic neurons, supporting its neuroprotective potential in PD.

To characterize the molecular correlates of CRSJ-mediated immunomodulation in Parkinson’s disease, transcriptomic profiling was performed on striatal tissues from PD mice treated with mid-dose CRSJ. Principal component analysis (PCA) of variance-stabilized expression values showed clear separation between the model (MT) and CRSJ-treated (CT) groups along the first principal component, accounting for 55% of total variance (Figure 5A). Consistently, sample correlation analysis demonstrated high intra-group similarity and distinct inter-group clustering, indicating treatment-associated transcriptional differences with minimal technical variability (Figure 5B). Analysis of Th17/Treg-related immune markers revealed a coordinated shift toward a regulatory transcriptional profile following CRSJ treatment. Treg-associated components, including Tgfb1, Smad2/3, and Stat5 family members, were relatively upregulated, whereas Th17-associated regulators (Rorc, Rora, Stat3) and immune activation markers were attenuated (Figure 5C), indicating restoration of Th17/Treg-associated transcriptional balance. Pathway-level assessment using single-sample GSEA–like scoring further demonstrated coordinated pathway activity shifts, including suppression of Th17-related programs and the IL-6–STAT3 axis, accompanied by enhancement of Treg-associated signatures and the TGF-β–SMAD signaling pathway, together with reduced pro-inflammatory and microglial activation signatures in CRSJ-treated mice (Figure 5D). Among these pathways, TGF-β signaling emerged as a consistently altered feature across multiple analytical layers. Consistent with the transcriptomic results, Western blot analysis confirmed increased protein expression of TGF-β and SMAD3 in the striatum of CRSJ-treated mice compared with the PD model group (P < 0.05) (Figure 5E). Collectively, these data indicate that CRSJ treatment is associated with restoration of Th17/Treg-related immune programs, accompanied by consistent modulation of the TGF-β/SMAD3 signaling axis.

Given the established links between Th17/Treg imbalance, dysregulated TGF-β/SMAD3 signaling, and microglial activation, we next examined whether CRSJ modulates microglial M1/M2 polarization in a PD mouse model. Western blot analysis showed that PD model mice exhibited significantly increased expression of the M1 marker inducible nitric oxide synthase (iNOS) and reduced expression of the M2 marker arginase-1 (Arg1) compared with normal controls (P < 0.05) (Figure 6C). CRSJ treatment markedly reversed this polarization shift, with decreased iNOS and increased Arg1 levels across medium-, and high-dose groups (P < 0.05) (Figure 6C). Immunofluorescence double staining further confirmed these effects at the cellular level. The proportion of IBA1⁺CD86⁺ microglia was significantly increased in the model group, whereas IBA1⁺CD206⁺ cells were reduced (P < 0.05) (Figures 6A,B). CRSJ treatment significantly decreased IBA1⁺CD86⁺ co-expression in CRSJ-M and CRSJ-H, but not in the low-dose group (P > 0.05) (Figure 6A). In contrast, IBA1⁺CD206⁺ polarization was significantly enhanced only in the high-dose group, with no significant differences observed in the low- or medium-dose groups compared with the model group (P > 0.05) (Figures 6A,B). The Madopar group showed partial improvement. Together, these findings indicate that CRSJ shifts microglial polarization from a pro-inflammatory M1 phenotype toward an anti-inflammatory M2 state in the PD brain.

Thirty one cytokines and chemokines was quantified to characterize systemic immune alterations. The Model group showed broad pro-inflammatory activation, with notable increases in neuroimmune chemokines (CX3CL1, CXCL10, CXCL12, CCL4) and interleukins such as IL-1β, IL-16, and IL-4, confirming heightened microglial and peripheral immune activity. CRSJ treatment partially normalized these abnormalities, reducing CX3CL1, CXCL10, CXCL12, TNF-α, IL-1β, and recruitment-related chemokines (CCL2, CCL3, CCL22), while markers such as CCL7, CCL12, and CCL20 remained largely unchanged (Figures 7A,B). Consistent with these trends, IL-6 was significantly elevated and IL-10 reduced in PD mice (P < 0.05), indicating Th17/Treg imbalance; CRSJ effectively reversed both changes (P < 0.05) (Figure 7B). Together, these results demonstrate that CRSJ attenuates PD-associated cytokine dysregulation and restores key neuroimmune regulatory pathways.

To assess CRSJ’s immunomodulatory effects, we examined Th17/Treg-associated transcription factors and cell populations. In PD mice, RORγt expression was increased and Foxp3 was decreased in the mesencephalic region, indicating Th17-skewed immune differentiation (Figure 8A). CRSJ treatment significantly reduced RORγt expression across all doses, whereas Foxp3 expression and the RORγt/Foxp3 ratio were significantly corrected only in the medium- and high-dose groups but not in the low-dose group compared with the model group (P > 0.05), with similar effects observed in the Madopar group (Figure 8B). Flow cytometry further confirmed systemic immune imbalance: model mice displayed increased Th17 (CD4⁺IL-17A⁺) and decreased Treg (CD4⁺CD25⁺Foxp3⁺) populations, resulting in a disrupted Th17/Treg ratio (Figure 9B). Mid- and high-dose CRSJ effectively normalized these proportions (P < 0.05). Correspondingly, Western blot analysis showed striatum of upregulated RORγt and downregulated Foxp3 in model mice, both of which were significantly corrected by CRSJ-M, CRSJ-H, and Madopar (Figure 9A). Together with cytokine profiling and immunofluorescence data, these results demonstrate that CRSJ restores Th17/Treg homeostasis by modulating transcriptional programs and T-cell differentiation at multiple regulatory levels.

To examine whether CRSJ regulates chemokine CX3CL1 and Th17-cell–related immune responses in PD mice, we quantified CX3CL1 expression and key Th17-associated indicators. Among these, CX3CL1 was significantly elevated in the Model group compared with controls (P < 0.05), and was markedly reduced following CRSJ-M and CRSJ-H treatment (P < 0.05) (Figure 7B). To assess downstream mediators, striatal expression of CX3CR1—the cognate receptor of CX3CL1—and IL-17A, a key Th17 effector molecule, was examined. Western blotting revealed significant upregulation of both CX3CR1 and IL-17A in the Model group, which were significantly suppressed by CRSJ-M, CRSJ-H, and Madopar treatments (P < 0.05); IL-17A reductions in the CRSJ-L group did not reach statistical significance (P > 0.05) (Figure 10A). Dual immunofluorescence co-localization analysis further demonstrated increased CX3CL1–IL-17A co-expression in the Model group (P < 0.05), which was significantly attenuated by all CRSJ doses and Madopar (P < 0.05) (Figure 10B). Collectively, these results show that CRSJ effectively modulates CX3CL1/CX3CR1 signaling and Th17-related activity, thereby contributing to restoration of Th17/Treg balance and exerting neuroprotective effects in PD.