Animals and reagents

Sprague Dawley rats, aged 8 weeks and weighed 180 ± 20 g, were obtained from the animal experimental center at Shanghai University of TCM (license No. SYXK (Hu) 2020-0009). The SH-SY5Y cell line were obtained from the Shanghai Cell Bank, Chinese Academy of Sciences (Shanghai). The compounds 6-Hydroxydopamine hydrobromide (6-OHDA, MKBP0832V), Apomorphine (APO, SLBF6369V), and L-Ascorbic acid (063K1082) were obtained from Sigma Chemicals (St Louis, USA). Pentobarbital sodium (WS20130112) was procured from Shanghai Chinese and Western Pharmaceutical Co., Ltd. MPP+ (HY-W008719) from Med Chem Express (New Jersey, USA). The following antibodies were purchased from Proteintech (Wuhan Biotechnology Co., Ltd, China): Transferrin Polyclonal antibody (Cat No. 17435-1-AP), ACSL4/FACL4 Polyclonal antibody (Cat No. 22401-1-AP), COX2/PTGS2 Monoclonal antibody (Cat No. 66351-1-Ig), GPX4 Monoclonal antibody (Cat No. 67763-1-Ig), Ferritin light chain Polyclonal antibody (Cat No. 10727-1-AP), TH Polyclonal antibody (Cat No. 25859-1-AP), and Alpha Actin Polyclonal antibody (Cat No. 23660-1-AP). Cell Signaling Technology was the source of the Anti-mouse IgG (#5257) and Anti-rabbit IgG (#5151) antibodies. MDA detection kits, ROS detection kits, GSH detection kits, and Iron ion detection kits (Shanghai Weiao Biotechnology Co., Ltd, China).

CDG preparation

CDG is made up of seven herbs: Rehmannia glutinosa (Gaertn.) DC. (Voucher numbers CDG01-130306), Paeonia lactiflora Pall (CDG02-DH2012071703), Uncis Uncaria rhynchophylla (Miq.) Miq. ex Havil (CDG03-HY2012102204), Hyriopsis cumingii (Lea) (CDG04-HY2012040501), Salvia miltiorrhiza Bge (CDG05-YT2012091506), Acorus tatarinowii Schott (CDG06-LY2012080321) and Buthus martensii Karsch (CDG07-YT2012092411). CDG, with the lot number 20220701, made by Shanghai Wanshicheng Pharmaceutical Co., Ltd. To summarize, finely chop the herbs and immerse them in distilled water for the duration of about 30 min. Then, the items were routinely subjected to two rounds of boiling, each lasting one hour. Begin by immersing them in a solution consisting of 10 volumes of distilled water, followed by a second round in a solution consisting of eight volumes of distilled and deionized water. Samples were gathered, refined and then condensed into a cream with a specific gravity of 1.3. The preparation undergoes a process of drying, sieving, and subsequent pulverization into the CDG. As described in the 2010 edition of China Pharmacopoeia, all of these were compliant with the standard.

Identification of major chemical components of CDG by LC–MS

The quality of CDG was evaluated by LC–MS [41]. We used a Thermo Scientific Hypersil Gold C18 chromatographic column (2.1 mm × 100 mm × 1.9 m). A mobile phase contains water and 0.1% formic acid; B is acetonitrile and 0.1% formic acid, with an elution gradient of 0–5 min and 5–15% B; 5–8 min, 15–17% B; 8–16 min, 17–20% B; 16–18 min, 20–95% B; 18–19 min, 95% B; 19–19.1 min, 95–5% B; 19.1–20 min, 5% B; Column temperature 35 ℃, injection volume 5 μL. Mass spectrometry condition: electric spray ion source, scanning mode: negative ion scanning (ESI−, m/z 50–1000); heater temperature: 300 ℃; Sheath gas flow rate 45 psi (1 psi ≈ 6.895 kPa); Auxiliary airflow speed 5 L/min; Exhaust gas flow rate of 0.3 L/min; Electric spray voltage − 3.2 kV; The capillary temperature is 350 ℃.

Prediction of putative CDG prescription targets

The chemical constituents of CDG are from HERB 2.0 database (http://47.92.70.12/) and the Batman 2.0 (http://bionet.ncpsb.org.cn/batman-tcm/). The compounds in above database are intersected with the compounds measured by LC–MS to get the candidate compounds of CDG. Typical SMILES structures corresponding to the active components were obtained from the organic small molecule biological activity database PubChem (https://PubChem.ncbi.nlm.nih.gov/). SMILES structure obtained from PubChem was imported into Swiss adme (http://www.swissadme.ch/) database, and the drug structure was predicted, so as to obtain the target of candidate components.

PD-associated therapeutic targets

With "Parkinson's Disease" as the search term, PD-related targets were collected from the following six databases. GeneCards (Relevance score ≥ 2) (https://www.genecards.org/), Drugbank (https://go.drugbank.com/#), CTD (Inference Score ≥ 50) (https://ctdbase.org/), Disgenet (score_gda ≥ 0.01) (https://www.disgenet.org/), pharmgkb (https://www.pharmgkb.org/), OMIM (https://omim.org/), after deleting duplicates, use UniProt database for correction.

Network pharmacology analysis

In order to explore the potential effect of CDG on PD, the candidate targets of CDG and the potential therapeutic targets of PD were intersected, and the common targets were obtained by Venn diagram. To evaluate the importance of these targets, STRING (https://cn.string-db.org/) was used to import these potential targets. We defined the species as "Homo sapiens", defined the interaction score as > 0.900, and removed the single targets without interaction. Eigenvector, Betweenness and Closeness are calculated by using CytoNCA plug-in of Cytoscape 3.10.2, and the median of the results is taken as the screening threshold, and the targets exceeding the threshold are regarded as key targets.

Functional enrichment analysis

We introduced the candidate components of CDG into Metascape database (https://metascape.org//) for pathway enrichment. Select gene ontology, GO), Kyoto encyclopedia of genes and genomes, KEGG), Hallmark Gene set, Reactome Gene set, WikiPath, Oncogenic Signatures, Transcription Factor Targets, DisGeNET database are analyzed, and the obtained paths are visualized by R language package ggplot2.

Animal grouping and model establishment

The male Sprague Dawley rats employed in this investigation were raised in a research facility located at Shanghai University of TCM. The animals were housed individually in cages containing 6–8 individuals. They had unrestricted access to food and water. The humidity was preserved at 60–65%, the temperature was kept at 23 ± 2 °C, and they were maintained under a 12-h dark/light cycle.

The rats were deprived of water for 12 h before surgery, and no abnormal neurobehavioral tests were confirmed before surgery. Concerning previous studies, two coordinates of the right substantia nigra were determined (5.2 mm behind the fontanel, 1.0 mm right of the median line, 9.0 mm subdural; 5.2 mm posterior fontanel, 2.5 mm right median line, 8.5 mm subdural). Rats anesthetized with 3% pentobarbital sodium (50 mg/kg) were fixed in a brain stereoscope, shaved and disinfected at the cranial top, incised along the median line, peeled periosteum, exposed the cranial top, drilled holes in the skull, and inject 6-OHDA (dissolved in saline containing 0.02% ascorbic acid, 2 μg/μL) into the hole with a micro sampler (injection speed 1.0 mm/min), 3 μL per hole, the injection speed was 1 μL/min, the needle stayed in place for a 5-min interval before being retracted at 1.0 mm/min. The cranial hole was filled with medical gelatin sponge, the incision was sutured, and 200,000 units of gentamicin were injected intramuscularly for 7 days to prevent intracranial infection, and the rats were put into a feeding cage after waking up. The Sham group received no treatment except fixed rats. Ten days after surgery, the rats were induced to rotate to the healthy side by intraperitoneal injection of APO (0.5 mg/kg), and the number of rotations from the beginning of rotation to the end of 30 min was recorded, and patients with > 7 rotations/min were considered as successful animal models of PD [26].

Firstly, we randomly assigned 44 successful model rats to four groups: Model, CDG, Erastin + CDG, and Fer-1. Additionally, 11 normal rats were included in the Sham group. The final group was divided as follows: (1) Sham group: received gavage of normal saline (1 mL/100 g) and intraperitoneal injection of a 10% DMSO solution. (2) Model group: received gavage of normal saline (1 mL/100 g) and intraperitoneal injection of a 10% DMSO solution. (3) CDG group: received intragastric administration of CDG (7 g/kg/d) and intraperitoneal injection of a 10% DMSO solution. (4) Erastin + CDG group: received intragastric administration of Erastin (20 mg/kg/d) and CDG (7 g/kg/d) and intraperitoneal injection of a 10% DMSO solution. (5) Fer-1 group: received intraperitoneal injection of Fer-1 (2.5 μmol/kg/d) and intragastric administration of normal saline (1 mL/100 g). All rats were treated continuously for 4 weeks.

Secondly, for evaluating the mechanism of CDG, 66 rats with successful Model were randomly divided into Model group, CDG group, ML385 + CDG group, Znpp + CDG group, Oltipraz group, Hemin group, and 11 normal rats were included as Sham group. Normal saline (1 mL/100 g) was given by gavage to the rats in Sham group and Model group; and rats in CDG group were given CDG (7 g/kg/d) by gavage, rats in ML385 + CDG group were oral administrated of ML385 (20 mg/kg/d) and CDG (7 g/kg/d); Similarly, Znpp (10 mg/kg/d) and CDG (7 g/kg/d) were intragastrically given to the rats in Znpp + CDG group; Oltipraz (15 mg/kg/d) was given by gavage to the rats in Oltipraz group; Hemin (50 mg/kg/d) was given by gavage to the rats in Hemin group; and 10% DMSO solution was injected intraperitoneally to all rats and treated continuously for 4 weeks.

Behavioral testRotation test

The changes in healthy side rotation behavior induced by APO (0.5 mg/kg, sc.c.) were measured at 0, 14, and 28 days after treatment, respectively, and the number of rotation laps from the beginning of rotation to the end of 30 min was recorded.

Suspension test

On the 28th day of the intervention, suspension experiments were performed on the rats of each group at the same time, 24 h after gavage [27]. Hang the rat's front PAWS on a metal wire placed horizontally 30 cm above the ground, record the time from the beginning of hanging to the landing, and score the score as follows: the duration of approximately 0–5 s is 0 point, 6–10 s is 1 point, 11–15 s is 2 points, 16–20 s is 3 points, 21–25 s is 4 points, 26–30 s is 5 points, and more than 30 s is 6 points. A total of 3 detection times are taken as an average, and the interval of each detection is about 2 min.

Pole climbing experiment

A foam ball measuring 2 cm in diameter is securely attached to the apex of a wooden pole that is 50 cm long and has a diameter of 1 cm. To prevent slippage, encase the hardwood pole in two layers of gauze. Hold the model rat by its tail, place its head on top of the pole (ensuring that the rat's hind legs are on the ball), and allow it to climb down naturally. The rat climbs completely from standing on the top of the pole to the platform at the bottom of the pole with two front legs. On the 28th day of the intervention, the rats were tested 24 h after intragastric administration, and the climbing time and climbing score were recorded, respectively. Scoring criteria: use all four limbs; a successful climb from the pole is 0 point; Step-by-step spiral downward crawling with sliding hind legs behavior is 0.5 point; After intermittent pause several times after climbing down, but can hold the metal pole is 1 point: sliding after falling is 1.5 points; Can not grab the pole, drop directly to 2.0 points. The exercise score and the overall exercise duration from the start of the exercise to crawling to the bottom of the rod were recorded, and the average value was measured 3 times, each time with an interval of 5 min. If the rats stopped or reversed crawling in the middle of the course, they were not recorded and re-measured [28].

Cell culture

SH-SY5Y neuroblastoma cells were obtained from the Shanghai Institute of Cell Biology. 43% Ham's F-12 Nutrient Mixture (ICELL, Shanghai, China), 43% culture medium, 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 1% non-essential amino acids, 1% Sodium Pyruvate, 1% GlutaMAX-1, and 1% penicillin–streptomycin (BIOSHARP, Anhui, China). Humidified incubator conditions comprised 5% CO2 and 37 °C.

Nrf2/HMOX1 knockdown in SH-SY5Y cells

When SH-SY5Y cells grew to 80%–90%, the cells were digested and counted, and SH-SY5Y cells were inoculated into 12-well plates with 2 × 105 cells/well. 24 h later, the viral stock was extracted from the – 80 ℃ freezer and thawed in the ice bath. The stock virus stock was diluted with 800 μL complete medium according to MOI = 10, the original medium of the treatment group was absorbed, and the 500 μL medium containing the diluent of chronic disease venom was added to the cells of the treatment group. After 24 h, the culture medium was changed, and following 16 h of infection, the culture medium containing lentivirus was completely changed into a 1 mL complete culture medium. After 48 h, the suitable eukaryotic resistance screening cells (total lethal concentration of Blasticidin 20 μg/mL, maintenance concentration of 10 μg/mL) were selected, and the cells were rendered stable after two cycles of comprehensive drug screening, with each cycle lasting for three days, as established by an antibiotic pre-test. After stabilization, the basal maintenance cells were completely cultured with F12K + 10% FBS + 1% P.S + 10 μg/mL Blasticidin (Sigma 15205 St Louis, USA). After the cells were overgrown, the digestive cells were expanded and cultured, and part of the cells were extracted for western blot identification.

Overexpression of Nrf2/HMOX1 in SH-SY5Y cells

H_NRF2/H_HMOX1 SH-SY5Y cells were inoculated with 2 × 105 cells/well in a 12-well plate. 24 h later, the viral stock was extracted from the − 80 ℃ refrigerator and thawed in the ice bath. The stock virus stock was diluted with 800 μL complete medium according to MOI = 10, the original medium of the treatment group was absorbed, and the 500 μL medium containing the diluent of chronic disease venom was added to the cells of the treatment group. After 24 h, the culture medium underwent a change, and after 16 h of infection, the culture medium containing lentivirus was completely changed into a 1 mL complete culture medium. After 48 h, we selected eukaryotic cells that were suitable for resistance screening. The fatal value of Blasticidin was 20 μg/mL, and the concentration was 10 μg/mL. 0.5 μg/mL puromycin was added, and the maintenance dose was 0.25 μg/mL. We performed two cycles of comprehensive drug screening, with each cycle lasting for 2 days, as specified, in order to stabilize the cells. The cells were fully sustained after stabilization using a culture medium consisting of F12K enriched with 10% FBS, 1% PS, 10 μg/mL Blasticidin, and 0.25 μg/mL Puromycin.

Cell treatment

Cultured SH-SY5Y cells were subjected to MPP+ at various doses for 24 h, then, the CCK-8 assay was conducted to establish the optimal dosage concentration for the damage model. Subsequently, in the coculture of MPP+, different concentrations of CDG were infused with the culture medium for 24 h, and CCK-8 was determined. Finally, the cells are grouped as follows: (1) Sham group (PBS), (2) Model group: 2 mM/mL MPP+, (3) CDG group: MPP+ (2 mM/mL), CDG (200 μg/mL); (4) Fer-1 group: 2 mM/mL MPP+  + 10 μm/L Fer-1, (5) DFO group: 2 mM/mL MPP +  + 50 μm/L DFO. All cells were incubated at 37 ℃ with 5% CO2 for 24 h.

Grouped the cells as follows: (1) Sham group (PBS), (2) Model group (2 mM/mL MPP+), (3) CDG group: MPP+ (2 mM/mL), CDG (200 μg/mL); (4)sh-Nrf2 + CDG group: MPP+ (2 mM/mL), CDG (200 μg/mL); (5) sh-HMOX1 + CDG group: MPP+ (2 mM/mL), CDG (200 μg/mL);(6) OE-Nrf2 group (2 mM/mL MPP+), (7) OE-HMOX1 group (2 mM/mL MPP+). All cells were incubated at 37 ℃ with 5% CO2 for 24 h.

Transmission electron microscopy (TEM)

The pathological changes of mitochondria in the rat striatum were observed under an electron microscope. After the rats were anesthetized, fresh rat striatal tissue (1 mm3) was taken; the striatal tissue was washed with phosphate buffer solution (PBS) and then preserved overnight at 4 °C using a 2.5% glutaraldehyde solution. The specimens were rinsed with PBS for a duration of 30 min, then fixed with a 1% osmium tetroxide solution for a period of 2 h, and then subjected to dehydration using a gradient of ethanol, impregnated with epoxy resin, embedded, and then ultrathin sections (60 nm) stained with lead citrate and uranium, and finally observed using TEM, images were collected and analyzed.

The pathological changes of mitochondria in SH-SY5Y cells were observed under an electron microscope. After the treatment of each group of cells, the cells with the size of rice grains were collected by digestion and centrifugation with pancreatic enzyme, fixed at 4 ℃ overnight with propylene glycol, rinsed with PBS, and then the samples were embedded after gradient dehydration with acetone and immersion with acetone and resin. The ultrathin sections were prepared, stained by lead-uranium, observed under an electron microscope, and photographed.

Immunohistochemistry (IHC)

The level of TH of rats was quantified by immunohistochemistry. The brains of rats were injected into the heart under anesthesia, fixed with 4% paraformaldehyde solution, and the mesencephalon nigra and striatum tissue with a thickness of about 2 mm in coronal position was taken. The tissue was dehydrated, transparent, impregnated with wax, and then embedded in paraffin wax. A continuous coronal section with a thickness of about 5 μm was made by a paraffin micro-slicing machine. After antigen repair, sealing, antibody incubation, color rendering, sealing, and microscopic examination, in each section, the dense area of TH-positive cells was selected at low magnification, and then 3 non-adjacent fields were randomly selected and photographed at 400 times magnification. The average optical density was analyzed and averaged using Image-Proplus 6.0 software.

RNA-sequencing analysis

The extraction of total RNA from the rat striatum was performed employing Trizol reagent (thermofisher, 15596018) following the manufacturer's recommendations. The amount and purity of the total RNA were ascertained by employing the Bioanalyzer 2100 and the RNA 6000 Nano LabChip Kit from Agilent (CA, USA, 5067-1511). Subsequently, sequencing libraries were prepared to employ high-quality RNA samples with RIN values of more than 7.0. Ultimately, we conducted paired-end sequencing (PE150) with 2 × 150 bp read length using the Novaseq 6000 platform (Illumina) (LC-Bio Technology CO, Ltd, Hangzhou, China). Analyze the data via Unicawa Bio's complimentary online omicstudio (www.omicstudio.cn) or R program. A fold change (FC) value ≥ 2 and an adjusted p value of ≤ 0.01 were the criteria for identifying differentially expressed genes (DEGs).

Immunofluorescence (IF)

The expression colocalization of Nrf2, HMOX1, and TH in substantia nigra was detected by immunofluorescence. In the in vivo experiment, the brain slices that were pre-cut for immunohistochemistry were rinsed with PBS three times. Subsequently, each section was treated with 1% BSA for blocking for a duration of 1 h. Following that, primary antibodies targeting Nrf2 (1:200) or HMOX1 (1:100) and TH (1:500) were introduced and left to incubate at a temperature of 4 ℃ overnight in addition to 1 h at room temperature for the second antibody; after washing, a long-acting anti-fluorescence quencher containing DAPI was added, and the slide was observed under the Olympus BA51 microscope. SH-SY5Y cells were inoculated on 24-well plate slides in vitro. After culture for 48 h, drug intervention for 24 h, PBS washing for 3 times, methanol fixation for 15 min, 1% BSA sealing for 30 min. Primary antibodies Nrf2 (1:1000), HMOX1 (1:1000), and TH (1:3000) were incubated at 4 ℃ overnight. The second antibody (1:1000) was incubated at a temperature of 37 ℃ for 60 min on the next day, washed with PBS 3 times (3 min each time), and then followed by staining with DAPI for 10 min at 37 °C. Cells were detected by IF.

Biochemical index detection

In the in vivo experiment, the supernatant extracted from rat striate homogenate was stored at − 80 ℃. ROS (ER9407M), Fe2+ (ER9292M), MDA (ER9405M), and GSH(ER9290M) in rat striate tissue samples were quantified using a commercial kit based on the manufacturer's recommendations (Shanghai Weiao BioEngineering, China). In the in vitro experiment, the cells were gathered into a centrifuge tube. After being spun in a centrifuge, the supernatant was removed. Then, 1 mL of PBS was added, and the cells were disrupted using ultrasonic waves (ice bath, power 200 W, ultrasonic 3 s, interval 10 s, repeated 10 times). Centrifuge 12,000 r for 10 min at 4 ℃, take the supernatant, and put it on the ice to be measured. The contents of Fe2+ (BYSH-1212W), MDA (BYSH-0109W), and GSH(BYSH-0206F) were determined according to the instructions of the manufacturer (Nanjing Bo Yan Biotechnology Co., Ltd., China) for testing cell samples with commercial kits.

Live/dead cell staining

Cell viability was measured using live/dead staining. The reagents in tubes A and B were mixed according to the reagent instructions, mixed into the cells to be tested in a single culture at 1:9, placed at 37 ℃ for 30 min, and then photographed under a fluorescence microscope. Living and dead cells fluoresce green and red, respectively.

ROS detection

Intracellular ROS were detected using DCFH-DA fluorescent probes (Dojindo, R252, Shanghai, China). The cells were first inoculated in 96-well plates and cultured in incubators for 48 h. After incubation in the incubator for 24 h, the supernatant was removed, the cells were washed with HBSS twice, and then DCFH-DA fluorescent working liquid was added into the hole and cultured in the incubator for 30 min. Remove the working solution, wash the cells with HBSS twice, then add HBSS, and observe and take photos with a fluorescence microscope.

Western blot analysis

Rat striatum tissue or SH-SY5Y cells were cleaved by adding 1 × RIPA cleavage buffer containing a mixture of protease inhibitors. Preparation of denatured protein samples, and based on the manufacturer's recommendations (Beyotime, P0001, Shanghai, China) determination of protein concentration. The sample (20 μg/lane) was separated via SDS-PAGE gel electrophoresis and then transferred onto a polyvinylidene fluoride (PVDF) membrane, and which was sealed at ambient temperature for 1 h, employing a Tris-buffered brine solution with 0.1% Tween®20 detergent (TBST) buffer with 5% skim milk. Subsequently, the specimens were subjected to overnight incubation at 4 ℃ with antibodies targeting TH, GPX4, ACSL4, TF, PTGS2, Nrf2, HMOX1, FTH, and GAPDH. Afterward, the membrane and the secondary antibody were placed in a controlled environment at room temperature for a duration of 1 h. Finally, 1 × TBST was used to wash the membrane, and an enhanced chemiluminescence (ECL) detection reagent was used to show the protein bands. Data were analyzed using Gel-Proanalyzer4.0 software (GelMediaSystem, China).

Statistical analysis

SPSS statistical software was used for statistical analysis. The measured values are expressed as Mean ± SD A one-way analysis of variance (ANOVA) was employed to compare the groups, and a paired comparison was performed using the least significant difference (LSD) and Dunnett test. P < 0.05 manifests a significant difference.