Mechanisms of Intervertebral Disc Degeneration Treatment with Deer Antlers Based on Network Pharmacology and Molecular Docking

Background With the aging of the population, the prevalence of IVDD increases preoperatively. How to better treat IVDD has become an important clinical issue. Deer antlers proved to have a great effect on the treatment of IVDD in many studies, but the molecular mechanism has not been clarified. Objective To investigate the molecular mechanism and target of deer antlers in the treatment of IVDD. Methods Compounds from deer antlers were collected and targets were predicted using HERB, TCMSP, TCMID, SwissADME, and SwissTargetPrediction. Collection of disease targets for IVDD was done using GeneCards, TTD, DrugBank, DisGeNET, and OMIM. Cytoscape 3.7.2, AutoDock Vina (v1.1.2), and R software were used for data analysis and the construction of network diagrams. Results A total of 5 active compounds from deer antlers were screened and 104 therapeutic targets were predicted. A total of 1023 IVDD disease targets were collected. Subsequently, PPI network prediction analysis was performed for disease and treatment targets, and 112 core targets were collected after screening. After obtaining the core target, we used the clusterProfiler software package of R software to carry out GO and KEGG enrichment analyses for the core target and plot the bubble maps. According to the GO enrichment results, the main biological processes of IVDD treatment by deer antlers lie in the rhythmic process, mRNA catabolic process, and G1/S transition of the mitotic cell cycle. KEGG results were mainly related to the PI3K-Akt signaling pathway, thyroid hormone signaling pathway, and Notch signaling pathway. Molecular docking results showed that estrone had the best docking results on ESR1. Conclusion Deer antlers are rich in various compounds that can prevent the development of IVDD by upregulating the PI3K-Akt signaling pathway and Notch signaling pathway. Its key compounds estradiol and estrone can reduce the inflammatory response and oxidative stress in tissues and organs, thus slowing down the progression of IVDD. Estrone, the active compound in deer antlers, was found by molecular docking to have good results against ESR1, the target of the disease, which may be a potential site for drug therapy.


Introduction
Deer antlers (Cervi Cornu Pantotrichum) refer to the outgrowths on the forehead of the male of sika deer and red deer, which are covered with a layer of velvety fur [1]. Deer antlers, owning a beneficial effect of tonifying "kidneyyang," are normally used to treat low back pain, soreness of the knee, morbid vaginal discharge, and so on [2]. e efficiency of deer antlers is dependent on their bioactive components. Recent studies reported that the deer antlers' significant ingredients are polysaccharides, polypeptides, and free amino acids [3]. e nucleus pulposus, cartilage endplates, and annulus fibrosus constitute the intervertebral disc, which can keep the spine stable and flexible [4]. One of the main factors contributing to low back pain is intervertebral disc degeneration (IVDD) [5]. With the aging of the population, the prevalence of IVDD is increasing in proportion [6]. One study showed that the incidence of IVDD in the whole lumbar spine was 31.6% in men and 44.7% in women [7]. People have thought that inflammation and oxidative stress are closely related to IVDD. Polysaccharides have good antioxidant properties [8]. Furthermore, deer antler peptide, exhibiting significant anti-inflammatory and antioxidative effects, can effectively protect osteoblasts [9][10][11]. Meanwhile, proteins contained in deer antlers protect against oxidative stress and inflammation [3,12].
Network pharmacology, which is based on the concept of a multilevel and multiangle interaction network among diseases, genes, targets, and drugs, can observe the interventional mechanism and influence of drugs on the disease network systematically and comprehensively [13]. It can generate complex networks of interactions according to target molecules, biological functions, and bioactive compounds to clarify the mechanism of action of TCM prescriptions at the molecular level [14]. Up to now, network pharmacology has been used in many studies of Chinese    [15]. Molecular docking is an effective tool in structural molecular biology and computer-assisted drug design that predicts a ligand's major binding mode(s) with a target protein [16]. Due to the unique therapeutic properties, deer antlers have attracted much research interest. So far, nevertheless, the target and mechanism of deer antlers in the treatment of IVDD have not been elucidated in the literature, and people know little about it, which limits the further development and application of deer antlers to some extent. is study aims to explore the potential mechanism of deer antlers action from the perspective of network pharmacology.

Core Target Screening.
Using the BisoGenet plug-in in Cytoscape 3.7.2, protein interaction networks were predicted separately for drug component targets and disease targets, and the two networks were merged to collect their intersecting targets. Next, the CytoNCA plug-in was used to analyze the topology of the intersection network and extract the HUB targets based on the values of "Betweenness," "Degree," and "Closeness" in the results as filtering criteria.

GO and KEGG Enrichment
Analyses. GO enrichment analysis is able to discover the link between genes and gene product features across all species. KEGG enrichment analysis is useful in clarifying in vivo comprehensive inferences of reactions. e R software packages "pathview" and "clusterProfiler" were used for KEGG and GO enrichment analyses of intersection targets. Finally, the "ggplot" software package was used to visualize the results.

Molecular Docking.
Five core target proteins in the PPI network and significant components in the "componenttarget" network diagram were selected for molecular docking verification. e 3D structures of the target protein were downloaded from the PDB website (https://www.rcsb. org/), and the 3D structures of the components were obtained from the ZINC website (https://zinc.docking.org/). First, the intrinsic small ligand of the protein was docked to the protein structure, and the binding energy was calculated. Second, the target proteins and components were docked in AutoDock Vina (v1.1.2), and the binding energy was also calculated. e binding energy of the intrinsic small ligand to the protein was the standard for verifying the binding conditions of the new compounds. Ultimately, PyMOL (v2.5) was utilized to present a visual analysis of the molecular structure. Figure 1 shows the brief flowchart with network pharmacology and molecular docking.

Component-Target Network Diagram Construction.
A total of 5 active ingredients were obtained from the screening of deer antlers (Table 1). A total of 104 targets were  Evidence-Based Complementary and Alternative Medicine obtained after aggregation and deweighting. e data were imported into Cytoscape 3.7.2 to construct a "componenttarget" network diagram, which showed a total of 104 nodes and 238 component-target interactions (Figure 2). From the figure, we can find that alpha-estradiol and 17-beta-estradiol interact with the most target genes and are potential key compounds. Based on the results of the topological analysis, we attained the degree values for the five active ingredients ( Table 2).

Results of the Core Target Screening.
A total of 1023 disease targets were obtained after the search summary, and after intersection mapping with drug action targets, the intersection network was topologically analyzed using the CytoNCA plug-in. Subsequently, the "Degree" value in the results was used as a filtering criterion to screen targets with a "Degree" value greater than or equal to 2 times the median (degree ≥68), and a total of 2579 targets were obtained, that is, because the degree value can reflect the importance degree of a compound in deer antlers. Afterward, the new results were analyzed according to the three indicators "Betweenness," "Degree," and "Closeness," and targets greater than or equal to the median were screened, resulting in 112 core targets (Figure 3, Table 3).

GO and KEGG Enrichment Analyses Results.
A total of 1061 GO entries were obtained after enrichment, containing biological process (BP), cellular component (CC), and molecular function (MF), with a P value screening for each component.
e main results are the rhythmic process, mRNA catabolic process, and G1/S transition of the mitotic cell cycle (Figures 4-6). Meanwhile, the core targets were subjected to the KEGG pathway enrichment analysis to screen for pathways with P < 0.05, and a total of six were obtained, such as the PI3K-Akt signaling pathway, the thyroid hormone signaling pathway, the Notch signaling pathway, the FoxO signaling pathway, the estrogen signaling pathway, and the HIF-1 signaling pathway (Figure 7), among which the specific mechanism of deer antler treating intervertebral disc degeneration in the PI3K-AKT signaling pathway is shown in Figure 8.

Molecular Docking Analysis.
As can be found from the PPI interaction analysis diagram, NTRK1, ESR1, CDK2, CUL3, and TP53 were the proteins with the highest degree values, which were also selected as our core target proteins. We listed the basic information of the target protein and the binding energy of the intrinsic ligand and the protein given in Table 4 (CUL3 had no intrinsic small ligand, so its binding  energy was empty). e binding energies of each component to each target protein were listed in Table 5. e target proteins with the best binding energy were given in Table 6, and the number of hydrogen bonds formed was also recorded. e molecular docking results were presented in Figure 9. e binding energy of estrone with CDK2 was −5.9 kcal/mol. As can be seen from Figure 9, the residues THR-14, THR-158, and estrone formed two hydrogen bonds. Estrone and ILE-35, PRO-45, and PHE-152 formed alkyl interactions (Figure 9(a)). e binding energy of 17beta-estradiol with NTRK1 was −5.5 kcal/mol. ere was no hydrogen bond formed between 17-beta-estradiol with NTRK1. e residue ARG-574 and 17-beta-estradiol formed one Pi-alkyl interaction. 17-Beta-estradiol and HIS-503, LEU-532, and GLN-568 formed alkyl interactions (Figure 9(b)). e binding energy between alpha-estradiol and TP53 was −6.85 kcal/mol. LYS-164 of TP53 and alphaestradiol formed a hydrogen bond. Alpha-estradiol and GLN-100, SER-166, and Mer-169 formed alkyl interactions. Alpha-estradiol and the residue GLN-167 formed one Pialkyl interaction (Figure 9(c)). e binding energy of estrone with CUL3 was −4.42 kcal/mol. CUL3 and the residues SER270 and PRO259 formed two hydrogen bonds. e residues ARG266, VAL263, and estrone formed two Pi-cation bonds. Estrone and the residues PRO307 and GLU258 formed alkyl interactions (Figure 9(d)). e binding energy between estrone and ESR1 was −6.46 kcal/mol. Estrone and the residue TYP213 formed two Pi-Pi interactions. Estrone and HIS206, GLU210 formed alkyl interactions (Figure 9(e)).

Discussion
First recorded in the Shen Nong Ben Cao Jing, deer antlers are true bone growth found in pairs on the heads of male deer and are commonly utilized as Chinese medicine to tonify "kidney-yang," which is believed to have the effect of strengthening the muscles and bones. Modern pharmacology has found through various experimental studies that deer antlers have significant anti-inflammatory and antioxidant effects, which protect osteoblasts, and this may be the mechanism by which deer antlers treat degenerative disc degeneration. However, few studies explored the molecular mechanism of deer antlers in the treatment of IVDD.
rough the network pharmacology and molecular docking analysis of deer antlers for the treatment of IVDD, we have made a preliminary exploration of its molecular mechanism. e mechanisms by which IVDD occurs are complex, and current studies mainly focus on oxidative stress [17],  Evidence-Based Complementary and Alternative Medicine inflammatory irritation [18,19], nutritional deficiencies [20], and DNA damage [21]. In the screening results of the small molecule active compounds of deer antlers, we found that the active compounds such as estradiol and estrone are all steroid hormones. It has been found that both α-estradiol and 17-β-estradiol can increase the activity of anti-inflammatory markers such as IL-6 receptors through the action of estrogen receptors, thereby reducing the secretion of inflammatory factors TNF-α and IL-6 and inhibiting inflammatory stimulation [22,23]. It has also been shown that estradiol protects tissues by inhibiting oxidative stress in organ tissues [24,25]. e specific mechanism of action is through binding to the estrogen receptor. It is particularly noteworthy that some researchers have found that intervertebral discs and their surrounding tissues have a large number of estrogen receptors and that the use of 17-betaestradiol is effective in slowing down the process of IVDD, which may be a mechanism for the treatment of IVDD with deer antlers [26][27][28]. All six pathways are crucial to the occurrence of IVDD. e PI3K-Akt signaling pathway plays a critical role in IVDD [29]. After PI3K-Akt activation, it can help promote the apoptosis of nucleus pulposus cells [30]. It was reported by Krupkova that, through promoting Akt phosphorylation, epigallocatechin 3-gallate prevented nucleus pulposus cells from oxidative stress [31]. e thyroid signal pathway is also an important factor. It has been reported that thyroid hormone can regulate microRNAs to induce an antioxidative stress effect [32]. Several studies reported that the Notch signaling pathway can stimulate chondrogenesis and cartilage development, which has an effect on curing IVDD [33]. It is also reported that the activated Notch signaling pathway reduced the growth arrest and apoptosis of nucleus pulposus cells and promoted the regeneration and proliferation of nucleus pulposus cells [34]. e expression of FoxO protein can induce several kinds of antioxidant enzymes and play the role of antioxidation [35,36]. Alvarez-Garcia O et al. found that IVDD is associated with an obvious decrease in the number of effectors that receive FoxO signals, which affects the expression of FoxO protein [37]. So, we can deduce that Foxo is an important regulatory protein for antiaging and antioxidation. Maintaining the normal expression of FoxO is essential in delaying IVDD. Estrogen secretion can effectively reduce the incidence of IVDD. Some studies have shown that with a reduction in estrogen secretion, postmenopausal women's incidence of IVDD also increased [38]. Other studies have found that the presence of estrogen helps maintain the extracellular matrix [39]. HIF-1 keeps nucleus pulposus cells alive and synthesizes and maintains the extracellular matrix [40]. At the same time, Hif-1 can regulate the stabilization of oxygen by regulating a variety of enzymes [41].
Molecular docking was utilized to determine if the four components had an affinity for the five target proteins. With the results listed, it indicates that these components could bind tightly to the target proteins. Given that the binding affinity of alpha-estradiol on TP53 was slightly lower than the original ligand, we can conclude that estrone on ESR1 had the best docking result. We can speculate that estrone-ESR1 may have a potential antidenaturation effect.

Conclusion
In summary, we initially explored the molecular mechanism of deer antlers for the treatment of IVDD through network pharmacology and molecular docking in this study, predicting that the key therapeutic mechanism is anti-inflammatory and antioxidant. We also found that estrone showed the best docking results against ESR1 and predicted that it could be used as a potential drug treatment site. Although the effective compounds from this study screen scored highly, it cannot be determined that the remaining compounds are not clinically effective, especially large-molecule compounds such as peptides. Furthermore, experimental validation of the results of this study is still required due to algorithmic limitations and database sources limited to published research reports.

Abbreviations
IVDD: Intervertebral disc degeneration TCMSP: Traditional Chinese medicine systems pharmacology PPI: Protein-protein interaction GO: Gene ontology KEGG: Kyoto Encyclopedia of Genes and Genomes TCM: Traditional Chinese medicine.

Data Availability
e data used to support the conclusions of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that there are no conflicts of interest.

Authors' Contributions
Rui Weng and Wenchao Li designed and conceptualized the study and revised the manuscript. Rui Weng, Hongheng Lin, and Wenchao Li wrote the manuscript. Xiaoxiao Lin, Zhenyu Zhang, Qiqi Chen, and Yiqi Yao were responsible for extracting data. Rui Weng, Zhuoyao Li, and Daman Chen carried out the molecular docking analysis.