Compound-target-biolabel link profile of HL treating OA construction
The association network of compound targets and both biolabels assessed by STRING database is shown in Supplementary Figure S1. Based on the in-house database and the association network, the compound-target-biolabel link profile of HL treating OA was established (Figure 3). Five compounds from HL (myricetin, fisetin, esculetin, 7-hydroxycoumarin-4-acetic acid, and caffeic acid) synergistically regulated both biolabels (Itga2b and Itgb3) through eleven targets (CBFB, FYN, HDAC1, HSP90AA1, ITGA4, LCK, MAPK1, PIK3CA, PTPN1, RUNX1, and SYK). These five compounds may be the active ingredients of HL treating OA.
OA model for the validation of the active ingredients analysis results
Joint swelling is one clinical symptom of OA reflecting the presence of synovitis (Berenbaum, 2013) (Figure 4A). Compared to the joint diameter in the control group, that in MIA model group was increased by 47.6%. Compared to the joint diameter in MIA model group, that in MIA + DSS group was decreased by 17.2%; those in MIA + 14, 28, and 56 mg/kg myricetin groups were decreased by 21.9, 20.2, and 18.7%, respectively; those in MIA + 14, 28, and 56 mg/kg fisetin groups were decreased by 22.8, 20.6, and 21.2%, respectively; those in MIA + 14, 28, and 56 mg/kg esculetin groups were decreased by 23.7, 19.5, and 19.6%, respectively; those in MIA + 14, 28, and 56 mg/kg 7-hydroxycoumarin-4-acetic acid groups were decreased by 18.4, 16.9, and 20.0%, respectively; those in MIA + 14, 28, and 56 mg/kg caffeic acid groups were decreased by 18.0, 17.0, and 14.6%, respectively.
The change of microstructure can directly reflect the synovial damage (Figure 4B and Supplementary File S2). Synoviocytes in the control group were monolayer and arranged compactly and regularly, and there was no abnormal inflammatory cell in synovial tissue. A serious inflammatory damage occurred in the MIA model rats, and irregular synoviocytes arrangement, synovial hyperplasia, and inflammatory cells infiltration were found in synovial tissue. DSS and the herbal compounds showed the tendency to repair the synovial tissue damaged by MIA.
Itga2b and Itgb3 are abundantly and predominantly expressed in platelets and promote their aggregation. The levels of both subunits can represent the number of platelets (Li et al., 2020). In immunohistochemical analysis (Figure 5 and Supplementary File S3), compared to the percent areas of Itga2b/Itgb3 in the control group, those in the MIA model group were increased by 95.5/75.6%. Compared to the percent areas of Itga2b/Itgb3 in MIA model group, those in MIA + DSS group were decreased by 28.6/19.7%; those in MIA + 14, 28, and 56 mg/kg myricetin groups were decreased by 31.3/22.6, 37.5/31.9, and 40.8/41.8%, respectively; those in MIA + 14, 28, and 56 mg/kg fisetin groups were decreased by 37.2/27.9, 25.4/22.1, and 28.0/36.5%, respectively; those in MIA + 14, 28, and 56 mg/kg esculetin groups were decreased by 44.0/36.8, 37.2/23.1, and 31.7/22.3%, respectively; those in MIA + 14, 28, and 56 mg/kg 7-hydroxycoumarin-4-acetic acid groups were decreased by 31.8/34.6, 41.0/25.7, and 23.7/28.1%, respectively; those in MIA + 14, 28, and 56 mg/kg caffeic acid groups were decreased by 27.6/37.6, 31.2/35.3, and 29.4/22.6%, respectively.
In ELISA analysis (Figure 6), compared to the levels of Itga2b/Itgb3 in the control group, those in the MIA model group were increased by 111.3/113.0%. Compared to the levels of Itga2b/Itgb3 in MIA model group, those in MIA + DSS group were decreased by 37.7/35.5%; those in MIA + 14, 28, and 56 mg/kg myricetin groups were decreased by 28.7/46.0, 40.3/51.8, and 26.4/51.1%, respectively; those in MIA + 14, 28, and 56 mg/kg fisetin groups were decreased by 38.4/49.1, 23.9/29.6, and 26.1/40.3%, respectively; those in MIA + 14, 28, and 56 mg/kg esculetin groups were decreased by 43.0/45.0, 36.2/29.9, and 25.3/25.2%, respectively; those in MIA + 14, 28, and 56 mg/kg 7-hydroxycoumarin-4-acetic acid groups were decreased by 70.8/28.7, 34.4/27.9, and 25.3/44.8%, respectively; those in MIA + 14, 28, and 56 mg/kg caffeic acid groups were decreased by 25.1/24.4, 25.3/17.7, and 23.9/21.6 %, respectively.