Mechanism of Guilu Erxian Gum in the treatment of osteoporosis based on network pharmacology

Peng Liu, Dong-Xiang Yang, Dong-Dong Yu, Xiu-Ting Wang

1. Liaoning University of Traditional Chinese Medicine,Shenyang 110847,China

2. Affiliated Hospital of Liaoning University of Traditional Chinese Medicine,Shenyang 110032,China

Keywords:Chinese traditional medicine Network pharmacology Guilu Erxian Gum Osteoporosis Mechanism of action

ABSTRACT Objective: This article uses the method of network pharmacology to study the related mechanism of Guilu Erxian Gum in the treatment of osteoporosis. Methods: Based on the TCMSP database, ETCM database, chemistry database, and DrugBank database, the potential active ingredients and related targets of Guilu Erxian Gum were obtained. The known therapeutic targets of osteoporosis were obtained from OMIM and Genecards databases, and the STRING database was used. A protein interaction network (PPI) of active ingredientsdisease targets was established, and the topological parameters of the network were analyzed using Cytoscape 3.8.0 software to obtain key active ingredients and their targets. In R4.0.2,the Bioconductor data package was used to analyze the GO biological function and KEGG pathway analysis of key targets, and obtain the effective ingredients and targets of Guilu Erxian Gum for treating osteoporosis. Results: The prediction results show that Guilu Erxian Gum has 87 active ingredients and 2305 targets, and there are 4141 known therapeutic targets for osteoporosis. The two act together to obtain a total of 71 PPI core genes. The GO biological process analysis yielded 95 entries, and the KEGG pathway analysis yielded 115 pathways.Conclusion: The analysis results show that Guilu Erxian Gum may play an anti-osteoporosis effect by regulating inflammatory factors, promoting osteoblast differentiation, inhibiting osteoclast formation, and improving microcirculation. The pathways involved include TNF signaling pathways, IL-17 signaling pathway, NF-κB signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway, PI3K-Akt signaling pathway, etc. This study provides a theoretical basis for further elucidating the pharmacological mechanism of Guilu Erxian Gum in the treatment of osteoporosis.

1. Introduction

Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass and destruction of bone tissue microstructure,resulting in increased bone fragility and prone to fractures[1].According to statistics, more than 200 million women in the world over the age of 60 suffer from OP, and more than 9 million people suffer fractures due to OP every year[2]. The pathogenesis of OP is more complicated and closely related to age, gender and lifestyle.The current research on the pathogenesis of OP mainly focuses on cell differentiation and apoptosis, osteogenesis/osteoclast related signaling pathways and gene polymorphisms[3]. Western medicine treatment mainly uses bone resorption inhibitors, bone formation promoters, bone mineralization drugs, etc. Many side effects of drugs and drug interactions are relatively large. For example, longterm use of hormone drugs can increase the risk of breast cancer and cardiovascular events[4]. Therefore, the clinical treatment and management plan for OP needs to be further optimized.According to the clinical manifestations of OP, it can be attributed to the categories of "bone fistula", "gubi" and "bone dryness" in traditional Chinese medicine. "Suwen·Ai Lun" mentioned that "the kidney governs the bone marrow of the body... the kidney qi is hot,the waist and spine are not lifted, the bone is dry and the marrow is reduced, and the hair is bony failure"; "Lingshu·Meridian" says:"feet less If the Yin Qi is extinct, the bones are dry. Therefore,the pathogenesis of OP is closely related to the deficiency of kidney essence. Guilu Erxian Collagen is contained in "Medicine Circumstances" written by Wang Sancai of the Ming Dynasty. In the prescription, ginseng can nourish vital energy, goji berry nourishes the kidney and nourish essence. Deer and turtle two gums can not only nourish qi, nourish kidney yin, and strengthen kidney yang ,And can nourish the spleen and stomach to help the "source of qi and blood biochemistry". The whole prescription has the effects of nourishing the kidney, replenishing essence, and strengthening the bones. Qi Pengkun and others [5] used Guilu Erxian Decoction and Alendronate to treat postmenopausal osteoporosis. After 6 months of treatment, the bone mineral density and low back pain VAS score of patients were significantly improved, and the effect was better than that of Western medicine alone. . Wang Xinyan et al. [6] found that Guilu Erxian Gum can increase the level of serum bone sclerostin and reduce serum β-gelled degradation products (β-CTX),osteocalcin (N-MID) and the N-terminal of total type I procollagen.Propeptide (T-PINP) levels, thereby reducing bone resorption and improving bone quality.

Network pharmacology is the first research method proposed by Hop-kins. This method first uses public database and literature search, combined with bioinformatics technology to screen out known drug ingredients and specific disease target information,and then uses network visualization tools to construct " Active ingredient-target-pathway-disease" network model, and finally through multi-level analysis to find out the key nodes related to the active ingredient and disease, thereby predicting the pharmacological mechanism of drug prevention and treatment of diseases[7], which reflects the multi- The characteristics of "components, multiple targets and multiple pathways" are in line with the understanding of the nature of diseases in Chinese medicine. This study uses the research method of network pharmacology to explore the possible targets and related signal pathways of Guilu Erxian Gum in the treatment of OP, in order to provide corresponding ideas and basis for the clinical application of Guilu Erxian Gum and the follow-up study of OP.

2. Materials and methods

2.1 Screening of Guilu Erxian Gum Compounds and Active Ingredients

Utilizing the TCM System Pharmacology Technology Platform(TCMSP, http://tcmspw.com/tcmsp.php), the ETCM database(http://www.tcmip.cn/), and the chemistry database (http://www.organchem. csdb.cn/) and DrugBank database (https://www.drugbank.ca/) to retrieve the compounds contained in tortoise shell gum, antler gum, ginseng, and wolfberry in Guilu Erxian Gum.Among them, the compounds contained in ginseng and wolfberry are searched in the TCMSP database, while the compounds contained in tortoise shell gum and antler gum cannot be searched in TCMSP, so search in the ETCM database, chemical professional database and DrugBank database. According to the characteristics of drug ADME(absorption, distribution, metabolism, excretion), oral bioavailability(OB) and drug-likeness (DL) are used as screening indicators, and the screening threshold is OB≥30%, DL≥0.18, The active ingredients that meet the conditions were screened. Although the compounds contained in tortoise shell gum and antler gum did not meet the ADME screening criteria, it was found through literature review that the tortoise shell contained compounds that could promote the differentiation of MSCs into osteoblasts [8] and antler gum contained There are a variety of amino acids, and there are compounds that can promote calcium absorption and bone mineralization[9], so these compounds were included as active ingredients of tortoise shell gum and antler gum for analysis.

2.2 Screening of potential targets of Guilu Erxian Gum and OP-related disease targets

Based on the TCMSP database, the active ingredients of tortoise shell, antler gum, ginseng, and wolfberry are matched to potential targets one by one, and the UniprotKB search function in the Uniprot database (http://www.uniprot.org/) is used to enter the protein name and limit the species to " human", correct all the retrieved proteins to their official names, which are the predicted targets of active ingredients.

Obtaining known therapeutic targets of OP, using "osteoporosis" as the keyword, respectively, retrieved from the OMIM (https://www.omim.org/) and Genecards (https://www.genecards.org/) databases,the obtained targets It is a known therapeutic target for OP.

2.3 The interaction network between the active ingredient targets of Guilu Erxian Gum and OP-related therapeutic targets

Input the results of the active ingredient genes and OP disease genes of Guilu Erxian Gum into the online Venn Diagram platform(http://bioinfogp.cnb.csic.es/tools/venny/index.html) to obtain the intersection genes and Wayne Figure, and use Cytoscape 3.8.0 software to build a visual image. Use the STRING platform (https://string-db.rog/) to map the OP’s known therapeutic target to the target of the active ingredient of Guilu Erxian Gum. The species select Homosapiens, hide free protein, and construct protein-protein The interaction network (protein-protein interaction, PPI), and download the PPI protein interaction relationship diagram, screen the first 30 PPI network core genes, and construct a histogram.

2.4 Analysis of GO function and KEGG pathway enrichment

Using the Bioconductor biological data package of R 4.0.2 for gene GO function and KEGG pathway enrichment analysis, according to statistical significance, set P value to 0.05 and q value to 0.05 for GO biological enrichment analysis and KEGG pathway enrichment analysis , Output the results and draw barplot histograms and bubble charts, and finally analyze the results to discuss the treatment mechanism of Guilu Erxian Gum for OP.

3. Results

3.1 Collection and screening of Guilu Erxian gum compounds, active ingredients and potential targets

Using TCMSP database to screen according to the criteria of OB≥30%, DL≥0.18, 22 active ingredients of ginseng and 45 active ingredients of wolfberry fruit were obtained. Tortoise shell and antler gum could not be retrieved in TCMSP, so we passed ETCM database, chemical database and After searching the literature, 15 active ingredients of tortoise shell gum and 14 active ingredients of antler gum were obtained after screening. A total of 96 active compounds were obtained in the whole recipe, and 87 active ingredients remained after excluding duplicate ingredients. Based on the TCMSP database, the active ingredients are paired with potential targets one by one, and the non-target active ingredients are eliminated. Finally, there are 748 targets for ginseng, 1202 targets for medlar, 761 targets for tortoise shell gum-antler gum, a total of 2711 targets point. The target name was corrected to the official name through the Uniprot database and duplicates were eliminated. In the end, Guilu Erxian Gum had 87 active ingredients and 2305 targets.

3.2 Screening of known disease targets for osteoporosis

A total of 4278 OP-related known therapeutic targets were retrieved from OMIM and Genecards databases, and a total of 4141 targets were deleted after duplicates were deleted.

3.3 Intersection genes of the active ingredient target of Guilu Erxian Gum and the target of osteoporosis

Import the target genes of the active ingredient of Guilu Erxian Gum and OP target genes into Excel to obtain the common target of Guilu Erxian Gum-osteoporosis. Use the online Venn diagram platform to draw the Venn diagram and record the common target.Corresponding to the key compounds of osteoporosis, remove irrelevant compounds. Finally, 50 compounds related to Guilu Erxian Gum-osteoporosis and 71 common targets were obtained.

Figure 1 Venny map of Guilu Erxian gum-osteoporosis intersection gene

Table1 Intersection gene of Guilu Erxian gum in the treatment of osteoporosis

3.4 Construction of a visual network of Guilu Erxian glueosteoporosis targets

Import the obtained intersection genes into Cytoscape 3.8.0 software to construct the Guilu Erxian Gum-osteoporosis visualization network (Figure 2). There are 123 nodes in the network(71 genes, 50 key compounds, and 1 disease name). , 1 drug name),in which the red rectangle indicates 50 key active ingredients, which include steroids and flavonoids. The blue diamonds represent 71 intersection genes. Sorted by node degree, the top four chemical components are: quercetin (54), beta-sitosterol (11), glycine (11),stigmasterol (9), the top The four targets are PGR, PTGS1, NCOA2,GABRA1.

Figure 2 Guilu Erxian gum-osteoporosis visualization network

3.5 PPI network construction

Enter the obtained 71 intersection genes into the STRING software to get Figure 3, where each node represents a related protein, and each edge represents the protein-protein interaction relationship. The number of edges is positively correlated with the core degree of the related protein, and the selection ranks top To construct a histogram of the 30 proteins (Figure 4), the names of the first 30 proteins are: ALB, IL6, VEGFA, CASP3, MAPK8, EGFR, ESR1, MYC,CCND1, FOS, AR, CTNNB1, ERBB2, PPARG, RELA , CASP8,AHR, NOS3, PGR, HIF1A, ICAM1, CASP9, CAV1, CCNB1,ESR2, RB1, VCAM1, PARP1, HSPB1, NFKBIA. These proteins play a key role in the treatment of osteoporosis.

Figure 3 Core net work of interaction between Guilu Erxian gum and osteoporosis protein

Figure 4 Core protein histogram

3.6 GO enrichment analysis

Figure 5 shows the GO function enrichment analysis results of Guilu Erxianjiao treatment of OP core target genes. The GO function of the core target genes of Guilu Erxian Gum for OP treatment mainly involves nuclear receptor activity, ligand-activated transcription factor activity, DNA binding transcription factor binding, RNA polymerase II specific DNA binding transcription factor binding, and DNA binding transcription Activator activity,steroid hormone receptor binding, nuclear hormone receptor binding,estrogen receptor binding, etc.

Figure 5 Barplot histogram of gobiological processen richment analysis of key Targets of Guilu Erxian gum in the treatment of osteoporosis

3.7 KEGG pathway enrichment analysis

The results of KEGG pathway enrichment analysis of the core target genes of Guilu Erxian glue in the treatment of OP are shown in Figure 6. The KEGG function of the core target genes of Guilu Erxian glue in the treatment of OP mainly involves: TNF signaling pathway, apoptosis signaling pathway, IL-17 signaling pathway,endocrine resistance, p53 signaling pathway, NF-κB signaling pathway, HIF-1 signaling pathway, MAPK signaling pathway, PI3KAkt signaling pathway, Toll-like receptor signaling pathway, C-type lectin receptor signaling pathway , EGFR tyrosine kinase inhibitor resistance, osteoclast differentiation, VEGF signaling pathway, etc.,are sorted according to the P value from large to small. Table 2 is obtained by selecting the first 15 key pathways.

Table 2 Key signaling pathways of Guilu Erxian gum in the treatment of osteoporosis

Figure 6 Barplothistogram of signal pathway of key target enrichment of Guilu Erxian gum in the treatment of osteoporosis

4. Discussion

The onset of OP is based on the deficiency of kidney essence [10].Guilu Erxian Gum is a classic remedy for invigorating the kidney.It has the effects of invigorating the kidney, replenishing essence,replenishing vital energy and strengthening bones, and plays an important role in improving bone metabolism and increasing bone density [11]. Pharmacological studies have shown [12] that the medicated serum of Guilu Erxian Gum can significantly promote the proliferation of bone marrow mesenchymal stem cells (BMSCs) and differentiation into osteoblasts. The collagen, peptides and amino acids contained in tortoise shell and antler gum have significant bone-strengthening effects[13-15]. However, there is still a lack of related experimental studies on the treatment of OP by Guilu Erxian Gum, so the preliminary discussion is carried out using network pharmacology.

In this study, 50 active ingredients and 71 key targets of Guilu Erxian Gum for OP treatment were screened. The network analysis of "drug-active ingredient-disease-target" shows that quercetin,β-sitosterol, stigmasterol and glycine may be the key active ingredients of Guilu Erxian Gum for OP treatment. Previous experiments have shown [16] that quercetin can promote osteogenic differentiation by up-regulating the expression of BMP2 and Smad4, and play an anti-OP therapeutic effect. In addition, in vitro experiments[17] also proved that quercetin can promote the proliferation and osteogenic differentiation of BMSCs, increase alkaline phosphatase (ALP) activity and the number of mineralized nodules; β-sitosterol can stimulate ovarian granules Cells secrete estradiol (E2) and increase the ratio of osteoblastic osteoprotective factor (OPG) to osteoclast differentiation factor (ODF), thereby enhancing the osteogenic effect[18]; a variety of amino acids such as glycine in deer bone meal can Increase the bone density of OP rats, improve the ratio of urine calcium/creatinine and serum ALP level[19]. For stigmasterol, it may be an important effective ingredient of Guilu Erxian Gum in the treatment of OP. There is currently no relevant experimental study and further verification is needed.

This study shows that the main active ingredients of Guilu Erxian Gum can affect the pathogenesis of OP through multiple target genes.The core target genes of Guilu Erxian Gum for OP treatment mainly include ALB, IL-6, VEGFA, CASP3, MAPK8, etc. These genes can regulate the occurrence of OP by mediating bone resorption and bone formation. At present, malnutrition is considered to be an important risk factor for the onset of OP[20], and serum albumin(ALB) is an important indicator to measure the nutritional status of the body. Clinical and experimental studies have shown that[21-22], the reduction of serum ALB level and bone density The decline was significantly positively correlated, suggesting that low ALB levels are closely related to the incidence of OP. Caspase 3 (CASP3)is a key protein in the cellular mitochondrial apoptosis pathway. It exists in an inactive form under normal circumstances. When the apoptosis program is initiated, CASP3 is activated by proteolytic enzymes and interacts with downstream proteins. A cascade reaction occurs and apoptosis occurs[23]. Therefore, regulating the apoptosis of osteoblasts and osteoclasts is an important mechanism for many Chinese medicines to treat OP[24]. Interleukin-6 (IL-6) is closely related to the onset of OP. The increase in serum IL-6 is an important risk factor for the onset of OP[25]. IL-6 can promote the release of interleukin-1 (IL-1) from osteoblasts , Prostaglandin E2 (PGE2)and other downstream signaling molecules, reduce OPG expression and increase RANKL expression, thereby promoting osteoclast formation and enhancing bone resorption activity[26]. Xu Jun[27]reported that Fangfeng polysaccharide can reduce the serum IL-6 and tumor necrosis factor-α (TNF-α) levels in ovariectomized rats by regulating cytokines, thereby reducing bone resorption in OP rats.Vascular endothelial growth factor A (VEGFA) is a key cytokine in the VEGF family that induces angiogenesis and increases vascular permeability. It is also VEGF in the traditional sense. Studies have found[28] that the expression of VEGF in normal healthy people is much higher than that in OP people, suggesting that it plays an important role in maintaining bone metabolism. Mitogen-activated protein kinase 8 (MAPK8) is one of the important members of the MAPKs family. It can phosphorylate and activate activator protein-1(AP-1), thereby activating the expression of downstream genes,which can be used in cell proliferation, differentiation, and survival.It plays an important regulatory role in the process[29]. Current studies have found that it is mainly closely related to the onset of cancer, but there are few studies on MAPK8 in bone metabolism.Therefore, further studies are needed to confirm the potential role of MAPK8 in the pathogenesis and treatment of OP.

There are many KEGG pathways for the core target genes of Guilu Erxian Gum in the treatment of OP. Through the above pathway analysis, it can be seen that the related pathways of Guilu Erxian Gum in the treatment of OP mainly involve: (1) Regulation of inflammatory factors: research findings[30], Th17 cells can secrete interleukin-17 (IL-17), interleukin-22 (IL-22) and other inflammatory factors to promote the formation of osteoclasts and accelerate bone resorption. IL-17 can enhance the ability of osteoclasts to degrade bone matrix by up-regulating the expression of cathepsin k (CathepsinK) and matrix metalloproteinase 9 (MMP9)[31]. Liu Jinyong et al.[32] observed the clinical efficacy of Guilu Bugu Decoction in the treatment of postmenopausal osteoporosis,and explored its effect on Th17/Treg cytokines. The experimental results showed that Guilu Bugu Decoction can reverse Th17/Treg imbalance, Reduce IL-17, IL-22 and other pro-inflammatory factors, thereby reducing bone loss and improving bone density.(2) Regulating osteoblast/osteoclast differentiation: PI3K/Akt signaling pathway is closely related to bone metabolism, and it is an important pathway for regulating bone constituent cells[33].Studies have found[34] that the application of LY294002 to target inhibition of PI3K can significantly reduce the number of osteoclast differentiation, and down-regulate the proto-oncogene (c-fos) protein and gene expression levels in the p38MAPK pathway, thereby reducing osteoclast resorption; The NF-kB signaling pathway plays an important role in regulating osteoblasts and osteoclast differentiation and maturation [35]. Studies have found[36] that by knocking out mouse NF-KB1 and NF-kB2 genes, the ability of osteoclast precursor cells to differentiate into osteoclasts can be significantly reduced, while inhibiting the osteoclast-related factor anti-tartrate acid phosphatase (TRAP ), expression of calcitonin receptors. Zhang Yuzhuo et al.[37] found that the water extract of tortoise shell can significantly promote the differentiation of MC3T3-E1 into osteoblasts, and reduce the expression of NF-kBp65, NFkBp105 and IL-6 during the differentiation process, suggesting that it promotes osteogenic differentiation mechanism and Inhibiting the NF-kB signaling pathway and reducing inflammation are related;studies have found[38] that the level of HIF-1α in osteoclasts and bone resorption index CTX-1 in ovariectomized mice increase,which can be improved by the intervention of HIF-1α inhibitors Obviously reverse this situation, indicating that the occurrence of OP is closely related to the HIF-1 signaling pathway, and inhibiting this pathway can play an anti-OP therapeutic effect. (3) Improve microcirculation: VEGF has a strong ability to induce angiogenesis and is an important cytokine that regulates the vascular-osteogenesis coupling. On the one hand, it can directly promote osteogenesis and reduce bone resorption. By cooperating with VEGFR-2 and VEGFR-1 to regulate endothelial cell activity, promote the formation of intraosseous vascular network, thereby improving bone metabolism and increasing bone density[39]. Wang Xiaoyan et al.[40] found that the bone marrow microcirculation perfusion and the expression of vascular endothelial markers CD31 and VEGF were significantly reduced after castration in rats. This situation can be effectively improved after the intervention of Qing'e Pill,which proves that Qing'e Pill can improve Treatment of OP with intramedullary microcirculation. (4) Promote osteoclast apoptosis:Yu Dongdong et al. [41] found that Lujiao capsules can promote osteoclasts by promoting AKT phosphorylation, regulating Bcl-2-Bax complex, and increasing Caspase-3 lysis. Apoptosis, and the mechanism of action can be reversed by PI3K/Akt pathway inhibitors, suggesting that Lujiao capsules promote osteoclast apoptosis is closely related to PI3-K/Akt signaling pathway.

In summary, Guilu Erxian Gum mainly regulates bone metabolism by inhibiting inflammation, regulating osteoblast/osteoclast differentiation, improving intraosseous microcirculation, and promoting osteoclast apoptosis to treat OP. The biological network of Guilu Erxian Gum is complex and huge. In this study, the core active ingredients and targets of Guilu Erxian Gum were predicted by constructing an "active ingredient-target" network, and bioinformatics data analysis was performed to predict the tortoise and deer. The related mechanism of Erxian Gum in the treatment of OP, citing existing research results, provides a new idea for the follow-up study on the pharmacological mechanism of Guilu Erxian Gum.