Inhibition of Postmenopausal Osteoporosis in Ovariectomized Mice by Huo Xue Tong Luo Capsule Using Network Pharmacology-based Mechanism Prediction and Pharmacological Validation

Abstract

Background and objectives: Huo Xue Tong Luo Capsule (HXTL) has been clinically used to treat osteonecrosis of the femoral head, osteoporosis, and other bone and joint diseases with promising effects. Our previous study has shown that HXTL can promote osteogenesis in mesenchymal stem cells by inhibiting lncRNA-Miat expression through histone modifications. However, the mechanism by which HXTL treats postmenopausal osteoporosis (PMOP) remains unclear. In this study, we used network pharmacology-based mechanism prediction, molecular docking, and pharmacological validation to investigate the mechanism of HXTL in treating PMOP.

Methods: The key candidate targets and relevant signaling pathways of HXTL for PMOP treatment were predicted using network pharmacology and molecular docking analysis. RAW264.7 cells were used for Western blot to validate the predicted mechanistic pathways. The ovaries of mice were surgically removed to simulate PMOP. The effect of HXTL on PMOP was evaluated using tartrate-resistant acid phosphatase staining and immunohistochemical assays in vivo.

Results: Network pharmacology analysis suggested that HXTL interacted with 215 key targets linked to PMOP, primarily affecting the PI3K-AKT signaling pathway. Molecular docking showed that the main components of HXTL exhibited strong binding affinity to NFATc1, p-PI3K, and p-AKT1. Furthermore, our in vitro results confirmed that HXTL suppressed the PI3K-AKT signaling pathway. In vivo, HE and tartrate-resistant acid phosphatase staining results showed that HXTL inhibited osteoclast formation and protected bone mass.

Conclusions: This research demonstrated that HXTL could inhibit osteoclast formation and prevent bone loss induced by ovariectomy in mice by inhibiting the PI3K-AKT signaling pathway. These findings provide important evidence for the clinical application of HXTL in treating PMOP.

Keywords

Huo Xue Tong Luo Capsule / Post-menopausal osteoporosis / Network pharmacology / Molecular docking / PI3K-AKT signaling pathway / Osteoclastic differentiation

References

[1] Mu L, Gao J, Zhang Q, Kong F, Lian Y, Li X, et al. Mechanism of action of Sambucus williamsii Hance var. miquelii in the treatment of osteoporosis analyzed by UHPLC-HRMS/MS combined network pharmacology and experimental validation. Fitoterapia 2024;176:106003. doi:10.1016/j.fitote.2024.106003, PMID:38729247.

[2] Li D, Zhao Z, Zhu L, Feng H, Song J, Fu J, et al. 7,8-DHF inhibits BMSC oxidative stress via the TRKB/PI3K/AKT/NRF2 pathway to improve symptoms of postmenopausal osteoporosis. Free Radic Biol Med 2024; 223:413-429. doi:10.1016/j.freeradbiomed.2024.08.014, PMID:391 55025.

[3] Wong CH, Kan AKC, Tsoi KH, Chan SSY, Jiang NS, Loong CHN, et al. Clinical characteristics, densitometric parameters and outcomes of patients with atypical femoral fractures related to bisphosphonate treatment for osteoporosis. Endocrine 2024; 84(1):223-235. doi:10.1007/s12020-023-03608-z, PMID:37985574.

[4] Jiang T, Li C, Li Y, Hu W, Guo J, Du X, et al. Multi-omics and bioinformatics for the investigation of therapeutic mechanism of roucongrong pill against postmenopausal osteoporosis. J Ethnopharmacol 2025; 337(Pt 2):118873. doi:10.1016/j.jep.2024.118873, PMID:393 62330.

[5] Wang Y, Feng Y, Li M, Yang M, Shi G, Xuan Z, et al. Traditional Chinese Medicine in the Treatment of Chronic Kidney Diseases: Theories, Applications, and Mechanisms. Front Pharmacol 2022;13:917975. doi:10.3389/fphar.2022.917975, PMID:35924053.

[6] Xu W, Jiang T, Ding L, Jiang Y, Zhang L, Xia T, et al. Bajitianwan formula extract ameliorates bone loss induced by iron overload via activating RAGE/PI3K/AKT pathway based on network pharmacology and transcriptomic analysis. J Nat Med 2024; 78(3):488-504. doi:10.1007/s11418-024-01779-1, PMID:38530577.

[7] Sun J, Yang XG, Hu YC. Efficacy of Jintiange Capsules in the Treatment of Osteoporosis: A Network Meta-analysis. Orthop Surg 2019; 11(2):176-186. doi:10.1111/os.12439, PMID:30854796.

[8] Yang Z, Yuan ZZ, Ma XL. Network Pharmacology-Based Strategy and Molecular Docking to Explore the Potential Mechanism of Jintiange Capsule for Treating Osteoporosis. Evid Based Complement Alternat Med 2021; 2021:5338182. doi:10.1155/2021/5338182, PMID: 34899951.

[9] Cheng BR, Wu RY, Gao QY, Jiang KX, Li SS, Qi SH, et al. Chinese Proprietary Medicine Xianling Gubao Capsule for Osteoporosis: A Systematic Review and Meta-Analysis of Randomized Clinical Trials. Front Endocrinol (Lausanne) 2022;13:870277. doi:10.3389/fendo.2022.870277, PMID:35464071.

[10] Luo MH, Zhao JL, Xu NJ, Xiao X, Feng WX, Li ZP, et al. Comparative Efficacy of Xianling Gubao Capsules in Improving Bone Mineral Density in Postmenopausal Osteoporosis: A Network Meta-Analysis. Front Endocrinol (Lausanne) 2022;13:839885. doi:10.3389/fendo. 2022.839885, PMID:35250888.

[11] Yin X, Zhao S, Xiang N, Chen J, Xu J, Zhang Y. Efficacy of Bushen Jiangu therapy in the treatment of glucocorticoid-induced osteoporosis: A systematic review and meta-analysis of randomized controlled trials. Medicine (Baltimore) 2023; 102(11):e33278. doi:10.1097/ MD.0000000000033278, PMID:36930100.

[12] Yu JS, Cui W. Proliferation, survival and metabolism: the role of PI3K/ AKT/mTOR signalling in pluripotency and cell fate determination. Development 2016; 143(17):3050-3060. doi:10.1242/dev.137075, PMID:27578176.

[13] Amjadi-Moheb F, Akhavan-Niaki H. Wnt signaling pathway in osteoporosis: Epigenetic regulation, interaction with other signaling pathways, and therapeutic promises. J Cell Physiol 2019; 234(9):14641-14650. doi:10.1002/jcp.28207, PMID:30693508.

[14] Liu F, Zhao Y, Pei Y, Lian F, Lin H. Role of the NF-kB signalling pathway in heterotopic ossification: biological and therapeutic significance. Cell Commun Signal 2024; 22(1):159. doi:10.1186/s12964-024- 01533-w, PMID:38439078.

[15] Lu J, Shi X, Fu Q, Han Y, Zhu L, Zhou Z, et al. New mechanistic understanding of osteoclast differentiation and bone resorption mediated by P2X7 receptors and PI3K-Akt-GSK3β signaling. Cell Mol Biol Lett 2024; 29(1):100. doi:10.1186/s11658-024-00614-5, PMID:38977961.

[16] Wei QS, Hong GJ, Yuan YJ, Chen ZQ, Zhang QW, He W. Huo Xue Tong Luo capsule, a vasoactive herbal formula prevents progression of asymptomatic osteonecrosis of femoral head: A prospective study. J Orthop Translat 2019; 18:65-73. doi:10.1016/j.jot.2018.11.002, PMID:31508309.

[17] Fang B, Li Y, Chen C, Wei Q, Zheng J, Liu Y, et al. Huo Xue Tong Luo capsule ameliorates osteonecrosis of femoral head through inhibiting lncRNA-Miat. J Ethnopharmacol 2019;238:111862. doi:10.1016/j. jep.2019.111862, PMID:30970282.

[18] Nair A, Morsy MA, Jacob S. Dose translation between laboratory animals and human in preclinical and clinical phases of drug development. Drug Dev Res 2018; 79(8):373-382. doi:10.1002/ddr.21461, PMID:30343496.

[19] Dong Y, Duan L, Chen HW, Liu YM, Zhang Y, Wang J. Network Pharmacology- Based Prediction and Verification of the Targets and Mechanism for Panax Notoginseng Saponins against Coronary Heart Disease. Evid Based Complement Alternat Med 2019; 2019:6503752. doi:10.1155/2019/6503752, PMID:31354855.

[20] Tang R, Tian RH, Cai JZ, Wu JH, Shen XL, Hu YJ. Acute and sub-chronic toxicity of Cajanus cajan leaf extracts. Pharm Biol 2017; 55(1):1740-1746. doi:10.1080/13880209.2017.1309556, PMID:28494681.

[21] Guo Z, Zhu P, He X, Yan T, Liang X. Components identification and isomers differentiation in pigeon pea (Cajanus cajan L.) leaves by LCMS. J Sep Sci 2021; 44(13):2510-2523. doi:10.1002/jssc.202001194, PMID:33830649.

[22] Liu W, Kong Y, Zu Y, Fu Y, Luo M, Zhang L, et al. Determination and quantification of active phenolic compounds in pigeon pea leaves and its medicinal product using liquid chromatography-tandem mass spectrometry. J Chromatogr A 2010; 1217(28):4723-4731. doi:10.1016/j.chroma.2010.05.020, PMID:20965080.

[23] Liu W, Fu Y, Zu Y, Kong Y, Zhang L, Zu B, et al. Negative-pressure cavitation extraction for the determination of flavonoids in pigeon pea leaves by liquid chromatography-tandem mass spectrometry. J Chromatogr A 2009; 1216(18):3841-3850. doi:10.1016/j.chroma. 2009.02.073, PMID:19303077.

[24] He X, Zhang H, Liang X. Separation of six compounds from pigeon pea leaves by elution-extrusion counter-current chromatography. J Sep Sci 2019; 42(6):1202-1209. doi:10.1002/jssc.201801111, PMID:30653252.

[25] UniProt Consortium. UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res 2021; 49(D1):D480-D489. doi:10.1093/ nar/gkaa1100, PMID:33237286.

[26] Liang G, Zhao J, Dou Y, Yang Y, Zhao D, Zhou Z, et al. Mechanism and Experimental Verification of Luteolin for the Treatment of Osteoporosis Based on Network Pharmacology. Front Endocrinol (Lausanne) 2022;13:866641. doi:10.3389/fendo.2022.866641, PMID:35355555.

[27] Zhang Y, Li Z, Yang M, Wang D, Yu L, Guo C, et al. Identification of GRB2 and GAB1 coexpression as an unfavorable prognostic factor for hepatocellular carcinoma by a combination of expression profile and network analysis. PLoS One 2013; 8(12):e85170. doi:10.1371/journal. pone.0085170, PMID:24391994.

[28] Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc 2009; 4(1):44-57. doi:10.1038/nprot.2008.211, PMID:19131956.

[29] Trott O, Olson AJ. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010; 31(2):455-461. doi:10.1002/ jcc.21334, PMID:19499576.

[30] Cai XY, Zhang ZJ, Xiong JL, Yang M, Wang ZT. Experimental and molecular docking studies of estrogen-like and anti-osteoporosis activity of compounds in Fructus Psoraleae. J Ethnopharmacol 2021;276:114044. doi:10.1016/j.jep.2021.114044, PMID:33775805.

[31] Hu Y, Han J, Ding S, Liu S, Wang H. Identification of ferroptosisassociated biomarkers for the potential diagnosis and treatment of postmenopausal osteoporosis. Front Endocrinol (Lausanne) 2022;13:986384. doi:10.3389/fendo.2022.986384, PMID:36105394.

[32] Nogués X, Martinez-Laguna D. Update on osteoporosis treatment. Med Clin (Barc) 2018; 150(12):479-486. doi:10.1016/j.medcli. 2017.10.019, PMID:29179892.

[33] Xie W, Tang C, Zhang Y, Fan W, Qin J, Xiao H, et al. Effect of stigmasterol and polyglycerol polyricinoleate concentrations on the preparation and properties of rapeseed oil-based gel emulsions. Food Chem X 2024;23:101636. doi:10.1016/j.fochx.2024.101636, PMID:39113734.

[34] Zhao Y, Ning J, Teng H, Deng Y, Sheldon M, Shi L, et al. Long noncoding RNA Malat1 protects against osteoporosis and bone metastasis. Nat Commun 2024; 15(1):2384. doi:10.1038/s41467-024-46602-3, PMID:38493144.

[35] Zheng H, Liu Y, Deng Y, Li Y, Liu S, Yang Y, et al. Recent advances of NFATc1 in rheumatoid arthritis-related bone destruction: mechanisms and potential therapeutic targets. Mol Med 2024; 30(1):20. doi:10.1186/s10020-024-00788-w, PMID:38310228.

[36] Kitazawa R, Haraguchi R, Kohara Y, Kitazawa S. RANK- NFATc1 signaling forms positive feedback loop on rank gene expression via functional NFATc1 responsive element in rank gene promoter. Biochem Biophys Res Commun 2021; 572:86-91. doi:10.1016/j.bbrc.2021.07.100, PMID:34358968.

[37] Murakami A, Matsuda M, Harada Y, Hirata M. Phospholipase C-related, but catalytically inactive protein (PRIP) up-regulates osteoclast differentiation via calcium-calcineurin-NFATc1 signaling. J Biol Chem 2017; 292(19):7994-8006. doi:10.1074/jbc.M117.784777, PMID:283 41745.

[38] Forsprecher J, Wang Z, Goldberg HA, Kaartinen MT. Transglutaminase- mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein. Cell Adh Migr 2011; 5(1):65-72. doi:10.4161/cam.5.1.13369, PMID:20864802.

[39] Wong BR, Besser D, Kim N, Arron JR, Vologodskaia M, Hanafusa H, et al. TRANCE, a TNF family member, activates Akt/PKB through a signaling complex involving TRAF6 and c-Src. Mol Cell 1999;4(6):1041-1049. doi:10.1016/s1097-2765(00)80232-4, PMID:10635328.

[40] Moon JB, Kim JH, Kim K, Youn BU, Ko A, Lee SY, et al. Akt induces osteoclast differentiation through regulating the GSK3β/NFATc1 signaling cascade. J Immunol 2012; 188(1):163-169. doi:10.4049/jimmunol. 1101254, PMID:22131333.

[41] Poulsen RC, Kruger MC. Soy phytoestrogens: impact on postmenopausal bone loss and mechanisms of action. Nutr Rev 2008; 66(7):359-374. doi:10.1111/j.1753-4887.2008.00046.x, PMID:18667012.

[42] Dai J, Li Y, Zhou H, Chen J, Chen M, Xiao Z. Genistein promotion of osteogenic differentiation through BMP2/SMAD5/RUNX2 signaling. Int J Biol Sci 2013; 9(10):1089-1098. doi:10.7150/ijbs.7367, PMID:24339730.

[43] King TJ, Shandala T, Lee AM, Foster BK, Chen KM, Howe PR, et al. Potential Effects of Phytoestrogen Genistein in Modulating Acute Methotrexate Chemotherapy-Induced Osteoclastogenesis and Bone Damage in Rats. Int J Mol Sci 2015; 16(8):18293-18311. doi:10.3390/ijms160818293, PMID:26258775.

[44] Gholami M, Tarverdi A, Gholami A. The Effect of Vanillic Acid on Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in Wistar Male Rats. Biol Bull Russ Acad Sci 2022; 49(3):117-124. doi:10.1134/S1062359022030074.

[45] Wang YG, Jiang LB, Gou B. PROTECTIVE EFFECT OF VANILLIC ACID ON OVARIECTOMY-INDUCED OSTEOPOROSIS IN RATS. Afr J Tradit Complement Altern Med 2017; 14(4):31-38. doi:10.21010/ajtcam. v14i4.4, PMID:28638864.

[46] Onishi S, Tebayashi S, Hikichi Y, Sawada H, Ishii Y, Kim CS. Inhibitory effects of luteolin and its derivatives on osteoclast differentiation and differences in luteolin production by Capsicum annuum varieties. Biosci Biotechnol Biochem 2021; 85(11):2224-2231. doi:10.1093/bbb/zbab149, PMID:34435616.