Mechanism of Erzhiwan in treating osteoporosis based on molecular docking technology and molecular dynamics simulation

This experiment was a network pharmacology research based on the theoretical system of traditional Chinese medicine. TCMSP database, PubChem database, RCSB database, and SwissTargetPrediction database were used to study the effective chemical constituents of Ligustri lucidi Fructus and Ecliptae Herba in Erzhiwan, a traditional prescription for nourishing the liver and kidney. Then Genecards database, OMIM database, OMIM Gene Map, and Metascape database were used to study the therapeutic targets of osteoporosis. At last, Cytoscape 3.6.0 software, its built-in Bisogenet and CytoNCA, AutoDockTools-1.5.6 software, PYMOL-2.2.0 software, and Gromacs software, by drawing the relationship diagram between chemical components and disease targets, PPI network of disease, semi-flexible molecular docking technology, evaluation and analysis of enrichment pathway, and molecular dynamics simulation, were used to study the therapeutic mechanism of Erzhiwan on osteoporosis. It is found that the intervention and regulation of Erzhiwan on osteoporosis were mainly realized through multiple targets of active ingredients and multiple pathways, which provided support for the continued development of Erzhiwan.


Introduction
Erzhiwan is derived from Fu Shou Jing Fang (1530) written by Wu Manji in the Ming Dynasty. The prescription includes two kinds of medicinal materials: Ligustri lucidi Fructus and Ecliptae Herba. The winter solstice is the shortest day in the Northern Hemisphere; at this time, the fruit of Ligustrum lucidum Ait. is mature, and the color turns black and dark purple. Therefore, traditional Chinese medicine believes that the winter solstice is the best time to harvest the fruit of Ligustrum lucidum Ait. The original plant of Ecliptae Herba is Eclipta prostrata L. It is an annual herb and likes a cloudy, wet, and humid climate. The climate environment before the summer solstice is most suitable for the growth of Eclipta prostrata L. Therefore, traditional Chinese medicine believes that the summer solstice is the best time to harvest the herba of Eclipta prostrata L.
Modern research also found that the content of therapeutic chemicals in Ligustri lucidi Fructus [1] and Ecliptae Herba [2] is higher in the winter solstice and summer solstice. Traditional Chinese medicine believes that Erzhiwan belongs to the formula of nourishing yin and tonifying kidney. Both Ligustri lucidi Fructus and Ecliptae Herba belong to the liver and kidney meridians and are used for the surface syndrome of liver and kidney yin deficiency. Therefore, the clinical application of Erzhiwan can tonify the liver and kidney. Clinical application and modern research have found that Erzhiwan has good effects of protecting the liver and kidney [3][4][5] and regulating human hormone secretion [6,7]. The reason may be that Erzhiwan is rich in many special chemical components such as iridoids, phenylethanol, triterpenoids, coumarins, flavonoids, and organic acids [8,9]. Modern medical research believes that the pathogenesis 1 3 of osteoporosis (OP) is mainly divided into three aspects [10][11][12]: first, the amount of the bone in the human body is reduced, which is easy to feel tired and affect normal life when it is serious; the second is the degradation of bone microstructure, that is, the destruction of bone molecular structure of newly formed bone and old bone is easy to cause pain, and in severe cases, it will lead to systemic bone disease with low traumatic fracture; third, bone fragility increases, and the density and quality of new and old bone decrease significantly, which leads to fracture.
This study started from the network pharmacology-molecular docking-molecular dynamics to explain the compatibility law and the action mechanism of Erzhiwan in the treatment of osteoporosis from the perspective of multi-target interaction, so as to construct the action chain of "traditional Chinese Medicine-effective components of drugs-target proteintherapeutic effect" and reveal the effect of Erzhiwan on the regulation of hormone secretion of osteoporosis.

Experimental design
Firstly, the internal chemical components of Ligustri lucidi Fructus and Ecliptae Herba in Erzhiwan were studied through TCMSP database and PubChem database, and then the targets were screened through SwissTargetPrediction database, and the target and gene were transformed; using Cytoscape 3.6.0 software construct the network of traditional Chinese medicine-drug effective component-target protein disease and analyze its correlation degree; the target genes related to osteoporosis can be screened by Genecards database, OMIM database, and OMIM gene map, and then combined with the corresponding targets, to screen the target of Erzhiwan in the treatment of diseases, the Bisogenet and CytoNCA in Cytoscape 3.6.0 software was used to construct PPI network of Erzhiwan for disease target interaction; download the threedimensional structure of small molecule chemical components and macromolecular target proteins through PubChem database and RCSB database, and use AutoDockTools-1.5.6 software for small molecule torsion semi flexible molecular docking, and then PyMOL-2.2.0 software draws the docking results into three-dimensional pictures and calculates the key positions; finally, the one-stop analysis of Metascape database was used to perform GO function enrichment analysis and KEGG signal pathway analysis.

Screening of active ingredients and target prediction of Erzhiwan
Pharmacology database of Chinese medicine systems and Analysis platform TCMSP (https:// tcmspw. com/ index. php) can inquire the chemical components contained, and the corresponding physicochemical properties of Chinese medicines enter the names of Chinese medicines Ligustri lucidi Fructus and Ecliptae Herba; you can get the chemical composition and corresponding physicochemical properties of the Ligustri lucidi Fructus and Ecliptae Herba. According to the Lipinski five-fold rule setting molecular weight (MW) ≤ 500, lipid-water partition coefficient (Clog P) ≤ 5, hydrogen bond acceptor (N'S + O'S) ≤ 10, hydrogen-bond donor (NH'S + OH'S) ≤ 5, dr uglikeness(DL) ≥ 0.18, and oral bioavailability(OB) ≥ 30%, the chemical composition that meets the above conditions is the active chemical ingredient.
The TCMSP database is used to query the drug targets of the active chemical ingredients of the Erzhiwan, and the targets are entered into the database of SwissTarget-Prediction (http:// www. Swiss targe tpred iction. ch), verify all the active chemical ingredients of the Erzhiwan, draw the chemical formulas, then obtain the corresponding smiles chemical formula of the active medicinal ingredients of the Erzhiwan. Finally, using the smiles chemical formula to search for the targets of therapeutic disease on the database of SwissTargetPrediction and limit it to "homosapiens," you can obtain the target genes of therapeutic disease and discard the low score content; we can obtain the target genes of therapeutic disease of the active chemical components of the Ligustri lucidi Fructus and the Ecliptae Herba.

Construct the network of traditional Chinese medicine-active ingredient-target protein
First of all, the active ingredient of the Erzhiwan should correspond to the target gene, make a date file in ececel format, and make a trpe file in ececel format corresponding to the active ingredients and target genes, then import separately the date file and the trpe file into Cytoscape_ v3.6.0, establish a network diagram of "Active Ingredient of Traditional Chinese Medicine-Predicted Target Gene" of Erzhiwan; traditional Chinese medicine, active chemical ingredients of traditional Chinese medicine, target genes for treating diseases as network nodes; different classifications of nodes will be connected by lines according to the scores of the SwissTargetPrediction database to represent their network relationships, with "nodes" represent the target protein molecules, the "edges" represent interrelationships, through the analysis function of the software to obtain Degree, Neighborhood Connectivity and Closeness Centrality data, using the degree of correlation to represent the size of the node, and then according to the data, the correlation between the chemical composition of the Erzhiwan and the target gene can be analyzed.

Acquisition and screening of osteoporosis-related target genes
Osteoporosis-related target genes need to use the Genecards database (https:// www. genec ards. org/) and the OMIM database (https:// omim. org/) and use "osteoporosis" as the keyword to search for target genes. The Genecards database can score the correlation of disease target genes and select the target genes with a correlation score (Relevance score) ≥ 10, the related disease target genes of the OMIM database and OMIM Gene Map were summarized, and the query results of the two databases were combined and screened to obtain the target genes of osteoporosis.

Construction of the interaction network between target gene and disease therapeutic gene
The potential target of the Erzhiwan in the treatment of osteoporosis is imported into the Bisogenet plug-in of Cytoscape_v 3.6.0, and the CytoNCA plug-in can run normally in the background, the Bisogenet plug-in is mainly responsible for the operation of the interaction network, but the CytoNCA plug-in can calculate data for it, run the plug-in to get the corresponding PPI network, and filter out all the nodes whose degree value is greater than twice the median in the data. CytoNCA plug-in of Cytoscape was used to analyze the betweenness (B), closeness (C), eigenvector (E), local average connectivity-based method (LAC), and neighborhood connectivity (NC) and export the analysis results.

Molecular docking to verify the correlation between drugs and action targets
Autodocktools-1.5.6 software is used to dock the threedimensional structure of different compounds with the three-dimensional structure of macromolecular protein, so as to predict the calculation amount and model, also known as semi-flexible molecular docking. When using autodocktools-1.5.6 for docking, after determining the docking box, run 50 times of docking, and appropriately increase the number of molecular docking runs according to the structure of different compounds, and appropriately increase the amount of random numbers generated, to ensure that the optimal binding sites and intermolecular interactions that support the stability of complex structures are found.
Download the three-dimensional structure of the main chemical components for the treatment of osteoporosis through the PubChem database (https:// pubch em. ncbi. nlm. nih. gov/). Download the three-dimensional protein structure of the core target protein through the RCSB database (https:// www. rcsb. org/). The three-dimensional models of main chemical components and target proteins first need to be treated with autodocktools-1.5.6 software for water removal, hydrogenation, and impurity removal. Finally, the format conversion software is used to convert the three-dimensional models of main chemical components and target proteins into pdbqt format, set the grid box and adjust the docking parameters, run Autogrid for molecular docking, analyze the docking results, and record their binding free energy; the level of binding free energy is closely related to the binding ability between chemical components and target. When the binding free energy between chemical components and target proteins is lower, the possibility of action is greater, and the docking results are more accurate. The pdbqt format file is exported. Using PyMOL-2.2.0 software arranges the analysis results in ascending order from small to large and selects the chemical components with the smallest binding energy and the best docking effect to visualize the docking results. Then draw the data results of molecular docking into a rectangular clustering heat map. The advantage of this map is that it is displayed in color according to the binding energy of molecular docking, which can more intuitively compare the binding energy differences of different molecular docking.

GO function enrichment and KEGG signal pathway analysis
Forty-one predicted target genes were input to Metascape database for GO biological processes (BP), GO cellular components (CC), GO molecular functions (MF), and KEGG pathway analysis.
Ececel format date files are made according to the enrichment pathways and action genes, and ececel format trpe files are made according to the enrichment pathways and action genes and their classification. The date files and trpe files are imported into Cytoscape_v3.6.0, and according to the color discrimination of trpe editing for enrichment pathway and action gene, the network diagram of Gene Ontology and KEGG signal pathway is established. The enrichment pathway and action gene are taken as network nodes, and the network relationship between nodes is connected in the form of lines, and the mutual relationship is represented by "edge."

Molecular dynamics studies
On the basis of molecular docking and other processes, we verified the results by molecular dynamics simulation, carried out MD simulation with Gromacs software, set the physical conditions as constant temperature (310 K) and constant pressure (101kpa) and periodic boundary conditions, and used TIP3P water model to simulate the human environment in 0.145 mol/L neutral sodium chloride solution. After the state balance of all environments, we used the screened component target complex system for 50 ns MD simulation, in which the conformation was stored and calculated every 10 ps. The RMSD (root mean square deviation) and RG (radius of gyration) analysis and visualization of MD simulation results were carried out using Gromacs embedded program and VMD. Therefore, taking the binding energy ≤ − 7.6 kcal/mol as the limit, we selected the first four docking systems for molecular dynamics simulation. In order to make these data more intuitive in front of us, we visualized their output data.

Screening of active ingredients and target prediction of Erzhiwan
Filter in TCMSP database according to Lipinski five-fold and oral bioavailability, and obtain 13 main active ingredients of Ligustri lucidi Fructus and 10 main active ingredients in Ecliptae Herba (Table 1).
Query the smiles formula of the main active ingredient of Erzhiwan, and enter the miles formula into the SwissTargetPrediction database to query-related targets. A total of 231 targets of Ligustri lucidi Fructus and 229 targets of Ecliptae Herba were obtained, including 160 intersection targets, 71 unique targets of Ligustri lucidi Fructus, and 69 unique targets of Ecliptae Herba (Fig. 1).

Construction the network of traditional Chinese medicine-drug active ingredient-target protein
Use Cytoscape software to draw the network diagram of traditional Chinese medicine-drug active ingredient-target protein of Erzhiwan, and analyze the data. This network includes a total of 319 nodes, 2 traditional Chinese medicine network nodes: Ligustri lucidi Fructus (NZZ) and Ecliptae Herba (MHL), and 17 chemical composition nodes of traditional Chinese medicine (7 chemical constituents of Ligustri lucidi Fructus, 8 chemical constituents of Ecliptae Herba, and 2 repeated components), 300 target gene nodes, and 1216 edges.

Screening and analysis of disease-related targets
Osteoporosis-related target genes were searched through Genecards database and OMIM database and target genes with Relevance Score ≥ 20 from Genecards database, combined the results of the two databases, query, and screened to obtain a total of 215 osteoporosis-related targets. Compared with the predicted targets of Erzhiwan, 41 potential targets were related to the treatment of osteoporosis ( Table 2).

Construction of PPI network based on Bisogenet plug-in and CytoNCA plug-in
The drug genes and disease target genes were introduced into the Bisogenet plug-in of Cytoscape_v3. 6 In the figure, the size of the degree value is positively correlated with the size of the node. Among the target genes of Erzhiwan in the treatment of osteoporosis, the 10 targets with better degree values are as follows: NTRK1  APP The correlation is 585; the TP53 correlation is 521; the CUL3 correlation is 505; the EGFR correlation is 487; the XPO1 correlation is 461; the TRAF6 correlation is 454; the FN1 correlation is 452; and the SRC correlation is 448 ( Fig. 3; Table 3).

Molecular docking to verify the correlation between drug and target
According to the evaluation standard of the free binding energy of semi-flexible molecular docking, it is considered that the binding free energy of chemical components with less than − 5.0 kcal/mol has a good activity; the order of the binding free energy from small to large is as follows:    (Fig. 7).

GO function enrichment and KEGG signal path analysis
In terms of molecular biological function. The results were remarkable:   (Fig. 9).

Results of molecular dynamics studies
From the RMSD results of MD simulation, it can be seen that in the four MD systems of component and target complexes, the overall performance of the blue broken line, the complex system of NTRK1, and acacetin was poor, including large fluctuations in RMSD and radius of gyration, which means that the system cannot show a good therapeutic effect on osteoporosis.
For the two complex systems (red and green broken lines) formed by TRAF6, TP53, and beta-sitosterol, we can observed that in the RMSD and Rg experimental results, the system formed by TRAF6 and beta-sitosterol had better protein hydrophobicity and more stable tertiary structure than the system formed by TP53 and beta-sitosterol, but the performance of these two systems was good, which was very intuitive. The binding energy data of these two systems were also very ideal, and TRAF6 and TP53 were possible  as therapeutic targets for osteoporosis. We had made it clear that the chemical component beta-sitosterol of Erzhiwan can well target TRAF6 and TP53 proteins. In terms of RMSD and Rg, the molecular dynamics system (purple broken line) formed by ESR1 and betasitosterol showed an excellent state; the system can exist stably without affecting its own and other protein structures (Fig. 10).

Discussion
This experiment design principle was based on the theoretical system of traditional Chinese medicine; osteoporosis in TCM treatment system was called "bone impotence" and "kidney impotence." Chinese medicine dialectically considered that the etiology and pathogenesis were as follows: kidney dominates the bone of body, and kidney influenced human bone development through menstruation, which was a sex hormone secreted by the human body or other hormones promoting bone formation. The function of the liver was to control the generation of Qi and blood, and they had a common source; therefore, people with abnormal liver and kidney function had a higher risk of osteoporosis. Therefore, traditional Chinese medicine considered that the location of osteoporosis was in the kidney and liver, and the main treatment directions are nourishing Yin and tonifying the kidney and liver. Erzhiwan was a traditional prescription of nourishing Yin and tonifying the liver and kidney; it was widely used in clinic. Based on the theory of traditional Chinese medicine, Erzhiwan in the clinical application and modern research continuously discovered new effect, and the study of the chemical composition had been deep.

The analysis of the chemical composition in Erzhiwan
In the study on the effective components of Ligustri lucidi Fructus and Ecliptae Herba in the prescription of Erzhiwan for the treatment of diseases through TCMSP database and PubChem database, according to Lipinski five-fold rule and oral bioavailability, 21 qualified chemical components were screened. Through Swis-sTargetPrediction database, 21 chemical components were screened for binding targets, and a total of 300 targets were obtained. The above results were analyzed by Cytoscape drawing, and the rank of chemical components rich in action targets were as follows: luteolin, quercetin, acacetin, kaempferol, butin, eriodictyol, 3′-O-Methylorobol, pratensein, demethylwedelolactone, 1,3,8,9-tetrahydroxybenzofurano[3,2-c]chroen-6-one, linarin, Olitoriside_qt, lucidumoside D.
Most of the chemical composition had fully studied the effect of the treatment of disease. For example, quercetin and luteolin were not only the common chemical components of Ligustri lucidi Fructus and Ecliptae Herba, but also contained the most therapeutic target genes. Luteolin was a natural flavonoid compound with a variety of pharmacological activities, such as antiinflammatory [13,14], anti-allergic [15], reducing uric acid [16], and anti-tumor [17,18]. Quercetin had good expectorant and antitussive effect [19]; it can also reduce blood pressure [20,21], reduce blood lipids, expand coronary artery [22], and increase coronary blood flow [22].    Eriodictyol in Ligustri lucidi Fructus and butin in Ecliptae Herba also had good effects on treating osteoporosis. Butin can inhibit AMPK pathway, upregulate MITF, and regulate the expression of melanin synthesis-related proteins [23]. Although some of these chemical elements had not to research the therapeutic effect, it is rich in therapeutic targets, and this kind of chemical composition may be the focus of future research.

Analysis of therapeutic target genes for osteoporosis
A total of 215 target genes related to osteoporosis were searched by Genecards database and OMIM database, among which 41 potential targets of Erzhiwan were related to osteoporosis. PPI network was constructed by Bisogenet plug-in and CytoNCA plug-in of Cytoscape software, and a core network with 118 nodes and 1706 edges was obtained. The target genes with the highest correlation degree were NTRK1, ESR1, APP, TP53, CUL3, EGFR, XPO1, TRAF6, FN1, and SRC. Among them, the NTRK gene is mainly related to the occurrence of tumors, for which some studies have proposed the possibility of Erzhiwan for the treatment of the liver and kidney tumors [24,25]. Recent studies have found that the mutation of NTRK1 will lead to diseases mainly manifested by repeated fractures [26], and patients with congenital orthopedic diseases without sweat and pain due to the mutation of NTRK1.
At present, there had been a lot of research on the effect of ESR1 on osteoporosis [27][28][29]; it directly affected the gene expression levels of estrogen in women. The estrogen content in women also directly affected the role of bone cells, especially on the activity of osteoblasts and osteoclasts; estrogen can increase bone formation and inhibit the differentiation of osteoclasts, so as to avoid the occurrence of osteoporosis; clinical studies also support this view [30]. EGFR had been found to play an important role in the growth and development of the bone. The expression of EGFR played an important role in the endochondral ossification and bone formation of human cartilage [31]. The fundamental reason may be that EGFR had a significant impact on osteoclast. of TRAF6(1LB4) and ESR1(1GWP) was the highest, and beta-sitosterol was the most significant chemical component. TRAF6 and ESR1 are also proved to be important target genes for the treatment of osteoporosis [27][28][29]32]; it had important guiding significance for the treatment of osteoporosis. The binding free energy content of NTRK1(1WWW) and EGFR(1YY9) were higher of the target proteins, and acacetin, demethylwedelolactone, and beta-sitosterol were the highest and most significant chemical component. These two genes had been studied in the treatment of osteoporosis, but not attracted widespread attention [26,31,33]. With the further study of its gene expression, a new therapeutic approach of Erzhiwan for osteoporosis may be found. The binding free energy content of APP(1TAW) and TP53(3TG5) were higher of the target proteins, among which beta-sitosterol was the highest binding free energy. However, there were few studies on APP(1TAW) in relation to osteoporosis, so it may be of great significance to study Erzhiwan in the treatment of its gene-related diseases. The binding free energy content of CUL3(4AP2) was not significant, among which beta-sitosterol was the highest binding free energy. However, there are few studies on the gene expression of CUL3(4AP2); it may be of significance to study Erzhiwan for the treatment of its gene-related diseases.

Study on GO function enrichment and KEGG signal path
By inputting 41 potential targets of Erzhiwan for osteoporosis into Metascape database for GOBP analysis, it was found that the research results with good enrichment were as follows: the result of gland morphogenesis (0,022,612) of GO enrichment was most significant, which had important research significance in terms of affecting estrogen expression [34]; ossification (0,001,503), skeletal system development (0,001,501), cellular response to organic cyclic compound (0,071,407), and gland development (0,048,732) of GO enrichment were outstanding; The response to hormone (0,009,725) of GO enrichment was better; the odontogenesis (0,042,476) and bone development (0,060,348) of GO enrichment were average; the cell population proliferation (0,008,283) and cellular response to lipid (0,071,396) of GO enrichment were poor. Among them, ossification (0,001,503) [35], skeletal system development (0,001,501) [36], and bone development (0,060,348) [37] were a biological process directly related to the growth and development of bone, so they had important research significance. The gland morphogenesis (0,022,612), gland development (0,048,732), and response to hormone (0,009,725) of Erzhiwan were found strongly correlated with the expression of estrogen receptor gene in molecular docking analysis. The estrogen content also affected the formation of the bone [38]; there was a great significance to study the biological processes related to hormone glands. The cellular response to organic cyclic compound (0,071,407), cell population proliferation (0,008,283), and cellular response to lipid (0,071,396) may be related to the pathway of cellular absorption promoting bone formation, which need further research.
KEGG pathway signal pathway analysis results showed that osteoclast differentiation pathway (hsa04380) was most relevant to the treatment of osteoporosis. In the results of molecular docking, EGFR and TRAF6 genes related to osteoclast differentiation had high binding free energy, which proved that Erzhiwan was likely to affect osteoclast differentiation in the treatment of osteoporosis.

Molecular dynamics simulation analysis
Molecular dynamics (MD) was a comprehensive molecular dynamics simulation method combining physics, mathematics, and chemistry. It was a means to simulate the movement of small molecules in the body environment by computer. The result data of molecular dynamics such as RMSD and RG value were to measure the stability of the complex system of components and proteins, the stability of the tertiary structure of proteins combined with small molecules, and the hydrophobicity of amino acid residues.
As we knew, the convolution radius RG of protein reflected the volume and structural state of protein macromolecules. The larger the RG value of the same system, the expansion of the system occurred in the MD process. According to the analysis results, the functional study of NTRK1 may be related to human bone resorption and bone mineral density [33], which has important research prospects. Erzhiwan may play a role in the treatment of bone diseases by acting on NTRK1; the research on TP53 mainly focuses on its inhibitory effect on cancer and its therapeutic effect on lung cancer [39], but there was also research finding that TP53 Arg72Pro polymorphisms may contribute to the risk of osteoporosis [40]. The low concentration of estrogen in postmenopausal women leads to the increase of the risk of osteoporosis [27,29], Erzhiwan is strongly correlated with ESR1, and Erzhiwan acted on ESR1 expression, which made it possible to treat osteoporosis caused by estrogen deficiency in postmenopausal women; the study on TRAF6 had found that TRAF6 was a gene that can affect osteoclast differentiation, which was an important factor in bone formation [32]. The effect of Erzhiwan on TRAF6 expression had become the theoretical basis in the treatment of osteoporosis.

Conclusion
This study was based on the network pharmacology-molecular docking technology-molecular dynamics to research the mechanism of Erzhiwan in the treatment of osteoporosis. EGFR and TRAF6 genes related to osteoclast differentiation in molecular docking had high binding free energy, which proved that Erzhiwan was likely to treat osteoporosis by affecting osteoclast differentiation. According to the molecular dynamics, betasitosterol and acacetin of Erzhiwan played a role in improving or treating osteoporosis to some extent after acting on the four target proteins of ESR1, TRAF6, TP53, and NTRK1.
It is found that the intervention and regulation of Erzhiwan on osteoporosis were mainly realized through multiple targets of active ingredients and multiple pathways, which provided support for the continued development of Erzhiwan. However, this study adopted network pharmacology to reveal the mechanism of Erzhiwan in treating osteoporosis, which had certain limitations. For example, the impact of processing on drug components was not considered. Therefore, in the follow-up study, in vivo and in vitro experiments can be carried out for verification.
Author contribution Yanling Li and Weijuan Han were responsible for designing the project and obtaining funds; Ziliang Li, Tongsheng Ye, and Fuqi Hao were responsible for data collation and statistical analysis; Yichi Wang and Wenqian Li carried out the molecular dynamics simulation experiments; Qingfeng Yan and Huawei Shi prepared the first draft. All authors had read and approved the final manuscript.
Funding This study was supported by the Henan Science and Technology Research Project (212400410206) and the Cadre Teacher Training Program of the Sanquan College of Xinxiang Medical University (SQ2021GGJS06).
Data availability N/A. All data generated or analyzed during this study were included in this published article.

Competing interests
The authors declare no competing interests.

Conflict of interest
The authors declare no competing interests.