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Research on Molecular Mechanism of Fructus Ligustri Lucidi against Osteoporosis based on Network Pharmacology

Abstract

TCMSP platform of systematic pharmacology of traditional Chinese medicine

This study aimed to investigate the molecular mechanism of Fructus Ligustri Lucidi (NZZ, Chinese abbreviation) against osteoporosis (OP) by means of network pharmacology.ChemDraw Professional 15.1 software and Molinspiration Smiles database were used to draw the chemical formulas of the components. The active ingredients and related target proteins of NZZ were searched in platform of systematic pharmacology of traditional Chinese medicine database, Drugbank, Therapeutic Target Database, SymMap and other databases. Gene Ontology(GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were carried out on the selected target through Enrichr and KEGG Automatic Annotation databases, and their mechanism was studied. A total of 29 compounds and 140 corresponding targets, including 14 key targets and 14 protein factors in protein-protein interaction core network were obtained. The key targets were tumor necrosis factor(TNF), interleukin(IL)-6R and sestrogen receptor alpha. The number of GO items was 466 (P<0.05), including 399 items of biological process (BP), 54 items of cell composition (MF) and 13 items of molecular function (CC). KEGG pathway enrichment screened 85 signaling pathways (P<0.05), including the IL-17 signaling pathway, TNF signaling pathway, advanced glycation end products and their receptors signaling pathway and cAMP signaling pathway. The active ingredients of NZZ. exert their anti-OP effects through multi-components, multi-targets and multi-pathways, which can provide new evidence for further study of their anti-OP mechanism.

Keywords:
Fructus Ligustri Lucidi; Traditional Chinese medicine; Network pharmacology; Osteoporosis; Target

INTRODUCTION

Osteoporosis(OP) is a metabolic osteopathy characterized by abnormal bone mass and bone tissue microstructures. Its mechanism is extremely complex and involves many unrelated pathogeneses. OP occurs in different genders and at any age, but mostly in postmenopausal women and middle-aged and elderly men. The main features of OP are pain and easy to fracture skeleton. OP can be divided into primary and secondary types and affects more than 200 million people in the world, thus greatly increasing the proportion of human abnormal death. (Kastner et al., 2014Kastner M, Sawka AM, Hamid J, Chen M, Thorpe K, Chignell M, et al. A knowledge translation tool improved osteoporosis disease management in primary care: an interrupted time series analysis. Implement SCI. 2014;(9):109.)

Traditional Chinese medicine (TCM), includes the medicines originating in China. TCM differs from Western medicine, and its components are mixed. A TCM often has numerous components, that can act on many disease targets. The multi-component and multi-target functions of TCM make it important in the treatment of difficult and complicated diseases.

Fructus Ligustri Lucidi(NZZ) belongs to the fruit of NZZ, which contains desirable anti-OP active ingredients, which is popular among people. In addition, NZZ has anti-inflammatory, anti-tumor, hypolipidemic, hypoglycemic and immunomodulatory active ingredients (Ngo et al., 2017Ngo QT, Lee HS, Nguyen VT, Kim JA, Woo MH, Min BS. Chemical constituents from the fruits of Ligustrum japonicum and their inhibitory effects on T cell activation. Phytochemistry. 2017;(141):147-155.). The research on the chemical constituents and pharmacological effects of NZZ has been increasing in China, which has gradually attracting the interest of clinical researchers toward the anti-OP mechanism of NZZ(Gan et al., 2019Gan D, Xu X, Chen D, Feng P, Xu Z. Network Pharmacology-Based Pharmacological Mechanism of the Chinese Medicine Rhizoma drynariae Against Osteoporosis. Med Sci Monit. 2019;(25):5700-5716.; Liu et al., 2015Liu R, Kang X, Xu L, Nian H, Yang X, Shi H, et al. Effect of the combined extracts of herba epimedii and fructus ligustri lucidi on sex hormone functional levels in osteoporosis rats. Evid-based Complementary Altern. 2015;2015:1-13.). However, although the research on anti-OP targets is ongoing, a target or drug that can cure anti-OP completely remains unavailable. The number of drugs used to treat OP in clinic is limited, and their adverse reactions are serious. Therefore, we need to further study the pathogenesis and therapeutic of OP.

To understand the anti-OP mechanism of NZZ, based on systematic pharmacology and network pharmacology, we obtained the possible anti-OP targets and related genes of NZZ, determined the relationship between components and targets through target component interaction network, and identified the core targets through a protein protein interaction (PPI) network. Finally, the paths and potential mechanisms of NZZ were analyzed by pathology and physiology.

MATERIAL AND METHODS

Relevant Data Collection

All known active ingredients of NZZ were obtained on the database and analysis Platform of Systematic Pharmacology of Traditional Chinese Medicine (http://lsp.nwu.edu.cn/, TCMSP). In accordance with the requirement of TCMSP for Chinese medicines with high utilization value, eligible compounds were screened from the above active ingredients. ChemDraw Professional 15.1 software was used to draw the chemical formulas of the selected components and save them in smiles format. The files saved in smiles format were imported into Molinspiration Smiles (https://www.molinspiration.com) database and the qualified compounds were screened following Linpinski’s five-fold rule. Disease information in the TCMSP database, which can be queried and downloaded originated from the Therapeutic Target Database (TTD) and Pharmacogenomics Knowledgebase (PharmGKB). PharmGKB uniquely provided pharmacokinetic information for each compound. Users can select compounds with good drug-like and ADME (absorption, distribution, metabolism, and excretion) characteristics for further research.

Target Prediction and Construction of the Component Target Network

The Swiss Target Prediction (http://www.swisstargetprediction.ch) database was used to predict the active targets of the above active ingredients. We used Cytoscape 3.6.1 software to construct protein-protein (PPI) and component-target interaction networks. Target protein molecules are represented by “nodes” and the interrelationships by “edges”. With the excellent visual interface of Cytoscape 3.6.1, which is the most trustworthy software at present, the interaction between components and targets can be displayed.

Retrieval of OP Targets Used in Clinical Therapy

With “OP” as the key word, we used TTD (http://bid.nus.edu.sg/BIDD-Databases /TTD/TTD.asp) database and DrugBank (http://www.drugbank.ca) database to search and screen known OP-related targets, and found their corresponding gene names in UniProt database.

Gene Ontology (GO) Enrichment Analysis

The obtained targets of compounds into were imported into the Enrichr gene enrichment database. (http://amp.pharm.mssm.edu/Enrichr/, Enrichr). We obtained several GO items (P<0.05), including biological process (BP), cellular component (CC), and molecular function (MF).

Using Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Analysis

Using KEGG automatic annotation database (https://www.kegg.jp/blastkoala/) was used to analyze the obtained targets and obtain OP-related signaling pathways (P<0.05).

Molecular Docking Technologies

The ligands and water molecules in the target proteins were removed by Pymol software, and the active components of NZZ were transformed into mol2 format by OpenBabel software. Swissdock(http://www.swissdock.ch/) was used to connect the structure files of the target proteins and small molecules.

RESULTS AND DISCUSSION

Active Constituents of NZZ

A total of 119 active ingredients of NZZ were obtained from TCMSP database,. In accordance with the requirement of TCMSP for Chinese medicines with high utilization value, 34 active ingredients were screened out of 119 active ingredients based on the principle of negative logarithmic value of lipid water partition coefficient (-lg(P)>1.3013) and class DL (>0.18). Using Molinspiration Smiles database and Linpinski’s five-fold rule, 29 components including salidroside, daidzein, luteolin, quercetin, kaempferol and hydroxy tyrosine were obtained.

TABLE I
Linpinski Five Screening Components of NZZ

Target Prediction and Network Graph Analysis

Using the Swiss Target Prediction database, 14 OP-related targets were predicted, including estrogenreceptor alpha (ESR1), prostaglandin G/H synthase 2 (PTGS2), and beta-2 adrenergic receptor (ADRB2). Using Cytoscape to construct the PPI network and component target interaction networks, we observed that 140 PPI-related targets were mapped after 14 protein factors have interacted. Based on the PPI enrichment P value: <1.0e-16, three of the most interacting nodes were transcription factor AP-1 (JUN), mitogen-activated protein kinase 14 (MAPK14) and tumor necrosis factor(TNF). TNF receptor-related factor 6 MAPK8 and ESR1 were the most potent factors. The component-target interaction network showed that 29 OP-related active ingredients interacted with 14 gene targets. On the basis of the effect, the targets was arranged from the strongest to the weakest as follows, in order of PTGS2, ADRB2, TNF, JUN, ESR1, arachidonate 5-lipoxygenase (ALOX5), interleukin receptor 6 (IL6R), and cytochrome P450 1A2 (CYP1A2).

TABLE II
Predicted targets for NZZ

FIGURE 1
Protein-Protein Interaction Network (PPI)

FIGURE 2
Components-Targets Interaction Network

Anti-OP Targets Used Clinically

With “osteoporosis” as a key word, we used TTD and DrugBank database to search and screen the known OP-related targets, and find the corresponding gene names in UniProt database. We have retrieved 34 clinical targets for OP or related symptoms. These targets included the nuclear factor kappa B receptor activating factor ligand (RANKL), parathyroid hormone receptor and WNTL signaling pathway.

Intersection of NZZ Action and Clinical Targets

By comparing the targets of the active components of NZZ with the current anti-OP targets in clinic, we observed that the targets of direct intersection (the same) were beta-2 adrenergic receptor (ADRB2) and ESR1; the targets of intersection were PTGS2, TNF, IL6R and cGMP inhibition. The non-intersecting targets were MAPK14, ALOX5, JUN, interstitial collagenase (matrix metalloproteinase (MMP) 1), estrogen-1 (MMP3), C-C chemokine 2, collagen alpha-1 (I) chain and CYP1A2.

SUPPLEMENTAL TABLE I
Anti-OP targets used clinically. Red indicates intersection

Results of GO Enrichment Analysis

Using Enrichr database for GO enrichment analysis, we obtained 466 GO entries (such as type I-II diabetes mellitus.P<0.05), of which 399 belong to BP, including cytokine-mediated signaling pathway, positive regulation of cell differentiation, positive regulation of acute inflammatory response, intracellular ESR signaling pathway, positive regulation of MAPK cascade, positive regulation of protein serine/threonine kinase activity, and positive regulation of protein serine/threonine kinase activity. A total of 21 key processes were observed in the regulation of vitamin D biosynthesis process; The MF entries included 12 key functions, such as oxidoreductase activity, metal endopeptidase activity, heme binding, DNA binding in transcription regulatory region, adrenergic receptor activity, and CC comprised 13 items, including 11 key components such as cytoplasmic cavity, fibrous gel protein enriched granule, nuclear chromatin and endoplasmic reticulum cavity, and so on.

FIGURE 3
Cellular component

FIGURE 4
Molecular functions

FIGURE 5
Biological process

Results of KEGG Pathway Analysis

We used KEGG database to obtain 85 OP-related signaling pathways (P<0.05), including IL17 signaling pathway, TNF signaling pathway, rheumatoid arthritis pathway, advanced glycation end products and their receptors (AGE-RAGE) signaling pathway of diabetic complications, osteoclast differentiation, estrogen signaling pathway, cGMP-PKG signaling pathway, asthma, and so on.

FIGURE 6
KEGG pathway

SUPPLEMENTAL TABLE II
The glossary in this list

Results of molecular docking technologies

SUPPLEMENTAL TABLE
III The results of molecular docking technologies in this list

DISCUSSION

Nearly 20 years of history has passed since human beings entered the post-genomic era. Detection of gene sequences is no longer an important direction of human research on diseases, but focus should shift to the study of gene function, gene drugs, and bioinformatics. The mechanism of NZZ in the treatment of OP based on network pharmacology is the core of bioinformatics research in the post-genomic era. (Chen et al., 2015Chen HS, Hutter CM, Mechanic LE, Amos CI, Bafna V, Hauser ER, et al. Genetic simulation tools for post-genome wide association studies of complex diseases. Genet Epidemiol. 2015;39(1):11-19.; Ouni et al., 2019Ouni E, Vertommen D, Amorim CA. The human ovary and future of fertility assessment in the Post-Genome Era. Int J Mol Sci. 2019;17(20).)

NZZ has a notably distinct anti-OP effect, which is mainly reflected in its active ingredients, including luciferin D_qt, salidroside, quercetin, daidzein, sinapyl alcohol, caffeic acid, coniferol and eugenol, which can act on ADRB2 and ESR1 receptors(Chen et al., 2017Chen B, Wang L, Li L, Zhu R, Liu H, Liu C, et al. Fructus Ligustri Lucidi in Osteoporosis: A Review of its Pharmacology, phytochemistry, pharmacokinetics and safety. Molecules. 2017;22(9):1469.). Furthermore, ESRs can be expressed in all cells related to bone resorption and bone formation, especially when ESR1 receptor is inhibited, these receptors can directly reduce the number of osteoclasts. (Bukhari et al., 2019Bukhari S, Hussain F, Thu HE, Hussain Z. Synergistic effects of combined therapy of curcumin and Fructus Ligustri Lucidi for treatment of osteoporosis: cellular and molecular evidence of enhanced bone formation. J Integr Med. 2019;17(1):38-45.; Liu et al., 2015Liu RH, Kang X, Xu LP, Nian HL, Yang XW, Shi HT, et al. Effects of the combined extracts of Herba Epimedii and Fructus Ligustri Lucidi on bone mineral content and bone turnover in osteoporotic rats. BMC Complement Altern Med. 2015;15(1):1-18.; Song et al., 2015Song L, Xie XB, Peng LK, Yu SJ, Peng YT. Mechanism and Treatment Strategy of Osteoporosis after Transplantation. INT J Endocrinol. 2015;(2015):280164.)

Lei’s research showed that patients with endocrine and metabolic diseases (diabetes, and hyperthyroidism, etc.), rheumatoid diseases (rheumatoid arthritis, etc.), hematological diseases (anemia, leukemia, etc), and kidney diseases (renal failure, chronic nephritis, etc) are all at risk of OP. However, it is interesting to note that NZZ may also have some therapeutic effects on secondary OP (Lei MM 2018Lei MM. LZGW Pathogenesis of secondary osteoporosis. Chin J Osteopor. 2018;24(11):1514-1520.).

On the basis of PPI network and components-targets networks, we performed GO enrichment analysis, and compared the results with the clinical anti-OP targets. The results showed that NZZ can act on OP and related diseases through ESR1, ADRB2 and PTGS2 targets. Moreover, it is gratifying that these targets are mutual and directly correspond to salidroside and daidzein in NZZ.

Estrogen is an important factor to improving bone density and prevent bone loss after menopause. ESR (estrogen receptor) is found on the surface of human osteoblasts and osteoclasts, which are involved in estrogen signal transduction pathway. Given the considerable number of ADRB2 receptors in bone tissue, nuclear factor-κB signaling pathway can be used to treat OP by inhibiting it through the reduction of its expression level. PTGS2_(or COX-2), is abundant in inflammatory cells and can reduce inflammation by inhibiting the IL-17 signaling pathway. Coincidentally, the above conclusions are consistent with the results of KEGG pathway analysis.

ACKNOWLEDGMENTS

This study was supported by the scientific and technological innovation team (STD201602, New Drug Research and Development Scientific and Technological Innovation Team) of Sanquan College of Xinxiang Medical University.

REFERENCES

  • Bukhari S, Hussain F, Thu HE, Hussain Z. Synergistic effects of combined therapy of curcumin and Fructus Ligustri Lucidi for treatment of osteoporosis: cellular and molecular evidence of enhanced bone formation. J Integr Med. 2019;17(1):38-45.
  • Chen B, Wang L, Li L, Zhu R, Liu H, Liu C, et al. Fructus Ligustri Lucidi in Osteoporosis: A Review of its Pharmacology, phytochemistry, pharmacokinetics and safety. Molecules. 2017;22(9):1469.
  • Chen HS, Hutter CM, Mechanic LE, Amos CI, Bafna V, Hauser ER, et al. Genetic simulation tools for post-genome wide association studies of complex diseases. Genet Epidemiol. 2015;39(1):11-19.
  • Gan D, Xu X, Chen D, Feng P, Xu Z. Network Pharmacology-Based Pharmacological Mechanism of the Chinese Medicine Rhizoma drynariae Against Osteoporosis. Med Sci Monit. 2019;(25):5700-5716.
  • Kastner M, Sawka AM, Hamid J, Chen M, Thorpe K, Chignell M, et al. A knowledge translation tool improved osteoporosis disease management in primary care: an interrupted time series analysis. Implement SCI. 2014;(9):109.
  • Lei MM. LZGW Pathogenesis of secondary osteoporosis. Chin J Osteopor. 2018;24(11):1514-1520.
  • Liu R, Kang X, Xu L, Nian H, Yang X, Shi H, et al. Effect of the combined extracts of herba epimedii and fructus ligustri lucidi on sex hormone functional levels in osteoporosis rats. Evid-based Complementary Altern. 2015;2015:1-13.
  • Liu RH, Kang X, Xu LP, Nian HL, Yang XW, Shi HT, et al. Effects of the combined extracts of Herba Epimedii and Fructus Ligustri Lucidi on bone mineral content and bone turnover in osteoporotic rats. BMC Complement Altern Med. 2015;15(1):1-18.
  • Ngo QT, Lee HS, Nguyen VT, Kim JA, Woo MH, Min BS. Chemical constituents from the fruits of Ligustrum japonicum and their inhibitory effects on T cell activation. Phytochemistry. 2017;(141):147-155.
  • Ouni E, Vertommen D, Amorim CA. The human ovary and future of fertility assessment in the Post-Genome Era. Int J Mol Sci. 2019;17(20).
  • Song L, Xie XB, Peng LK, Yu SJ, Peng YT. Mechanism and Treatment Strategy of Osteoporosis after Transplantation. INT J Endocrinol. 2015;(2015):280164.

Publication Dates

  • Publication in this collection
    13 July 2022
  • Date of issue
    2022

History

  • Received
    02 Dec 2019
  • Accepted
    16 July 2020
Universidade de São Paulo, Faculdade de Ciências Farmacêuticas Av. Prof. Lineu Prestes, n. 580, 05508-000 S. Paulo/SP Brasil, Tel.: (55 11) 3091-3824 - São Paulo - SP - Brazil
E-mail: bjps@usp.br