Preparation of samples and solutions
Samples of WJT powder were provided by Jilin Wantong Pharmacy Group Company (Tonghua, China). The traditional Chinese medicines containing in WJT were shown in Table S1. An alcohol-water dual extraction method was applied to ensure complete extraction. First, a 2.5 kg sample was dissolved in 25 L of 75% ethanol for 7 days, and the sample was then extracted via reflux to obtain filtrates. This above procedure was repeated, and the filtrates were then combined. The collected filtrates were concentrated under vacuum conditions until there was no scent of alcohol. The final extracts were packed separately, sealed, and stored at –80 °C. Extracts were dissolved with 0.1% DMSO at different concentrations prior to experiments.
For pharmaceutical analysis, a sample extract (1 g) was placed in a 25 mL methanol solution. Then, the solution was passed through a 0.22 µm filter prior to ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UHPLC-QTOF-MS) analysis.
UHPLC–QTOF-MS
UHPLC–QTOF-MS was conducted on an Agilent 1290 ultra-high definition accurate mass QTOF spectrometer with UHPLC (Agilent Technologies, AB Sciex, CA, USA). A UPLC C18-column (2.1 mm × 100 mm, ID 1.7 μm, ACQUITY UPLC® BEH; Waters, Milford, MA, USA) was used for separation, together with a C18-pre-column (2.1 mm × 5 mm, ID 1.7 μm, VanGuardTM BEH; Waters) at room temperature (20 °C). The mobile phase consisted of 0.1% formic-acid–water (A) and 0.1% formic-acid–acetonitrile (B), with the following optimized linear gradient elution: 0–3.5 min, 5% B; 3.5–6 min, 1% B; 6–12 min, 30% B; 12–12.5 min, 70% B; 12.5–22 min, 100% B. The injection volume was 5 μL and the flow rate was of 400 μL/min. Mass spectra were acquired in positive mode and negative mode. Data analysis was performed using Progenesis QI. Retention time correction, peak identification, peak extraction, peak integration, and peak alignment were optimized by the software. The corresponding Chinese medicine metabolism library was also queried to identify compounds. Then, matching between the self-built secondary mass spectrometry database and the corresponding fragmentation regularity as used to identify the peaks based on MS/MS data.
Cell culture
Human RA‐FLSs were obtained from BeNa Culture Collection (BeiJin, China) and were cultured in high glucose Dulbecco's Modified Eagle's medium (DMEM; Hyclone, Logan, Utah, USA) that was supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 100 U/mL penicillin (MRC, Jintan, China), and 100 mg/mL streptomycin (MRC) at 37°C and 5% CO2. RA-FLSs obtained from passages three to six were used for experiments.
Cell viability and proliferation assays
RA-FLSs were seeded in 96-well plates 4 × 103 /cells. After overnight, RA-FLSs were treated with various concentrations (0, 1, 2, 3, or 4 mg/mL) of WJT extract for different time (0, 12, 24, 36, 48, and 72 h). Then, cell viability was measured by the Cell Counting Kit (CCK-8; Beyotime, Shanghai, USA) according to manufacturer's instructions. Controls were treated with 0 mg/mL WJT extract. In addition, RA-FLSs were plated in 6-well plates 0.5 × 103 /cells. After incubation with WJT extract for 24 h and 48 h, cells were cultured for another 2 weeks. Then, cells were stained with Giemsa’s dye solution and the number of colonies was counted using Image J software (Version 1.8.0). For the 5-ethynyl-2-deoxyuridine (EdU) incorporation assay (Solarbio, Beijing, China), cell proliferation was assessed following the manufacturer's protocol after treatment with WJT extract.
Cell morphology
The cell morphology was examined using an optical microscope. In addition, cells were collected by centrifugation (1000 rpm for 5 min) and then fixed in a 2.5% glutaraldehyde solution at 4 °C for 10 h. The fixed cells were rinsed three times with PBS, and then dehydrated in a graded series of ethanol solutions (30%, 50%, 70%, 90% and 100%) at 1 min intervals. After 12 h of freeze-drying, the cells were covered by cathodic spraying, for observation by scanning electron microscopy (SEM).
Cell apoptosis assay
Enzyme linked immunosorbent assay (ELISA) kits (Abcom, Cambridge, UK) were utilized to measure the activity of caspase-3 and caspase-9 following the manufacturer's guidelines. Protein levels of Bax and Bcl-2 were evaluated by werstern blotting. And primary antibodies against Bax and Bcl-2 were from Abcom.
Profiling of mRNA expression
After incubation with 3 mg/mL WJT extract for 24 h and 48 h, RNA in RA-FLSs was extracted for sequencing. The NEBNext® UltraTM RNA Library Prep Kit for Illumina® (NEB, USA) was used to profile the expression of mRNAs. Quality control and quantification of gene expression were also performed. The DESeq2 R package (1.16.1) was used to perform the differential expression analysis of the different treatment groups (3 replicates per group). The Benjamini-Hochberg approach was used to adjust p-values and decrease the false discovery rate. Subsequently, the significant DEGs (adjusted p < 0.05 and |log2(fold-change)| > 1) were identified by DESeq2. The STEM software (version 1.3.12) was utilized to conduct the Series-Cluster analysis of expression profiles of DEGs. A co-expression network was created by WGCNA using the R package (Version 1.68). Then, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs were determined using the clusterProfiler R package. The protein-protein interaction (PPI) networks were constructed using a search tool for the retrieval of interacting genes (STRING) (http://www. string-db.org/). These results were visualized using Cytoscape software (version 3.6.0).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from RA-FLSs using a Total RNA Extraction Kit (Solarbo, Beijing, China), and reverse transcription was performed using a first-strand cDNA synthesis kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s guidelines. Premix Ex Taq SYBR Green PCR (TaKaRa, Dalian, China) was used to conduct real-time PCR on an ABI PRISM 7300 (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s protocols. Table 1 shows the sequences of the primers, and GAPDH was used as the internal control.
Western blot analysis
After extraction of total protein from RA-FLSs, the protein concentration was determined using the Bradford method (Beyotime). Equal amounts of protein lysates (20 μg per lane) were separated by 8-12% SDS-PAGE gels, and the proteins were then transferred to 0.45 µm polyvinylidene fluoride (PVDF) membranes (ThermoFisher, Waltham, MA, USA). The membranes were blocked using 5% BSA (Solarbio) and incubated with primary antibodies (all from Abcom) at 4 °C overnight. Then, the PVDF membranes were treated with horseradish peroxidase-conjugated secondary antibody (Bioss) at room temperature for 2 h. Protein bands were visualized using a Tanon 5200 (Tanon, Shanghai, China).
Statistical analysis
Data are presented as means ± standard deviations (SDs). Student's t-test was used to compare groups, and all statistical analyses were conducted using SPSS version 20.0. All experiments were performed at least three independent times. The significance of statistical differences are expressed as * (P < 0.05), ** (P < 0.01), and *** (P < 0.001).