Puerarin targets

The target of puerarin was searched in TCMSP (https://old.tcmsp-e.com/tcmsp.php/) using “puerarin” as the key word. Canonical SMILES and chemical structural formulae of puerarin were obtained in Pubchem (https://pubchem.ncbi.nlm.nih.gov/).

The targets of puerarin were searched through Pharmmappe (http://www.lilab-ecust.cn/pharmmapper/), SwissTargetPrediction (http://www.swisstargetprediction.ch/), and Targetnet databases (http://targetnet.scbdd.com/). The targets of puerarin were screened according to the principle of probability >0 in SwissTargetPrediction database and Prob>0 in Targetnet database, and the targets were supplemented in combination with literature in PubMed database. After merging, duplicate data were removed, and UniPort protein standardization database (https://www.uniprot.org/) was used to uniformly transform puerarin related targets.

OA targets

The GSE12021, GSE55235, and GSE82107 datasets were downloaded from the GEO database (https://www.ncbi.nlm.nih.gov/geo/), which included 30 OA groups and 26 control groups. Batch effect is eliminated through the “SVA” package of R software. The limma package of R software was used for data analysis, the absolute value of logFC was set to be > 1, and p value was < 0.05 after correction. Genes with differential expression in the chip data were screened out, and visualized in the form of volcano map and heat map.

Common genes of puerarin, ferroptosis, and osteoarthritis

Genes related to ferroptosis, such as “Driver,” “Suppressor,” and “Marker,” were retrieved from FerrDb database (http://www.zhounan.org/ferrdb/). The acquired puerarin target genes, differentially expressed genes, and ferroptosis related genes were interposed, and Veen was used to make the intersection and draw Venn diagram. The intersection genes were the common genes of puerarin, ferroptosis, and osteoarthritis.

GO enrichment analysis and KEGG enrichment analysis

The GO and KEGG enrichment analysis of intersection genes was performed by the “clusterProfiler” package of R software, and the significant enrichment results were visualized by the ggplot2 package.

Molecular docking

The common genes obtained in “1.3” were imported into the Uniprot database to obtain gene numbers, and the numbers were imported into the RCSB PDB database to obtain the protein structures encoded by the genes, and the receptors were selected and downloaded in PDB format.

Pymol software (input instruction: remove solvent; remove organic) was then used to remove water molecules and small molecules. The 3D structure of puerarin was obtained from the PubChem database and saved in SDF format. The spatial structure was optimized by Chemoffice software and saved in PDB format. The common genes of puerarin, ferroptosis, and OA were hydrogenated and docking boxes were created by AutoDock 1.5.6, and the docking of these genes with puerarin was performed by vina.

In vitro experimentsCell lines

Human articular chondrocytes (batch number REXFRHPZWA) were purchased from Procell Life Science &Technology Co., Ltd.

Drugs and reagents

Puerarin (batch number 27535) and celecoxib (batch number 19552) were purchased from MedChemexpress Biotechnology Inc. IL-1β (batch number 0606B95-1) and sterile ultrapure water (batch number A20353) were purchased from Multisciences (Lianke) Biotech, Co., Ltd. Human articular chondrocytes complete medium (batch number WH01112208SP08), 0.25% trypsin solution (batch number WH1622G051), and PBS buffer (batch number WH0022A071) were purchased from Procell Life Science &Technology Co., Ltd. CCK8 proliferation cell viability assay kit (batch number KL0306TV0989) was purchased from Elabscience Biotechnology Co., Ltd. Human IL-1β, IL-6, and TNF-α ELISA kit (batch number 10/2022) were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd.

Instrument

MB-530 multifunctional microplate analyzer (Shenzhen Huisong Technology Development Co., LTD.), Heal Force HF151-CO2 carbon dioxide incubator (Shanghai Ding International Trading Co., LTD.)

Experimental verification that puerarin has anti-osteoarthritis effectCulture and passage of articular chondrocytes

Human articular chondrocytes were first cultured at 37 ℃ with 5% CO2. The cells in logarithmic phase were cultured; when the cells reached 70–80% confluence, the articular chondrocytes were treated with complete culture medium containing IL-1β (10 ng/mL) for 24 h. Human articular chondrocytes were cultured in 5% CO2, 20% O2, 37 ℃ incubator for 2 days, and then changed to the medium. The cells were passaged after the fusion degree reached 70–80%. When the fusion degree of the second-generation chondrocytes reached 70–80%, the supernatant was discarded, and 1 mL of 0.25% trypsin was added, rinsed with PBS, and centrifuged at 1000 r/min for 5min. After mixing, the cells were divided into 25-cm2 cell culture flasks, placed into 5% CO2 incubator at 37 ℃ for routine culture and passage, and observed regularly under an inverted microscope.

Cell activity was detected by CCK-8 assay

The effects of different concentrations of puerarin on the proliferation of articular chondrocytes were detected by CCK-8 kit. A total of 5×103 Wells were seeded in 96-well plates and divided into blank control group, different concentrations of puerarin group (5 μM, 25 μM, 50 μM, 100 μM, 200 μM) and celecoxib group (5 μM, 10 μM, 20 μM, 100 μM, 200 μM), with 6 multiple Wells in each group. After 24 h of cell adherence, the cells were replaced with new medium and the non-adherent cells were discarded, and then the cells were cultured for 24 h and 48 h. Then, 10 μL CCK-8 solution was added to each well, gently shaken and mixed, and then routinely cultured for 2 h. The optical density value at 450-nm wavelength was detected by microplate reader to determine the proliferation of articular chondrocytes with different concentrations of puerarin and celecoxib.

Experimental grouping

The second generation of articular chondrocytes were divided into blank control group, model group (10 ng/mL IL-1β), puerarin group (10 ng/mL IL-1β+10uM puerarin), and celecoxib group (10 ng/mL IL-1β+10uM celecoxib) according to the different cultures. After 24 h of intervention, 10 multiple holes were set in each group.

ELISA was used to detect the related inflammatory factors

The operation was carried out according to the instructions of the ELISA kit, cell culture medium was added to the blank well, and the test samples were added to the sample well. Each group had 10 repeated detection samples, and the 450-nm wavelength was detected by microplate reader.

Statistical analysis

SPSS 22.0 software was used for processing, measurement data in line with normal distribution were expressed as (x±s), t test was used for comparison between two groups, one-way analysis of variance was used for comparison between multiple groups, and LSD-t test was used for pairwise comparison between groups. p < 0.05 was considered statistically significant.