Exploration of LP component targets
As mentioned above, the active constituents of LP were obtained: Fuling, Mudanpi, Jiuyurou, Shanyao, Shanzhuyu, Shudihuang and Zexie . The ingredient screening conditions were oral bioavailability (OB) ≥ 30% and drug-like index (DL) ≥ 0.18. The prediction of targets was by TCMSP (https://tcmspw.com/tcmsp.php). And the non-human targets were removed after correction by the UniProt database (https://www.uniprot.org/).
Exploration of targets in PMOP
With “PMOP” as the keyword, the related human genes were explored in GeneCards (http://www.genecards.org/) , National Center for Biotechnology Information (NCBI) gene (https://ncbi.nlm.nih.gov/)  and Online Mendelian Inheritance in Man (OMIM®) (http://omim.org) . The median screening was performed in the GeneCards database according to the target scores for obtaining more relevant targets.
Manufacture of Venn diagrams
The screened LP targets and PMOP targets were input into the Venn diagram creation software Venny 2.1, and 117 common targets were obtained.
Protein–protein interaction (PPI) analysis
The above 117 common targets were entered into the STRING database (https://string-db.org/cgi/input.pl) to establish the PPI network, and the biological species was set as “Homo sapiens” and the confidence score > 0.7, the PPI network was obtained.
The PPI network was imported into Cytoscape 3.8.0, the NetworkAnalyzer performed topological analysis, and genes with a score greater than the average were selected as key targets by degree sorting. A total of 46 key targets were screened, and the top 20 were screened. The target points were drawn using R 4.0.3.
Molecular complex detection (MCODE) cluster analysis
The PPI network was imported into Cytoscape 3.8.0, and the MCODE module was opened for gene cluster analysis and core target screening. A total of 6 gene clusters and 5 core genes were obtained.
Gene Ontology (GO) analysis
Common targets of LP and PMOP were subjected to GO enrichment pathway analysis using R4.0.3. Referring to the STRING database, items with a corrected P value < 0.05 were screened. Bubble charts were drawn with the clusterProfiler package.
Kyoto Encyclopedia of Genes and Genomes (KEGG)
Traditionally, KEGG pathway analysis was one strategy for discovering biological functions and pathways. KEGG analysis was applied using R 4.0.3. After installing and referencing the clusterProfiler package, bubble charts were drawn.
Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) of LP
First, LP was dissolved in an extractant of 40% methanol + 40% acetonitrile + 20% H2O2. After standing for 10 min, it was sonicated at 4 °C. Let it stand for 1 h at -20 °C. Discard the pellet. The supernatant was drained with a freeze concentrator. The samples were redissolved in 50% acetonitrile + 50% H2O2. LP was analyzed using a Shimadzu Prominence UPLC system (Nexera UHPLC LC-30A, Kyoto, Japan) with QTRAP MS (SCIEX, QTRAP 5500). 3 μL sample was drawn and loaded onto a T3 column at 40 °C. The mobile phases comprised 0.1% aqueous formate (mobile phase A) and acetonitrile (mobile phase B). The flow rate of the mobile phase was set to 0.3 mL/min. Data were collected using Analyst 1.7.1 software (SCIEX), and relative amounts of quercetin were analyzed using MultiQuant 3.0.3 software (SCIEX).
Preparation of LP medicated serum
Five male SD rats of 220–280 g (Hunan SJA Laboratory Animal Co., Ltd.) were acclimated for 1 week. Rats were gavaged 5.4 g/kg of LP twice daily for 5 days. Two hours after the last administration, the rats were anesthetized with 2% (w/v) pentobarbital (40 mg/kg), and the rats were killed.
Cell culture and treatment
Mouse osteoblasts (MC3T3-E1) were bought from the ATCC (USA). In addition, cells were placed in Dulbecco's modified eagle medium (DMEM) (containing 10% FBS) with penicillin (100 U/mL, Gibco) at 37 °C in the condition of 5% CO2.
For establishing in vitro model of PMOP, cells were exposed to ferric ammonium citrate (FAC) (100 μmol/L, Sigma, USA) for 48 h.
To detect the function of quercetin in PMOP, cells were exposed to quercetin (Sigma, USA) (10/25/50 μM) for 48 h . The cells in LP group were treated with 10% LP Medicated Serum for 48 h .
Osteoblasts (3 × 105 cells/well) were cultured. When cells reached 70% confluence, they were transfected with pcDNA3.1-FOS (oe-FOS, Beyotime, China) or FOS siRNA (si-FOS, Beyotime, China) for 48 h using Lipofectamine 2000 (Invitrogen) in line with the protocol of the manufacturer.
Alizarin red staining
The osteogenic differentiation in cells was assessed by alizarin red staining. Cells were seeded overnight. Polyformaldehyde (5%, 500 μL, Beyotime, China) and alizarin (200 μL, 30 min, Beyotime, China) were added to the medium, and the microscope was applied to observe the calcium nodules.
Western blot detection
RIPA was applied to extract protein from cells. BCA was applied to assess the protein concentration. Subsequently, SDS-PAGE gel (10%) was applied to separate the proteins, and then proteins were transferred onto PVDF membranes. Primary antibodies were applied to incubate the membranes overnight after blocking for 1 h. Afterward, a secondary anti-rabbit antibody (SA00001-1; Proteintech, USA, 1:5000) was applied to incubate the membranes for 1 h. The primary antibodies were as follows: anti-Bax (ab32503, Abcam, Cambridge, MA, USA; 1:1000), anti-Caspase-3 (ab32351, Abcam; 1:5000), anti-Bcl-2 (ab59348, Abcam; 1:1000), anti-p-PI3K (ab182651, Abcam; 1:1000), anti-PI3K (ab227204, Abcam; 1:1000), anti-p-AKT (ab81283, Abcam; 1:5000), anti-AKT (ab108266, Abcam; 1:1000), anti-p-mTOR (ab109268, Abcam; 1:1000), anti-FOS (ab208942, Abcam; 1:1000), anti-JUN (66313-1-Ig, Proteintech; 1:1000), anti-TGFB1 (21898-1-AP, Proteintech; 1:1000), anti-PPARD (ab178866, Abcam; 1:1000), anti-mTOR (ab2732, Abcam; 1:2000) and anti-β-actin (66009-1-Ig, Proteintech; 1:5000). β-actin was applied for normalization.
Cell Counting kit-8 (CCK8)
Cells (5 × 103) were cultured overnight. Subsequently, cells were treated for 12, 24 or 48 h. Subsequently, cells were exposed to CCK8 (10 μl, Beyotime) for 2 h. A microplate reader was applied to assess the absorbance (450 nm).
Cells were trypsinized, washed and re-suspended in binding buffer. Subsequently, APC-A (5 μl) and propidium (PI, 5 μl) were applied to stain the cells with no light for 15 min. Flow cytometer (BD, USA) was applied to calculate the cell apoptosis rate.
SPSS v18.0 (SPSS, USA) was applied to analyze the data. Means ± SEM was applied to show the data. ANOVA (followed by the Tukey–Kramer post hoc test) was applied to analyze the differences among multiple groups, and independent samples t test was applied to analyze the differences between the two groups. P < 0.05 was considered significant.