Network Pharmacology-Based Analysis on the Action Mechanism of Oleanolic Acid to Alleviate Osteoporosis
Authors: Qin Li
Li-Jie Gao
Ying-Sai Fan
Ye Chen
Yi Wu
Journal: ACS Omega (2021)
Discipline: CHEMISTRY
Keywords: Oleanolic acid; antiosteoporosis; network pharmacology; molecular docking; targets
Affiliations: College of Life Sciences and Food Engineering, Hebei University of Engineering, 056038 Handan, China, College of Animal Science and Technology, Hebei Agricultural University, 071000 Baoding, China, College of Traditional Chinese Veterinary Medicine, Hebei Agricultural University, 071000 Baoding, China

Abstract:  Oleanolic acid (OA) is a triterpenoid commonly found in plants and has shown extensive pharmaceutical activities. This study aimed to investigate the underlying mechanism of antiosteoporosis (OP) action of OA by utilizing the network pharmacology approach and molecular docking methods. First, the targets of OA were identified using the GeneCards, Stitch, and Swisstarget databases, and the targets related to OP were mined using the NCBI, Genecards, and DisGeNet databases. The overlapped targets of OA and OP were regarded as candidate targets, and the String database was used to obtain the protein-protein interactions among the targets. Then, Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway enrichment pathways of the candidate targets were performed using the DAVID database. In addition, the top 16 targets in the protein interaction network were used for molecular docking. Finally, an animal model constructed using d-galactose-induced oxidative stress and a low-calcium diet with accelerated bone loss was used to verify the in vivo effects of OA on osteoporotic mice. A total of 42 candidate targets for OA to treat OP were obtained. According to the protein-protein interaction network, MAPK1 showed the highest connectivity with other proteins. Additionally, GO analysis identified the top 20 biological processes, 9 cellular components, and top 20 molecular functions. Moreover, the candidate targets were mainly involved in 13 signaling pathways such as TNF signaling pathway, insulin resistance, MAPK signaling pathway, apoptosis, and PI3K-Akt signaling pathways. Furthermore, molecular docking revealed that OA has a high degree of connections with 16 key proteins. In addition, the anti-OP effects of OA are further validated through the in vivo model. Altogether, our study elucidated the candidate targets for OA to alleviate OP, explored the protein-protein interactions and related signaling pathways of the targets, and validated the anti-OP effects of OA. It could provide a better understanding of the action mechanism in OA to treat OP. 

Retracted: No

PubPeer comments:
 The following 22 papers are a subset of [a larger group of 30-odd papers] , all from the Traditional Chinese Medicine genre, and all citing the work of C. Zhao and colleagues, under the misapprehension that this work is about extracting active phytochemical ingredients from herbs. What singles out these 22 entries is the presence of identical passages, to contain these irrelevant citations. This is consistent with some kind of citation manipulation. It would be helpful if the authors of any of these papers could explain whether the references and repeated passages were their own idea, and if not, who suggested them. The irrelevance is particularly obvious in the case of Ma et al (#8 in the list), which purports to apply herbal-ingredient extraction methods to a mineral substance, Realgar. -------------------------- The relevant canon of C. Zhao papers is - A. Hassan, M., Umar, M., Ding, W., Mehryar, E., Zhao, C. (2017). Methane enhancement through co-digestion of chicken manure and oxidative cleaved wheat straw: Stability performance and kinetic modeling perspectives. Energy, 141, 2314u20132320. - B. Qiao, X. L., Zhao, C., Shao, Q. J., Hassan, M., Energy Fuel 2018, 32, 6022-6030.  Structural characterization of corn stover lignin after hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment - C. Zhang, M. Q., Zhao, C., Shao, Q. J., Yang, Z. D., Int. J. Agr. Biol. Eng. 2019, 12, 143-148.  Determination of water content in corn stover silage using near-infrared spectroscopy - D. Zhao, C., Shao, Q. J., Ma, Z. Q., Li, B., Zhao, X. J., Ind. Crop. Prod. 2016, 83, 86-93.  Physical and chemical characterizations of corn stalk resulting from hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment - E. Zhao, C., Cao, Y., Ma, Z. Q., Shao, Q. J., Biomass Bioenergy 2017, 98, 61-69.  Optimization of liquid ammonia pretreatment conditions for maximizing sugar release from giant reed (*Arundo donax* L.). - F. C. Zhao, X.L. Qiao, Y. Cao, Q.J. Shao, Methane enhancement through co-digestion of chicken manure and oxidative cleaved wheat straw: stability performance and kinetic modeling perspectives, Energy 141 (2017) 2314u20132320. - G. Zhao, C., Qiao, X. L., Cao, Y., Shao, Q. L., Fuel 2017, 205, 184-191.  Application of hydrogen peroxide presoaking prior to ammonia fiber expansion pretreatment of energy crops - H. Zhao, C., Qiao, X., Shao, Q., Hassan, M., Ma, Z., 2020a, Evolution of the lignin chemical structure during the bioethanol production process and its inhibition to enzymatic hydrolysis, Energ. Fuel., 2020, 34, 5938-5947. - I. Zhao, C., Qiao, X. L., Shao, Q. J., Hassan, M., Ma, Z. Q., Yao, L. J., Ind. Crop. Prod. 2020, 146, 112177.  Synergistic effect of hydrogen peroxide and ammonia on lignin ----------------------------------------------- 1. [Wang et al (2020)] .  Simultaneous determination of 20 bioactive components in Chuanxiong Rhizoma from different production origins in Sichuan province by ultrau2010highu2010performance liquid chromatography coupled with triple quadrupole mass spectrometry combined with multivariate statistical analysis . -  TCM is usually prepared by boiling herbs in water, and it is impossible to obtain all the chemical components of herbs. ... Meanwhile, The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide [36, 37] and pre-soaking [38-40] may bring unexpected insights into the material basis of TCM. -  In addition, Chuanxiong Rhizoma contains water, and the determination of water content may be performed before extraction [41], which contribute to accurate determination of active components in future research. 2. [Li et al (2020)] .  Metabonomics approach reveals the vital role of Huangqi in Huangqi Jianzhong tang against chronic atrophic gastritis coupled with molecular docking and BAWP . -  Additionally, TCM was usually prepared by reflowing the herbs in water, which could not obtain their whole chemical constitutes. The emerging potential approaches to pre-treat and extract the active compounds with presoaking [32], CO2 extraction [33], ammonia and hydrogen peroxide, etc [34u201336]. might introduce unexpected insights into its chemicalome and material basis research for TCM, which depicted the compound compatibility environment of single herb in a whole TCM formula. 3. [Wang et al (2021)] .  Geographical traceability of Eucommia ulmoides leaves using attenuated total reflection Fourier transform infrared and ultraviolet-visible spectroscopy combined with chemometrics and data fusion . -  The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide (Zhao et al., 2020a; Zhao et al., 2017a; Zhao et al., 2020b) and pre-soaking (Zhao et al., 2016; Zhao et al., 2017b) may bring unexpected insights into the material basis of *E. ulmoides*. -  The EULs may contain water, and the determination of water content may be performed before experiment (Zhang et al., 2019), which contribute to accurate determination of active components in future research. 4. [Liu et al (2020)] .  UHPLC Q-Exactive MS-based spleen metabolomics and lipidomics to explore the effect mechanisms of Danggui Buxue Decoction in anemia mice . -  However, the sample pretreatment also might be potential hot spots for increasing the metabolic coverage of the tissue samples, which might better reveal the pathological location and specific metabolic mechanism of DBD. The emerging potential approaches to pre-treat and extract the active compounds with Solid Phase Extraction (SPE) [26], supercritical fluid extraction [27], ammonia and hydrogen peroxide, etc [28]., etc. from tissue sample might introduce unexpected insights into its metabolic mechanism of TCM [29]. 5. [Liu, Ma Cai (2020)] .  Fecal metabonomics study of raw and bran-fried Atractylodis Rhizoma in spleen-deficiency rats . -  In this study, because of the uncertainty of material basis, we used the powder solution of traditional Chinese medicine. It has been reported that the emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide [24-26] and pre-soaking [27-29] may bring unexpected insights into the material basis of TCM. Novel extraction method may be helpful for our future material basis research. -  It also should be noted that AR contains water, and the determination of water content may be performed before extraction [30], which contribute to accurate determination of active components in future research. 6. [Zhao et al (2021)] .  Tissue-specific metabolite profiling of Fallopia multiflora (Heshouwu) and Fallopia multiflora var. angulata by mass spectrometry imaging and laser microdissection combined with UPLC-Q TOF-MS . -  However,the secondary metabolites identified from *F. multiflora* in this study are not as abundant as previously reported [30], which may be related to samples volume and or the extraction method. The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide and presoaking [34] may bring unexpected insights into the material basis of *F. multiflora* and *F. multiflora var. angulata*. 7. [Cheng et al (2021)] .  Involvement of mTOR Survivin signaling pathway in TUA(2u03b2, 3u03b2, 23-trihydroxy-urs-12-ene-28-olic acid)-induced apoptosis in human gastric cancer cell line BGC823 cells . -  In the study, the *A. fulvicoma* Hance extracts were extracted by 95 % ethanol, and it may not be the most effective method for extracting TUA from the herb. The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide (Zhao,et al.,2020; Zhao, et al.,2017; Zhao, et al.,2020)and pre-soaking (Zhao, et al.,2017)may bring unexpected insights into the material basis of the herb. -  The herbal material of *A. fulvicoma* Hance contained water, and the determination of water content may be performed before extraction (Zhang et al.,2019), which contribute to accurate analysis of TUA in future research. 8. [Ma et al (2021)] .  Realgar increases defenses against infection by Enterococcus faecalis in Caenorhabditis elegans . -  In present work, the realgar is prepared by 0.1 M NaOH, and it may not be the most effective method for extracting active components from the realgar. The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide (Zhao et al., 2017a; 2020a, b) and pre-soaking (Zhao et al., 2017b) may bring unexpected insights into the effects of realgar on immunity responses. 9. [Fu et al (2021)] .  Deciphering the metabolic profile and pharmacological mechanisms of Achyranthes bidentata blume saponins using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry coupled with network pharmacology-based investigation . -  Meanwhile, the development of instrumentations and postacquisition data mining platforms (Wu et al., 2015, 2020; Yu et al., 2016) as well as the emerging potential methods of pretreatment and extraction of active components (Zhao et al., 2020) may bring unexpected insights into the material basis of herbal medicine. 10. [Liu et al (2020)] .  Urinary metabolomics research for Huangqi Jianzhong Tang against chronic atrophic gastritis rats based on H NMR and UPLC-Q TOF MS -  In addition, HQJZ was prepared by reflowing the herbs in water with a TCM classical method. The emerging potential approaches to pretreat and extract the active compounds with CO2 extraction, ammonia and hydrogen peroxide, etc. from herbs might introduce unexpected insights into its chemical constituents and material basis research in future.[40,41] 11. [Guo et al (2020)] .  Chemical Comparison of White Ginseng before and after Extrusion by UHPLC-Q-Orbitrap-MS MS and Multivariate Statistical Analysis . -  Meanwhile, the sample of ginseng contains water, and the determination of water content may be performed before extraction [35, 36], which contribute to accurate determination of polysaccharide in future research. 12. [Li et al (2020)] .  Quantification Analysis of 13 Organic Components and 8 Inorganic Elements in Angelica Sinensis Radix and Its Different Parts Combined with Chemical Recognition Pattern . -  TCM is usually prepared by boiling herbs in water, and it is impossible to obtain all the chemical components of herbs. The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide [25u201327] and presoaking [28u201330] may bring unexpected insights into the material basis of TCM -  In addition, TCM contains water, and the determination of water content may be performed before extraction [32], which contributes to accurate determination of active components in future research. 13. [Lia et al (2021)] .  Study on the Spectrum-Effect Relationship of the Traditional Effect of Saponins in Glycyrrhiza uralensis Fisch . -  At first, the enriched GS is usually extracted and prepared in ethanol by sonication, and it may not be the most effective method for extracting active components from the *Glycyrrhiza uralensis*. The emerging potential methods of pretreatment and extraction of active components [30] may bring unexpected insights into the material basis of *Glycyrrhiza uralensis*. 14. [Ma et al (2021)] .  Study on the Bioassay of Anti-Inflammatory Effects of Fuke Qianjin Capsule Based on COX-2 Inhibiting Activity . -  In addition, FKQJ is usually prepared by boiling the eight kinds of TCM in water, and it may not be the most effective method for extracting active components from the TCM. The emerging potential methods of pretreatment and extraction of active components with hydrogen peroxide presoaking may bring unexpected insights into the material basis of FKQJ [21]. 15. [Liu et el (2021)] .  Assessing the Quality of Calyx of Physalis alkekengi L. var. franchetii Based on Quantitative Analysis of Q-Marker Combined with Chemometrics and Machine Learning Algorithms . -  Furthermore, the emerging potential methods of pretreatment and extraction of active components [34u201336] may bring unexpected insights into the material basis of PALF. -  Determination of water content in the experiment material PALF was carried out using some methods before the test [24u201326], which will help the test results to be more accurate 16. [Wu et al (2021)] . ** Network Pharmacology-Based Analysis on the Action Mechanism of Oleanolic Acid to Alleviate Osteoporosis **. -  The emerging potential methods of pretreatment and extraction of active components may provide guarantee for the sustainable supply of OA in a large scale. (23,24) -  Future studies may explore whether a certain method to measure the water content in OA prior to the experiment would contribute to more accurate results. (50) 17. [Li et al (2022)] .  Mulberry leaves ameliorate diabetes via regulating metabolic profiling and AGEs RAGE and p38 MAPK NF-u03baB pathway . -  The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide and pre-soaking may bring unexpected insights into the... 18. [Wang et al (2022)] .  Shengmai Yin formula exerts cardioprotective effects on rats with chronic heart failure via regulating Linoleic Acid metabolism -  Emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide and pre-soaking [25,26] may bring unexpected insights into the u2026 19. [Chen et al (2021)] .  Application of UV-Vis and Infrared Spectroscopy on Wild Edible Bolete Mushrooms Discrimination and Evaluation: A Review . -  The emerging potential methods of pretreatment and extraction of active components with ammonia and hydrogen peroxide and presoaking may bring unexpected insights into the u2026 20. [Zhao et al (2021)] .  Monascus ruber fermented Panax ginseng ameliorates lipid metabolism disorders and modulate gut microbiota in rats fed a high-fat diet . -  The emerging potential new methods, such as ammonia pretreatment, enzymatic hydrolysis and so on (Zhao et al., 2020), may bring unexpected insights into the material basis of ginseng. 21. [Hu et al (2021)] .  Determination and analysis of monosaccharides in Polygonatum cyrtonema Hua polysaccharides from different areas by ultrau2010highu2010performance liquid chromatography quadrupole trap tandem mass spectrometry . -  The emerging potential methods of pretreatment and extraction [42] may provide unexpected insights into the active material bases of herbal medicine. 22. Gong et al (2021)] .  Anti-angiogenesis Function of Ononin via Suppressing the MEK Erk Signaling Pathway . -  The emerging potential methods of pretreatment and extraction of ononin with ammonia and hydrogen peroxide and presoaking may bring unexpected insights into the material basis of the herbs.52 ,