淫羊藿苷通过增强HOTTIP表达促进间充质干细胞成骨分化

doi:10.5246/jcps.2026.06.038

中国药学(英文版) ›› 2026, Vol. 35 ›› Issue (6): 538-551.DOI: 10.5246/jcps.2026.06.038

• 【研究论文】 • 上一篇

淫羊藿苷通过增强HOTTIP表达促进间充质干细胞成骨分化

邵将¹^,², 张锦芳³, 张雅歌³^,^*(), 詹冬香¹^,⁴^,⁵^,⁶^,^*()

^1. 广州中医药大学第一附属医院，广东广州 510405
^2. 山东中医药大学附属医院，山东济南 250011
^3. 广州中医药大学深圳医院(福田)，广东深圳 518034
^4. 广东省中医临床研究院，广东广州 510405
^5. 医疗机构中药制剂与中药新药转化广东省工程研究中心，广东广州 510405
^6. 广东省岭南特色医院制剂工程技术研究中心，广东广州 510405

收稿日期:2026-03-07 修回日期:2026-03-25 接受日期:2026-04-11 出版日期:2026-07-05 发布日期:2026-07-05
通讯作者: 张雅歌, 詹冬香

Icariin promotes osteogenic differentiation of mesenchymal stem cells through upregulating HOTTIP

Jiang Shao¹^,², Jinfang Zhang³, Yage Zhang³^,^*(), Dongxiang Zhan¹^,⁴^,⁵^,⁶^,^*()

^1. The First Affiliated Hospital of Guangzhou University of Chinese Medicine, Guangzhou 510405, Guangdong, China
^2. Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Jinan 250011, Shandong, China
^3. Shenzhen Hospital of Guangzhou University of Traditional Chinese Medicine (Futian), Shenzhen 518034, Guangdong, China
^4. Guangdong Clinical Research Academy of Chinese Medicine, Guangzhou 510405, Guangdong, China
^5. Guangdong Engineering Research Center of Commercialization of Medical Institution Preparations and Traditional Chinese Medicines, Guangzhou 510405, Guangdong, China
^6. Guangdong Engineering Technology Research Center of Commercialization of Lingnan Special Medical Institution Preparations, Guangzhou 510405, Guangdong, China

Received:2026-03-07 Revised:2026-03-25 Accepted:2026-04-11 Online:2026-07-05 Published:2026-07-05
Contact: Yage Zhang, Dongxiang Zhan
Supported by:
The Medical Scientific Research Foundation of Guangdong Province, China (Grant No. B2024108).

摘要/Abstract

摘要：

间充质干细胞(MSCs)由于其多向分化的能力而成为组织修复工程的一种有前途的策略。在控制间质干细胞分化的调控分子中，长链非编码RNA (lncRNA) HOTTIP已被确定为成骨分化和骨稳态维持的关键调节剂。在机制上，HOTTIP协调染色质重塑和成骨基因的转录激活，表明它是骨再生策略的一个有希望的治疗靶点。淫羊藿苷(Icariin, ICA)是一种从淫羊藿中提取的类黄酮苷，具有促进成骨分化和促进骨代谢的作用。然而，淫羊藿苷和LncRNA介导的表观遗传调控之间的精确串扰仍然是难以捉摸的。它们在骨组织工程中的协同潜力有待进一步研究。研究发现在浓度低于50 μM时，ICA对骨髓间充质干细胞无明显的细胞毒性。在5和10 μM淫羊藿苷干预下，ALP活性和ARS染色明显增强，成骨相关基因表达明显增加，表明ICA促进了成骨分化。进一步研究发现ICA增加了lncRNA HOTTIP的剂量依赖性；在HOTTIP敲除的MSCs中，ICA对MSCs成骨的影响被成功逆转。此外，还发现HOTTIP KO MSCs内Wnt信号通路核心因子β-catenin的表达水平下降，ICA促进WT小鼠MSCs β-catenin表达，对HOTTIP小鼠MSCs β-catenin表达无促进作用。综上所述，ICA通过增加lncRNA HOTTIP的表达调控β-catenin来促进MSCs的成骨分化。

关键词: 淫羊藿苷, 长链非编码 RNA HOTTIP, 骨髓间充质干细胞, 成骨分化, Wnt/β-catenin

Abstract:

Mesenchymal stem cells (MSCs) have emerged as a highly promising strategy in tissue repair engineering owing to their robust self-renewal capacity and multidirectional differentiation potential. Among the regulatory factors governing MSC fate determination, the long non-coding RNA (lncRNA) HOTTIP has been identified as a pivotal modulator of osteogenic differentiation and the maintenance of bone homeostasis. Mechanistically, HOTTIP regulates osteogenic commitment through orchestrating chromatin remodeling and transcriptional activation of key osteogenesis-related genes, highlighting its potential value as a therapeutic target in bone regeneration strategies. Icariin (ICA), a bioactive flavonoid glycoside isolated from Epimedium, has been reported to promote osteogenic differentiation and enhance bone metabolic activity. However, the precise molecular interplay between ICA and lncRNA-mediated epigenetic regulation during osteogenesis remains largely unexplored, and further investigation is required to elucidate their potential synergistic roles in bone tissue engineering. In the present study, our results demonstrated that ICA exerted no significant cytotoxic effects on bone marrow-derived mesenchymal stem cells (BMSCs) at concentrations below 50 μM. Treatment with 5 and 10 μM ICA markedly enhanced alkaline phosphatase (ALP) activity and Alizarin Red S (ARS) staining intensity, accompanied by a significant upregulation of osteogenic-related gene expression, collectively indicating that ICA effectively promoted osteogenic differentiation. Notably, we further observed that lncRNA HOTTIP expression was upregulated in a dose-dependent manner following ICA treatment, and the pro-osteogenic effects of ICA were effectively abolished in HOTTIP-depleted MSCs. In addition, the expression level of β-catenin, a core component of the canonical Wnt/β-catenin signaling pathway, was markedly reduced in HOTTIP-depleted MSCs. While ICA significantly promoted β-catenin expression in MSCs from wild-type (WT) mice, this stimulatory effect was absent in HOTTIP-deficient MSCs. Taken together, these findings indicated that ICA promoted osteogenic differentiation of MSCs through the upregulation of β-catenin signaling via enhanced expression of lncRNA HOTTIP, thereby revealing a previously unrecognized lncRNA-mediated epigenetic mechanism underlying ICA-induced osteogenesis.

Key words: Icariin, LncRNA HOTTIP, Bone marrow mesenchymal stem cells, Osteogenic differentiation, Wnt/β-catenin

Supporting:

邵将, 张锦芳, 张雅歌, 詹冬香. 淫羊藿苷通过增强HOTTIP表达促进间充质干细胞成骨分化[J]. 中国药学(英文版), 2026, 35(6): 538-551.

Jiang Shao, Jinfang Zhang, Yage Zhang, Dongxiang Zhan. Icariin promotes osteogenic differentiation of mesenchymal stem cells through upregulating HOTTIP[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(6): 538-551.

图/表 7

Table 1. Primer sequences.

Figure 1. Characterization of mouse BMSCs. (A−B) Phase-contrast microscopy of cultured BMSCs at day 3 post-isolation (A) and at day 5 post-isolation (B). (C−D) Morphological characteristics of BMSCs at primary passage (P1) (C) and at passage 3 (P3) (D). (E) Flow cytometric analysis of standard MSCs surface markers (e.g., CD90, CD44, CD34, CD45).

Figure 2. The effects of ICA on the proliferation of BMSCs. (A) Representative photographic image of ICA. (B) Chemical structural formula of ICA. (C) Three-dimensional (3D) molecular structure of ICA. (D−F) The proliferative effects of ICA at different concentrations on BMSCs were evaluated using the MTT assay at 24-, 48-, and 72-h post-treatment. All results are presented as fold change relative to the DMSO-treated control group. All experiments were performed in triplicate, and data were presented as means ± SD, n = 3 per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. ICA promotes osteogenic differentiation of BMSCs. (A&B) Representative photomicrographs of ALP staining and quantitative measurement of ALP activity in BMSCs treated with ICA. (C&D) Representative images of ARS-stained calcium nodules with ICA treatment (C), and quantitative assessment of ARS staining intensity (D). (E) qRT-PCR examination of osteogenesis-related genes’ expression in ICA-treated BMSCs. All results are presented as fold change relative to the DMSO-treated control group. All results are relative to the control. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. HOTTIP is regulated by ICA in BMSCs. (A) qRT-PCR was employed to quantify HOTTIP expression levels in BMSCs treated with ICA. (B) The dynamic expression of HOTTIP was monitored throughout the osteogenic differentiation process of BMSCs. The dynamic expression of the HOTTIP gene was monitored on days 2, 4, 6, and 8 during the osteogenic differentiation process of BMSCs. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. HOTTIP depletion reverses the effects of ICA on osteogenic differentiation of BMSCs. (A−B) Representative photomicrographs of ALP staining and quantitative measurement of ALP activity in BMSCs derived from HOTTIP G-KO mice treated with ICA. (C−D) Representative images of ARS-stained calcium nodules and quantification of calcium deposition in ICA-treated BMSCs. (E) The expression of osteogenic marker genes in ICA-treated BMSCs was examined by qRT-PCR. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. ICA promotes osteogenic differentiation through HOTTIP/β-catenin axis. (A) Western blotting analysis of β-catenin expression in BMSCs derived from WT and HOTTIP G-KO mice. (B) Western blotting analysis of β-catenin expression in BMSCs derived from WT mice subjected to ICA intervention during osteogenic induction. (C) Western blotting analysis of β-catenin expression in BMSCs derived from HOTTIP G-KO mice subjected to ICA intervention during osteogenic induction.

参考文献 38

[1]	Baccaro, L.F.; Conde, D.; Costa-Paiva, L.; Pinto-Neto, A.M. The epidemiology and management of postmenopausal osteoporosis: a viewpoint from Brazil. Clin. Interv. Aging. 2015, 583.
[2]	Kostenuik, P.; Mirza, F.M. Fracture healing physiology and the quest for therapies for delayed healing and nonunion. J. Orthop. Res. 2017, 35, 213–223.
[3]	Lerner, U.H.; Kindstedt, E.; Lundberg, P. The critical interplay between bone resorbing and bone forming cells. J. Clin. Periodontol. 2019, 46, 33–51.
[4]	Runyan, C.M.; Gabrick, K.S. Biology of bone formation, fracture healing, and distraction osteogenesis. J. Craniofacial Surg. 2017, 28, 1380–1389.
[5]	Zhang, X.Z.; Du, X.M.; Xiang, J.; Shu, Q.F.; Huang, H.Y.; Guo, Y.; Wang, T.; Hu, Y.M.; He, J.Z. Effective treatment of non-union after internal fixation of femoral neck fracture using integrated traditional Chinese and western medicine. Explore. 2025, 21, 103095.
[6]	Hu, S.J.; Chen, G.C.; Wang, F.Y.; Fang, Y.Q.; Wang, S.Q.; Song, Z.L.; Zhao, Z.H.; Zhang, Q.L.; Meng, X.Y.; Zhang, Q.Y.; Qin, L.P. Network pharmacology analysis uncovers the mechanism of Shudihuang-Shanzhuyu herb pair in prevention and treatment of diabetic osteoporosis via PI3K/AKT pathway. J. Ethnopharmacol. 2025, 345, 119581.
[7]	Li, B.; Lima, M.R.M.; Nie, Y.H.; Xu, L.; Liu, X.; Yuan, H.C.; Chen, C.; Dias, A.C.; Zhang, X.Y. HPLC-DAD fingerprints combined with multivariate analysis of epimedii folium from major producing areas in eastern Asia: effect of geographical origin and species. Front. Pharmacol. 2021, 12, 761551.
[8]	Xiao, Y.P.; Zeng, J.; Jiao, L.N.; Xu, X.Y. Review for treatment effect and signaling pathway regulation of kidney-tonifying traditional Chinese medicine on osteoporosis. China J. Chin. Mater. Med. 2018, 43, 21–30.
[9]	Ma, Z.T.; Tang, X.F.; Chen, Y.H.; Wang, H.; Li, Y.M.; Long, Y.T.; Liu, R.H. Epimedii folium and ligustri lucidi fructus promote osteoblastogenesis and inhibit osteoclastogenesis against osteoporosis via acting on osteoblast-osteoclast communication. Oxid. Med. Cell Longev. 2023, 2023, 7212642.
[10]	Ding, S.C.; Shi, C.J.; Pang, F.X.; Wen, R.J.; Li, N.; Mai, Y.X.; Zhou, S.T.; Zhang, J.F. HOTTIP suppresses ferroptosis via mediating DGCR8/miR-214-3p/GPX4 regulatory axis in osteosarcoma. Oncol. Rep. 2025, 54, 1–13.
[11]	Li, Z.P.; Mai, Y.X.; Zhou, S.T.; Shi, C.J.; Shao, J.; Liang, P.P.; Liang, W.C.; Zhang, J.F. Long noncoding RNA hottip maintained skeletal homeostasis via suppressing the enhancer of zeste homolog 2 (Ezh2)/histone methylation regulatory axis. Non Coding RNA Res. 2025, 12, 141–151.
[12]	Abdallah, B.M.; Khattab, H.M. Recent approaches to isolating and culturing mouse bone marrowderived mesenchymal stromal stem cells. Curr. Stem Cell Res. Ther. 2021, 16, 599–607.
[13]	Smajilagić, A.; Aljičević, M.; Redžić, A.; Filipović, S.; Lagumdžija, A.C. Rat bone marrow stem cells isolation and culture as a bone formative experimental system. Bosnian J. Basic Med. Sci. 2013, 13, 27.
[14]	Kisiel, A.H.; McDuffee, L.A.; Masaoud, E.; Bailey, T.R.; Esparza Gonzalez, B.P.; Nino-Fong, R. Isolation, characterization, and in vitro proliferation of canine mesenchymal stem cells derived from bone marrow, adipose tissue, muscle, and periosteum. Am. J. Vet. Res. 2012, 73, 1305–1317.
[15]	Ye, J.; Gong, P.; Zhou, F.J.; Li, G.D.; Ye, C.; Sung, H.; Mo, A.C. Culture and identification of human bone marrow mesenchymal stem cells from alveolar ridge dental implant site. J. Craniofacial Surg. 2013, 24, 1539–1543.
[16]	Mai, Y.X.; Li, Z.P.; Pang, F.X.; Zhou, S.T.; Li, N.; Wang, Y.Y.; Zhang, J.F. Aucubin promotes osteogenic differentiation and facilitates bone formation through the lncRNA-H19 driven Wnt/β-catenin signaling regulatory axis. Stem Cells Int. 2024, 2024, 5388064.
[17]	Pan, F.F.; Shao, J.; Shi, C.J.; Li, Z.P.; Fu, W.M.; Zhang, J.F. Apigenin promotes osteogenic differentiation of mesenchymal stem cells and accelerates bone fracture healing via activating Wnt/β-catenin signaling. Am. J. Physiol. Endocrinol. Metab. 2021, 320, E760–E771.
[18]	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. 2021, 2, e580–e592.
[19]	Khan, S.; Peracha, A.; Abro, A.A.; Sufyan, M.; Najjad, M.K.R.; Aziz, S.; Khan, M.M. Clinical studies investigating the role of mesenchymal stem cells in healing of fracture non-unions: a systematic review. J. Pak. Med. Assoc. 2023, 73, S26–S31.
[20]	Wang, D.L.; Fang, F.F.; Liu, C.S.; Su, J.C. Expert consensus on integrated traditional Chinese and western medicine diagnosis and treatment for osteoporotic fractures. China J. Orthop. Trauma. 2024, 37, 242–250.
[21]	Chen, H.; Xue, C.Y.; Chen, S.; Chen, Z.T.; Tang, T.; Liu, X.; Xi, Z.P.; Kang, R.; Xie, L. Cervical spondylosis: innovative understanding from traditional Chinese medicine and treatment by classic formulas. China J. Chin. Mater. Med. 2025, 50, 2596–2604.
[22]	Jiao, Y.; Wang, X.; Wang, Q.; Geng, Q.S.; Cao, X.X.; Zhang, M.X.; Zhao, L.; Deng, T.T.; Xu, Y.; Xiao, C. Mechanisms by which kidney-tonifying Chinese herbs inhibit osteoclastogenesis: Emphasis on immune cells. Front. Pharmacol. 2023, 14, 1077796.
[23]	Jiang, J.; He, J.J.; Xiao, S.C.; Shenyuan, J.Y.; Chen, T.; Pei, D. Screening of superior anti-osteoporotic flavonoids from Epimedii Folium with dual effects of reversing iron overload and promoting osteogenesis. Biomed. Chromatogr. 2023, 37, e5686.
[24]	Zhang, X.Y.; Chen, Y.P.; Zhang, C.; Zhang, X.; Xia, T.; Han, J.; Song, S.L.; Xu, C.H.; Chen, F. Effects of icariin on the fracture healing in young and old rats and its mechanism. Pharm. Biol. 2021, 59, 1243–1253.
[25]	Fan, J.J.; Cao, L.G.; Wu, T.; Wang, D.X.; Jin, D.; Jiang, S.; Zhang, Z.Y.; Bi, L.; Pei, G.X. The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells. Molecules. 2011, 16, 10123–10133.
[26]	Bridges, M.C.; Daulagala, A.C.; Kourtidis, A. LNCcation: lncRNA localization and function. J. Cell Biol. 2021, 220, e202009045.
[27]	Herman, A.B.; Tsitsipatis, D.; Gorospe, M. Integrated lncRNA function upon genomic and epigenomic regulation. Mol. Cell. 2022, 82, 2252–2266.
[28]	Pierce, J.B.; Zhou, H.Y.; Simion, V.; Feinberg, M.W. Long noncoding RNAs as therapeutic targets. Long Noncoding RNA. Cham: Springer International Publishing. 2022, 161–175.
[29]	Lanzillotti, C.; De Mattei, M.; Mazziotta, C.; Taraballi, F.; Rotondo, J.C.; Tognon, M.; Martini, F. Long non-coding RNAs and microRNAs interplay in osteogenic differentiation of mesenchymal stem cells. Front. Cell Dev. Biol. 2021, 9, 646032.
[30]	Wierzbicki, A.T. The role of long non-coding RNA in transcriptional gene silencing. Curr. Opin. Plant Biol. 2012, 15, 517–522.
[31]	Yang, Y.; Wang, Y.J.; Wang, F.; Yuan, L.H.; Guo, Z.Q.; Wei, Z.C.; Wang, Z.R.; Wang, S.W. The roles of miRNA, lncRNA and circRNA in the development of osteoporosis. Biol. Res. 2020, 53, 40.
[32]	El-Sisi, M.G.; Radwan, S.M.; Ali, S.S.; Mostafa, M.Y.; Hamdy, N.M. The long antisense non-coding RNA hoxa transcript at the distal tip (LncRNA hottip) in health and disease: a comprehensive review and in silico analysis. Naunyn Schmiedeberg's Arch. Pharmacol. 2025, 398, 16537–16575.
[33]	Yao, X.Y.; Liu, J.F.; Luo, Y.; Xu, X.Z.; Bu, J. LncRNA HOTTIP facilitates cell proliferation, invasion, and migration in osteosarcoma by interaction with PTBP1 to promote KHSRP level. Cell Cycle. 2021, 20, 283–297.
[34]	Gopinathan, G.; Zhang, X.M.; Luan, X.H.; Diekwisch, T.G.H. Changes in hox gene chromatin organization during odontogenic lineage specification. Genes. 2023, 14, 198.
[35]	Wang, G.; Xu, Y.L.; Zhang, X.H.; Tang, L.; Li, Y. LncRNA HOTTIP regulates TLR4 promoter methylation by recruiting H3K4 methyltransferase MLL1 to affect apoptosis and inflammatory response of fibroblast-like synoviocyte in rheumatoid arthritis. Kaohsiung J. Med. Sci. 2024, 40, 335–347.
[36]	Li, Z.H.; Zhao, X.H.; Zhou, Y.; Liu, Y.M.; Zhou, Q.B.; Ye, H.L.; Wang, Y.X.; Zeng, J.L.; Song, Y.D.; Gao, W.C.; Zheng, S.Y.; Zhuang, B.X.; Chen, H.M.; Li, W.Z.; Li, H.G.; Li, H.F.; Fu, Z.Q.; Chen, R.F. The long non-coding RNA HOTTIP promotes progression and gemcitabine resistance by regulating HOXA13 in pancreatic cancer. J. Transl. Med. 2015, 13, 84.
[37]	Wang, F.; Tang, Z.Q.; Shao, H.L.; Guo, J.; Tan, T.; Dong, Y.; Lin, L.B. Long noncoding RNA HOTTIP cooperates with CCCTC-binding factor to coordinate HOXA gene expression. Biochem. Biophys. Res. Commun. 2018, 500, 852–859.
[38]	Xiong, L.; Tang, Y.; Tang, J.; Liu, Z.Y.; Wang, X.Z. Downregulation of lncRNA HOTTIP suppresses the proliferation, migration, and invasion of oral tongue squamous cell carcinoma by regulation of HMGA2-mediated Wnt/β-catenin pathway. Cancer Biother. Radiopharm. 2020, 35, 720–730.

淫羊藿苷通过增强HOTTIP表达促进间充质干细胞成骨分化

Icariin promotes osteogenic differentiation of mesenchymal stem cells through upregulating HOTTIP

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