3.1 Active ingredients of BSHX formula
After screening the TCMID and TCMSP databases with the ADME thresholds of OB ≧ 30% and DL ≧ 0.18, a total of 218 related ingredients were identified in BSHX formula, including 87 ingredients (40.0%) in GC, 36 (16.5%) in GQZ, 26 (11.9%) in DZ, 18 (8.2%) in TR, 17 (7.8%) in HH, 14 (6.4%) in SZY, 12 (5.5%) in SY, 5 (2.3%) in FZ, 2 (0.9%) in SDH, 0 (0%) in RG. As shown in Fig. 1, a Herb-Ingredient network were further constructed using Cytoscape software. According to the descending order of edge number in the Herb-Ingredient network, we listed and analyzed the top four ingredients that were beta-sitosterol (MOL000358, DL = 0.75, OB = 36.91, found in DZ, GQZ, HH, TR, SZY), stigmasterol (MOL000449, DL = 0.76, OB = 43.83, found in GQZ, SY, SDH, HH, SZY), quercetin (MOL000098, DL = 0.28, OB = 46.43, found in DZ, GC, GQZ, HH) and sitosterol (MOL000359, DL = 0.75, OB = 36.91, found in FZ, GC, SZY, SDH).
3.2 Target prediction and PPI network analysis
Target fishing on the 218 active ingredients was conducted in Uniprot databases, and we obtained 274 related targets of BSHX formula among which there were 220 in GC, 206 in DZ, 201 in HH, 190 in GQZ, 67 in SY, 46 in TR, 51 in SZY, 10 in, FZ 29 in SDH. Then, 292 PMOP related targets were obtained through GeneCards and DisGeNet databases. After the establishment of Venn diagram, 64 overlapping genes between BSHX formula and PMOP were identified and considered as the key therapeutic targets through which BSHX formula exerts the anti-PMOP effects (Fig. 2).
A Protein-Protein Interaction (PPI) network of these 64 overlapping genes was built in the the String database, which contained 64 nodes and 1110 edges (Fig. 3). Then, we used three main parameters, “degree”, “betweenness” and “closeness”, as the screening thresholds to select the central target genes. After the first screening round of degree ≥ 12, betweenness ≥ 0.002 and closeness ≥ 0.566, 41 nodes and 634 edges were obtained. Through the second screening round of degree ≥ 24, betweenness ≥ 0.008 and closeness ≥ 0.700, only 21 nodes and 202 edges were identified (Fig. 4). The information of these hub nodes were listed in Table 2.
Table 2
Information of 21 hub targets.
Uniprot ID
|
Gene Symbol
|
Description
|
P31749
|
AKT1
|
RAC-alpha serine/threonine-protein kinase
|
P15692
|
VEGFA
|
Vascular endothelial growth factor A
|
P01375
|
TNF
|
Tumor necrosis factor
|
P27361
|
MAPK3
|
Mitogen-activated protein kinase 3
|
P14780
|
MMP9
|
Matrix metalloproteinase-9
|
P04637
|
TP53
|
Cellular tumor antigen p53
|
P05412
|
JUN
|
Transcription factor AP-1
|
P01133
|
EGF
|
Pro-epidermal growth factor
|
P35354
|
PTGS2
|
Prostaglandin G/H synthase 2
|
P40763
|
STAT3
|
Signal transducer and activator of transcription 3
|
P05121
|
SERPINE1
|
Plasminogen activator inhibitor 1
|
P22301
|
IL10
|
Interleukin-10
|
P01584
|
IL1B
|
Interleukin-1 beta
|
P10145
|
CXCL8
|
Interleukin-8
|
P35222
|
CTNNB1
|
Catenin beta-1
|
P37231
|
PPARG
|
Peroxisome proliferator-activated receptor gamma
|
P01100
|
FOS
|
Proto-oncogene c-Fos
|
Q15848
|
ADIPOQ
|
Adiponectin
|
P03372
|
ESR1
|
Estrogen receptor
|
P02741
|
CRP
|
C-reactive protein
|
P28482
|
MAPK1
|
Mitogen-activated protein kinase 1
|
3.3 GO enrichment analysis
Go enrichment analysis was performed on these 64 overlapping genes by using DAVID database. Based on the filter of FDR < 0.01, a total of 55 GO items were obtained, including 38 BP terms, 4 CC terms and 13 MF terms (Fig. 5A). As BP played a dominant role, we further build a bubble diagram for these BP terms according to the ascending order of log P-value (Fig. 5B). The results of diagram showed that 38 BP were mainly concentrated in two categories, angiogenesis and cell proliferation. There are 6 BP terms in the category of angiogenesis, including positive regulation of angiogenesis (GO:0045766), angiogenesis (GO:0001525), cellular response to hypoxia (GO:0071456), positive regulation of endothelial cell proliferation (GO:0001938), response to hypoxia (GO:0001666) and positive regulation of vascular endothelial growth factor production (GO:0010575). For cell proliferation, we found 4 BP terms that were positive regulation of cell proliferation (GO:0008284), negative regulation of cell proliferation (GO:0008285), positive regulation of smooth muscle cell proliferation (GO:0048661) and positive regulation of endothelial cell proliferation (GO:0001938).
3.4 KEGG enrichment analysis
To determine the potential pathways of the anti-PMOP effects of BSHX formula, KEGG enrichment analysis was conducted on the 64 overlapping genes. Based on the threshold of Number ≥ 6, we screened a total of 99 pathways (20 of these listed in Fig. 6), among which VEGF signaling (hsa04370) directly regulates angiogenesis and β-catenin signaling (hsa04310) controls cell proliferation.
3.5 BSHX formula preserves bone mass in OVX mice
To further carry out an animal experimental validation, C57BL/6 J mice were subjected to an OVX surgery and continuously treated with BSHX formula for 8 weeks. The 3D images of µCT showed severe bone loss in the OVX mice compared to the sham ones, and BSHX formula effectively alleviated bone loss (Fig. 7A). We also found that bone microstructure parameters were significantly improved after treatment of BSHX formula, including the increase of BMD, BV/TV, Tb.Th and Tb.N and the decrease of Tb.Sp (Fig. 7B–7F). These results indicated that BSHX formula could preserve bone mass in the OVX mice.
3.6 BSHX formula promotes bone and blood vessel formation in OVX mice
The pathological staining showed that the OVX mice presented sparse and thin trabeculae, massive lipid droplets and decreased blood vessels in the bone marrow (Fig. 8A, boxed areas a-c). After treated with BSHX formula for 8 weeks, both trabecular bone area and blood vessel number were significantly increased, while the area of lipid droplet was drastically decreased (Fig. 8B-8D). Bone homeostasis requires the balance of osteoblast-mediated bone formation and osteoclast-mediated bone resorption (Tella and Gallagher 2014). Our previous study have revealed that BSHX formula has little effect on osteoclasts, and the enhanced trabecular bone are relayed mainly on the osteoblastic activities (Xia et al. 2020). Blood vessels contain a layer flattened endothelial cells that not only participate in angiogenesis but also can regulate osteoblast proliferation for bone formation (Tong et al. 2019). Combined with the data of GO enrichment analysis, we found that BSHX formula prevented bone loss mainly through promoting osteoblast proliferation and angiogenesis.
3.7 OVX-induced down-regulation of β-catenin, ALP, VEGF and CD31 are restored by BSHX formula
Furthermore, we evaluated the expressions of β-catenin signaling and VEGF signaling that have been identified by KEGG enrichment analysis. CD31, expressed in vascular endothelial cells, can specifically reflect the formation of blood vessel. ALP, synthesized by osteoblasts, plays a fundamental role in osteogenesis. The levels of β-catenin, ALP, VEGF and CD31, were significantly deceased in the OVX mice compared to the sham ones (Fig. 9A-9D). Mice treated with BSHX formula presented the improvement of VEGF, CD31, β-catenin and ALP, indicating that BSHX formula could restore the inhibited β-catenin and VEGF sinaling caused by OVX surgery.