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.