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.