Gene expression profiling and differential data analysis

To obtain the RA-related genes (differentially expressed genes (DEGs) of AIA-M model group and the normal group) and the BHGZD effective genes (DEGs of BHGZD administration group and the AIA-M model group), we downloaded the transcriptomic data of rat synovial tissue and peripheral blood cells that had been uploaded to the GEO database of NCBI by our group earlier (GSE189942. GSE190523). Significant DEGs were identified based on the criteria of P value < 0.05 and |log twofold change (FC)|> 1.0.

Network construction and analysis

Based on ETCM 2.0 database (http://www.tcmip.cn/ETCM2/front/#/) [13], we obtained the chemical composition of BHGZD and predicted the candidate target profile referring to our previous studies [11]. The interaction information between genes in the String database (version 10.0, http://string-db.org/) [14] was used to extract the interactions between BHGZD effective genes from both transcriptomic data and TCMIP v2.0 platform, and AIA-M-related genes from transcriptomic data, and then to construct the ‘‘ disease gene-drug target ’’ interactions network. The core nodes were selected by calculating node’s degree, betweenness and closeness, and functional enrichment analysis was performed for the above core genes based on KEGG (version 96.0, https://www.kegg.jp/) [15] and Reactome database (https://reactome.org/) [16], and further hypothesized that the core pathway of BHGZD intervention in RA.

Molecular docking stimulation

To investigate the binding abilities of two BACs in BHGZD to the target proteins, molecular docking was performed using both AutoDock Vina (The Scripps Research Institute, version 1.2.0) and Ledock (The Scripps Research Institute, version 1.0) docking software. Mol 2 files or pdb files of MG and GA were downloaded from the ZINC (version 20, http://zinc.docking.org/) [17], DrugBank (version 5.1.8, released 2021-01-03, https://www.drugbank.ca/) [18] and PubChem (last update in 2023-04-21, https://pubchem.nc-bi.nlm.nih.gov/) [19] databases, and the crystal structures of target proteins downloaded from PDB (The Protein Data Bank, https://www.rcsb.org/)[20] database. After removing the ligands and water molecules from protein structures using Discovery Studio (version 4.5) software, corresponding active pocket information of proteins molecules were predicted in PyMOL (version 2.5, https://pymol.org/2/) via GETBOX plug-in, subsequently followed by docking performed. The ligand is considered to exert strong binding affinities with receptor if the absolute value of docking score is higher than six.

SPR detection

SPR assay was performed using Biacore 8K Biomolecular Interaction Assay System for validating the binding affinities of putative representative pharmacophores (MG and GA) to candidate targets (vascular endothelial growth factor receptor 2 antibody (VEGFR2) and proto-oncogene tyrosine-protein kinase Src antibody (SRC) in accordance with similar routine to our previous studies, with the KD value less than 10−7 mol representing a strong binding affinity [10].

Preparation of experimental drugs

Raw herbal materials of BHGZD were purchased from Beijing Tong Ren Tang Pharmacy, and decocted by referring to the Pharmacopoeia of the People's Republic of China (2020 edition). BACs that composed of MG (599.79 μg/kg of BHGZD, Lot No. 4773-96-0, Purity. 99.72%, Shanghai Standard Technology Co., Ltd., Shanghai, China) and GA (1185.43 μg/kg of BHGZD, Lot No. 1405-86-3, Purity. 98.00%, Shanghai Standard Technology Co., Ltd., Shanghai, China), as well methotrexate (MTX) were dissolved by dimethylsulfoxide, and diluted at 1g/mL prior to use.

Animals

Male Lewis rats of specific-pathogen free (n = 25, 200 ± 20 g in weight, 6 ~ 8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Production License No. SCXK 2021-0017, Beijing, China), and housed in the Experimental Animal Center, Institute of Basic Theory for Chinese Medicine, Chinese Academy of Traditional Chinese Medicine. All experimental operations conformed to the relevant regulations on experimental animal welfare and ethics of the Ethics Committee of the Institute of Basic Theory for Chinese Medicine, Chinese Academy of Traditional Chinese Medicine (No. IBTCMCACMS21-2105-01). All animals were allowed to drink and eat freely with a constant temperature of 24 ± 1 °C and a light/dark cycle of 12 h.

RA modeling, grouping, and treatment

A total of 25 Lewis rats were randomly divided into five groups on average: (1) normal control group; (2) adjuvant induced arthritis (AIA) -wind-hot-dampness stimulation modified model group (AIA-M); (3) AIA-M-BHGZD treatment group (21.4 g/kg, 2 times of the RA patients clinical dosage, which had been proved to exert the most prominent therapeutic effects against RA among 0.5, 1 and 2 times the daily RA patient dosage in our previous studies) [10, 11, 21]; (4) AIA-M-BACs treatment group (MG, 0.6 mg/kg; GA, 1.185 mg/kg, comparable to the corresponding content in 21.4 g/kg BHGZD); (5) AIA-MTX treatment group (0.2 mg/kg).

The AIA-M rat model was constructed in consistent with our previous studies [21], briefly, combining classical AIA model and external environmental stimulation by placing animals in the model box (production license No: RXZ-380A) for 2 h daily with certain wind velocity (6 m/s), temperature (37℃) and humidity (90%) for a period of 15 days from the day of primary immunization. Rats in normal control were simultaneously instilled with equal volume of saline solution at the same injection region. Dosing began on the day of initial immunization and continued for a total of 30 days. On the 31st day before sacrifice, rats were anesthetized by intraperitoneal injection of sodium pentobarbital, and blood was obtained from the abdominal aorta. After centrifuging, the supernatant was divided and frozen at − 80 ℃ for subsequent enzyme linked immunosorbent assay (ELISA). Bilateral ankle and knees of the hind limbs in rats were taken by removing skin and excess muscle tissues around the joints, part of which were fixed in 4% paraformaldehyde for subsequent histopathological examination, and the rest stored immediately at − 80 ℃ for western blot assay.

Assessment of arthritis severity

The severity of arthritis in rats of different groups was evaluated by assessing arthritis prevalence, arthritis score and limb swelling using our established and proved methods [21], and please refer to the details in Additional file 1: Section S1 Severity assessment of arthritis.

Histopathological evaluation

Samples from the hind limbs of rats that completed decalcification were removed, paraffin-embedded, cut into 4 μm slices, and stained with hematoxylin and eosin according to the general procedure, then assessed under a light microscope for synovial inflammation as well as proliferation of synovial vascular opacities in the knee joint of rats. Scoring was calculated via double-blind method [22, 23], with scores ranging from 0 to 3. Higher scores indicate more severe disease severity.

Immunohistochemical evaluation

Paraffin sections of knee joints were taken and the expression of vascular endothelial growth factor A (VEGFA), platelet endothelial cell adhesion molecule-1 (CD31), a vascular marker in rat knee synovial tissues, was detected using Rabbit IgG immunohistochemical staining kit (Cat No. SA1028, Boster Biological Technology Co., Ltd., Wuhan, China). The sections were incubated with CD31, VEGFA antibodies at 4 °C overnight, respectively. The poly-HRP-labeled anti-rabbit IgG was used as the secondary antibody. Then, tissue sections were treated with fresh diaminobenzidine (DAB) solution until the brown positive stain developed. The integrated optical density (IOD) was measured by Image J (Image Progressing and Analysis in Java, version 1.42q, https://imagej.nih.gov/ij/) software. Detailed information of antibodies was provided in Additional file 1: Table S1 Detailed information of antibodies to CD31, VEGFA, VEGFR2, SRC, PI3K, AKT1, p-VEGFR2, p-SRC, p-PI3K, p-AKT and GAPDH proteins.

Cell culture and treatment

Human umbilical vein endothelial cells (HUVEC, Shanghai Zishi Biotechnology Co., Ltd., Shanghai, China) and arthritis synovial fibroblasts (MH7A, Riken Cell Bank, Japan) at the generation of four to eight were chosen for in vitro experiments. Both cells were incubated in ECM medium, supplemented with 10% ~ 15% fetal bovine serum, 100 U/mL each of penicillin and streptomycin at 37 °C in a 5% CO2 incubator.

For drug treatment, both of HUVEC and MH7A cells were divided into seven groups as follows: (1) normal control group (isovolume medium); (2) model group (isovolume medium); (3) BHGZD treatment group (BHGZD, 7.135 μg/mL); (4) BACs treatment group (MG 0.2 ng/ mL, GA 0.4 ng/mL, as the same content of that in 7.135 μg/mL BHGZD); (5) MG treatment group (MG, 0.2 ng/mL); (6) GA treatment group (GA, 0.4 ng/mL); (7) SU5416 treatment group (SU5416, 0.238 μg/mL).

Transwell co-culture migration assay

MH7A cells at the logarithmic growth stage were digested, resuspended in DMEM medium with 10% FBS, and inoculated in 24-well plates (600 μL, 5 × 104/well). MH7A, which was seeded in advance of the plate for 24 h, was used as a model group to induce HUVEC cell migration. After 24 h of seeding, HUVEC cells at the same stage were also taken, digested. Transwell chambers were placed in the 24-well plate of advance seeding plate MH7A, and the upper chamber was added with drug and 100 μL of HUVEC cells resuspended with serum-free ECM medium (5 × 104/well). After 6 h of cell migration, the chambers were fixed in 4% paraformaldehyde for 15 min, stained with 0.1% crystal violet for 15 min, washed three times with PBS, and the unmigrated cells in the upper chamber of the transwell were swabbed away with a moistened cotton swab. Cells were observed under a light microscope and photographed, and the number of cells migrating to the lower surface of the chambers was automatically analyzed using Image J software.

Transwell migration assay

To detect the effect of BHGZD and BACs on the migration ability of HUVEC cells and MH7A cells, cells in the logarithmic growth phase were digested separately, and 100 μL of 3 × 104 HUVEC cells resuspended in serum-free ECM medium, as well 100 μL of 5 × 104 MH7A cells resuspended in serum-free DMEM medium. Drugs were then added to the upper chamber of the transwell. ECM medium with 15% FBS and/or a final concentration of 20 ng/mL VEGF for HUVEC cells, and DMEM medium with 10% FBS and/or a final concentration of 20 ng/mL TNF-α for MH7A cells were added to the lower chamber, respectively, followed by conventional method as in the “Transwell co-culture migration experiment”.

Transwell invasion assay

To assay the influence of BHGZD and BACs on the invasion ability of HUVEC cells and MH7A cells, the matrix gel was melted overnight at 4 °C ahead of schedule. The bottom of the upper chamber of the transwell was wrapped with 40 μL of matrix gel per well on ice and remained for 3 h at 37 °C. HUVEC or MH7A cells containing drugs were added to the upper chamber of the transwell. ECM medium containing 20% FBS and/or a final concentration of 20 ng/mL VEGF for HUVEC cells and DMEM medium containing 20% FBS and/or a final concentration of 20 ng/mL TNF-α for MH7A cells were appended to the lower chamber, respectively. After cell invasion (20 h for HUVEC, and 24 h for MH7A), chambers were fixed in 4% paraformaldehyde for 20 min, followed by conventional method as in the “Transwell co-culture migration experiment”.

Tube lumen formation assay

The matrix gel was melted in advance overnight at 4 °C, and 100 μL of matrix gel was added to each well of the 48-well plate on ice and then placed at 37 °C for 2 h to solidify. HUVEC cells (100 μL, 2 × 104) were resuspended in ECM medium containing 5% FBS, inoculated into the 48-well plate that covered with matrix gel, and a final concentration of 20 ng/mL VEGF was added and treated for 0.5 h. After incubation in an incubator at 37 ℃ for 5 h, the cells were observed under a light microscope and photographed, and the number of branching points and branch length of the formed lumen were automatically analyzed by Image J software.

ELISA assay

The levels of VEGF, Endostatin and nitric oxide (NO) in serum and the enzymatic activity of endothelial nitric oxide synthase (eNOS) (ml064294, ml003042, ml016840, ml003124, Shanghai Enzyme Linked Biotechnology Co., Ltd., Shanghai, China) in rats were measured using ELISA, and the regulatory effects of BHGZD, BACs and MTX on vascular neovascularization factors in rats were also investigated. In terms of MH7A cells, cells were first inoculated in 10 cm culture dishes, and 20 ng/mL TNF-α was added for reacting1 h. Cell supernatants were then collected after 24 h of drug treatment, and the levels of VEGF and NO as well as the enzymatic activity of eNOS (ml064255, ml016840, ml025095, Shanghai Enzyme Linked Biotechnology Co., Ltd., Shanghai, China) were measured to investigate the effects of BHGZD, BACs and SU5416 on the regulation of vascular neovascularization factors. Subsequently, the ratio of VEGF/ Endostatin was calculated. The absorbance was measured by Multiskan™ GO microplate spectrophotometer (Thermo Fisher Scientific, US).

Western blot analysis

The expression of VEGFR2, SRC, phosphatidylinositol 3-kinase 3 antibody (PI3K), RAC-alpha serine/threonine-protein kinase antibody (AKT1), phosphorylated phospho-VEGF receptor 2(p-VEGFR2), phospho-SRC antibody (p-SRC), phospho-PI3K antibody (p-PI3K) and phospho-AKT1/2/3 antibody (p-AKT) proteins in the articular cartilage of each group of rats and HUVEC cells was detected by western blot, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal reference protein. In terms of HUVEC cells, cells were first inoculated in 10 cm culture medium, and after 24 h of drug treatment, VEGF at a final concentration of 20 ng/mL was added for 5 min for induction in all groups except the normal group, where an equal volume of medium was added. Western blot analysis was performed according to previous studies [11]. Detailed information of antibodies was provided in Additional file 1: Table S1 Detailed information of antibodies to CD31, VEGFA, VEGFR2, SRC, PI3K, AKT1, p-VEGFR2, p-SRC, p-PI3K, p-AKT and GAPDH proteins.

Statistical analyses

GraphPad Prism (version 8.0.1, San Diego, CA, USA) software was used to calculate and analysis the experimental data, and the differences between groups were analyzed by one-way ANOVA, and the experimental data were expressed as mean ± standard deviation (x̅ ± s). Differences were considered statistically significant when the P value was less than 0.05.