Section of Network PharmacologyThe potential components screening and target prediction of active ingredients of CR

Traditional Chinese Medicine Systems Pharmacology (TCMSP) is a unique pharmacological platform for traditional Chinese medicine systems, where we can understand the relationships between drugs, targets, and diseases(https://old.tcmsp-e.com/tcmsp.php). Using "Huanglian" as the search term, the active chemical constituents of traditional Chinese medicine CR were searched in the TCMSP database. OB ≥ 30%, DL ≥ 0.18 [20, 21] were screened to obtain the potential active ingredients of CR.

The SMILES structures of the potential active components of CR were sequentially entered into the Swiss Target Prediction database (http://swisstargetprediction.ch/), and the “Probility > 0” was used as the screening condition to obtain the predicted targets of CR-related active ingredients.

Screening of target genes for periodontitis disease

Genecards(https://www.genecards.org/) is a comprehensive database of searchable genes, where we can obtain information on almost all known human genes. Using "periodontitis" as the search term in GeneCards, TTD (http://db.idrblab.net/ttd/), OMIM (https://omim.org/), DrugBank (https://go.drugbank.com) database was searched for disease-related target genes. Merge the results from the four databases and remove duplicates.

Construction of the gene network map of the intersection of CR and periodontitis

Using the Venny 2.1.0 platform (https://bioinfogp.cnb.csic.es/tools/venny/index.html), the drug targets and disease targets predicted by the above screening were crossed to obtain a common target set. These are the potential targets of CR for the treatment of periodontitis. Import the above potential targets into String (https://cn.string-db.org/), and select "Homo sapiens" for analysis. Finally, the analysis results were saved, and the results were visualized in Cytoscape3.7.1 software.

GO and KEGG enrichment analysis

The screened common target genes were imported into the Metascape platform (https://metascape.org/gp/index.html), then analyzed and the data was saved. The results were visualized by using the R language package clusterProfiler to obtain the top 10 GO enrichment analysis and KEGG signaling pathway according to the P value.

Molecular docking

Download the 3D structure of BBR through the Pubchem database, and obtain 30 cross target receptor proteins from the Uniprot database. Obtain the corresponding PDB IDs through the RCSB PDB database to obtain the 3D structure of the proteins. The specific method is to first search for the target protein in the RCSB PDB database, and secondly narrow down the range based on the conditions on the left: (1) What experimental method was used to obtain the protein structure, among which X-ray crystallography is still the main method for obtaining the protein tertiary structure; (2) Resolution: Å is a commonly used unit of measurement for the length of light waves and the diameter of molecules. The smaller the value, the higher the resolution, and the more accurate the structure. Therefore, we prioritize choosing the smaller value; (3) Time to obtain protein structure: Generally speaking, the newer the year, the better. Finally, select the PDB ID of the optimal protein for subsequent experiments. Use Discovery Studio software to prepare ligands for BBR components, and then perform dehydration, ligand removal, and hydrogen supplementation treatments on 30 receptor proteins one by one. Macromolecular docking is performed in the "LibDock" module. Calculate the combination of BBR molecules and 30 protein receptors to sort according to small to large. Generally speaking, the lower the binding, the more stable, and the results of several sets of ligands are visualized. These graphics can display the interaction between the receptor protein and the small molecule of the ligand through the number of hydrogen bonds [22].

Section of in vitro experimentIn vitro experimental materials Cell source

The study is approved by Ethics Committee of Guiyang Dental Hospital (ethical review batch number: GYSKLL-KY-20111210–01). Peripheral blood of healthy people and peripheral blood of patients with chronic periodontitis are provided by volunteer patients and volunteer employees who will be treated in Guiyang Stomatological Hospital, Nanming District, Guiyang City, Guizhou Province from December 2021 to July 2022. All participants provided written informed consent, and the procedure complied with the requirements of the Ethics Committee of Guiyang Stomatological Hospital.

Inclusion criteria: Chinese; adults and about 30–40 years old; at least 20 teeth in the whole mouth that can be evaluated for periodontal evaluation and diagnosed as patients with chronic periodontitis; no smoking history; medical history and physical examination within one year; no immune-related diseases; no orthodontic treatment history; no antibiotics and immunosuppressants within 3 months; no systemic periodontal treatment; no scaling within half a year; women without pregnancy and breastfeeding. Exclusion criteria: After excluding occlusal trauma factors, food impaction factors, and periodontal-pulp combined disease factors, at least one tooth in each quadrant has a periodontal pocket > 4 mm and attachment loss (CAL) ≥ 3 mm [23].

Separation of PBMCs

Collect 5 ml of venous blood at the elbow with heparin sodium anticoagulation from each normal volunteer and chronic periodontitis patient, and the lymphocyte separation method is based on the research protocol of Dagur PK et al. [24]. Add equal volume of PBS(company: Biological Industries) to dilute the blood. Ficoll-Hypaque(company: Solarbio) was added to the centrifuge tube and the diluted blood was slowly layered on top. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient centrifugation. Carefully suck out the mononuclear cell layer into a conical tube, add PBS and mix, centrifuge at 1000 rpm for 5 min, discard the supernatant, and repeat twice to obtain the final extraction of PBMC.

CCK-8

The main components of CR for experimental medicine: BBR hydrochloride hydrate (Item No.: B107342, purity 98%), purchased from Aladdin Reagent Co., Ltd.

The lymphocyte proliferation experiment was carried out by CCK-8 method, and the optimal LPS concentration and CR concentration were screened out as the subsequent in vitro experimental conditions. PBMCs were suspended in DMEM medium(company: HyClone) containing 10% calf serum(company: Every green) and added to a 96-well plate (about 5000 cells/well). Set up different experimental groups and add different concentration gradients of lipopolysaccharide or BBR solutions to stimulate. The blank group was added with the same volume of 10% calf serum DMEM medium. 4.5 h before the end of each cell culture period, 10μL/well of CCK-8 solution was added and incubated with the cells until the end time. Finally, use a microplate reader to measure the absorbance at 450 nm.

Flow cytometry

Flow cytometry is widely used to analyze the expression of molecules on cell surfaces and within cells, identify and determine different cell types in heterogeneous cell populations, evaluate the purity of isolated subpopulations, and analyze cell size and volume. The method of determining T cell subsets refers to the research protocol of Falay M et al. [25]. Take 100ul of anticoagulant from the participants, add 10ul of CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC(company: Agilent) mixed monoclonal fluorescent antibody, and incubate at room temperature for 15 min in the dark. Add 500 μl of red blood cell lysate, mix by gently pipetting, and incubate at room temperature for 10 min in the dark. Add 1 ml of PBS for washing, centrifuge at 1500 rpm for 5 min, and discard the supernatant. Finally, 0.5 ml of PBS was added to resuspend the cells, and the cells were detected and analyzed on the machine.

Collect about 5 × 105 lymphocytes in the experimental group after 72 h of culture, add 100 μl PBS and mix well. Then add 10 μl CD3-FITC/CD8-PE/CD45-PerCP/CD4-APC mixed monoclonal fluorescent antibody and repeat the above steps. After antibody incubation, PBS was added for washing. The stained cells were resuspended in 0.5 ml PBS, and the cells were detected and analyzed on the machine.

Suspend the cells in PBS, adjust the concentration to 1–2 × 107 cells/ml, and add 2 μl of Cell Activation Coctail (with Brefeldin A) per 1 ml of cell suspension. It was spread into a 12-well plate, and 1 ml of serum-free DMEM medium was added to each well, followed by culturing for 6 h in a 37 °C, 5% CO2 incubatorCells were harvested and suspended in 100 μl of PBS. Add CD4 antibody(APC anti-human CD4, Clone RPA-T4, company: Biolegend) to each tube of cells and incubate at room temperature for 15 min in the dark. Fix the cells by adding Fixation Buffer(company: Biolegend) and incubate at room temperature for 20 min in the dark. Add 1 ml of 1 × Permeabilization Wash Buffer(company: Biolegend) working solution to each tube, wash and centrifuge. Add 100ul of Permeabilization Wash Buffer to resuspend the cells, then add IL-17 antibody(PE anti-human IL-17A, Clone BL168, company: Biolegend), and incubate for 30 min at room temperature in the dark. Finally, the cells were washed, centrifuged, and the supernatant was discarded. The stained cells were resuspended and fixed in 0.5 ml PBS, and detected and analyzed on the machine.

Elisa

The serum of all subjects was collected, sealed and stored in a -80 °C refrigerator for later detection. The lymphocyte culture supernatants stimulated by lipopolysaccharide for 72 h, lipopolysaccharide and CR for 72 h were collected, sealed and stored in a -80 °C refrigerator for later detection. The secretion levels of IL-6, IL-8, IL-17 cytokines in the samples were determined using the relevant ELISA kits according to the manufacturer's instructions(HumanIL-17A ELISA Kit, Human IL-6 ELISA Kit, Human IL-8 ELISA Kit, All purchased from Elabscience, 96 T).

Statistical analysis

The data were expressed as mean ± SD, and the results were retained to two or three decimal places. The parameters between the two groups were compared by two independent sample t-test, and the parameters between multiple groups were compared by one-way analysis of variance. All data were evaluated by IBM SPSS statistics 20 and graphpad prism V.9 software, P < 0.05 was significant, P < 0.01 was extremely significant.