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Systematic Pharmacological Methodology to Explore the Pharmacological Mechanism of Siwu Decoction for Osteoporosis

Background Osteoporosis is an important health problem worldwide. Siwu decoction (SWD) and its modification have a good clinical effect on osteoporosis. However, the molecular mechanism of SWD on osteoporosis has not been thoroughly explained. Material/Methods A systematic pharmacological methodology was utilized to predict the active compounds and potential targets of SWD, collect the genes of osteoporosis and the known targets of SWD, and analyze the osteoporosis and SWD’s network. Results Five networks were constructed and analyzed: (1) Osteoporosis genes’ protein–protein interaction (PPI) network; (2) Compound-compound target network of SWD; (3) SWD-osteoporosis PPI network; (4) Compound-known target network of SWD; and (5) SWD known target- osteoporosis PPI network. Several osteoporosis and treatment-related targets (eg.,. HSP90AB1, FGFR1, HRAS, GRB2, and PGF), clusters, biological processes, and signaling pathways (e.g., PI3K-Akt signaling pathway, insulin signaling pathway, MAPK signaling pathway and FoxO signaling pathway) were found. Conclusions The therapeutic effect of SWD on osteoporosis may be achieved by interfering with the biological processes and signaling pathways related to the development of osteoporosis.


Background
Osteoporosis is an important health problem worldwide, the incidence of which is closely related to the increase of the elderly population and the decline of quality of life [1,2]. This systemic bone disease is mainly characterized by low bone mass and bone microstructural damage, which leads to increased bone fragility and fracture [1,2]. The primary goal of osteoporosis treatment is to prevent fractures, maintain or increase bone mineral density (BMD), and improve bone physiology [3,4]. Fracture prevention is a global public health priority [4], and current methods include non-drug management and drug intervention [4]. Non-drug management methods include nutritional improvement, calcium, vitamin D supplements, and exercise [4,5]. Drug interventions mainly include anti-bone resorption drugs (such as bisphosphonates) and osteogenic drugs (e.g., parathyroid hormone and other estrogen hormone therapy [raloxifene]) and other bone metabolism regulation drugs, such as teriparatide, zoledronic acid, ibandronate, and strontium ranelate [4,6]. However, long-term use of these drugs often causes adverse events; for example, anti-bone resorption drugs have liver and kidney toxicity, and it can also lead to gastrointestinal complications, musculoskeletal pain, and osteonecrosis of the jaw [7]. The adverse effects of estrogen hormone therapy are mainly endometrial hyperplasia and uterine bleeding [8]. The adverse effects of calcitonin include flushing, nausea, vomiting, dizziness, and mild facial redness, and continued application may diminish its effect; bone density will gradually decrease after stopping the drug [9].
Recent research shows that Siwu decoction (SWD) and its modification have a good clinical effect on senile osteoporosis and bone fractures [10][11][12]. SWD is an oral decoction, that is, a liquid dosage form prepared by soaking and boiling its 4 herbs in water and then removing the juice from the residue. Studies also show that SWD has estrogen-like effects in both in vitro and in vivo [13][14][15]. SWD extracts can stimulate bone formation through the PI3K/Akt/NF-kB signaling pathway in osteoblasts [16]. These facts suggest that SWD may be an alternative treatment for osteoporosis. SWD is composed of 4 herbs: Rehmanniae Radix Praeparata (Shu Di Huang), Angelicae Sinensis Radix (Dang Gui), Paeoniae Radix Alba (Bai Shao), and Chuanxiong Rhizoma (Chuan Xiong). At present, research on the effect of SWD on osteoporosis is mostly applied by traditional pharmacological methods, which is limited by the perspective of "single compound-single target-single pathway". Therefore, this study used a systematic pharmacology approach to explore the pharmacological mechanism of SWD in treatment of osteoporosis.

Exceptional molecules
Since the application of biological models to predict SWD compounds has limitations [22], in order to avoid missing active compounds during the pre-screening process, we searched a large number of references and selected oral absorbable compounds with pharmacological activity.

Compound targets and known targets for SWD
To assess the compound targets and known targets for SWD, we collected the molecular structure of each compound by searching the SciFinder (http://scifinder.cas.org), drew them in ChemBioDraw, and saved them as "mol2" file format. We input the "mol2" files of each compound into PharmMapper (http://lilab-ecust.cn/pharmmapper) to predict their potential targets [30]. The known targets were collected from the TCMSP [18].

Protein name correction
The UniProtKB (http://www.uniprot.org/), a database containing the accurate annotation of proteins and other substances, was used for the correction of protein's names and the collection of official symbols with the species limited to "Homo sapiens". The corrected target is detailed in the supplementary materials (Supplementary Tables 1, 2).

Osteoporosis genes
OMIM database and Genecards were utilized to collect the osteoporosis-related disease genes and targets [19][20][21]. OMIM database (http://omim.org/) is the database that catalogs all known diseases with a genetic component [31]. Genecards (http://www.genecards.org) is "a database about genes, their products and biomedical applications maintained by Israel's Weizmann Institute of Science" [19][20][21]. Relevance scores greater than 5 were included in the network construction and analysis. The osteoporosis-related genes and their relevance scores are shown in Supplementary Table 3.

Cluster
The definition and the methodology of acquisition of clusters were described in our previous work "Exploring the Pharmacological Mechanism of Danzhi Xiaoyao Powder on ER-Positive Breast Cancer by a Network Pharmacology Approach" [19].

Osteoporosis network
We input 357 genes into the String database to collect the PPI information so as to build this network. It contains 357 nodes and 33770 edges ( Figure 1).

Clusters of osteoporosis network
After analyzing the osteoporosis network by MCODE, 7 clusters were returned (Table 1, Figure 2), and these were subjected to GO enrichment analysis. Several biological processes were obtained. Cluster 16 and 18 did not return any human biological processes. Cluster 2,4,5,11,13,14,15, and 17 did not return any osteoporosis-related biological processes. Taking Supplementary Table 4.

Pathway of osteoporosis network
After the pathway enrichment analysis, 17 osteoporosis-related pathways were obtained ( Figure 3). The PI3K-Akt signaling pathway had the largest number of genes in this analysis (30 genes); the MAPK signaling pathway includes 24 genes; Osteoclast differentiation has 22 genes; Jak-STAT signaling pathway contains 17 genes; Wnt signaling pathway has 16 genes, and so on. The details of each pathway are shown in Supplementary Table 5.
These pathways with so many osteoporosis genes may be the key pathways involved in the development of osteoporosis. Early intervention in these signaling pathways may indicate a strategy for future treatment and prevention of osteoporosis.

SWD-osteoporosis network
To integrate the osteoporosis network and SWD network, the SWD-Osteoporosis network was formed. This network contains 671 nodes and 10698 edges. There are 317 nodes that are compound targets, 31 nodes are SWD-osteoporosis targets, and 323 nodes are osteoporosis genes ( Figure 5).

Clusters of SWD-osteoporosis metwork
Twenty-one clusters were collected after being analyzed by MCODE. There were some osteoporosis-related genes in clusters (Blue circles), and these genes may be the key genes of SWD that are active in treating osteoporosis (Table 2, Figure 6). These clusters were subjected to GO enrichment analysis. Cluster 5,11,12,13,14,15,16,17,18,19,20 did not return osteoporosis-related biological processes. As an example, in cluster 1 there were many biological processes, includ-  Supplementary Table 6.

Compound-known target network
This network contains 125 nodes (103 compound targets node and 22 compound nodes) and 250 edges. This network is smaller than the compound-compound target network and has fewer targets. Some of the targets in this network have also appeared in previous networks ( Figure 8).

SWD known target-osteoporosis network
The SWD known target-osteoporosis network is composed of 440 nodes and 6262 edges ( Figure 9). This network is much smaller than the SWD-osteoporosis network, but some targets that appear in Figure 5 can be found in this network, such as SWD's predicted targets and SWD-osteoporosis targets. There are also some new targets in these areas, and these new targets may be indirectly regulated by SWD's predicted targets.

Cluster and pathway of SWD known target-osteoporosis network
In analyzing the network by MCODE, 11 clusters were returned. (Table 3, Figure 10). Most of them are the same as that in the clusters of the SWD-Osteoporosis network, which indirectly confirm SWD's effects.
These clusters were subjected to GO enrichment analysis. Cluster 5, 9, 10 did not return osteoporosis-related biological processes. As an example: Cluster 1 also has many biological processes, including GO: 0000165, GO: 0038128, GO: 0042346, GO: 0043065, GO: 0043123, GO: 0043410, GO: 0046850, GO: 0048010, GO: 0071392, and GO: 0098868. The details of each cluster and biological process are shown in Supplementary Table 8.
We obtained 15 osteoporosis-related pathways after pathway enrichment analysis. They are the same as pathways in Figures 3 and 7, suggesting that these pathways may be the crucial pathways in osteoporosis development ( Figure 11). The PI3K-AKT signaling pathway in Figures 7 and 11 contains the most targets and genes; these targets and genes are shown in Figure 12 (red squares) as an example. Some of the red squares in Figure 12B overlap with those in Figure 12A, which further demonstrates the reliability of the predicted network. The details are described in Supplementary Table 9.

8157
In this compound-known target network, we found some signaling pathways and biological processes that have appeared in previous networks, such as: hsa04380, hsa04210, hsa04910, GO: 0070374, GO: 0045453, and GO: 0001957. Several biological processes are also similar in functions. This somewhat verifies the reliability of the predicted target network. However, because this method failed to directly reflect the impact of compound content on biological processes, more research is required to test the effects of SWD and compounds in relation to osteoporosis and to confirm their pharmacological and molecular mechanisms.

Discussion
Three main networks (Osteoporosis genes PPI network, SWD-osteoporosis PPI network, and SWD known target-osteoporosis PPI network) were constructed and analyzed in this research. The first network uncovered the possible mechanism of osteoporosis. The second network explored the potential mechanism of SWD treating osteoporosis. The third network confirmed the feasibility of the second network.

8158
According to the network analysis, the compounds with high degree (with many connected predicted targets) are thought to be the core compounds of an herb; the pathways containing many targets linked by core compounds are considered to be the ones regulated by those core compounds. Based on this, the core compounds of Rehmanniae Radix Praeparata are acteoside, benzoic acid, and 5-hydroxymethylfurfural. The top 3 pathways regulated by acteoside are insulin signaling pathway, PI3K-Akt signaling pathway, and osteoclast differentiation. The top 3 pathways controlled by benzoic acid are PI3K-Akt signaling pathway, FoxO signaling pathway, and insulin signaling pathway. The top 3 pathways regulated by   The development of osteoporosis is the result of a combination of multiple factors. At the molecular level, the process of bone formation and bone remodeling involves a variety of signaling pathways, and they interact with each other to play a fine regulatory role in complex regulatory network systems. Studies have shown that Wnt/b-catenin signaling pathway [36], BMP-2 signaling pathway [37], and OPG/RANKL signaling pathway [38] play important regulatory roles in bone formation and bone remodeling [39]. The BMP-2 signaling pathway process has 2 roles in the cell. One is to transfer the external growthpromoting signals to the nucleus via Smads to promote osteogenic differentiation [37]. The other is the MAPK signaling pathway, which also includes 3 signaling pathways: the extracellular signal-regulated kinase (ERK) signaling pathway, the c-Jun N-terminal kinase (JNK) signaling pathway, and the p38 signaling pathway [40][41][42]. These BMP-2 signaling pathways act through phosphorylation, which in turn regulates transcription of downstream target genes such as RUNX2 and Osterix, so as to promote osteogenesis [40][41][42]. In summary, existing studies have found that the interaction of multiple signaling pathways mediates the development of osteoporosis. Changes in the expression of these signaling pathways may reduce osteoblast formation, proliferation, and differentiation, ultimately leading to osteoporosis.

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From a cytological point of view, osteoporosis is closely related to abnormal osteoclastogenesis [43]. Osteoclasts are derived from the monocyte-macrophage lineage and play an important role in normal development and in some diseases [44]. Osteoclastogenesis in vivo is controlled by various cytokines and signaling pathways [45], wherein macrophage colony-stimulating factor (M-CSF) and the receptor activator NF-kB ligand (RANKL) are the 2 most important factors 8163 enrichment analysis, such as hsa04010, hsa04310, hsa04668, GO: 0070374, GO: 0000165, and GO: 0030514.
Because the OPG/RANKL/RANK signaling pathway, Wnt/b-catenin signaling pathway, and BMPs signaling pathway play an important role in regulating osteogenesis and osteoclast lineage function, as well as in regulating bone mass and bone remodeling, some protein molecules in these signaling pathways have become new targets for osteoporosis treatment. Targeted therapeutics for these targets have also entered clinical trials, but they are generally more expensive and have potential adverse effects [49][50][51][52][53][54]. This research shows that the active components of SWD can directly (purple circles in Figure 5) or indirectly (through pink circles to blue circles in Figure 5) regulate osteoporosis-related targets, thereby regulating "(hsa04010) MAPK signaling pathway", "(hsa04668) TNF signaling pathway", and "(hsa04064) NF-kappa B signaling pathway", as well as "(GO: 0071773) cellular response to BMP stimulus", "(GO: 0043410) positive regulation of MAPK cascade", "(GO: 0060070) canonical Wnt signaling pathway", and "(GO: 0046330) positive regulation of JNK cascade".

8165
For endocrine therapy, estrogen is a bone resorption inhibitor that directly regulates bone metabolism, reduces bone resorption after menopause, relieves bone and joint pain, and reduces the incidence of fractures [49]. Therefore, selective estrogen receptor modulators play an important role as a classic drug for the treatment of osteoporosis. Our study found that SWD can regulate estrogen-related biological processes and signaling pathways to exert anti-osteoporosis effects, such as "(hsa04915) Estrogen signaling pathway", "(GO: 0032355) response to estradiol", and "(GO: 0043627) response to estrogen". Liu et al. used microarray transcriptional analysis to demonstrate the discovery of phytoestrogens in SWD [15]. Wen et al. also identified SWD as Nrf2 activator and phytoestrogens [14].  Lu et al. found that SWD drug-containing serum showed an estrogen-like effect, and in vitro experiments further confirmed that SWD drug-containing serum can activate ER signaling pathways [13]. The long-term use of estrogen is likely to lead to an increased risk of estrogen-related tumors and cardiovascular adverse events. Meanwhile, because of its two-way regulation, phytoestrogens can alleviate the symptoms of low estrogen after menopause, while avoiding the risk of reproductive system tumors caused by medical treatment of estrogen replacement therapy. The estrogen-like effects of phytoestrogens affect hormone secretion, metabolic biological activity, protein synthesis, and growth factor activity. It has different functions under different physiological conditions. For example, in the case of strong estrogen physiological activity, phytoestrogens can act as antiestrogens and reduce the risk of estrogen-regulated cancers such as breast cancer. Recent studies in breast cancer have also shown a 21% reduction in mortality among patients with low intake (³0.3 mg/d) vs. the high-intake breast cancer group (³1.5 mg/d). This suggests that phytoestrogens are safe even in the treatment of high-risk breast cancer [55]. When estrogen has low physiological activity (such as after female menopause), phytoestrogens can enhance estrogen and reduce various symptoms caused by low estrogen (especially osteoporosis). Recent research has also shown that phytoestrogens can better prevent osteoporosis while reducing other risks associated with exogenous estrogen [56,57]. Therefore, SWD has an estrogen-like effect and may be a potential alternative treatment option [49].
In vitamin D and calcium supplementation, vitamin D is closely related to calcium and phosphorus metabolism. Vitamin D is an important hormone regulating bone metabolism in the body. It can affect bone formation through indirect and direct effects on osteoblasts [58,59]. Therefore, vitamin D plays an important role in the prevention of osteoporosis [58,59]. The active metabolite 1a,25-dihydroxyvitamin D [1,25(OH)2D] of vitamin D in the body can act on bone formation indirectly by promoting intestinal calcium absorption [60]. In addition, osteoblasts express vitamin D receptor (VDR), which is also an important target cell for 1,25(OH)2D action. In vitro osteoblast culture showed that physiological amounts of 1,25(OH)2D can stimulate osteoblast differentiation and promote bone formation [60,61]. Bone calcium stores calcium and plays a very important role in maintaining blood calcium stability. When the blood calcium concentration increases or decreases, the body will mobilize a variety of mechanisms to deal with it by adjusting the "inventory" to ensure normal blood calcium. When the blood calcium concentration is increased, calcium is sent to the bone for storage. This process is called bone formation. When the blood calcium concentration is lowered, calcium is dissolved from the bone to supplement the deficiency of blood calcium. This process is called bone resorption [62,63]. These 2 effects are regulated by hormones such as parathyroid hormone, vitamin D, and calcitonin. When people reach old age, various hormone secretions are reduced, bone metabolism is disordered, and the absorbed calcium cannot compensate for excreted calcium, and the body is in a state of negative calcium balance [64]. Hence, calcium preparations and vitamin D are often used as basic medicines for the treatment of osteoporosis. This research shows that SWD slows the progression of osteoporosis by regulating the biological processes of calcium metabolism, such as "(GO: 0035630) bone mineralization involved in bone maturation", and "(GO: 0030278) regulation of ossification", as well as vitamin D biological processes "(GO: 0060557) positive regulation of vitamin D biosynthetic process" and "(GO: 0042359) vitamin D metabolic process". In terms of bone formation, Wu et al. showed that NF-kB activation contributes to SWD extract-induced bone mineralization and expression of ALP, BMP-2 and OPN in osteoblasts in vitro [14]. In addition, there are also some biological processes directly related to the development of osteoporosis in Supplementary Tables 4 and 6, such as "(GO: 0030316) osteoclast differentiation", "(GO: 0045669) positive regulation of osteoblast differentiation", and "(GO: 0045453) bone resorption". This suggests that SWD may directly regulate these biological processes to achieve therapeutic effects.
Last but not least, because this method failed to directly reflect the impact of compound content on targets, pathways, and biological processes, further experimental research is required to test the effects of SWD and compounds in relation to osteoporosis and to confirm their pharmacological and molecular mechanisms. However, the present study did not produce negative results. Instead, it suggests that systemic pharmacology can save time and effort by predicting targets, signaling pathways, and biological processes to provide direction for subsequent in-depth experimental research.

Conclusions
The therapeutic effect of SWD on osteoporosis may be achieved by interfering with the above-mentioned targets (such as HSP90AB1, FGFR1, HRAS, GRB2, and PGF), biological processes, and signaling pathways (such as PI3K-Akt signaling pathway, insulin signaling pathway, MAPK signaling pathway, and FoxO signaling pathway) related to the development of osteoporosis.

Conflict of interest
None.