1. Bioinformatic Data Analysis of Res treatment OP
1.1 Acquisition of Res Prediction Targets
The Traditional Chinese Medicine Systematic Pharmacology Database (TCMSP, http://lsp.nwu.edu.cn/tcmp.php) was used to compile information on the chemical constituents and targets of action associated to Res(Ru et al., 2014). Standardize the targets' "protein names" to their official names using the Uniprot database (http://www.uniprot.org/) (2021b).
1.2 Acquisition of Res Therapeutic OP Action Targets
Screening for disease targets with the human genetic database GeneCards (https://www.genecards.org/)(Barshir et al., 2021), to find the appropriate targets for OP, enter the keyword "osteoporosis." Potential therapeutic targets for Res treatment of OP were determined by plotting the anticipated targets in "1.1" against the OP-related targets in the Venn diagram.
1.3 Construction and Analysis of PPI network
The STRING database (https://stringdb.org) was used in this study to examine the protein interaction network of prospective treatment targets for Res on OP(Sneha et al., 2018). Cytoscape 3.9.1 software was used to visualize and analyze the data after the prospective therapeutic targets were loaded into the STRING database, the species was set to human, and a moderate interaction value of "0.4" was taken to obtain protein interactions. The program was configured to represent the change in Degree size by setting the node size and color to reflect the topological qualities of possible therapeutic targets. The more nodes and the darker the Degree value, the higher the Degree value. To obtain the primary target points, filter the target points greater than or equal to one-half of the Degree value(Sneha et al., 2018).
1.4 Drug-Target-Disease Network Construction and Analysis
Using Cytoscape 3.9.1 software, combine the drug "Res" with the "potential therapeutic target" and illness name "OP" discovered in "1.2" to produce a "drug-target-disease network diagram"(De Marinis et al., 2021).
1.5 Biological Processes and Pathway Enrichment
Import the potential target genes of Res therapeutic OP into the Bioeasy Cloud Platform, then select Enrichment Analysis in the Tool Center and limit the species to "H. sapiens". In the shared parameters, enter the gene ID of the core target and submit. Finally, we obtained the enrichment analysis results of GO, KEGG, and Reactome for the core targets of Res therapeutic OP. The final outcomes are visually represented(Kanehisa et al., 2017, Jassal et al., 2020).
1.6 Molecular Docking to Validate Key Target Binding Capabilities
(1) Ligand processing: obtain the 3D structure of the proposed docking target in mol2 format from the Pubchem database, open the small ligand molecule with AutodockTools 1.5.6, hydrogenate, charge, detect the ligand root, search and define the rotatable bond, and then save it as a pdbqt file(Kim et al., 2021).
(2) Receptor processing: Download the core 3D structure of the target protein from the RCSB protein database (www .rcsb.org/) as a docking protein. Add all hydrogen atoms in AutodockTools 1.5.6 to open, calculate Gasteiger charge, bind non-polar hydrogen, define as receptor, and save as pdbqt file(Kuriakose et al., 2022).
(3) Docking parameter setting: determine the coordinates and box size of Vina molecular docking, set the parameter exhaustiveness to 15, and take the default value for other parameters.
(4) Operation and output. Autodockvina 1.1.2 was used for semi-flexible docking, and the conformation with the best affinity was selected as the final docked conformation.
2 Res Suppression of Apoptosis Promotes Proliferation and Differentiation into MC3T3-E1 Cells in Vitro.
2.1 Experimental Cell and Reagent Selection
Mouse pre-cranial osteoblast subclone 14 (MC3T3-E1 Subclone 14) was purchased from the cell bank of Chinese Academy of Sciences. Res was purchased from Aladdin Reagent Company, China, HPLC grade (≥ 94%). α-MEM medium was purchased from HyClone, USA. Fetal bovine serum (FBS) was purchased from Gibco, USA. TNF-a, CASP 3, IL-6 antibodies were purchased from Proteintech. CCK-8 kit (cell proliferation and toxicity assay kit) was purchased from Beijing Solabao Technology Co. Ltd.
2.2 Experimental Method
2.2.1 Preparation of Res solution : 10 mg Res was dissolved in 438 μl DMSO to form a 100 mmol/L Res stock solution, which was split and refrigerated at -20 °C. The Res stock solution was diluted with α-MEM medium (including 10% fetal bovine serum by volume) to the following concentrations: 0.01, 0.1, 1, 10, 100 mol/L.
2.2.2 Cell Culture : MC3T3-E1 cells were inoculated in α-MEM medium (containing 10% fetal bovine serum by volume) and cultured at 37 °C in an incubator with 5% CO2 by volume before being digested and passaged when the cells reached the logarithmic phase. When the cell growth fusion rate reached 80%, the cells were passaged once and the third and fourth generation cells were used for the experiment.
2.2.3 CCK-8 detection : MC3T3-E1 cells were grown to 80% fusion, digested with digestion solution containing 0.25% trypsin, made into cell suspension, and inoculated with 3000 cells/well in a 96-well plate and cultured at 37 ℃ in a 5% CO2 incubator. After 24 h of cell wall attachment, 1 μg/ml of lipopolysaccharide (LPS) was added to stimulate the cells for 24 h. After 24 h of cell wall attachment, 200 μL of α-MEM culture medium containing different concentrations of Res (0, 0.01, 0.1, 1, 10, 100 μmol/L, respectively) was replaced, and the cells were incubated for 24 h and 48 h. After 20 μL of CCK-8 solution was added to each well, the cells were incubated for 2 h in the incubator, and the OD values at 450 nm were read with a multifunctional enzyme marker. The OD value at 450 nm was read by multifunctional enzyme marker.
2.2.4 Alkaline phosphatase staining (ALP staining) : MC3T3-E1 cells were blown and mixed, and the plates were evenly spread with 12-well plates set at 5×104 cells/well, and after 24 hours of LSP intervention, they were divided into blank control groups and 0.01, 0.1, 1, 10, and 100 μmol/L Res groups. Each group was treated with different doses of osteogenic induction solution containing 50 mg/L ascorbic acid and 10 mol/L sodium β-glycerophosphate for 3 weeks. After observing the cell status, the medium was aspirated, the cells were washed twice with PBS, 4% paraformaldehyde was soaked for 30 min to fix the cell morphology, PBS was washed twice to remove paraformaldehyde, NBT-BCIP staining solution was added to stain the cells for 30 min at 37 ℃ to avoid light, and the stained cells were rinsed 3 times with distilled water and photographed under microscopic observation.
2.2.5 Alizarin red staining : MC3T3-E1 cells were blown and mixed, and the plates were evenly spread with 12-well plates set at 5×104 cells/well, and after 24 hours of LSP intervention, they were divided into blank control groups and 0.01, 0.1, 1, 10, and 100 μmol/L Res groups. Each group was treated with different doses of osteogenic induction solution containing 50 mg/L ascorbic acid and 10 mol/L sodium β-glycerophosphate for 3 weeks. After observing the cell status, the medium was aspirated and the cells were washed twice with PBS, and the cells were fixed by soaking in 4% paraformaldehyde for 30 min. The cells were stained with 0.2% volume fraction of alizarin red staining solution for 30 min at room temperature. After staining, the cells were washed 3 times with distilled water. The cells were observed for the presence of orange-red precipitates and mineralized nodules under the microscope.
2.2.6 Expression of bone development genes (qt-PCR) : For 48 hours following LPS stimulation, MC3T3-E1 cells were grown in blank control, 0.01, 0.1, 1, 10, and 100 mol/L Res groups to examine the expression of pre-developmental-specific genes in osteoblasts. Phosphate-buffered saline (PBS) solution was used to wash the cells twice after they had been isolated using 0.25% trypsin-edta. Cultured cells were treated with 1 mL of TRIZOL reagent to extract the total RNA (Invitrogen, USA). According to the manufacturer's instructions, a rigorous protocol was followed. UV spectrophotometry at A260/A280 was used to quantify total RNA. In the presence of reverse transcriptase, cDNA was produced after 30 minutes at 55°C, 5 minutes at 85°C, and 10 minutes at 4°C. The following cycling settings were used for qPCR on an ABIStepPnePlus machine (Thermo Fisher Scientific, USA): 50°C for 2 minutes, 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 1 minute (40 cycles). The RUNX2 and OPG primer sequences for PCR are shown in the Supplementary Material. All gene expression levels were normalized by GAPDH gene expression.
2.2.7 Screening Res Optimal Concentration to Validate Related Proteins :After LPS pretreatment for 24 h, CCK8 assay was performed to obtain the optimal concentration of Res, and then divided into blank control group and Res optimal concentration group to verify the apoptosis-related proteins TNF-a, CASP 3, and IL-6. MC3T3-E1 cells were blown and mixed, and 6-well plates were set at 1×105 cells/well to spread the plates evenly, and after LPS pretreatment for 24 h, they were divided into blank control group and Res optimal concentration group. After 24 hours of LPS pretreatment, the cells were divided into blank control group and Res optimal concentration group. After 48 hours of incubation, MC3T3-E1 cells were washed twice with PBS after the intervention, and the cells were lysed with RIPA lysis buffer to extract proteins. The proteins were separated by SDS-PAGE, transferred to PVDF membranes, and incubated with the corresponding primary and secondary antibodies for detection. The antibodies used included TNF-a, CASP 3, IL-6, primary antibodies and corresponding secondary antibodies. The membranes were developed in a chemiluminescence imaging system, and the results were analyzed using ImageJ software to calculate the grayscale values of each band.
2.3 Statistical Analysis
SPSS 20.0 software was used for statistical analysis, and the experimental data were expressed as x±s. All experiments were repeated three times. The means between two groups were compared by independent samples t-test, and the means between multiple groups were compared by one-way ANOVA, and further two-by-two comparisons were performed by LSD test. All statistical analysis results were considered significant at P<0.05.