Research on the Mechanism of Liuwei Dihuang Decoction for Osteoporosis Based on Systematic Biological Strategies

Background Osteoporosis is an important health problem worldwide. Liuwei Dihuang Decoction (LDD) and its main ingredients may have a good clinical effect on osteoporosis. Meanwhile, its mechanism for treating osteoporosis needs to be further revealed in order to provide a basis for future drug development. Methods A systematic biological methodology was utilized to construct and analyze the LDD-osteoporosis network. After that, the human transcription data of LDD intervention in patients with osteoporosis and protein arrays data of LDD intervention in osteoporosis rats were collected. The human transcription data analysis, protein arrays data analysis, and molecular docking were performed to validate the findings of the prediction network (LDD-osteoporosis PPI network). Finally, animal experiments were conducted to verify the prediction results of systematic pharmacology. Results (1) LDD-osteoporosis PPI network shows the potential compounds, potential targets (such as ALB, IGF1, SRC, and ESR1), clusters, biological processes (such as positive regulation of calmodulin 1-monooxygenase activity, estrogen metabolism, and endothelial cell proliferation), and signaling and Reactome pathways (such as JAK-STAT signaling pathway, osteoclast differentiation, and degradation of the extracellular matrix) of LDD intervention in osteoporosis. (2) Human transcriptomics data and protein arrays data validated the findings of the LDD-osteoporosis PPI network. (3) The animal experiments showed that LDD can improve bone mineral density (BMD), increase serum estradiol (E2) and alkaline phosphatase (ALP) levels, and upregulate Wnt3a and β-catenin mRNA expression (P < 0.05). (4) Molecular docking results showed that alisol A, dioscin, loganin, oleanolic acid, pachymic acid, and ursolic acid may stably bind to JAK2, ESR1, and CTNNB1. Conclusion LDD may have a therapeutic effect on osteoporosis through regulating the targets (such as ALB, IGF1, SRC, and ESR1), biological processes (such as positive regulation of calmodulin 1-monooxygenase activity, estrogen metabolism, and endothelial cell proliferation), and pathways (such as JAK-STAT signaling pathway, osteoclast differentiation, and degradation of the extracellular matrix) found in this research.


Introduction
Osteoporosis is a common systemic metabolic bone disease with a decrease in bone density and bone quality and bone microstructural damage caused by various reasons [1]. e serious clinical outcome of osteoporosis is osteoporotic fractures (fragility fractures), which lead to a significant increase in morbidity and mortality in patients with osteoporosis [2]. e treatments for osteoporosis include the following: (1) basic prevention, such as lifestyle adjustment (diet and outdoor sports) and basic bone health supplements (calcium and vitamin D); (2) drug interventions, such as antibone resorption drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators (SERMs), and estrogen); (3) drugs that promote bone formation, such as targeted drugs [2][3][4]. However, recent studies showed that the preventive and therapeutic effects of the above drugs are still controversial, such as vitamin D [5], while antibone resorption drugs increase the burden of medical resources and reduce patient compliance due to their high price [6].
Natural plant products have become the direction of new drug development due to their multicompound, multitarget features and cheap price [7,8].
Traditional Chinese medicine (TCM), as a traditional medicine applied for thousands of years, has gradually highlighted its therapeutic advantages in osteoporosis through long medical clinical practice [8]. Liuwei Dihuang Decoction (LDD) as a representative of TCM for the treatment of osteoporosis, comes from Jingyue Quanshu.
is . Current clinical studies showed that LDD alone or in combination with other antiosteoporosis drugs (alendronate, salmon calcitonin) for osteoporosis has a certain effect: it can effectively improve the patient's bone density (in the lumbar spine, the femoral neck, the forearm, the distal third of the junction, and the tibia bone density) and the clinical efficacy rate [9,10]. A systematic review and meta-analysis also showed that LDD can increase the bone mineral density of the hip, lumbar spine, ulna, and radius, and it has a good effect in improving the effective rate of clinical treatment of postmenopausal osteoporosis and reducing the pain caused by osteoporosis [11]. Its mechanism may be related to the regulation of hormone levels and oxidative stress through the Wnt/ β-catenin signaling pathway [12,13]. In addition, current pharmacological studies showed that LDD has the effect of inhibiting aging and prolonging the lifespan of C. elegans life-sustaining and natural aging mice [14]. However, its mechanism for treating osteoporosis needs to be further revealed in order to provide a basis for future drug development.
Systemic pharmacology is a discipline that studies the effects of drugs on disease at a system level, which combines multidisciplinary technologies such as bioinformatics and network pharmacology to bring new strategies for analyzing the mechanism of drugs [15,16]. is method can resolve multicompound and multitarget drugs from microscopic (molecular and biochemical network level) to macroscopic (tissue, organ, and overall level) levels [15,16]. Meanwhile, at present, researchers have explored the therapeutic mechanisms of natural products for disease through systematic pharmacology [17][18][19][20]. erefore, in this research, the systematic pharmacological methodology would be utilized to uncover the mechanism of LDD on osteoporosis. e process of this study is shown in Figure 1.

LDD's Potential Target Prediction and Osteoporosis Genes
Collection.
ese "sdf" files of each compound were input into PharmMapper (https://lilabecust.cn/pharmmapper/) to predict the potential targets [29]. e name of the target protein was imported into UniProtKB (https://www.uniprot.org/), and the species was limited to "Homo sapiens," so that the name of the protein was corrected to their official symbols (see Table S1 in Supplementary Materials). Meanwhile, the OMIM database (https://omim.org/) and GeneCards (https://www. genecards.org) were utilized to collect the osteoporosis-related disease genes and targets with the keyword "Osteoporosis" [17,18]. e osteoporosis-related genes and their relevance scores are shown in Table S2.

Human Transcriptome Data and Protein Arrays Data
Collection. Transcriptome data come from GEO (https:// www.ncbi.nlm.nih.gov/geo/). e data on LDD treating osteoporosis were obtained from GSE57273. e protein arrays data of LDD intervention in osteoporosis rats come from [30]. Differential expression analysis was performed using the R software package.

Network Construction and Analysis Methods.
e protein-protein interaction (PPI) data were collected from String database (https://string-db.org/) with medium confidence >0.4 and the IntAct database (https://www.ebi.ac.uk/ intact/) [31,32]. e LDD targets, osteoporosis genes, and their PPI data were input into Cytoscape 3.7.0 (https:// cytoscape.org/) for network construction and analysis [33]. In the PPI network, there are dense areas of some molecular complexes, which are defined as clusters [33]. Clusters can be viewed as functional modules, and drugs may treat diseases by regulating these functional modules. e cluster was obtained by analyzing the network using MCODE, a plug-in of Cytoscape software.

Gene Ontology (GO), Signaling Pathway, and Reactome Pathway Enrichment Analysis.
e LDD targets, osteoporosis genes, human transcriptome data, and protein arrays data were input into the DAVID database ver. 6.8 (https:// david-d.ncifcrf.gov) for GO enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment analysis [34]. GO enrichment analysis includes biological processes, cell components, and molecular function. Meanwhile, those data were input into the Reactome Pathway Database (https://reactome.org/) for Reactome pathway enrichment analysis [35].  Evidence-Based Complementary and Alternative Medicine 3 500 r/min for 15 minutes.

Experimental
e serum was separated and stored in a refrigerator at −80°C. e right femur of the rat was stored at −20°C for bone density detection and Quantitative Real-time PCR (qRT-PCR). e left femoral head of the rat was fixed with 4% paraformaldehyde for HE staining.

Serum E2, ALP Level, and Bone Mineral Density
(BMD) Detection. Serum E2 and ALP were detected by ELISA. BMD was measured by a dual-energy X-ray bone densitometer.

Histopathological Observation.
e left femoral head was fixed with a volume fraction of 4% paraformaldehyde for 72 h and then decalcified with 10% EDTA decalcification solution, and the decalcification solution was changed every 3 days. After decalcification was completed, it was rinsed with PBS, dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin, and sliced for HE staining. e histopathological changes in the femoral head were observed under a light microscope.

Osteoporosis Genes and the Potential Targets of LDD.
Two thousand and nine hundred and eighty-three osteoporosis genes were obtained from GeneCards and OMIM database. e osteoporosis genes with a relevance score ≥5.0 were selected to construct the LDD-osteoporosis PPI network (see Table S2). A total of 423 LDD potential targets were predicted from PharmMapper. e relationship between LDD compounds and LDD potential targets is shown in Figure 1, which consists of 423 compound targets, 68 compounds, and 10009 edges. In Figure 2, targets near the center are regulated by more compounds, whereas targets near the periphery are regulated by fewer compounds. For example, HSP90AA1, CDK2, GSTP1, AKR1B1, BACE1, CA2, F2, LCK, and GSTA1 are regulated by all compounds; ACE2 can be only regulated by chlorogenic acid.

LDD-Osteoporosis PPI Network.
e target shared by LDD potential target and osteoporosis genes is LDD-osteoporosis targets. e LDD-osteoporosis PPI network contains 743 nodes (379 LDD target nodes, 35 LDDosteoporosis nodes, and 329 osteoporosis genes nodes) and 8081 edges ( Figure 3). After analysis of the LDD-osteoporosis PPI network, it was found that LDD can directly and indirectly regulate the core target of osteoporosis. e top 20

Clusters of LDD-Osteoporosis PPI Network.
e Cytoscape's plug-in MCODE was utilized to analyze the LDD-osteoporosis PPI network, and 13 clusters were obtained (Table 3 and Figure 4). e targets and genes in clusters were input into the DAVID database to undergo GO enrichment analysis.
Cluster 1 is mainly involved in several signaling pathways in osteoporosis (such as MAPK, PI3K, and JAK-STAT signaling pathway) and positive regulation of calmodulin 1-monooxygenase activity, estrogen metabolism, endothelial cell proliferation, and neovascularization during bone remodeling. Cluster 2 is mainly involved in endothelial cell proliferation in bone remodeling, differentiation of osteoblasts and chondrocytes, bone resorption, and some signaling pathways (such as PI3K, MAPK, NF-κB, TGF-beta/SMAD, BMP, and Wnt signaling pathway). Cluster 3 is associated with bone resorption and reconstruction, mesenchymal cell differentiation, osteoblast differentiation, osteogenic matrix, and osteoporosis-related signaling pathways (BMP, Wnt, and TGF-β signaling pathway). Cluster 4 is mainly involved in endothelial cell proliferation in bone remodeling and differentiation of osteoblasts and chondrocytes. Cluster 6 is mainly related to steroid metabolism. Cluster 10 is related to steroid metabolism. Cluster 15 failed to return any human biological processes. Clusters 5,7,8,9,11,13,14, 16 and 17 do not return any osteoporosis-related biological processes. e details of each cluster and biological processes are described in Table S3.
Since cluster 1 is the most important one, it is used as an example to show its main biological processes on the bubble chart ( Figure 5(a)).

Cell Components, Molecular Functions, and Signaling Pathway of LDD-Osteoporosis PPI Network.
e LDD targets and osteoporosis in the LDD-osteoporosis PPI network were input into DAVID to collect cell component, molecular functions, and signaling pathways. e top 10 cell components were as follows: extracellular region, extracellular Evidence-Based Complementary and Alternative Medicine space, extracellular exosome, cytosol, ficolin-1-rich granule lumen, endoplasmic reticulum lumen, cytoplasm, secretory granule lumen, receptor complex, and membrane ( Figure 5(b)). e top 10 molecular functions were as follows: identical protein binding, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, enzyme binding, zinc ion binding, transmembrane receptor protein tyrosine kinase activity, protein tyrosine kinase activity, protein homodimerization activity, protein binding, protein serine/threonine/tyrosine kinase activity, and ATP binding ( Figure 5(c)). e top 10 pathways were as follows: FoxO signaling pathway, estrogen signaling pathway, osteoclast differentiation, PI3K-Akt signaling pathway, Ras signaling pathway, T-cell receptor signaling pathway, GnRH signaling pathway, metabolic pathways, and insulin signaling pathway ( Figure 5(d)). e role of LDD in the Wnt signaling pathway is shown in Figure 5(e). e LDD targets are marked in red, the osteoporosis gene is marked in blue, and the LDD-osteoporosis is marked in purple (see Table S4).
Meanwhile, the compounds of Cornus officinalis Sieb. et Zucc. totally regulate 228 targets (which is the most), while those of Rhizoma Dioscoreae regulate 216 targets. e compounds of Rhizoma Dioscoreae and Rhizoma  Evidence-Based Complementary and Alternative Medicine Dioscoreae regulate 206 targets, respectively. is suggests that Cornus officinalis Sieb. et Zucc. and Rhizoma Dioscoreae play a major role in LDD ( Figure 6).

Reactome Pathway of LDD-Osteoporosis PPI Network.
e LDD targets and osteoporosis in the LDD-osteoporosis PPI network were input into Reactome Pathway Database to collect the Reactome pathways. ese osteoporosis-related Reactome pathways were arranged according to the P-value from small to large, and it is found that interleukin-4 and interleukin-13 signaling is ranked first. According to the sorting, the other Reactome pathways (top 10) are as follows: Cytokine Signaling in Immune system, Signaling by Receptor Tyrosine Kinases, Negative regulation of the PI3K/AKT network, PI5P, PP2A and IER3 Regulate PI3K/AKT Signaling, Interleukin-12 family signaling, SUMOylation of intracellular receptors, Interleukin-12 signaling, Extracellular matrix organization, and Degradation of the extracellular matrix. e top 30 Reactome pathways are shown in Figure 5(e). e details of each Reactome pathways are shown in Table S5 At present, it was found that LDD has obvious antiosteoporosis effect in ovariectomized rats, which is mainly achieved by activating the Wnt/β-catenin signaling pathway [40]. Ge et al. found that in patients with postmenopausal osteoporosis (PMOP) with Shen (Kidney) yin deficiency, the therapeutic effect of LDD may be mediated through upregulation of CLCF1 gene and IRF1 gene expression and activation of JAK/STAT signaling pathway [12,41]. Xu et al. found that LDD can significantly inhibit the methylation of ERα gene promoter and increase the expression of ERα mRNA and protein and promote the proliferation and differentiation of osteoblasts by AMPK and β-catenin signaling mediated by this pathway [42]. In addition, in exploring the plant estrogen activity of LDD, new studies have shown that in the ovariectomized rat model, enhanced : LDD-osteoporosis PPI network; blue circle stands for osteoporosis genes; orange circle stands for LDD potential targets; green circle stands for LDD-osteoporosis targets. e size of each node is related to its degree; the bigger nodes have a larger value of degree. e width of the line is associated with its edge betweenness; the wider lines have a larger value of edge betweenness. estrogen activity occurs when combined with the soy diet with LDD [43]. Moreover, a large number of studies confirmed that the active compounds in LDD's herbs play an antiosteoporosis role.

Human Transcriptomics Data.
e transcriptomics data from GSE57273 were collected from GEO. A total of 45220 genes with their data were obtained (Table S9). e genes with log2FC ≥1 or ≤−1 and P-value <0.05 were thought to be differentially expressed genes (Figure 7(a)). e gene expression matrix of osteoporosis-related genes is shown in Figure 7(b); take 50 genes as an example (Table S6).
To further analyze the transcriptomics data, the identified genes with log2FC ≥2 or ≤−2 and P-value <0.01 (significantly differentially expressed genes) were selected. After the selection, a total of 4031 genes were obtained. Finally, the top 3000 genes were selected for enrichment analysis.

Enrichment Analysis for Human Transcriptomics Data.
After the enrichment analysis, several biological processes, Reactome pathways, and signaling pathways are obtained. e results of GO enrichment are mainly related to insulin-like growth factor and its receptor signaling pathway (GO: 0048009), extracellular matrix (GO: 0098609), endothelial cell formation (GO:0061028), skeletal system morphogenesis (GO: 0048705), several osteoporosis-related signaling (such as hippo signaling, MAPK signaling, regulation of T-cell receptor signaling pathway), and so on (Figure 8(a)). e results returned by Reactome pathways enrichment show that LDD is able to regulate Cell-extracellular matrix interactions, Interleukin-7 signaling, SUMOylation of transcription cofactors, SUMOylation of intracellular receptors, Downregulation of TGF-beta receptor signaling, MET activates RAP1 and RAC1, Insulin-like Growth Factor-2 mRNA Binding Proteins (IGF2BPs/IMPs/VICKZs) bind RNA, Interleukin-6 signaling, Cohesin Loading onto Chromatin, Interleukin-12 family signaling, and so on (Figure 8(b)).
e results of signaling pathway enrichment are protein processing in the endoplasmic reticulum, TGF-beta signaling pathway, Phosphatidylinositol signaling system, Neurotrophin signaling pathway, yroid hormone signaling pathway, Focal adhesion, Signaling pathways regulating pluripotency of stem cells, FoxO signaling pathway, Estrogen signaling pathway, Insulin signaling pathway, and NF-kappa B signaling pathway (Figure 8c). e details of biological processes, Reactome pathways, and signaling pathways are described in Tables S7-S9.

Protein Arrays Data.
e osteoporosis-related protein arrays data comes from [30]. e network was constructed by Metascape (Figure 9).

Enrichment Analysis for Protein Arrays Data.
Protein arrays data were input into DAVID and Reactome for enrichment analysis and returned several biological processes, Reactome pathways, and signaling pathways. e results of GO enrichment are shown in Figures 10(a) (Figure 10(a)). e results of signaling pathway enrichment are related to bone homeostasis (such as hsa04630: Jak-STAT signaling pathway, hsa04380:Osteoclast differentiation, hsa04550:Signaling pathways regulating pluripotency of stem cells, and hsa04012: ErbB signaling pathway) and other pathways (such as hsa04060: Cytokine-cytokine receptor interaction) (Figure 10(b)). e results returned by Reactome pathways enrichment are associated with immunomodulatory (such as R-HSA-1280215, R-HSA-449147, R-HSA-6785807, R-HSA-168256, and R-HSA-8877330), cell growth, proliferation, and cell cycle (such as R-HSA-9617828 and R-HSA-452723), transcription factor and coactivated genes that interact with STAT proteins (R-HSA-2173796 and R-HSA-9006936), and other transcription factors and their coactivated genes (R-HSA-2173789 and R-HSA-76002) (Figure 10(c)). e details of biological processes, Reactome pathways, and signaling pathways are described in Tables S10-S12.

Evidence-Based Complementary and Alternative Medicine
In summary, we found that the mechanism of LDD in the treatment of osteoporosis is related to immunity, cell growth and proliferation, endocrine hormones and cytokines, and differentiation of osteoclasts and osteoblasts. ese pathways work together to regulate the activity of osteoclasts and balance the process of bone turnover through the interaction of crucial proteins. Next, animal experiments were conducted to verify the mechanism of LDD on osteoporosis.

Effect of LDD on BMD of Proximal Femur.
e BMD of the proximal femur in the model group was lower than that in the sham operation group (P < 0.01). e BMD of the proximal femur in the LDD group and the positive control group was higher than that in the model group (P < 0.01). Compared with the positive control group, the BMD of the proximal femur in the LDD group was not statistically significant (P > 0.05) (Figure 11).

Effect of LDD on Serum E2 and ALP.
Compared with the model group, the serum E2 and ALP of the LDD group were significantly improved (P < 0.05), suggesting that LDD can increase serum E2 and ALP levels in osteoporotic rats ( Figure 12).

Effect of LDD on Bone Histopathology.
In the sham operation group, dense and regular bone trabeculae were arranged in the bone tissue of the rats, and the morphological structure was complete, and the bone marrow cavity size was normal. Compared with the model group, the trabecular bones of the LDD group were regularly arranged, with few fractures, morphological structure close to normal, and the size of the bone marrow cavity was normal, similar to the sham operation group. It is suggested that LDD can improve the destruction of bone tissue structure in PMOP rats ( Figure 13).

Effect of LDD on Wnt3a and β-Catenin mRNA
Expression. Compared with the sham operation group, the Wnt3a and β-catenin mRNA expression of the model group decreased (P < 0.05). Compared with the model group, Wnt3a and β-catenin were significantly increased in the LDD group and the positive control group (P < 0.05), similar to the sham operation group. It is suggested that LDD can  negative regulation of toll-like receptor 3 signaling pathway regulation of T cell receptor signaling pathway microtubule anchoring at centrosome microvillus assembly protein targeting to Golgi establishment of endothelial barrier IGF receptor signaling pathway positive regulation of glucose import in response to insulin stimulus negative regulation of JUN kinase activity platelet-derived growth factor receptor signaling pathway skeletal system morphogenesis regulation of nitric-oxide synthase activity positive regulation of VEGF production hippo signaling activation of MAPKK activity reactive oxygen species metabolic process reactive regulation of I-kB kinase/NF-kB signaling nagative regulation of angiogenesis stem cell population maintenance positive regulation of fibroblast proliferation ubiquitin-dependent protein catabolic process response to endoplasmic reticulum stress retrograde vesicle-mediated transport, Golgi to ER mitotic nuclear division G2/M transition of mitotic cell cycle positive regulation of I-kB-signaling angiogenesis Evidence-Based Complementary and Alternative Medicine improve osteoporosis in rats by regulating the expression of Wnt3a and β-catenin ( Figure 14).

Molecular Docking Results of LDD Components and
Osteoporosis-Related Gene. Due to the limitations of the prediction database, this study used molecular docking technology to further explore whether the LDD components can directly interact with osteoporosis-related genes. e lowest binding energy between the LDD component and the target protein is less than −5 kJ/mol, indicating that the ligand and the receptor can bind spontaneously and stably. e results are shown in Table 4. e docking mode of LDD components with JAK2, ESR1, and CTNNB1 is shown in Figure 15.
Comparing the pathways of the LDD-osteoporosis PPI network and human transcriptomics data, it was found that the osteoporosis-related pathways they share are TGF-beta signaling pathway, Neurotrophin signaling pathway, yroid hormone signaling pathway, FoxO signaling pathway, Estrogen signaling pathway, Insulin signaling pathway, and NF-kappa B signaling pathway. e unique osteoporosisrelated pathways in enrichment results of human transcriptomics data were as follows: Protein processing in endoplasmic reticulum, Phosphatidylinositol signaling system, Focal adhesion, and Signaling pathways regulating pluripotency of stem cells. Comparing the pathways of protein arrays data network and LDD-osteoporosis PPI network, it is found that the osteoporosis-related pathways they share are Jak-STAT signaling pathway, TGF-beta signaling pathway, Osteoclast differentiation, ErbB signaling pathway, Estrogen signaling pathway, Cytokine-cytokine receptor interaction, and TNF signaling pathway. e unique osteoporosis-related pathways in enrichment results of protein arrays data were as follows: Prolactin signaling pathway and Signaling pathways regulating pluripotency of stem cells [80,81]. e skeletal structure undergoes subtle changes throughout life, requiring the involvement of osteoblasts and osteoclasts [82]. e two cells interact at the same site on the bone surface, in which osteoclasts are involved in bone resorption, which weakens bone strength and causes osteoporosis when bone resorption is more than bone formation [82]. e current intervention in osteoporosis is to inhibit bone resorption-osteoclast activity regulation [83]. Osteoclasts are matured from mononuclearmacrophage cell lines, mainly regulated by macrophage colony-stimulating factor (MCSF), nuclear stimulating factor-κB receptor ligand (RANKL), and osteoprotegerin (OPG) [84,85], of which MCSF is a key factor in the  Evidence-Based Complementary and Alternative Medicine regulation of osteoclast differentiation [86]. Bone biopsy data and in vitro studies have shown that osteoclasts that complete the bone resorption mission will automatically undergo apoptosis, which is one of the mechanisms for the body to maintain bone metabolism balance [86]. e regulation of these cytokines on osteoclast activity is mainly reflected in [87][88][89][90]: (1) the auto/paracrine effect of local cells directly regulates the proliferation and differentiation of immature osteoblasts and bone resorption activities; (2) they can indirectly increase the sensitivity of bone resorption associated with parathyroid hormone (PTH); (3) inhibition of osteoclast activity by the interaction of signaling pathways. Among them, the RANKL/ RANK system is the most irreplaceable regulation of bone cell unit activity, and various external factors play a role in changing the signaling pathway in this system [86]. For example, L-1a, IL-6, IL-11, IL-16, and PTH can activate the RANKL/RANK system to enhance RANKL expression, thus completing bone remodeling [91]. e signaling pathways associated with the RANKL/RANK system are mainly the NF-κB pathway, MAPK pathway, PI3K/AKt pathway, and CN/NFAT pathway [92]. Hence, the RANKL/RANK system plays a role similar to the common terminal pathway during osteoclast activation; and the drugs designed by the system, such as denosumab, have many advantages in the clinical practice of treating osteoporosis, such as fewer side effects [93].
MCSF is produced by various cells in the bone microenvironment and acts on the osteoclasts in a paracrine manner, resulting in increased expression of genes involved in osteoclast differentiation and increased osteoclast activity [94]. In addition, MCSF can also promote the formation of mature osteoclasts indirectly through the RANKL pathway [94]. Osteoprotegerin (OPG) is mainly expressed on the surface of osteoblasts and inhibits the activation of osteoclasts and its bone resorption activity by competitively inhibiting the binding of RANKL to RANK [95]. Meanwhile, prostaglandins (PGs), metastatic growth factors (TGF-β), interferon (INF), and insulin-like growth factor (IGF) are also associated with the activation of osteoclasts [96].
Hormones also play an important role in the process of bone regulation, especially the regulation of osteoclast activity [97]. For example, estrogen plays an important role in  inhibiting bone resorption, and various forms of hypogonadism that lack estrogen can lead to osteoporosis [97]. e mechanism is that estrogen can act on ERα of osteoclasts, stimulate OPG expression, inhibit the activation of osteoclast-associated cytokine expression, and ultimately reduce the rate of osteoclastogenesis through the RANKL signaling pathway [98]. For this target, the main drugs are selective estrogen receptor modulator (raloxifene) and artificial estrogen to inhibit bone resorption, treat osteoporosis, and at the same time relieve menopausal symptoms in Evidence-Based Complementary and Alternative Medicine menopausal women [99]. Other hormones, such as calcitonin, are also associated with inhibition of bone resorption, and their short-term efficacy in the treatment of osteoporosis is obvious, but long-term efficacy is poor [100]. e hormones that act indirectly on bone resorption are PTH and glucocorticoids [101]. e synthetic PTH analog, teriparatide, enhances osteoclast function by promoting the lysosomal enzyme production and acid synthesis in osteoclasts and through the AC/cAMP pathway [101]. rough the construction and enrichment analysis of the predicted target network of small molecule compounds in LDD, we can find that the small molecule compounds of LDD can regulate some biological processes. For example, they can regulate endothelial cell proliferation and neovascularization, bone formation, bone resorption and reconstruction, differentiation of mesenchymal stem cells, formation of extracellular matrix and collagen of bone formation, differentiation of osteoblasts, and osteoclasts, and so on. ese biological processes play an important role in the development of osteoporosis. Meanwhile, this study suggests that LDD can regulate some cytokines related to osteoporosis, such as TGF-β, INF, IGF, and calmodulin 1monooxygenase, which are closely associated with the activation of osteoclasts. [94,102].
e GO enrichment analysis of LDD has also shown that it can regulate the production and metabolism of hormones such as estrogen and glucocorticoids; these hormones can regulate osteoclast activity directly (estrogen) or indirectly (glucocorticoids), thereby regulating bone resorption [103]. e results of the pathway enrichment analysis show that LDD can regulate the FoxO signaling pathway, Estrogen signaling pathway, Osteoclast differentiation, PI3K-Akt signaling pathway, Ras signaling pathway, T-cell receptor signaling pathway, and so on. ese pathways are closely related and synergistic, acting together on the bone resorption process, regulating osteoclast activity [104], and ultimately regulating the core mechanism of osteoporosis-bone turnover. e current drug development of osteoporosis has also developed targeted drugs for the above multiple pathways, such as denosumab (for OPG/ RANKL/RANK pathway) [105], romosozumab (for Wnt signaling pathway) [106], and rapamycin (for mTOR signaling pathway) [107]. e most important signaling pathways associated with osteoporosis are the OPG/RANKL/RANK signaling pathway, which includes NF-κB signaling, MAPK signaling, PI3K/AKt signaling, and CN/NFAT signaling [92,96]. e MAPK family pathway mainly includes the extracellularregulated protein kinase (ERK) signaling pathway, Jun N-terminal kinase (JNK) signaling pathway, the ERK5/ macroton activating protein kinase signaling pathway, and the p38 signaling pathway [108]. In addition, the signaling pathway with osteoporosis includes PPAR-c [109], Wnt/ β-catenin [110], Hedgehog [111], BMP [112], Notch [113], JAK/STAT [114], and TGF-β/SMAD signaling pathway [115].
In conclusion, although this study explored the molecular mechanism of LDD in the treatment of osteoporosis through bioinformatics and systems pharmacology and transcriptomics and verified some related molecular mechanisms, its molecular mechanism still needs to be further elucidated in order to promote the thorough disclosure and elucidation of the molecular network mechanism of LDD in the treatment of osteoporosis.

Conclusion
LDD may have a therapeutic effect on osteoporosis through regulating the targets (such as ALB, IGF1, SRC, and ESR1), biological processes (such as positive regulation of calmodulin 1-monooxygenase activity, estrogen metabolism, and endothelial cell proliferation), and pathways (such as JAK-STAT signaling pathway, osteoclast differentiation, and degradation of the extracellular matrix) found in this research.
Data Availability e data that support the findings of this study are available in supplementary materials.

Disclosure
Long Zhi-yong, and Yu Gan-peng should be considered joint first authors.

Supplementary Materials
Table S1-1: components meeting the screening criteria. Table S1-2: compound targets for each compound of LDD. Table  S2: osteoporosis genes. Table S3: enrichment analysis of clusters based on Gene Ontology (GO) annotation of LDDosteoporosis PPI network. Table S4: pathway enrichment analysis of LDD-osteoporosis PPI network. Table S5: Reactome pathways of LDD-osteoporosis PPI network. Table S6: Human Transcriptomics Data. Table S7: the biological processes of Human Transcriptomics Data Network.