Zanthoxylum bungeanum Maxim (ZBM) is a traditional Chinese seasoning, and is often used as a Chinese herbal medicine in treating osteoarthritis (OA) and other diseases, but its active ingredients and pharmacological mechanisms are still unclear. We used network pharmacology, molecular docking, and molecular dynamics simulation (MDS) to explore the potential mechanisms of ZBM for the treatment of OA. A total of three potential active ingredients of ZBM, including quercetin, diosmetin, and beta-sitosterol, were screened from the TCMSP. The active ingredient targets were intersected with the OA-related targets obtained from GeneCards, OMIM, PharmGkb, and TTD databases, 43 common ZBM-OA targets were obtained. Hub genes (HIF1A, EGFR, CASP3, IL6, FOS and VEGFA) were obtained in the key target PPI network. GO and KEGG enrichment analysis showed that ZBM is involved in oxidative stress, inflammation, and apoptosis of chondrocytes mainly through regulating AGE-RAGE signaling pathway in diabetic complications, and thus plays a role in the treatment of OA; Molecular docking results showed that the key active compounds in ZBM could bind tightly to key target proteins; the MDS results showed that the active ingredient diosmetin could bind stably to EGFR. This study reveals the potential active ingredients and molecular mechanisms of ZBM for the treatment of OA.

Xin Liu, Ruolin Hou, Shuiqi Cai, Chengyi Liu, Dachong Sha, Qun Huang. Exploring the Mechanism of Zanthoxylum Bungeanum Maxim in the Treatment of Osteoarthritis Based on Network Pharmacology, Molecular Docking and Molecular Dynamics Simulation. Frontiers in Medical Science Research (2022) Vol. 4, Issue 12: 46-60. https://doi.org/10.25236/FMSR.2022.041208.

[1] Y. Chen, J. Zhao, S. Lao, et al., Association between bisphosphonate use and risk of undergoing knee replacement in patients with osteoarthritis, Annals of the rheumatic diseases. 78(2) (2019) e13-e13. https: //doi.org/10.1136/annrheumdis-2018-212998.

[2] J. Wei, High prevalence and burden of osteoarthritis in China, The Lancet Rheumatology. 2(3) (2020) e127-e128. https: //doi.org/https://doi.org/10.1016/S2665-9913(20)30034-5.

[3] W. Yue, L. Wei, S. Quanquan, et al., Research progress on the relationship between osteoarthritis and life-related genes, Chinese Jouranl of Endemiology. 37(11) (2018) 924-928. https: //doi.org/10. 3760/cma.j.issn.2095-4255.2018.11.017.

[4] M. Wang, L. Liu, C.S. Zhang, et al., Mechanism of Traditional Chinese Medicine in Treating Knee Osteoarthritis, Journal of pain research. 13 (2020) 1421-1429. https: //doi.org/10.2147/jpr.s247827.

[5] G. Huanhuan, X. Zhipeng, L. Taixian, et al., Analysis of Formulation Regularity of Chinese Patent Medicine for Knee Osteoarthritis, China Pharmacy. 30(15) (2019) 2096-2100. https: //doi.org/10. 6039/j.issn.1001-0408.2019.15.15.

[6] L. xianxiang, Research of Osteoarthritis Based on the Academic Thought of Chen Keji, Rehabilitation Medicine. 26(1) (2016) 2-5. https: //doi.org/10.3724/SPJ.1329.2016.01.002.

[7] Z. Mengjiao, S. Fangfang, C. Kai, et al., Research Progress of the Medicinal Value of Zanthoxylum bungeanum Maxim, Farm Products Processing. (1) (2020) 65-67,72. https: //doi.org/10. 16693/j. cnki.1671-9646(X).2020.01.018.

[8] M. Zhang, J. Wang, L. Zhu, et al., Zanthoxylum bungeanum Maxim. (Rutaceae): A Systematic Review of Its Traditional Uses, Botany, Phytochemistry, Pharmacology, Pharmacokinetics, and Toxicology, International journal of molecular sciences. 18(10) (2017). https: //doi.org/10.3390/ijms18102172.

[9] Y. Ma, Y. Wang, X. Li, et al., Sensory Characteristics and Antioxidant Activity of Zanthoxylum bungeanum Maxim. Pericarps, Chemistry & biodiversity. 16(2) (2019) e1800238. https: //doi.org/10. 1002/cbdv.201800238.

[10] L. Yu, W. Wu, Y. Pan, et al., Quality evaluation of different varieties of Zanthoxylum bungeanum Maxim. peels based on phenolic profiles, bioactivity, and HPLC fingerprint, Journal of food science. 85(4) (2020) 1090-1097. https: //doi.org/10.1111/1750-3841.15095.

[11] W. yinyin, Z. hong, Z. chunyan, et al., Based on data mining technology to analyze the prescription and medication rules of "Chinese Medical Dictionary" in the treatment of knee osteoarthritis, Modern Journal of Integrated Traditional Chinese and Western Medicine. 30(4) (2021) 394-397. https: //doi.org/10.3969/j.issn.1008-8849.2021.04.011.

[12] C.S. Zheng, X.J. Xu, H.Z. Ye, et al., Network pharmacology-based prediction of the multi-target capabilities of the compounds in Taohong Siwu decoction, and their application in osteoarthritis, Experimental and therapeutic medicine. 6(1) (2013) 125-132. https: //doi.org/10.3892/etm.2013.1106.

[13] J.L. Tao, S.C. Wang, Y.Z. Chen, et al., Review on the research in network pharmacology of traditional Chinese medicine compound, China Journal of Traditional Chinese Medicine and Pharmacy. 34(9) (2019) 3903-3907. https: //doi.org/CNKI:SUN:BXYY.0.2019-09-001.

[14] Y. Long, Z. Li, C. Huang, et al., Mechanism and Protective Effect of Smilax glabra Roxb on the Treatment of Heart Failure via Network Pharmacology Analysis and Vitro Verification, Frontiers in pharmacology. 13 (2022) 868680. https: //doi.org/10.3389/fphar.2022.868680.

[15] Y. Diao, Q. Ding, G. Xu, et al., Qingfei Litan Decoction Against Acute Lung Injury/Acute Respiratory Distress Syndrome: The Potential Roles of Anti-Inflammatory and Anti-Oxidative Effects, Frontiers in pharmacology. 13 (2022) 857502. https: //doi.org/10.3389/fphar.2022.857502.

[16] H. Shao, Q. Gan, Z. Chen, et al., Analysis of the Underlying Mechanism of the Jiu Wei Zhen Xin Formula for Treating Generalized Anxiety Disorder Based on Network Pharmacology of Traditional Chinese Medicine, Evidence-based complementary and alternative medicine : eCAM. 2022 (2022) 7761852. https: //doi.org/10.1155/2022/7761852.

[17] Z. Xia, L. Lv, X. Di, et al., The compatibility of six alkaloids in ermiao pill explored by a comparative pharmacokinetic and network pharmacological study, Biomedical chromatography : BMC. 33(5) (2019) e4509. https: //doi.org/10.1002/bmc.4509.

[18] X. Song, Y. Zhang, E. Dai, et al., Prediction of triptolide targets in rheumatoid arthritis using network pharmacology and molecular docking, International immunopharmacology. 80 (2020) 106179. https: //doi.org/10.1016/j.intimp.2019.106179.

[19] D.B. Kitchen, H. Decornez, J.R. Furr, et al., Docking and scoring in virtual screening for drug discovery: methods and applications, Nature reviews. Drug discovery. 3(11) (2004) 935-49. https: //doi.org/10.1038/nrd1549.

[20] K.G. Daniels, Y. Suo, T.G. Oas, Conformational kinetics reveals affinities of protein conformational states, Proceedings of the National Academy of Sciences of the United States of America. 112(30) (2015) 9352-7. https: //doi.org/10.1073/pnas.1502084112.

[21] A.M. Ruvinsky, T. Kirys, A.V. Tuzikov, et al., Structure fluctuations and conformational changes in protein binding, Journal of bioinformatics and computational biology. 10(2) (2012) 1241002. https: //doi.org/10.1142/s0219720012410028.

[22] V. Salmaso, S. Moro, Bridging Molecular Docking to Molecular Dynamics in Exploring Ligand-Protein Recognition Process: An Overview, Frontiers in pharmacology. 9 (2018) 923. https: //doi.org/10.3389/fphar.2018.00923.

[23] M. Karplus, J.A. McCammon, Molecular dynamics simulations of biomolecules, Nature structural biology. 9(9) (2002) 646-52. https: //doi.org/10.1038/nsb0902-646.

[24] S.K. Kim, S. Lee, M.K. Lee, et al., A systems pharmacology approach to investigate the mechanism of Oryeong-san formula for the treatment of hypertension, Journal of ethnopharmacology. 244 (2019) 112129. https: //doi.org/10.1016/j.jep.2019.112129.

[25] Q. Jin, J. Lu, R. Gao, et al., Systematically Deciphering the Pharmacological Mechanism of Fructus Aurantii via Network Pharmacology, Evidence-based complementary and alternative medicine : eCAM. 2021 (2021) 6236135. https: //doi.org/10.1155/2021/6236135.

[26] A. Basu, J. Schell, R.H. Scofield, Dietary fruits and arthritis, Food & function. 9(1) (2018) 70-77. https: //doi.org/10.1039/c7fo01435j.

[27] M. Mohamed, N. Zein, F. Yassin, Protective Study on Quercetin Extracted from Broccoli Via Blocking Hepatic Stellate Cells Activation and in CCL4 Fibrotic Mice, Bulletin of Faculty of Science Zagazig University.  (2021). https: //doi.org/10.21608/bfszu.2021.57487.1057.

[28] M. na, L. yajing, F. jiping, Research Progress on Pharmacological Action of Diosmetin, Journal of Liaoning University of Traditional Chinese Medicine. (9) (2018) 214-217. https: //doi.org/10.13194/j.issn.1673-842x.2018.09.062.

[29] G. Mo, Y. He, X. Zhang, et al., Diosmetin exerts cardioprotective effect on myocardial ischaemia injury in neonatal rats by decreasing oxidative stress and myocardial apoptosis, Clinical and experimental pharmacology & physiology. 47(10) (2020) 1713-1722. https: //doi.org/10.1111/1440-1681.13309.

[30] S. Lv, X. Wang, S. Jin, et al., Quercetin mediates TSC2-RHEB-mTOR pathway to regulate chondrocytes autophagy in knee osteoarthritis, Gene. 820 (2022) 146209. https: //doi.org/10.1016/j.gene.2022.146209.

[31] M. Heydari Nasrabadi, M. Parsivand, N. Mohammadi, et al., Comparison of Elaeagnus angustifolia L. extract and quercetin on mouse model of knee osteoarthritis, Journal of Ayurveda and integrative medicine. 13(2) (2021) 100529. https: //doi.org/10.1016/j.jaim.2021.10.001.

[32] W. Li, Y. Wang, Y. Tang, et al., Quercetin Alleviates Osteoarthritis Progression in Rats by Suppressing Inflammation and Apoptosis via Inhibition of IRAK1/NLRP3 Signaling, Journal of inflammation research. 14 (2021) 3393-3403. https: //doi.org/10.2147/jir.s311924.

[33] L. Hong, S. Jun, M. Jinghong, et al., Diosmetin relieves apoptosis and immune response in IL-1β-induced osteoarthritic chondrocytes of neonatal rat via NF-κB pathway, Chinese Journal of Immunology. 35(3) (2019) 292-297. https: //doi.org/10.3969/j.issn.1000-484X.2019.03.008.

[34] D. Hongzhi, M. Pei, X. Zhixing, et al., Feasibility study of diosmetin in the treatment of early osteoarthritis by inhibiting osteoclast differentiation, Practical Pharmacy and Clinical Remedies. 22(10) (2019) 7. https: //doi.org/10.14053/j.cnki.ppcr.201910004.

[35] P.C. Liao, M.H. Lai, K.P. Hsu, et al., Identification of β-Sitosterol as in Vitro Anti-Inflammatory Constituent in Moringa oleifera, Journal of agricultural and food chemistry. 66(41) (2018) 10748-10759. https: //doi.org/10.1021/acs.jafc.8b04555.

[36] Z. lipin, X. xianzhu, R. hua, et al., Regulation of β-sitosterol in Duzhongye on Homeostasis of Bone Metabolism, Lishizhen Medicine and Materia Medica Research. 23(5) (2012) 1051-1053. https: //doi.org/10.3969/j.issn.1008-0805.2012.05.001.

[37] G.L. Semenza, Regulation of mammalian O2 homeostasis by hypoxia-inducible factor 1, Annual review of cell and developmental biology. 15 (1999) 551-78. https: //doi.org/10. 1146/annurev. cellbio.15.1.551.

[38] R.H. Wenger, Mammalian oxygen sensing, signalling and gene regulation, The Journal of experimental biology. 203(Pt 8) (2000) 1253-63. https: //doi.org/10.1242/jeb.203.8.1253.

[39] T. Cramer, Y. Yamanishi, B.E. Clausen, et al., HIF-1alpha is essential for myeloid cell-mediated inflammation, Cell. 112(5) (2003) 645-57. https: //doi.org/10.1016/s0092-8674(03)00154-5.

[40] S. Hu, C. Zhang, L. Ni, et al., Stabilization of HIF-1α alleviates osteoarthritis via enhancing mitophagy, Cell death & disease. 11(6) (2020) 481. https: //doi.org/10.1038/s41419-020-2680-0.

[41] X. Li, W. Mei, Z. Huang, et al., Casticin suppresses monoiodoacetic acid-induced knee osteoarthritis through inhibiting HIF-1α/NLRP3 inflammasome signaling, International immunopharmacology. 86 (2020) 106745. https: //doi.org/10.1016/j.intimp.2020.106745.

[42] F. Yang, R. Huang, H. Ma, et al., miRNA-411 Regulates Chondrocyte Autophagy in Osteoarthritis by Targeting Hypoxia-Inducible Factor 1 alpha (HIF-1α), Medical science monitor : international medical journal of experimental and clinical research. 26 (2020) e921155. https: //doi.org/10. 12659/msm.921155.

[43] M. Zhou, D. Wang, J. Tang, Identification of the Resveratrol Potential Targets in the Treatment of Osteoarthritis, Evidence-based complementary and alternative medicine : eCAM. 2021 (2021) 9911286. https: //doi.org/10.1155/2021/9911286.

[44] L. Qin, F. Beier, EGFR Signaling: Friend or Foe for Cartilage?, JBMR plus. 3(2) (2019) e10177. https: //doi.org/10.1002/jbm4.10177.

[45] Y.W. Zhang, Y. Su, N. Lanning, et al., Targeted disruption of Mig-6 in the mouse genome leads to early onset degenerative joint disease, Proceedings of the National Academy of Sciences of the United States of America. 102(33) (2005) 11740-5. https: //doi.org/10.1073/pnas.0505171102.

[46] B. Staal, B.O. Williams, F. Beier, et al., Cartilage-specific deletion of Mig-6 results in osteoarthritis-like disorder with excessive articular chondrocyte proliferation, Proceedings of the National Academy of Sciences of the United States of America. 111(7) (2014) 2590-5. https: //doi.org/ 10.1073/ pnas.1400744111.

[47] Q. Lu, P. Liu, Z. Miao, et al., SIRT1 restoration enhances chondrocyte autophagy in osteoarthritis through PTEN-mediated EGFR ubiquitination, Cell death discovery. 8(1) (2022) 203. https: //doi.org/10.1038/s41420-022-00896-8.

[48] Y. Wei, X. Ma, H. Sun, et al., EGFR Signaling Is Required for Maintaining Adult Cartilage Homeostasis and Attenuating Osteoarthritis Progression, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research. 37(5) (2022) 1012-1023. https: //doi.org/10.1002/jbmr.4531.

[49] B.W. Wang, Y. Jiang, Z.L. Yao, et al., Aucubin Protects Chondrocytes Against IL-1β-Induced Apoptosis In Vitro And Inhibits Osteoarthritis In Mice Model, Drug design, development and therapy. 13 (2019) 3529-3538. https: //doi.org/10.2147/dddt.s210220.

[50] T.A. Zharova, E.A. Kogan, V.I. Makarov, et al., Correlation of synovial caspase-3 concentration and the photodynamic effectiveness in osteoarthritis treatment, Photodiagnosis and photodynamic therapy. 30 (2020) 101669. https: //doi.org/10.1016/j.pdpdt.2020.101669.

[51] Y. Lu, C. Zhang, S. Jiang, et al., Anti-Dlx5 Retards the Progression of Osteoarthritis through Inhibiting Chondrocyte Hypertrophy and Apoptosis, Evidence-based complementary and alternative medicine : eCAM. 2022 (2022) 5019920. https: //doi.org/10.1155/2022/5019920.

[52] A.M. Yassin, H.O. AbuBakr, A.I. Abdelgalil, et al., COL2A1 and Caspase-3 as Promising Biomarkers for Osteoarthritis Prognosis in an Equus asinus Model, Biomolecules. 10(3) (2020). https: //doi.org/10.3390/biom10030354.

[53] T. Tanaka, M. Narazaki, T. Kishimoto, IL-6 in inflammation, immunity, and disease, Cold Spring Harbor perspectives in biology. 6(10) (2014) a016295. https: //doi.org/10.1101/cshperspect.a016295.

[54] M. Laavola, T. Leppänen, M. Hämäläinen, et al., IL-6 in Osteoarthritis: Effects of Pine Stilbenoids, Molecules (Basel, Switzerland). 24(1) (2018). https: //doi.org/10.3390/molecules24010109.

[55] S. Glyn-Jones, A.J. Palmer, R. Agricola, et al., Osteoarthritis, Lancet (London, England). 386(9991) (2015) 376-87. https: //doi.org/10.1016/s0140-6736(14)60802-3.

[56] H. Park, H.R. Lee, H.J. Shin, et al., p16INK4a-siRNA nanoparticles attenuate cartilage degeneration in osteoarthritis by inhibiting inflammation in fibroblast-like synoviocytes, Biomaterials science. 10(12) (2022) 3223-3235. https: //doi.org/10.1039/d1bm01941d.

[57] L.X. Deng, J.H. Lin, Expression of c-fos gene associated with the proliferation and differentiation of cartilage, Chinese Journal of Clinical Rehabilitation. 10(1) (2006) 138-141. https: //doi.org/ 10.3321/j.issn:1673-8225.2006.01.058.

[58] H. Motomura, S. Seki, S. Shiozawa, et al., A selective c-Fos/AP-1 inhibitor prevents cartilage destruction and subsequent osteophyte formation, Biochemical and biophysical research communications. 497(2) (2018) 756-761. https: //doi.org/10.1016/j.bbrc.2018.02.147.

[59] A. Haseeb, D. Leigh, T.M. Haqqi, A small molecule harpagoside inhibits IL-1beta-induced expression of IL-6 by blocking the expression of C-FOS in primary human osteoarthritis chondrocytes, Osteoarthritis and Cartilage. 23 (2015) A155-A156. https: //doi.org/https://doi.org/ 10.1016/j. joca.2015.02.910.

[60] L.A. Fatima, R.S. Campello, R.S. Santos, et al., Estrogen receptor 1 (ESR1) regulates VEGFA in adipose tissue, Scientific reports. 7(1) (2017) 16716. https: //doi.org/10.1038/s41598-017-16686-7.

[61] M. Guan, Y. Zhu, B. Liao, et al., Low-intensity pulsed ultrasound inhibits VEGFA expression in chondrocytes and protects against cartilage degeneration in experimental osteoarthritis, FEBS open bio. 10(3) (2020) 434-443. https: //doi.org/10.1002/2211-5463.12801.

[62] Y. Bai, X. Gong, R. Dong, et al., Long non-coding RNA HCAR promotes endochondral bone repair by upregulating VEGF and MMP13 in hypertrophic chondrocyte through sponging miR-15b-5p, Genes & diseases. 9(2) (2022) 456-465. https: //doi.org/10.1016/j.gendis.2020.07.013.

[63] Y. Yamamoto, S. Yamagishi, H. Yonekura, et al., Roles of the AGE-RAGE system in vascular injury in diabetes, Annals of the New York Academy of Sciences. 902 (2000) 163-70; discussion 170-2. https: //doi.org/10.1111/j.1749-6632.2000.tb06311.x.

[64] W. Xinyuan, H. Xiabing, L. Juan, et al., Network pharmacology and molecular docking analysis on Taohong Siwu Decoction for rheumatoid arthritis and osteoarthritis based on the concept of "Treating Different Diseases with the Same Therapeutic Principle", Journal of Clinical Rehabilitative Tissue Engineering Research. 26(15) (2022) 2419-2425. https: //doi.org/10.12307/2022.601.

[65] Z. Qide, L. Haiqing, Study on the effect of Yiqi Shengjin Huoxue Granules on the content of IL-1,ET,NF-κB, TNF-α,and VEGF in myocardium of diabetic rats, Journal of Pharmaceutical Research. (7) (2018) 381-383. https: //doi.org/10.13506/j.cnki.jpr.2018.07.003.

[66] I. Kim, S.O. Moon, S.H. Kim, et al., Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells, The Journal of biological chemistry. 276(10) (2001) 7614-20. https: //doi.org/10.1074/jbc.M009705200.

[67] F. Zhang, X. Huang, Y. Qi, et al., Juglanin Inhibits Osteoclastogenesis in Ovariectomized Mice via the Suppression of NF-κB Signaling Pathways, Frontiers in pharmacology. 11 (2020) 596230. https: //doi.org/10.3389/fphar.2020.596230.