Compound-target-biolabel link profile of HL treating OA construction
The association network of compound targets and both biolabels was assessed by STRING v11 (https://string-db.org/). The “repacxn” of compound targets and the gene names of both biolabels were imported into the input-form as “multiple proteins” and “Homo sapiens” was selected as the reference organism. The compound targets associated with both biolabels (combined score more than 0.900) were screened from the association network. Based on these targets, the relevant herbal compounds were retrieved from the in-house compound-target database. The above information was used to establish the compound-target-biolabel link profile of HL treating OA.OA model for the validation ofthe active ingredients analysis resultsMale Sprague Dawley rats (270g~, SPF) from Tianjin Hospital of ITCWM Nankai Hospital (Tianjin, PR China) were randomly divided into control, MIA model, MIA + DSS, and MIA + herbal compounds-treated groups (MIA + low-, MIA + middle-, and MIA + high-dose), and there were 5 rats in each group. The protocol was approved by the Animal Ethical and Welfare Committee of Tianjin Hospital of ITCWM Nankai Hospital (approval No. NKYY-DWLL-2020-058) and followed by the Legislation on the Protection of Animals Used for Experiment Purposes (Directive 86/609/EEC). Rats were housed in groups in standard cages, kept under 12 h light/dark conditions, and had free access to food and water throughout. Animal studies followed with the recommendations made by the British Journal of Pharmacology.
MIA model, MIA + DSS, and MIA + herbal compounds-treated groups were anesthetized with 400 mg/kg chloral hydrate (i.p.). MIA (exactly 3 mg in 50 μl 0.9% sterile saline) was injected into the knee joint through the patellar ligament with a 26 G needle. Control rats received injections of 0.9% sterile saline after anesthesia. After 24 h of modeling, the MIA + low-, MIA + middle-, and MIA + high-dose groups were intragastrically administrated with the herbal compounds (in 0.5% carboxymethylcellulose (CMC)) at the dose of 14, 28, and 56 mg/kg in rat, respectively, once daily for 7 days. MIA + DSS group received 14 mg/kg DSS (in 0.5% CMC, p.o) once a day for 7 days. Control and MIA model groups were orally administrated with equal volume of 0.5% CMC once a day for 7 days.
The diameter of joints was measured at 24 h after the last treatment via using a digital vernier caliper (Preisser Products, Germany). Subsequently, the synovial samples were dissected from the joints for histopathological, immunohistochemical, and ELISA analyses.
Synovial sample was immediately fixed in 4% paraformaldehyde overnight, then embedded in paraffin, sectioned into 5 mm cross-sectional pieces by using a paraffin slicer (RM2016, LEICA, Shanghai, PR China). Paraffin sections were stained with hematoxylin-eosin for histopathological analysis and stained with primary and secondary (1:2000) antibodies for immunohistochemistry analysis. Primary antibodies were rabbit anti-rat Itga2b and Itgb3, at dilutions of 1:100 each. Each slide was observed in a microscope (XSP-C204, CIC, PR China). The percent areas of Itga2b and Itgb3 were assessed by ImageJ software (ver. 1.8.0; https://imagej.nih.gov/ij/). The experimental detail followed the Guidelines of British Journal of Pharmacology (Alexander et al., 2018).
Synovial samples were homogenized in ice-cold 0.9% saline. The homogenate was centrifuged at 3,000 rpm for 10 min at 4 ℃, and the supernatant was transferred to another tube. The levels of Itga2b and Itgb3 in the tissue homogenate was assessed with the ELISA kits according to the manufacturer’s instructions.
The statistical comparison of joint diameter, immunohistochemistry, and ELISA data was performed with Aov (ANOVA) and Tukey HSD (multiple comparison) in the R Programming Language (version 3.5.2). Statistical analysis data were presented as mean ± standard deviation and statistical significance was accepted if P < 0.05.