Exploring the mechanism of Bushenhuoxue formula acting on postmenopausal osteoporosis via network pharmacology and experimental validation

Background: Bushenhuoxue (BSHX) formula, a ten-compound herbal decoction, has been used to treat postmenopausal osteoporosis (PMOP) in China for decades. Our previous study also revealed the anti-PMOP effects of BSHX formula in ovariectomized (OVX) mice. However, its pharmacological mechanisms remain unknown. Methods: Network pharmacology followed by animal experiments was used to explore the action mechanism of BSHX formula. Firstly, we obtained the chemical compounds and potential targets of BSHX formula using TCMSP and TCMID databases. Simultaneously, GeneCards and DisGeNET databases were used to determine the targets in PMOP. Based on their overlapping genes between BSHX formula and PMOP, a protein-protein interaction (PPI) network was constructed for screening hub-targets. GO and KEGG enrichment analyses were further performed to identify the critical biological processes and signaling pathways. In vivo experimental study, an OVX mouse model was established to determine the anti-PMOP effects of BSHX formula via Micro-CT assay and ABH staining. The expressions of β-catenin, alkaline phosphatase (ALP), vascular endothelial growth factor (VEGF) and CD31 were detected by immunohistochemistry. Results: A total of 218 active ingredients and 274 related targets were identied in BSHX formula. After matching with 292 therapeutic targets of PMOP, 64 overlapping genes were obtained and further used to build a PPI network in which CTNNB1 and VEGFA played a central role. GO and KEGG enrichment analyses indicated that the anti-PMOP effects of BSHX formula were mainly associated with cell proliferation, angiogenesis and their regulated pathways including β-catenin signaling and VEGF signaling. In mouse experiments, we revealed that bone mass and blood vessels in the OVX mice were signicantly enhanced after treated with BSHX formula for 8 weeks. Moreover, down-regulations of β-catenin, ALP, VEGF and CD31 caused by OVX surgery were signicantly restored by BSHX formula. Conclusions: Through network pharmacology and an experimental validation, we have revealed that BSHX formula exerts anti-PMOP effects mainly through promoting β-catenin-mediated osteogenesis and VEGF-mediated angiogenesis. BSHX formula can be considered as a new option for the treatment of PMOP.


Introduction
Postmenopausal osteoporosis (PMOP) is a common bone disorder that was characterized by low bone mineral density and microstructure deterioration 1 . It is estimated that more than 15% of postmenopausal women over 50 years old are suffering from PMOP in United States 2 , which can cause poor clinical outcomes including low back pain, hunchback and fragility fractures 3 . Although there exit many antiosteoporosis drugs such as active vitamin D, estrogen receptor modulators, bisphosphonates and parathyroid hormone 4 , their application and e cacy remain limited because of various undesirable effects 5 . Currently, natural productions and herbs attract more and more attentions for their potential antiosteoporosis effects and relative safety 6 .
Traditional Chinese medicine (TCM) is used as a main alternative treatment worldwide. Bu-shen-huo-xue (BSHX) formula is a Chinese herbal decoction composed by ten herbs 7 . In China, BSHX formula has been used to treat PMOP since a long time, and gains satisfactory outcomes. Our recent study found that BSHX formula can attenuate bone loss in ovariectomized (OVX) mice, con rming its anti-PMOP effects 8 .
The theory of "kidney governing bones" has well clari ed its positive effects on PMOP in TCM perspective. Bone loss in PMOP is due to de ciency of kidney-qi and BSHX formula strengthens bone through tonifying kidney-qi 9 . However, it is still unclear about the pharmacological mechanism of BSHX formula.
For the multi-component properties of TCM formula, conventional animal or cellular research strategies can not meet the requirements to study massive molecular targets simultaneously. Network pharmacology is an emerging discipline that integrates pharmacology, bioinformatics, system biology and computer science 10,11 . Based on the concept of "Medicine -Target -Gene -Disease", network pharmacology provides a systematic and integrative viewpoint to explore the relationship between TCM formula and diseases 12,13 . Here, through network pharmacology, we comprehensively predicted the underlying molecular targets of BSHX formula in the treatment of PMOP.
In the network pharmacology study, related ingredients of BSHX formula originated from two databases were used to sh the potential targets. After matching with PMOP therapeutic targets obtained from GeneCards and DisGeNET databases, 64 overlapping target genes were identi ed and further used to build a protein-protein (PPI) network for analyzing their internal interactions. DAVID database and Kyoto Encyclopedia of Genes and Genomes (KEGG) database were utilized for gene ontology (GO) and pathway enrichment analysis, respectively. Based on the results of network pharmacology, animal experiments were performed to validate the key biological process and signaling pathway through which BSHX formula exerts the anti-PMOP effects.

PMOP targets screening
GeneCards Database (https://www.genecards.org/) and DisGeNET Database (https://www.disgenet.org/) were used to collect PMOP-associated target genes. After screening with the lter of score > 10 for GeneCards Database and score > 0.1 for DisGeNET Database (Zhu and Hou 2020), a total of 292 therapeutic targets were identi ed. Then, a Venn diagram was constructed to determine the overlapping genes between PMOP and active ingredients, which were terms as the potential therapeutic targets of BSHX formula for PMOP treatment. The network was constructed as follows: (1) The active ingredients obtained from BSHXF via TCMSP database and TCMID database were imported into Cytoscape3.8.0 software (http://www.cytoscape.org/) to establish a Herb-Ingredient network; (2) The overlapping targets were analyzed using the String database (https://string-db.org/) to build a PPI network. The PPI network topological feature was evaluate by three topological characteristics: Degree Centrality (DC), Betweenness Centrality (BC) and Closeness Centrality (CC).

Gene ontology and pathway enrichment analyses
The gene ontology (GO) enrichment analysis including biological process (BP), cellular component (CC), and molecular function (MF) terms were performed on the 64 overlapping targets using DAVID database (version 6.8, https://david.ncifcrf.gov/home.jsp). KEGG database (https://www.kegg.jp/) was used to identify the potential signaling pathways.

Experimental groups and OVX model
Ten-week-old female C57BL/6 J mice provided by Shanghai Laboratory Animal Center of Chinese Academy of Science (Shanghai, China) were randomly divided into three groups (n = 8 in each group): the sham group, the OVX group and the BSHX formula group. Mice in the latter two groups were subjected to bilateral ovariectomy, whereas a sham surgery only extracting the equal surrounding fatty tissues of ovaries were performed in the sham ones. BSHX formula was orally administered to the mice in BSHX formula group at a dosage of 0.2 mL/10 g body weight every two days 8 . The mice in the sham group and the OVX group were given same dosage of 0.9% normal saline. All animal experiments were approved by the Animal Ethics Committee of Zhejiang Chinese Medical University (LZ12H27001).

Micro-CT analysis
After administration with BSHX formula for 8 weeks, femur samples were obtained from the mice in each group for micro-computed tomography (Micro-CT) analysis. Three-dimensional (3D) images of femoral metaphysis were reconstruct using NRecon software. The parameters of bone microstructure including bone volume fraction (BV/TV, %), average trabecular number (Tb. N, 1/mm), average trabecular thickness (Tb. Th, mm), average trabecular separation (Tb. Sp, mm) and bone mineral density (BMD) was calculated as previously described 16 .

Histology and immunohistochemistry (IHC)
After Micro-CT scanning, femur samples were processed for para n sections at the thick of 3µm as previously described 17

Statistical analysis
All data were presented as mean ± standard deviation. One-way ANOVA followed by Dunnett's test were performed using SSPS 20.0 software. P < 0.001 was considered as statistical signi cance.
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 rst 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 identi ed (Fig. 4). The information of these hub nodes were listed in Table 2.

GO enrichment analysis
Go enrichment analysis was performed on these 64 overlapping genes by using DAVID database. Based on the lter 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.

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.

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 signi cantly 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.

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 signi cantly 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 ). Blood vessels contain a layer attened 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 identi ed by KEGG enrichment analysis. CD31, expressed in vascular endothelial cells, can speci cally re ect 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 signi cantly 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.

Discussion
With the progress of the aging population in society, PMOP has became a frequent public health disease 18 . BSHX formula, severing as a complementary and alternative medicine, present an anti-PMOP effects in clinic and can prevent bone loss in OVX mice 8 , but its mechanism remains unclear. Here, we aimed to explore the underlying mechanism of BSHX formula acting on PMOP through network pharmacology and experiment approaches.
Based on multi-database mining and co-targets screening between BSHX formula and PMOP, a total of 64 overlapping genes were preliminarily obtained and regarded as the potential action mechanism of BSHX formula. PPI network analysis conformed 21 central gene targets, including VEGFA and CTNNB1 that is the key component of VEGF signaling and β-catenin signaling, respectively. Furthermore, the results of GO and KEGG enrichment analyses focused mainly on angiogenesis and cell proliferation, and these two biological processes were directly controlled by VEGF signaling and β-catenin signaling. Therefore, by utilizing network pharmacology, we speculated that BSHX formula exacts anti-PMOP effects mainly through promoting angiogenesis and cell proliferation in a VEGF-and β-catenin-mediated manner.
The dynamic balance between bone formation and resorption maintains adult skeletal health 19 . Bone formation tends to weak due to the de ciency of estrogen, and PMOP occurs when bone formation directed by osteoblasts is slower than osteoclast-mediated bone resorption 20 . Osteoblasts, bone forming cells, play an important role in osteogenesis through self-proliferation and producing extracellular matrix 21 . Speci c proteins associated with osteoblast state, such as ALP, can be largely secreted during cell proliferation and matrix maturation, regarding as an early phenotypic marker for osteoblastogenesis 22 . It is well known that bone is a highly vascularized tissue with a wide network of capillaries and blood vessels. Bone formation needs a spatial and temporal involvement of vascularization to provide essential nutrients, oxygen and growth factors, which is named osteogenesisangiogenesis coupling 23,24 . More remarkably, recent researches demonstrated that new blood vessels can directly regulate osteoblast proliferation for osteogenesis 25,26 . Therefore, activating osteogenesisangiogenesis coupling in bone might be an underlying strategy in the prevention of PMOP.
In the experimental validation part, we observed that OVX-induced bone loss were signi cantly rescued by BSHX formula, as shown by the increased BMD, BV/TV, Tb.Th and Tb.N and decreased Tb.Sp in BSHX formula treated mice. The results of ABH staining showed sparse trabeculae and blood vessels in the bone marrows of OVX mice, while both trabecular bone area and blood vessel number were largely improved after treatment with BSHX formula. Furthermore, we found higher levels of ALP and CD31 in the BSHX formula treated mice compared to the OVX ones. All these data indicated that BSHX formula preserves bone mass mainly through promoting osteogenesis-angiogenesis coupling.
Indeed, multiple anabolic signaling pathways positively control osteogenesis and angiogenesis, especially WNT and VEGF pathways. Canonical WNT/β-catenin signaling has been reported to promote proliferation and differentiation of osteoblasts 27,28 . Its inhibition exerts critical effects in the development of PMOP 29 . VEGF, a major driver of angiogenesis, can activate a series of well-orchestral vascularization processes including endothelial cell proliferation, migration, sprouting vessel pruning and anastomosis, when it binds to the VEGF receptor on vascular endothelial cells 30 . Moreover, there is a close crosstalk between VEGF and β-catenin signaling in osteogenesis. The increased VEGF signaling can enhance βcatenin activity, resulting in excessively bone formation 31 . Thus, these two pathways are important molecular targets to prevent the progression of PMOP. Combined with KEGG enrichment analysis, we further determined the changes of β-catenin and VEGF signaling pathways in the OVX mice by IHC, and the results showed that the deceased β-catenin and VEGF proteins in the OVX mice were signi cantly restored by BSHX formula. All these data indicated that BSHX formula exerted anti-PMOP effects primarily through β-catenin-mediated osteogenesis and VEGF-mediated angiogenesis.

Conclusions
In summary, through network pharmacology and an animal experimental validation, our present study revealed that BSHX formula attenuates PMOP by promoting osteogenesis and angiogenesis in a VEGFand β-catenin-mediated manner. We also provided a new alternative for the clinical treatment of PMOP.

Declarations Con icts of interest
All authors state that they have no con icts of interest.

Funding statement
This study was supported by Traditional Chinese Medical Administration of Zhejiang Province (grant nos. 2021ZQ082, 2021ZA131). Figure 1 The network of Herb-Ingredient connection. The red square nodes represent herbs in BSHX formula, and the blue circles represent active ingredients. The edges represent the direct relationship between them.

Figure 4
The topological screening process of the PPI network. In the third image, the bigger size and more brilliant color represent higher DC value.