Osteoking exerts pro‑osteogenic and anti‑adipogenic effects in promoting bone fracture healing via EGF/EGFR/HDAC1/Wnt/&beta;‑catenin signaling

Zhang,Suya; Chen,Lin; Zhang,Chu; Gong,Chunzhu; He,Xiangxin; Zhong,Honggang; Liu,Chunfang; Cao,Zhigang; Chen,Weiheng; Lin,Na; Zhang,Yanqiong

doi:10.3892/ijmm.2025.5516

Osteoking exerts pro‑osteogenic and anti‑adipogenic effects in promoting bone fracture healing via EGF/EGFR/HDAC1/Wnt/β‑catenin signaling

Authors:
- Suya Zhang
- Lin Chen
- Chu Zhang
- Chunzhu Gong
- Xiangxin He
- Honggang Zhong
- Chunfang Liu
- Zhigang Cao
- Weiheng Chen
- Na Lin
- Yanqiong Zhang
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Affiliations: State Key Laboratory for Quality Ensurance and Sustainable Use of Dao‑di Herbs, Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, P.R. China, Department of Geriatric Orthopedics, Shenzhen Pingle Orthopedic Hospital (Shenzhen Pingshan Traditional Chinese Medicine Hospital), Shenzhen, Guangdong 518118, P.R. China, Laboratory of Biomechanics, Institute of Bone Injury, China Academy of Chinese Medical Sciences, Beijing 100010, P.R. China, Sailing Pharmaceutical Technology Group Co., Ltd., Yuxi, Yunnan 653103, P.R. China, Third Affiliated Hospital of Beijing University of Chinese Medicine, Beijing 100029, P.R. China
Published online on: March 7, 2025 https://doi.org/10.3892/ijmm.2025.5516
Article Number: 75
Copyright: © Zhang et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

Bone fractures, as a global public health issue, lead to disability and reduce the quality of life for patients. Chinese patent drug Osteoking has efficacy in bone fracture therapy. However, its therapeutic properties and underlying mechanisms remain unclear. In the present study, a rat model of bone fracture was established to evaluate the pharmacological effects of Osteoking by behavioral feature detection including mechanical pain threshold measurement, inclined plate and hindlimb weight‑bearing test and CatWalk XT gait analysis, as well as X‑ray scanning and micro‑computed tomography 3D reconstruction. Transcriptomics profiling, network analysis and in vivo western blotting, immunohistochemistry and immunofluorescence assessment were performed to determine the potential targets of Osteoking in promoting bone fracture healing. Osteoking effectively shortened the fracture healing time primarily by accelerating the process of endochondral ossification, decreasing the number of osteoclasts, increasing the levels of bone growth factors and bone formation biomarkers, and decreasing the level of bone resorption biomarkers. Following construction and analysis of the disease gene‑drug target network, it was hypothesized that EGF‑EGFR‑histone deacetylase 1 (HDAC1)‑Wnt/β‑catenin axis‑mediated adipogenesis‑angiogenesis‑osteogenesis crosstalk may be a candidate target of Osteoking in bone fracture. Osteoking significantly decreased expression levels of EGF, phosphorylated‑EGFR and HDAC1 protein and activated Wnt/β‑catenin signaling, which subsequently elevated the expression of VEGFA, Osterix (OSX) and CD31 proteins, increased the RUNX2/PPARγ ratio, decreased the receptor activator of nuclear factor κB ligand/osteoprotegerin ratio and reduced the serum levels of total cholesterol (TC), low‑density lipoprotein cholesterol (LDL‑C) and high‑density lipoprotein cholesterol (HDL‑C). There was a negative association between VEGFA, OSX, TC and LDL‑C levels. In conclusion, Osteoking may effectively reverse the disturbance of adipogenesis‑angiogenesis‑osteogenesis homeostasis and promote the fracture healing by regulating the EGF‑EGFR‑HDAC1‑Wnt/β‑catenin axis. These findings may offer guidance for the clinical application of Osteoking in bone fracture therapy.

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1	GBD 2019 Fracture Collaborators: Global, regional, and national burden of bone fractures in 204 countries and territories, 1990-2019: A systematic analysis from the global burden of disease study 2019. Lancet Healthy Longev. 2:e580–e592. 2021. View Article : Google Scholar : PubMed/NCBI
2	Zura R, Xiong Z, Einhorn T, Watson JT, Ostrum RF, Prayson MJ, Della Rocca GJ, Mehta S, McKinley T, Wang Z and Steen RG: Epidemiology of fracture nonunion in 18 human bones. JAMA Surg. 151:e1627752016. View Article : Google Scholar : PubMed/NCBI
3	Ekegren CL, Edwards ER, de Steiger R and Gabbe BJ: Incidence, costs and predictors of non-union, delayed union and mal-union following long bone fracture. Int J Environ Res Public Health. 15:28452018. View Article : Google Scholar : PubMed/NCBI
4	Hendrickx LAM, Virgin J, van den Bekerom MPJ, Doornberg JN, Kerkhoffs GMMJ and Jaarsma RL: Complications and subsequent surgery after intra-medullary nailing for tibial shaft fractures: Review of 8110 patients. Injury. 51:1647–1654. 2020. View Article : Google Scholar : PubMed/NCBI
5	Tian R, Zheng F, Zhao W, Zhang Y, Yuan J, Zhang B and Li L: Prevalence and influencing factors of nonunion in patients with tibial fracture: Systematic review and meta-analysis. J Orthop Surg Res. 15:3772020. View Article : Google Scholar : PubMed/NCBI
6	Antonova E, Le TK, Burge R and Mershon J: Tibia shaft fractures: Costly burden of nonunions. BMC Musculoskelet Disord. 14:422013. View Article : Google Scholar : PubMed/NCBI
7	Davis S, Martyn-St James M, Sanderson J, Stevens J, Goka E, Rawdin A, Sadler S, Wong R, Campbell F, Stevenson M, et al: A systematic review and economic evaluation of bisphosphonates for the prevention of fragility fractures. Health Technol Assess. 20:1–406. 2016. View Article : Google Scholar :
8	Hegde V, Jo JE, Andreopoulou P and Lane JM: Effect of osteoporosis medications on fracture healing. Osteoporos Int. 27:861–871. 2016. View Article : Google Scholar
9	Davis S, Simpson E, Hamilton J, James MMS, Rawdin A, Wong R, Goka E, Gittoes N and Selby P: Denosumab, raloxifene, romosozumab and teriparatide to prevent osteoporotic fragility fractures: A systematic review and economic evaluation. Health Technol Assess. 24:1–314. 2020. View Article : Google Scholar
10	Petrova NL, Donaldson NK, Bates M, Tang W, Jemmott T, Morris V, Dew T, Meacock L, Elias DA, Moniz CF and Edmonds ME: Effect of recombinant human parathyroid hormone (1-84) on resolution of active charcot neuro-osteoarthropathy in diabetes: A randomized, double-blind, placebo-controlled study. Diabetes Care. 44:1613–1621. 2021. View Article : Google Scholar : PubMed/NCBI
11	Aro HT, Govender S, Patel AD, Hernigou P, de Gregorio AP, Popescu GI, Golden JD, Christensen J and Valentin A: Recombinant human bone morphogenetic protein-2: A randomized trial in open tibial fractures treated with reamed nail fixation. J Bone Joint Surg Am. 93:801–808. 2011. View Article : Google Scholar : PubMed/NCBI
12	Ding W, Ji R, Yao S, Ruan P, Ye F, Lou X, Ni L, Ji Y, Chen J and Ji W: Hu'po Anshen decoction promotes fracture healing in mice with traumatic brain injury through BMP2-COX2-ATF4 signaling pathway. FASEB J. 37:e229522023. View Article : Google Scholar : PubMed/NCBI
13	Li Z, Li Y, Liu C, Gu Y and Han G: Research progress of the mechanisms and applications of ginsenosides in promoting bone formation. Phytomedicine. 129:1556042024. View Article : Google Scholar : PubMed/NCBI
14	Qian D, Zhang Q, He CX, Guo J, Huang XT, Zhao J, Zhang H, Xu C and Peng W: Hai-Honghua medicinal liquor is a reliable remedy for fracture by promotion of osteogenic differentiation via activation of PI3K/Akt pathway. J Ethnopharmacol. 330:1182342024. View Article : Google Scholar : PubMed/NCBI
15	Zhang L, Kuang H, Zhang Z, Rong K, Yuan Y, Peng Z, Zhao H, Liu K, Ou L and Kuang J: Efficacy and safety of Osteoking on fracture healing: A systematic review and meta-analysis. Front Pharmacol. 15:13634212024. View Article : Google Scholar : PubMed/NCBI
16	Xie L, Song X, Lei L, Chen C, Zhao H, Hu J, Yu Y, Bai X, Wu X, Li X, et al: Exploring the potential mechanism of Heng-Gu-Gu-Shang-Yu-He-Ji therapy for osteoporosis based on network pharmacology and transcriptomics. J Ethnopharmacol. 321:1174802024. View Article : Google Scholar
17	Luo X, Liu J, Wang X, Chen Q, Lei Y, He Z, Wang X, Ye Y, Na Q, Lao C, et al: Mechanism exploration of Osteoking in the treatment of lumbar disc herniation based on network pharmacology and molecular docking. J Orthop Surg Res. 19:882024. View Article : Google Scholar : PubMed/NCBI
18	Sun Y, Chen R, Zhu D, Shen ZQ, Zhao HB and Lee WH: Osteoking improves OP rat by enhancing HSP90-β expression. Int J Mol Med. 45:1543–1553. 2020.PubMed/NCBI
19	Zhou J, Zheng Z, Luo Y, Dong Y, Yan Y, Zhang Y, Tang K, Quan R, Lin J, Zhang K, et al: Clinical efficacy of Osteoking in knee osteoarthritis therapy: A prospective, multicenter, non-randomized controlled study in China. Front Pharmacol. 15:13819362024. View Article : Google Scholar : PubMed/NCBI
20	Ling H, Zeng Q, Ge Q, Chen J, Yuan W, Xu R, Shi Z, Xia H, Hu S, Jin H, et al: Osteoking decelerates cartilage degeneration in dmm-induced osteoarthritic mice model through TGF-β/smad-dependent manner. Front Pharmacol. 12:6788102021. View Article : Google Scholar
21	Zhang S, Liu Y, Ma Z, Gao S, Chen L, Zhong H, Zhang C, Li T, Chen W, Zhang Y and Lin N: Osteoking promotes bone formation and bone defect repair through ZBP1-STAT1-PKR-MLKL-mediated necroptosis. Chin Med. 19:132024. View Article : Google Scholar : PubMed/NCBI
22	Sun Z, Jin H, Zhou H, Yu L, Wan H and He Y: Guhong Injection promotes fracture healing by activating Wnt/beta-catenin signaling pathway in vivo and in vitro. Biomed Pharmacother. 120:1094362019. View Article : Google Scholar : PubMed/NCBI
23	Alentado VJ, Knox AM, Staut CA, McGuire AC, Chitwood JR, Mostardo SL, Shaikh MZ, Blosser RJ, Dadwal UC, Chu TMG, et al: Validation of the modified radiographic union score for tibia fractures (mRUST) in murine femoral fractures. Front Endocrinol (Lausanne). 13:9110582022. View Article : Google Scholar : PubMed/NCBI
24	Liu X, Mao X, Liu Y, Chen W, Li W, Lin N and Zhang Y: Preclinical efficacy of TZG in myofascial pain syndrome by impairing PI3K-RAC2 signaling-mediated neutrophil extracellular traps. iScience. 26:1080742023. View Article : Google Scholar : PubMed/NCBI
25	Garrick JM, Costa LG, Cole TB and Marsillach J: Evaluating gait and locomotion in rodents with the CatWalk. Curr Protoc. 1:e2202021. View Article : Google Scholar : PubMed/NCBI
26	Zhang Y, Wang X, Ding Z, Lin N and Zhang Y: Enhanced efficacy with reduced toxicity of tripterygium glycoside tablet by compatibility with total glucosides of paeony for rheumatoid arthritis therapy. Biomed Pharmacother. 166:1154172023. View Article : Google Scholar : PubMed/NCBI
27	Li W, Wang K, Liu Y, Wu H, He Y, Li C, Wang Q, Su X, Yan S, Su W, et al: A novel drug combination of mangiferin and cinnamic acid alleviates rheumatoid arthritis by inhibiting TLR4/NFκB/NLRP3 activation-induced pyroptosis. Front Immunol. 13:9129332022. View Article : Google Scholar
28	Zhang Y, Li X, Shi Y, Chen T, Xu Z, Wang P, Yu M, Chen W, Li B, Jing Z, et al: ETCM v2.0: An update with comprehensive resource and rich annotations for traditional Chinese medicine. Acta Pharm Sin B. 13:2559–2571. 2023. View Article : Google Scholar : PubMed/NCBI
29	Scardoni G, Tosadori G, Pratap S, Spoto F and Laudanna C: Finding the shortest path with PesCa: A tool for network reconstruction. F1000Res. 4:4842015. View Article : Google Scholar : PubMed/NCBI
30	Ma Z, Chen W, Liu Y, Yu L, Mao X, Guo X, Jiang F, Guo Q, Lin N and Zhang Y: Artesunate Sensitizes human hepatocellular carcinoma to sorafenib via exacerbating AFAP1L2-SRC-FUNDC1 axis-dependent mitophagy. Autophagy. 20:541–556. 2024. View Article : Google Scholar :
31	Li W, Mao X, Wu H, Guo M, Su X, Lu J, Guo Q, Li T, Wang X, Su W, et al: Deciphering the chemical profile and pharmacological mechanisms of Baihu-Guizhi decoction using ultra-fast liquid chromatography-quadrupole-time-of-flight tandem mass spectrometry coupled with network pharmacology-based investigation. Phytomedicine. 67:1531562020. View Article : Google Scholar : PubMed/NCBI
32	Mao X, Li W, Chen W, Li Y, Wang Q, Wang X, Pi Z, Wang D, Xu H, Guo Q, et al: Exploring and characterizing a novel combination of paeoniflorin and talatizidine for the treatment of rheumatoid arthritis. Pharmacol Res. 153:1046582020. View Article : Google Scholar : PubMed/NCBI
33	Fu R, Liu C, Yan Y, Li Q and Huang RL: Bone defect reconstruction via endochondral ossification: A developmental engineering strategy. J Tissue Eng. 12:204173142110042112021. View Article : Google Scholar : PubMed/NCBI
34	Min R: Analysis of therapeutic effect of henggu gushang healing agent combined with PFNA on intertrochanteric fracture of femur. J JIANGXI Univ CM. 35:43–46. 2023.
35	He P, Yue C, Chen J, Ma M, Yang G, Wang Q and Liu Y: Observation on the curative effect of Henggu Gushangyu Mixture in the treatment of femoral intertrochanteric fracture after internal fixation. Fujian J TCM. 53:60–62. 2022.
36	Liu Z, Zhao X, Luo W and Wang L: The effect of osteoking on postoperative fracture healing of femoral neck fracture. Int J Trad Chin Med. 44:1122–1126. 2022.
37	Einhorn TA and Gerstenfeld LC: Fracture healing: Mechanisms and interventions. Nat Rev Rheumatol. 11:45–54. 2015. View Article : Google Scholar :
38	Menger MM, Laschke MW, Nussler AK, Menger MD and Histing T: The vascularization paradox of non-union formation. Angiogenesis. 25:279–290. 2022. View Article : Google Scholar : PubMed/NCBI
39	Bixel MG, Sivaraj KK, Timmen M, Mohanakrishnan V, Aravamudhan A, Adams S, Koh BI, Jeong HW, Kruse K, Stange R and Adams RH: Angiogenesis is uncoupled from osteogenesis during calvarial bone regeneration. Nat Commun. 15:45752024. View Article : Google Scholar : PubMed/NCBI
40	Maes C, Kobayashi T, Selig MK, Torrekens S, Roth SI, Mackem S, Carmeliet G and Kronenberg HM: Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell. 19:329–344. 2010. View Article : Google Scholar : PubMed/NCBI
41	Huang C, Ness VP, Yang X, Chen H, Luo J, Brown EB and Zhang X: Spatiotemporal analyses of osteogenesis and angiogenesis via intravital imaging in cranial bone defect repair. J Bone Miner Res. 30:1217–1230. 2015. View Article : Google Scholar : PubMed/NCBI
42	Zhu S, Chen W, Masson A and Li YP: Cell signaling and transcriptional regulation of osteoblast lineage commitment, differentiation, bone formation, and homeostasis. Cell Discov. 10:712024. View Article : Google Scholar : PubMed/NCBI
43	Burger MG, Grosso A, Briquez PS, Born GME, Lunger A, Schrenk F, Todorov A, Sacchi V, Hubbell JA, Schaefer DJ, et al: Robust coupling of angiogenesis and osteogenesis by VEGF-decorated matrices for bone regeneration. Acta Biomater. 149:111–125. 2022. View Article : Google Scholar : PubMed/NCBI
44	Rauch A, Haakonsson AK, Madsen JGS, Larsen M, Forss I, Madsen MR, Van Hauwaert EL, Wiwie C, Jespersen NZ, Tencerova M, et al: Osteogenesis depends on commissioning of a network of stem cell transcription factors that act as repressors of adipogenesis. Nat Genet. 51:716–727. 2019. View Article : Google Scholar : PubMed/NCBI
45	Ambrosi TH, Scialdone A, Graja A, Gohlke S, Jank AM, Bocian C, Woelk L, Fan H, Logan DW, Schürmann A, et al: Adipocyte accumulation in the bone marrow during obesity and aging impairs stem cell-based hematopoietic and bone regeneration. Cell Stem Cell. 20:771–784.e6. 2017. View Article : Google Scholar : PubMed/NCBI
46	Schubert J, Lindahl B, Melhus H, Renlund H, Leosdottir M, Yari A, Ueda P, Jernberg T and Hagström E: Elevated low-density lipoprotein cholesterol: An inverse marker of morbidity and mortality in patients with myocardial infarction. J Intern Med. 294:616–627. 2023. View Article : Google Scholar : PubMed/NCBI
47	Poiana C, Radoi V, Carsote M and Bilezikian JP: New clues that may link osteoporosis to the circulating lipid profile. Bone Res. 1:260–266. 2013. View Article : Google Scholar : PubMed/NCBI
48	Mandal CC: High Cholesterol deteriorates bone health: New insights into molecular mechanisms. Front Endocrinol (Lausanne). 6:1652015. View Article : Google Scholar : PubMed/NCBI
49	Chang PY, Gold EB, Cauley JA, Johnson WO, Karvonen-Gutierrez C, Jackson EA, Ruppert KM and Lee JS: Triglyceride levels and fracture risk in midlife women: Study of women's health across the nation (SWAN). J Clin Endocrinol Metab. 101:3297–3305. 2016. View Article : Google Scholar : PubMed/NCBI
50	Fang L, Choi SH, Baek JS, Liu C, Almazan F, Ulrich F, Wiesner P, Taleb A, Deer E, Pattison J, et al: Control of angiogenesis by AIBP-mediated cholesterol efflux. Nature. 498:118–122. 2013. View Article : Google Scholar : PubMed/NCBI
51	Chen H, Shao Z, Gao Y, Yu X, Huang S and Zeng P: Are blood lipids risk factors for fracture? Integrative evidence from instrumental variable causal inference and mediation analysis using genetic data. Bone. 131:1151742020.
52	Barzilay JI, Buzkova P, Kuller LH, Cauley JA, Fink HA, Sheets K, Robbins JA, Carbone LD, Elam RE and Mukamal KJ: The association of lipids and lipoproteins with hip fracture risk: The cardiovascular health study. Am J Med. 135:1101–1108.e1. 2022.PubMed/NCBI
53	Baek WY and Kim JE: Transcriptional regulation of bone formation. Front Biosci (Schol Ed). 3:126–135. 2011.PubMed/NCBI
54	Kim HY, Jang HJ, Muthamil S, Shin UC, Lyu JH, Kim SW, Go Y, Park SH, Lee HG and Park JH: Novel insights into regulators and functional modulators of adipogenesis. Biomed Pharmacother. 177:1170732024.PubMed/NCBI
55	Huang X, Chen W, Gu C, Liu H, Hou M, Qin W, Zhu X, Chen X, Liu T, Yang H and He F: Melatonin suppresses bone marrow adiposity in ovariectomized rats by rescuing the imbalance between osteogenesis and adipogenesis through SIRT1 activation. J Orthop Translat. 38:84–97. 2022.PubMed/NCBI
56	Dong Y, Zhou H, Alhaskawi A, Wang Z, Lai J, Abdullah Ezzi SH, Kota VG, Abdulla Hasan Abdulla MH, Sun Z and Lu H: Alterations in bone fracture healing associated with TNFRSF signaling pathways. Front Pharmacol. 13:9055352022.PubMed/NCBI
57	Yu G, Fu X, Gong A, Gu J, Zou H, Yuan Y, Song R, Ma Y, Bian J, Liu Z and Tong X: Oligomeric proanthocyanidins ameliorates osteoclastogenesis through reducing OPG/RANKL ratio in chicken's embryos. Poult Sci. 103:1037062024.PubMed/NCBI
58	Zhu J, Shimizu E, Zhang X, Partridge NC and Qin L: EGFR signaling suppresses osteoblast differentiation and inhibits expression of master osteoblastic transcription factors Runx2 and osterix. J Cell Biochem. 112:1749–1760. 2011.PubMed/NCBI
59	Lees-Shepard JB, Flint K, Fisher M, Omi M, Richard K, Antony M, Chen PJ, Yadav S, Threadgill D, Maihle NJ and Dealy CN: Cross-talk between EGFR and BMP signals regulates chondrocyte maturation during endochondral ossification. Dev Dyn. 251:75–94. 2022.
60	Yi T, Lee HL, Cha JH, Ko SI, Kim HJ, Shin HI, Woo KM, Ryoo HM, Kim GS and Baek JH: Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling. J Cell Physiol. 217:409–422. 2008.PubMed/NCBI
61	Haberland M, Carrer M, Mokalled MH, Montgomery RL and Olson EN: Redundant control of adipogenesis by histone deacetylases 1 and 2. J Biol Chem. 285:14663–14670. 2010.PubMed/NCBI
62	Vishal M, Ajeetha R, Keerthana R and Selvamurugan N: Regulation of Runx2 by histone deacetylases in bone. Curr Protein Pept Sci. 17:343–351. 2016.PubMed/NCBI
63	Zhong YT, Liao HB, Ye ZQ, Jiang HS, Li JX, Ke LM, Hua JY, Wei B, Wu X and Cui L: Eurycomanone stimulates bone mineralization in zebrafish larvae and promotes osteogenic differentiation of mesenchymal stem cells by upregulating AKT/GSK-3β/β-catenin signaling. J Orthop Translat. 40:132–146. 2023.PubMed/NCBI
64	Kramer I, Halleux C, Keller H, Pegurri M, Gooi JH, Weber PB, Feng JQ, Bonewald LF and Kneissel M: Osteocyte Wnt/beta-catenin signaling is required for normal bone homeostasis. Mol Cell Biol. 30:3071–3085. 2010.PubMed/NCBI
65	Albers J, Keller J, Baranowsky A, Beil FT, Catala-Lehnen P, Schulze J, Amling M and Schinke T: Canonical Wnt signaling inhibits osteoclastogenesis independent of osteoprotegerin. J Cell Biol. 200:537–549. 2013.PubMed/NCBI
66	Takada I, Kouzmenko AP and Kato S: Wnt and PPARgamma signaling in osteoblastogenesis and adipogenesis. Nat Rev Rheumatol. 5:442–447. 2009.PubMed/NCBI
67	Takada I, Kouzmenko AP and Kato S: Molecular switching of osteoblastogenesis versus adipogenesis: Implications for targeted therapies. Expert Opin Ther Targets. 13:593–603. 2009.PubMed/NCBI
68	Olsen JJ, Pohl SÖG, Deshmukh A, Visweswaran M, Ward NC, Arfuso F, Agostino M and Dharmarajan A: The role of Wnt signalling in angiogenesis. Clin Biochem Rev. 38:131–142. 2017.