Study on the mechanism of Wuzi Yanzong pill in treating osteoporosis based on network pharmacology and molecular docking

Background: Based on network pharmacology and molecular docking, our study discussed the mechanism of Wuzi Yanzong pill in treating Osteoporosis (OP), which lays the foundation for drug development of OP. Methods: The chemical compounds and potential targets of Wuzi Yanzong pill were explored through TCMSP, PubChem, Swiss ADME and other databases. GeneCards, OMIM and Drugbank databases were used to obtain OP related targets. The intersection between the targets of Wuzi Yanzong pill and the related targets of OP was found by drawing a Venn diagram. PPI network was constructed with the STRING database and core targets were screened. The TCM-compound-action target-disease network was drawn using the Cytoscape software. The Metascape platform was used to find the pathways and functions for core target enrichment. Molecular docking validation of action compounds and core targets is completed by software such as Auto Dock Vina. Results: 59 compounds and 707 action targets of Wuzi Yanzong pill were found. 603 disease targets were selected, 106 intersection targets were found using a Venn diagram, and 37 core targets were screened. By enrichment analysis, 143 KEGG pathways, 1026 GO biological processes, 23 GO cell compositions and 60 GO molecular functions were obtained. The results of molecular docking showed that the effective compounds of Wuzi Yanzong pill, such as stigmasterol, quercetin, kaempferol and β -sitosterol, had high binding activity with STAT3, TNF and IL6 core target proteins. Conclusion: Wuzi Yanzong Pill may play a role in treating OP by regulating STAT3, TNF, IL-6, TP53, VEGFA, JUN, AKT1, IL-1B, SRC, MMP9 and other pathways, as well as cancer-correlation, rheumatoid arthritis-correlation, MAPK, Th17 cell differentiation, IL-17, TNF signaling pathway and so on, to interpret Wuzi Yanzong pill’s clinic.


Introduction
Osteoporosis (OP) is a common systemic bone disease characterized by low bone mass and destruction and damage of bone microstructure, leading to increased bone fragility and fracture [1].OP can occur in different sexes and ages, but it is more common in postmenopausal women and elder men.OP can be divided into two categories: primary OP includes postmenopausal OP, senile OP and idiopathic OP, and secondary OP refers to OP caused by any diseases, drugs and other definite causes that affect bone metabolism [2][3].With the aging of the global population, the medical and social economic burden caused by OP will further increase, and it has become an important public health problem facing China [4][5].Lifestyle modification, calcium and vitamin D supplementation, and anti-OP drugs are the primary clinical treatments for OP, which can significantly improve clinical symptoms and delay the onset of disease in patients, but there are still issues such as side effects and poor long-term compliance with their medication.
TCM has played a unique role in preventing and treating OP, aiming at the etiology, pathogenesis and syndrome types, thus improving clinical symptoms, relieving pathological pain, preventing and treating complications, and achieving good curative effect [6][7][8].According to the clinical manifestations and pathological features of OP, it belongs to the category of "bone flaccidity" and "bone arthralgia" in TCM.Its causes are deficiency of congenital endowment, maladjustment of acquired absorption and nourishment, and incursions of external evils, which lead to imbalances of qi (Qi is the origin of the universe and the most basic element that constitutes everything in the world.It is considered that qi is the most basic substance that constitutes the human body and also the most basic substance that maintains human life activities.Blood, a nutritious red liquid substance circulating in the veins, is one of the basic substances that constitute the human body and maintain human life activities) and blood, obstruction of meridian operations, failure of bone and dystrophy of bone marrow.The treatment is always based on the principles of tonifying kidney and replenishing essence, invigorating spleen and qi, promoting blood circulation and removing blood stasis.Wuzi Yanzong pill is a classic prescription for tonifying kidney and replenishing essence, which was first recorded in the Tang Dynasty's "Xuanjielu".Its compounds include five kinds of Chinese medicines, namely Lycii Fructus, Cuscuta Semen, Rubi Fructus, Schisandrae Chinensis Fructus and Plantaginis Semen.It has the effects of invigorating kidney qi (Kidney qi refers to the part that plays a role in consolidating and sealing the kidney) and replenishing essence marrow and is often used for kidney deficiency, impotence, spermatozoa and OP caused by kidney-yang deficiency.In recent years, Wuzi Yanzong pill and its modified formula have had a good therapeutic effect on oligozoospermia, mild cognitive impairment, OP and other diseases, which can significantly improve sperm motility and sperm density, improve patients' memory ability and relieve OP pain [9][10].
Wuzi Yanzong pill, a famous formula for tonifying kidney, has complex compounds, and its mechanism of action in treating OP is rarely studied, its pharmacological effect is still unclear, which has greatly hindered the modern research and clinical promotion of this medicine.Network pharmacology is a modern research method to screen potential active substances, action targets and pathways, and predict the mechanism and material basis of drug action [11][12].Molecular docking is a computer-aided method based on the combination of three-dimensional structures of receptor macromolecules and ligand macromolecules, evaluating the affinity relationship between receptor and drug molecules and drug design [13].The purpose of this study is to explore the molecular mechanisms of Wuzi Yanzong pill in treating OP, so as to provide a theoretical basis for its clinical promotion and ideas for further drug research.

Mining chemical compounds and targets of Wuzi Yanzong pill
Mining chemical compounds of single Chinese medicine of Lycii Fructus, Cuscuta Semen, Rubi Fructus, Schisandrae Chinensis Fructus and Plantaginis Semen in Wuzi Yanzong pill through TCMSP database, setting the OB ≥ 30% and the BL ≥ 0.18, and screening the action compounds and targets.In addition, reported chemical compounds and targets have been added by consulting the literature.The Uniprot database was used to transform the name of the protein, and the corresponding target gene was obtained after repeated removal.
Obtaining the potential targets of OP GeneCards, OMIM and Drugbank databases were searched with " Oste oporosis" as the keyword to obtain potential targets of OP.

Construction of PPI Network and Screening of Core Targets
The protein targets of Wuzi Yanzong pill and the potential targets of OP were compared, and the duplicated targets were screened to obtain the action targets of Wuzi Yanzong pill in treating OP.The action targets were introduced into the String Version 11.5 platform, and the species was selected as "Homo sapiens" to construct a PPI network.The high confidence interval with a score > 0.7 was selected, and the PPI network was constructed according to the targets with a degree of freedom ≥ 2 times the median to screen the core targets.

Construction of the TCM-compound-action target-disease network
The information of the five herbs, compounds and action targets of Wuzi Yanzong pill was imported into Cytoscape 3.8.0software to draw the network graph of TCM-compounds, and compounds-action targets.Finally, the network of TCM-compound-action target-disease was established by using the Merge function.

KEGG pathway and GO function enrichment analysis
In order to study the effect of Wuzi Yanzong pill on OP, the KEGG pathway and GO function enrichment analysis of core targets were carried out by using the Metascape platform, and the results were arranged in ascending order of P value.

Molecular docking verification
The main compounds with a number of targets in Wuzi Yanzong pill were selected, including Stigmasterol, Quercetin, Kaempferol and Beta-sitosterol, of which stigmasterol is an important compound in Rubi Fructus and Plantaginis Semen, and quercetin is an important compound in Lycii Fructus, Cuscuta Semen, Rubi Fructus and Plantaginis Semen.Kaempferol is an important compound of Cuscuta Semen and Rubi Fructus, and β-sitosterol is an important compound of Lycii Fructus, Cuscuta Semen and Rubi Fructus.Preliminary validation of molecular docking with core proteins was performed.Download the core compound and target files, Configure the config file, run Autodock vina1.1.2and calculate the molecular docking binding energy.The pdbqt format files were converted into pdb format file in Open Babel GUL software, and the pdb format files were opened in PyMOL.Finally, the visual analysis was carried out by the PLIP website.

Action compounds and targets of Wuzi Yanzong pill
Based on the comprehensive literature reports and the results collected by searching and screening in various databases, the main compounds of Wuzi Yanzong pill and their corresponding targets are as follows, including 36 kinds of Lycii Fructus, 11 kinds of Cuscuta Semen, 6 kinds of Rubi Fructus, 8 kinds of Schisandrae Chinensis Fructus and 7 kinds of Plantaginis Semen (Table 1).After removing duplicates, 59 compounds were obtained, involving 707 related action targets.

Potential targets of OP disease
4,641 OP-related targets were obtained by searching in the GeneCards database, and 547 potential targets were selected according to the Submit a manuscript: https://www.tmrjournals.com/praverage correlation score ≥ 2 times.104 and 22 OP-related targets were found in the OMIM and Drugbank databases, respectively.The sum of the three was the potential target of OP, and 603 disease targets were obtained by removing duplicates.

Construction of PPI network and screening of core targets
707 related action targets were intersected with potential targets of OP diseases, and 106 action targets of Wuzi Yanzong pill in treating OP were found (Figure 1).The core targets were screened by using the STRING platform, and 37 core targets were obtained according to the degree of freedom (Table 2).

Construction of the TCM-compound-action target-disease network
Five single Chinese medicines, 59 compounds and 106 action targets of Wuzi Yanzong pill were imported into Cytoscape to draw the network of TCM-compounds, compounds-action targets (Figure 2).Among them, the inverted triangle represents Wuzi Yanzong pill, the octagonal node represents single Chinese medicine, the hexagonal node represents compound components, the diamond node represents core target, and the triangle represents OP.
KEGG pathway and GO enrichment analysis 37 core targets were introduced into the Metascape platform, and 143 KEGG Pathways were obtained.The top-ranked pathways mainly included cancer, and atherosclerosis, rheumatoid arthritis, MAPK, IL-17, tumor necrosis factor signaling pathway, etc.The top 20 pathways were displayed according to P value, and the bubble diagram of enrichment analysis was drawn (Figure 3).The top 20 pathways and 37 core targets were imported into Cytoscape to draw the key pathway-core target network (Figure 4).At the same time, 37 core targets were introduced to the Metascape platform for GO BP, CC and MF enrichment analysis, resulting in 1026 BP, 23 CC and 60 MF items.According to the P value, the top 10 GO BP, the top 5 CC and the top 5 MF were selected to draw the bubble diagram for enrichment analysis (Figure 5).Enrichment analysis showed that BP was mainly involved in the cellular response to organic cyclic compound, response to hormone, regulation of MAPK cascade and positive regulation of locomotion.CC mainly comprised membrane raft, membrane microdomain, receptor complex, and serotonin receptor complex.MF mainly comprised cytokine receptor binding, nuclear receptor activity, ligand-activated transcription factor activity and steroid binding.

Molecular docking verification
Stigmasterol, Quercetin, Kaempferol and Beta-sitosterol, all of which have multiple targets in Wuzi Yanzong pill, were combined by molecular docking with the top three core targets STAT3, TNF and IL-6.The results of this study showed that four main compounds in Wuzi Yanzong pill can bind to core targets well (Table 3), among which β-sitosterol, stigmasterol and STAT3 protein had the best binding force, so β-sitosterol and stigmasterol with lower binding ability were selected for molecular docking pattern display (Figure 6).

Discussion
Wuzi Yanzong pill consists of five Chinese medicines.Lycii Fructus contains polysaccharides, alkaloids, flavonoids and other effective compounds, which have the effects of nourishing liver and kidney and benefiting smart eyes [14].Cuscuta Semen contains flavonoids, organic acids, and other effective compounds, which can tonify kidney yang (Kidney yang, also known as Yuanyang, Zhenyang and Zhenshui, is the foundation of human yang, and plays a role in promoting and warming the organs and tissues of the body) and kidney essence (Kidney essence, which comes from innate essence and is constantly nourished by acquired essence, is the material basis of renal function and the foundation of life activities of the body, and plays an extremely important role in various physiological activities of the body), resist aging and delay the loss of calcium and phosphorus [15].Rubi Fructus contains organic acids, flavonoids and other effective compounds, which have the effects of tonifying liver and kidney, and playing the roles of anti-aging, antioxidation and bacteriostasis [16].Schisandrae Chinensis Fructus contains lignans, polysaccharides, volatile oils and other effective compounds, which have the effects of tonifying the heart and kidney, arresting spermatorrhea, and playing an anti-oxidation and anti-aging role [17].Plantaginis Semen contains polysaccharides, phenylethanoid glycosides, flavonoids and other effective compounds, which have the effects of clearing away heat and diuresis, and eliminating dampness and quenching thirst [10].The whole formula "Wuzi" has the functions of tonifying kidney essence and replenishing marrow, and has the functions of anti-aging, anti-oxidation and delaying the loss of calcium and phosphorus.
In this study, 59 compounds of Wuzi Yanzong pill were screened out, involving 707 related targets.106 action targets and 37 core targets were excavated, and the PPI network of action targets and network diagram of TCM-compound-action targets-disease were constructed.The core targets were introduced into Metascape platform for analysis, and 143 KEGG pathways, 1026 GO BP, 23 GO CC and 60 GO MF were obtained.
Analysis of PPI network showed that core targets of Wuzi Yanzong pill in treating OP include STAT3, TNF, IL-6, TP53, VEGFA, JUN, AKT1, IL-1B, SRC and MMP9.IL-6 is closely related to the production of osteoblasts, stromal cells, osteoclasts and their precursors, and it mainly promotes the increase of osteoclasts in bone and bone marrow.Studies show that TCM plays an important role in the occurrence and development of OP by regulating the IL-6/JAK2/STAT3 signaling pathway, affecting the formation of bone trabecula, and thus reducing bone density [18].TNF-α is closely related to the occurrence of OP and is an important regulator of osteoclasts, in which TNF-α level is related to bone loss and the death of osteoblasts and osteoclasts [19][20].C-JUN is a proto-oncogene related to cell proliferation, and its products are closely related to the proliferation of osteoblasts and the differentiation, maturity and function of osteoclasts [21].Research by Li Haixia et al. shows that AKT1 gene is the key gene most related to OP in peripheral blood cells, which affects the function of osteoclasts and osteoblasts [22].Combined with enrichment analysis, Wuzi Yanzong pill may play a role through cancer, and atherosclerosis, rheumatoid arthritis, MAPK, IL-17, TNF signaling pathway, etc. Core targets are mainly involved in response to hormone, regulation of MAPK cascade, membrane raft, membrane microdomain, receptor complex, serotonin receptor complex and so on, thus affecting the occurrence and development of OP and playing a role in treating OP.
The results of molecular docking showed that docking energies of four main compounds in Wuzi Yanzong pill, namely stigmasterol, quercetin, kaempferol and β-sitosterol, with the core targets of STAT3, TNF and IL-6 were all less than −5 kcal/mol, which indicated that the action compounds stably combined with the core targets and played their role in treating OP.The results provided an initial validation of the reliability of the above-mentioned network pharmacology prediction and laid a foundation for the follow-up studies.
At the same time, studies show that stigmasterol has strong anti-inflammatory and anti-oxidation abilities, and can regulate inflammatory factors through MAPK signal transduction, effectively reduce inflammation, promote the repair of damaged cartilage and improve the joint environment [23].Quercetin can effectively remove the aging BMSCs, promote the proliferation and osteogenic differentiation, improve the structure and function of bone tissue, and have good preventive and therapeutic effects on aging, aging OP and postmenopausal OP [24,25].Kaempferol can effectively regulate the autophagy between osteoblasts and osteoclasts, increase the expression of osteogenic protein and osteopontin, promote the proliferation and differentiation of osteoblasts, improve osteogenic parameters, and maintain the dynamic balance between bone absorption and bone metabolism [26].β-sitosterol has an estrogen-like effect, which can combine with estrogen receptor to regulate bone metabolism balance, increase the ratio of osteoprotegerin to osteoclast differentiation factor, promote osteogenesis and inhibit osteoclast [27].

Conclusion
Based on network pharmacology and molecular docking, our study investigated Wuzi Yanzong pill, a well-known formula for tonifying the kidney, and found that it may have a significant role in treating OP through multiple compounds, multiple targets and multiple channels.Effective compounds such as stigmasterol, quercetin, kaempferol and β-sitosterol in Wuzi Yanzong pill can regulate the proliferation and differentiation of osteoblasts, bone absorption of osteoclasts and the proliferation of BMSCs in the process of bone metabolism through the core targets such as STAT3, TNF and IL-6 and pathways related to cancer, rheumatoid arthritis, MAPK and TNF.This enriches the treatment of OP with TCM and provides a reference for the basic research on TCM treatment of OP.However, there are some limitations in this study, and the core targets and pathways predicted by it need further validation by related fundamental studies.

Figure 2
Figure 2 Network diagram of TCM-compound-target-disease.

Figure 3 Figure 4
Figure 3 Bubble diagram for enrichment analysis

Figure 5
Figure 5 Enrichment analysis histogram for GO BP, CC, and MF

Table 3 Docking binding energy between main components and core targets of Wuzi Yanzong pill (kcal/mol)
Figure 6 Molecular docking diagram Submit a manuscript: https://www.tmrjournals.com/pr