Refer to a previous report (12) regarding the original PB-II recipe. We prepared slices of Chinese crude drugs from the pharmacy of Guangdong Province Hospital of Chinese medicine, which were decocted twice with 10 and 8 times of water, filtered, and concentrated in a water bath at 80°C to 1.6 kg/L (measured by crude drug weight/volume). The rats were administered PB-II twice daily at 32 g/kg (crude drug weight/body weight). After gavaging for 2 weeks, the rats under 30 mg/kg pentobarbital sodium anesthesia were bled from the arterial blood and separated into medicated serum according to a previously reported method (17). The treated and control sera were inactivated at 56°C for 30 min and stored at -80°C before use.

LC-MS was conducted using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) and a Q Exactive Orbitrap mass spectrometer. After screening, the analysis was performed using Waters (Milford, MA, USA) UPLCTM HSS T3 C18 (2.1 × 100 mm, 1.7 μm). Chromatographic conditions: Gradient elution was performed using acetonitrile (A) and -0.1% formic acid in water (B). The elution procedure was: 0 min, 10% A; 5 min, 20% A; 20 min, 60% A; 25 min, 90% A; 28 min 90% A; 29–33 min 10% A. The flow rate was 0.2 mL/min. Mass spectrometry conditions: Samples were ionized using an ESI ion source and analyzed using a Q Exactive Orbitrap high-resolution mass spectrometer. The main parameters of the ESI ion source were as follows: spray voltage 3500 V (Anion voltage, -3500 V); capillary temperature, 350°C; sheath gas, 40; auxiliary gas, 15. All other parameters were set to default values. A liquid eluent with a retention time of 0.5–29 min was selected for mass spectrometry analysis using an automatic switching valve.

The targets were predicted using network pharmacology after identifying the main compounds in PB-II-treated serum. Probable targets of the identified compounds were predicted using SwissTargetPrediction (http://www.swisstargetprediction.ch/) (18). Targets with a probability score >0 were used to analyze the Gene Ontology (GO) functional annotations and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using Metascape (https://metascape.org/gp/index.html#/main/step1) (19).

In our previous studies, we established multiple iPS cell lines from human fibroblasts (20). This study selected an iPS cell line preserved in our laboratory for research. Based on our previous reports (21), DAn were differentiated from iPSC using the necessary media and related cytokines. iPSCs were plated at a density of 4 × 10⁴ cells/cm² on Matrigel (BD)-coated tissue culture dishes for differentiation. Differentiation was performed in KSR medium. On day 3, the cells were nearly confluent (>80% confluency). The culture medium was gradually changed to N2 medium, supplemented on days 0–5 with SB431542 (5 μM) + LDN-193189 (100 nM) + Iwp2 (1 μM) to obtain retinal progenitors, and then split in a 1:3 ratio for the next six passages using Accutase. The cells were cultured using neural induction media supplemented with 3 μM CHIR99021 and 2 μM on X-ray-inactivated MEF feeders or Matrigel-coated plates. On days 6–10, induction factors were withdrawn, and PD173074 (0.2 μM) + DAPT (10 μM) was used for retinal ganglion cell induction. On days 10–15, the medium was replaced with N2, B27, 300 μg/mL cAMP (Sigma-Aldrich, St. Louis, MO, USA), 100 ng/mL SHH (C24 II), and 100 ng/mL FGF8b. Next, 10 ng/mL BDNF, 10 ng/mL GDNF, 10 ng/mL IGF-1, 1 ng/mL TGF-β, and 0.5 mM db-cAMP were added and the cells were cultured for 30 days. The culture medium for this stage was named DAn-modified Eagle’s medium (DAn-MEM), which was used for subsequent DAn cultures. The cells were collected and dopaminergic neuronal markers such as tyrosine hydroxylase (TH) and β3-Tubulin (TUJ1) were detected using immunofluorescence staining and RT-qPCR. TH antibody (CST-2791), TUJ1 (TU-20) antibody (CST-4466), and DAPI (Sigma-D9542) were used for immunofluorescence detection. The primers used for DAn-specific genes are listed in Table 2 . As described in our previous report (21), we utilized electrophysiological analysis to confirm the identity of human DAn.

The cells were fixed using 4% (v/v) paraformaldehyde (Alfa Aesar, Haverhill, MA, USA), washed three times with phosphate-buffer saline (PBS) containing 0.2% v/v Tween (PBST; Thermo Fisher Scientific), and permeabilized using 0.15% v/v TritionX-100 (Sigma-Aldrich) in PBS for 1 h at 25°C. After gentle removal of PBST, the cells were incubated with primary antibodies (Nrf2, TH and TUJ1) in PBST overnight at 4°C. Subsequently, the cells were washed three times with PBST and stained with the secondary antibody for 1 h at 37°C. The cells were thrice washed in PBST, stained with DAPI, and viewed under a laser scanning confocal microscope (Carl Zeiss-710). To observe the ratio of DAn, the proportion of TH⁺/TUJ1⁺ double-positive cells among all cells was statistically analyzed. The quantitative statistics of fluorescence intensity and localization of the related proteins were performed as we previously reported (21).

Cultured DAn were treated with 100 μM H₂O₂ for 12 h according to a previously reported protocol (22). After treatment, the apoptosis and reactive oxygen species (ROS) levels in the cells were examined with flow cytometry. Immunofluorescence (IF) data showed that the percentage of TH/TUJ1 double-positive cells in H₂O₂-treated cells was decreased significantly, which is considered a model of oxidative damage of DAn.

DAn culture were randomly divided into four groups: control (Ctrl), oxidative damage model (Model), model treated with blank serum (Blank Serum), and model treated with PB-II-treated serum group (PB-II Serum). Control cells were cultured under normal conditions (DAn-MEM), whereas the other cultures were treated separately. We added 10% rat serum to the Blank Serum group and 10% PB-II-treated medicated serum to the PB-II Serum group for 24 h. The following day, the three treatment groups (Blank Serum, PB-II Serum, and Model) were treated with 100 μM H₂O₂ for another 12 h. Finally, all cell samples were examined for apoptosis, DAn neuronal activity, ROS, and Nrf2 signaling pathway and associated gene expression.

Flow cytometry was used to analyze TH-positive cells, apoptosis, and ROS levels. Intracellular staining was performed to detect THs. The cells were digested, fixed with 4% paraformaldehyde, blocked with BSA, and incubated with anti-TH antibody (ab75875, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight- 488 goat anti-rabbit IgG (H+L) (ab96899) at a 1/500 dilution for 30 min at 22°C. A total of >5,000 events were acquired. For apoptosis detection, the KEYGEN apoptosis kit (^#KGA108–1) was used according to the manufacturer’s instructions. Annexin V-FITC/PI staining was performed using flow cytometry software (BD Biosciences, Franklin Lakes, NJ, USA). ROS were detected using the Reactive Oxygen Species kit (^#KGT010–1) according to the manufacturer’s instructions. ROS detection was based on the fluorescent probe DCFH-DA. Intracellular ROS can oxidize non-colored DCFH to fluorescent DCF. Thus, flow cytometry can be used to detect ROS fluorescence intensity.

Based on prior research in our lab, anti-oxidant responsive element (ARE)-luciferase plasmid was constructed by inserting a 39-bp ARE-containing sequence from the promoter region of the human NAD(P)H quinone oxidoreductase 1 (NQO1) gene into the cloning site of the pGL4.22[luc2CP/Puro] plasmid. We transfected ARE-luciferase plasmid into MDA-MB-231 cells and used 1.5μg/mL puromycin to selected the stable reporter cell lines. For the dual luciferase reporter gene assay, MDA-MB-231 cells were co-transfected with ARE-luciferase plasmid and Renilla luciferase plasmid. The transfected cells were treated with H₂O₂ or PB-II for 24 h. Luciferase activity was monitored with Dual-Luciferase Reporter Assay (Promega, J3082, USA) according to manufacturer’s instructions.

Sprague-Dawley rats (male, 220–250 g) were purchased from Guangdong Medical Laboratory Animal Center (Guangzhou, China). All animal experiments were approved by the Animal Review Board of Guangdong Provincial Hospital of Chinese Medicine (approval number: 2018009). Rats were housed under constant temperature (20–22°C) and a 12 h light-dark cycle, with free access to food and water. After one week of acclimation to the environment, the APO-induced rotation test (see section “APO-induced rotation test”) was performed before being used for modeling (23). Rats without rotation were used for stereotaxic injections.

Rats were separated into four groups (n = 5): control, sham, model, and PB-II. The rats in the control group were intragastrically administered with distilled water. The sham group was injected with vehicle and distilled water. The model group was injected with 6-OHDA (see the injection procedure) and distilled water. The PB-II group was injected with 6-OHDA and then administered with PB-II (32 g/kg). All intragastric treatments were continued for four weeks after modeling.

The stereotaxic injection procedure was as following: After being anesthesized with 3% pentobarbital sodium (50 mg/kg, i. p.), the rats were placed in a stereotaxic apparatus. 6-OHDA (3 µL, 5 mg/mL; 0.02% of ascorbic acid in normal saline) was injected unilaterally into each injection site of the left striatum (coordinates from bregma: site 1: antero-posterior: 1.0 mm, medio-lateral: 4.4 mm, dorso-ventral: -4.5 to -6.5 mm; site 2: antero-posterior: -1.2 mm, medio-lateral: 2.2 mm, dorso-ventral: -4.0 to -6.0 mm). The injection rate was 1 µL/min. Rats in the sham group were injected with 0.02% ascorbic acid in normal saline.

An APO-induced rotation test was conducted to evaluate motor function at weeks two, four, six, and eight post-6-OHDA injection. The APO was dissolved in normal saline containing 0.02% ascorbic acid. Rats were subcutaneously injected with 0.5 mg/kg APO (24). After 5 min, the number of contralateral rotations was recorded for 30 min.

The rats were anesthetized with 3% pentobarbital sodium (50 mg/kg, i. p.) and perfused with normal saline and 4% paraformaldehyde. Brains were isolated and fixed in 4% paraformaldehyde for 24 h. Brain tissues were dehydrated and embedded in paraffin. The coronal brain sections were prepared using a microtome. Sections were dewaxed, hydrated, and heated in citric acid buffer (pH 6.0) for 20 min. After incubation with 3% hydrogen peroxide for 15 min, the sections were washed with PBS and incubated with blocking solution for 30 min at 37°C. The primary antibody (TH 1:1000) was added to the sections and incubated overnight at 4°C. The sections were then washed with PBS and incubated with HRP-conjugated secondary antibody for 30 min at 37°C. Another three-wash step involved a DAB reaction for 5 min. Finally, sections were washed with water and dehydrated using an ethanol gradient. The images were captured using a BX61 microscope (Olympus, Tokyo, Japan).

The following human DAn ELISA kits were used: Monocyte Chemoattractant Protein-1 (MCP-1) (EHC113.96, Neobioscience, Shenzhen, China), tumor necrosis factor-α (TNF-α) (CSB-E09315h, Cusabio, Wuhan, China), interleukin 6 (IL-6) (CSB-E04638h, Cusabio, Wuhan, China), and interleukin 10 (IL-10) (CSB-E04593h, Cusabio, Wuhan, China). The following rat nigrostriatal region tissue ELISA kits were used: MCP-1 (CSB-E07429r, Cusabio, Wuhan, China), TNF-α (ERC102a.96, Neobioscience, Shenzhen, China), IL-6 (ERC003.96, Neobioscience, Shenzhen, China), and IL-10 (CSB-E04595r, Cusabio, Wuhan, China). Analyses were performed according to the manufacturer’s instructions.

Total RNA from rat midbrains was extracted with TRIzol^® Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and then enriched and purified using Oligo(dT) magnetic beads (Thermo Fisher Scientific) through direct targeted hybridization. mRNA-seq libraries were prepared and constructed using the VAHTS Stranded mRNA-seq Library Prep Kit (Vazyme Biotech, Jiangsu, China), following the manufacturer’s instructions. The raw sequence data reported in this paper have been deposited in the Genome Sequence Archive (Genomics, Proteomics & Bioinformatics 2021) at the National Genomics Data Center (Nucleic Acids Res 2022), China National Center for Bioinformation/Beijing Institute of Genomics, Chinese Academy of Sciences (GSA: CRA008871) and are publicly accessible at https://ngdc.cncb.ac.cn/gsa. Qualified libraries were sequenced using an Illumina NovaSeq 6000 system in a paired-end format. Reads were mapped to the reference genome (Rnor_6.0.104, ENSEMBL) using Hisat2 and feature counts were used to count the reads. Fragments per kilobase million were calculated after normalization to the trimmed mean of M values. The edgeR package was used to analyze significantly differentially expressed genes. Gene set enrichment analysis (GSEA) was performed using the pre-ranked method in GSEA Java implementation (25). The WIKI pathway database (MsigDB, http://software.broadinstitute.org/gsea/) was used for gene annotation, and a P-value <0.05 was considered statistically significant.

Western blotting was performed to detect the Nrf2 protein in cells and the midbrains of rats after various treatments. ImageJ software was used to analyze the protein gray values. RT-qPCR was used to detect the mRNAs of Nrf2 downstream genes, such as heme oxygenase-1 (HO-1), NQO1, multidrug resistance-associated Protein 2 (MRP2), and glutathione peroxidase 2 (GPX2). The primer sequences are shown in Table 2 .

Data are represented as the mean ± SEM unless otherwise indicated, and Student’s t-test was used to compare two groups. One-way ANOVA was used to compare multiple groups. GraphPad Prism 5 software was used for statistical analyses. Differences between two groups were considered significant when the P-value was <0.05.

LC-MS was used to analyze the active ingredients in the medicated serum and the six main compounds in the recipe, citric acid, hypaconitine, stilbene glucoside, glycyrrhizin, paeoniflorin, and Ginsenoside Rg1, were analyzed ( Figure 1 ). Identification and simultaneous detection were performed to provide a reference for the quality control of PB-II. A detailed map is shown in Figure 1 .

An iPSC line was reprogrammed from healthy human skin fibroblasts as previously described (20). iPS cells were cultured in ESC medium containing DMEM/F12 (11330; Gibco, Billings, MT, USA), KnockOut™ Serum Replacement (Gibco), MEM non-essential amino acids (Gibco), L-glutamine (Gibco), 55 mM β-mercaptoethanol (Gibco), and 20 ng bFGF (Gibco) on feeders made from CF1 mouse embryos and subjected to radioactive irradiation at a dose of 30 Gy. Colonies with the hESC morphology were observed between days 25 and 45. These cells were selected and expanded under hESC culture conditions. The pluripotency of the hiPSC line was confirmed by colony morphology, expression of the pluripotency markers OCT4 and TRA-1–81, and the formation of teratomas in NSG mice ( Figures 2A–C ). These data confirm the pluripotency of hiPSCs.

Using a previously described protocol (26), hiPSCs were differentiated into DAn. The presence of DAn in differentiating cultures was confirmed using DAn-specific markers. DAn-specific genes such as TH, TUBB3 (TUJ1), FOXA2, and Engrailed 1 (EN1) were significantly upregulated in iPSC-derived DAn ( Figure 2D ). In addition, cell morphology confirmed the morphological characteristics of DAn ( Figure 2E ). IF analysis demonstrated that approximately 60% of iPSC-derived cells expressed the neuron-specific marker TUJ1 and approximately 40% of iPSC-derived neurons were TH⁺/TUJ1⁺, confirming the presence of hiPSC-derived DAn ( Figure 2F ). Electrophysiological assays showed that the DAn possessed electrophysiological phenomena specific to human dopaminergic neurons ( Figures 2G–I ).

Immunofluorescence data showed more TUJ1⁺/TH⁺ neurons in the PB-II Serum samples than in the Model samples prepared using H₂O₂ damage and Blank Serum samples ( Figure 3A ). While TUJ1⁺/TH⁺ neurons decreased in the Model group after treatment with H₂O₂, PB-II Serum significantly increased the number of TUJ1⁺/TH⁺ neurons, indicating that PB-II Serum protected TUJ1⁺/TH⁺ neurons from oxidative stress ( Figure 3A ). In support of this conclusion, flow cytometric analysis indicated that the percentage of TH⁺ cells in the PB-II Serum group was significantly higher than that in the Model or Blank Serum groups ( Figures 3B, C ). The percentage of apoptotic cells was analyzed in each experimental group, indicating that PB-II Serum protected DAn from apoptosis after H₂O₂ treatment ( Figures 3D, E ). Furthermore, when compared to that in the Ctrl sample, the expression of Bcl-2 mRNA and protein in Model was significantly decreased, while the expression of Bax mRNA and protein was significantly increased ( Figures 3F-J ), indicating that apoptosis of DAn was significantly increased after oxidative stress. PB-II could significantly reduce the apoptosis of DAn after oxidative stress ( Figures 3D–J ).

To explore the mechanism by which PB-II protects DAn from oxidative stress and inflammation, the ROS levels and inflammatory cytokines in hiPSC-derived neuronal cultures were examined after various treatments. Although ROS levels were similar between the Model and Blank Serum treatment groups, PB-II Serum significantly decreased cellular ROS levels in DAn after oxidative stress, supporting the role of PB-II in reducing oxidative stress in DAn ( Figures 4A, B ; Supplementary Figures S1E, F ). Collectively, these findings support the hypothesis that PB-II protects DAn from oxidative stress by reducing cellular ROS levels.

As previously reported, ROS trigger the redox system by activating Nrf2 partly by promoting its nuclear translocation, leading to the increased expression of its downstream genes, such as HO-1, NQO1, MRP2, and GPX2. After treatment with H₂O₂ (100 μM) for 12 h, Nrf2 protein levels and downstream gene expression were similar to those of the Ctrl group ( Figures 4C–F ; Supplementary Figures S1A–D ). However, PB-II Serum treatment significantly increased the expression of Nrf2 protein and its downstream genes, such as NQO1 ( Figures 4C–F ; Supplementary Figures S1A–D ). PB-II Serum significantly induced Nrf2 nuclear translocation in oxidative stress DAn by H₂O₂ intervention with effects comparable to artemisitene (ATT), a known Nrf2 activator ( Supplementary Figures S2A–D ). Employing the MDA-MB-231 cells stably transfected with an ARE-luciferase reporter as a screening platform (27), we identified that PB-II could induce the expression of the ARE-dependent luciferase gene ( Figure 4G ). ELISA detection showed that H₂O₂ treatment significantly increased the expression of pro-inflammatory factors in DAn cells, such as MCP-1, TNF-α and IL-6, while inhibiting the expression of anti-inflammatory factors, IL-10. The treatment of PB-II could significantly reduce the inflammatory response of the Model group ( Figures 4H–K ; Supplementary Figures S1G-J ). Therefore, PB-II activates the Nrf2/ARE signaling pathway to protect DAn from oxidative stress and inflammation.

Network pharmacology analysis of the main identified compounds of PB-II-treated serum showed that several neuronal signalings were involved in the GO functions, such as “regulation of neurotransmitter levels” ( Figure 5A ), “oxidoreductase activity” ( Figure 5B ), “postsynapse”, and “neuronal cell body” ( Figure 5C ). The top 20 KEGG pathways of PB-II-treated serum also included neuron-associated pathways, such as “serotonergic synapse”, “alcoholism”, and “pathways of neurodegeneration-multiple diseases” ( Figure 5D ). Dopaminergic neuronal signaling has been implicated in neurotransmission, alcoholism, and neurodegenerative diseases. Transcriptome analysis was performed to better understand the mechanism of action of PB-II in PD. GSEA analysis showed that leading-edge genes associated with “dopaminergic neurogenesis” and “Parkinson’s disease” pathways were highly expressed, following PB-II treatment as compared to the model group ( Figures 5E, F ), suggesting that PB-II regulates dopaminergic neuronal signaling and the PD pathway.

To further validate that PB-II can activate the Nrf2/ARE signaling pathway to protect DAn from oxidative stress and inflammation, the effects of PB-II in PD rat models were tested by injecting 6-OHDA into the striatum of rats to induce midbrain DAn death (model group). The sham operation group was injected with an equal volume of normal saline (sham group). The PB-II group was administered 32 g/kg PB-II via gavage. During the four-week treatment course, the behavioral symptoms of the PD rats were measured weekly. Spinal behavior in PD rats was induced by subcutaneous injection of APO into the back of the neck, and the number of rotations was recorded within 30 min. During the initial stage of treatment (week 0), the rats in the control and sham groups showed no symptoms of in situ circles. However, the model and PB-II groups showed severe rotationary behavior, with more than 210 rotations in 30 min, and there was no significant difference between the two groups. During the third week of treatment, the number of rotations was significantly reduced in the PB-II group ( Figure 6B ). After four weeks of treatment, the rats were euthanized and tissues of the nigrostriatal region were obtained. The number of TH⁺ neurons in the substantia nigra of rats in the PB-II group was significantly higher than those in the sham and model groups ( Figure 6A ). In addition, transcriptome analysis showed that overall gene expression in the PB-II group was similar to that in the sham group ( Figure 6C ). GSEA revealed that the target genes associated with the Nrf2 pathway were highly expressed after PB-II treatment compared to those in the model group ( Figure 6D ). Furthermore, the expression of HO-1, NQO1, MRP2, and GPX2 in the midbrain of the PB-II group was also significantly increased, indicating the activation of the Nrf2/ARE signaling pathway by PB-II ( Figure 6E ). The Nrf2 and NQO1 protein levels in the midbrain DAn of rats in the PB-II group were significantly higher than those in the sham and model groups ( Figures 6F–H ). Meanwhile, the inflammatory response in the model group was intensified, while PB-II treatment significantly reduced the inflammation ( Figures 6I–L ). These data confirm that PB-II activates the Nrf2/ARE signaling pathway in the midbrain of PD rats by activating Nrf2 to reduce oxidative stress and inflammation in the DAn, thus protecting the DAn from oxidative stress and inflammation-induced apoptosis.