Metabolomics analysis of femoral tissue reveals the impact of Anemarrhenae Rhizoma on metabolic pathways in osteoporotic rats

doi:10.5246/jcps.2025.02.011

Abstract

Abstract:

This study aimed to assess the therapeutic potential of Anemarrhenae Rhizoma (AR) in osteoporotic rats and to elucidate the metabolic pathways involved in AR’s role in alleviating osteoporosis (OP). OP was induced in rats through ovariectomy (OVX), followed by oral administration of either high or low doses of AR, as well as estradiol valerate, over a 14-week period. Micro-computed tomography (Micro-CT) was employed to examine the femur tissue morphology, while enzyme-linked immunosorbent assay (ELISA) was adopted to measure serum levels of PINP and CTX-I to evaluate AR’s efficacy in treating OP. Additionally, metabolomic profiling of femur tissues was conducted using gas chromatography-mass spectrometry (GC-MS). The bioactive components of AR, along with its therapeutic targets for OP, were identified through UPLC-MS/MS and online database searches, and metabolic networks were established by integrating differential metabolites and potential targets. Furthermore, Western blotting analysis confirmed key molecular targets. The findings revealed that AR treatment significantly mitigated OVX-induced OP in rats. Metabolomic analysis indicated that AR exerted its effects by modulating the levels of 10 key metabolites (such as linoleic acid and inositol) and influencing five crucial metabolic pathways, including linoleic acid metabolism and the phosphoinositide signaling system. Among these, the linoleic acid metabolic pathway emerged as a pivotal focus for further investigation based on the constructed interaction network of differential metabolites and targets. Western blotting analysis demonstrated that AR reversed the up-regulation of CYP1A2 and CYP2C9, two targets associated with the linoleic acid metabolic pathway, in OP rats. In conclusion, AR appeared to ameliorate OP by modulating metabolite levels in OVX rats, with its mechanism of action likely centered on regulating the linoleic acid metabolic pathway.

Key words: Anemarrhenae Rhizoma, Osteoporosis, Rats, Metabolomics

Supporting:

Yuxin Wen, Qi Jiang, Zhuang Huang, Pengyu Chen, Xing Hong, Qiong Wang, Tao Huang, Lintao Han. Metabolomics analysis of femoral tissue reveals the impact of Anemarrhenae Rhizoma on metabolic pathways in osteoporotic rats[J]. Journal of Chinese Pharmaceutical Sciences, 2025, 34(2): 135-149.

Figures/Tables 6

References 34

[1]	Gopinath, V. Osteoporosis. Med. Clin. North Am. 2023, 107, 213–225.
[2]	Liu, Y.L.; Feng, Y.C.; Dang, X.; Liu, F.X.; Lin, Z.X.; Zhang, Y.K.; Che, Z.Y. Immune cell-mediated inflammation and osteoporosis: Mechanisms and pathways. Chin. J. Osteoporosis. 2024, 30, 863–868.
[3]	Qin, D.P.; Zhang, X.G.; Song, M.; Zhang, H.; Cao, L.Z.; Gao, G.D.; Zhao, W.T.; Wang, Z.P.; Xu, B.; Xu, S.W. Discussion on the prevention and treatment strategy of sarcopenia and osteoporosis with traditional Chinese medicine based on the theory of muscles and bones. China J. Tradit. Chin. Med. Pharm. 2019, 34, 4364–4369.
[4]	Lee, S.; Kim, G.J.; Kwon, H.; Nam, J.W.; Baek, J.Y.; Shim, S.H.; Choi, H.; Kang, K.S. Estrogenic effects of extracts and isolated compounds from belowground and aerial parts of Spartina anglica. Mar. Drugs. 2021, 19, 210.
[5]	Hagino, H.; Sugimoto, T.; Tanaka, S.; Sasaki, K.; Sone, T.; Nakamura, T.; Soen, S.; Mori, S. A randomized, controlled trial of once-weekly teriparatide injection versus alendronate in patients at high risk of osteoporotic fracture: primary results of the Japanese osteoporosis intervention trial-05. Osteoporos. Int. 2021, 32, 2301–2311.
[6]	Zeytinoglu, M.; Naaman, S.C.; Dickens, L.T. Denosumab discontinuation in patients treated for low bone density and osteoporosis. Endocrinol. Metab. Clin. N Am. 2021, 50, 205–222.
[7]	Duan, Y.; Su, Y.T.; Ren, J.; Zhou, Q.; Tang, M.; Li, J.; Li, S.X. Kidney tonifying traditional Chinese medicine: potential implications for the prevention and treatment of osteoporosis. Front. Pharmacol. 2023, 13, 1063899.
[8]	Wang, Y.L.; Dan, Y.; Yang, D.W.; Hu, Y.L.; Zhang, L.; Zhang, C.H.; Zhu, H.; Cui, Z.H.; Li, M.H.; Liu, Y.Z. The genus Anemarrhena Bunge: a review on ethnopharmacology, phytochemistry and pharmacology. J. Ethnopharmacol. 2014, 153, 42–60.
[9]	Lee, J.S.; Kim, M.H.; Lee, H.; Yang, W.M. Anemarrhena asphodeloides Bunge ameliorates osteoporosis by suppressing osteoclastogenesis. Int. J. Mol. Med. 2018, 42, 3613–3621.
[10]	Wang, N.N.; Xu, P.C.; Wang, X.P.; Yao, W.X.; Wang, B.J.; Wu, Y.Z.; Shou, D. Timosaponin AIII attenuates inflammatory injury in AGEs-induced osteoblast and alloxan-induced diabetic osteoporosis zebrafish by modulating the RAGE/MAPK signaling pathways. Phytomedicine. 2020, 75, 153247.
[11]	Wang, N.N.; Xu, P.C.; Wu, R.J.; Wang, X.P.; Wang, Y.J.; Shou, D.; Zhang, Y. Timosaponin BII improved osteoporosis caused by hyperglycemia through promoting autophagy of osteoblasts via suppressing the mTOR/NFκB signaling pathway. Free. Radic. Biol. Med. 2021, 171, 112–123.
[12]	Wilson, I.D.; Plumb, R.; Granger, J.; Major, H.; Williams, R.; Lenz, E.M. HPLC-MS-based methods for the study of metabonomics. J. Chromatogr. B. 2005, 817, 67–76.
[13]	Cao, H.X.; Zhang, A.H.; Zhang, H.M.; Sun, H.; Wang, X.J. The application of metabolomics in traditional Chinese medicine opens up a dialogue between Chinese and Western medicine. Phytother. Res. 2015, 29, 159–166.
[14]	Cao, S.; Han, L.T.; Li, Y.M.; Yao, S.Q.; Hou, S.H.; Ma, S.S.; Dai, W.Q.; Li, J.J.; Zhou, Z.X.; Wang, Q.; Huang, F. Integrative transcriptomics and metabolomics analyses provide hepatotoxicity mechanisms of asarum. Exp. Ther. Med. 2020, 20, 1359–1370.
[15]	Huang, P.; Duan, B.L.; Li, D.S.; Duan, Y.F.; Zhou, Z.X.; Han, L.T.; Li, J.J.; Wu, J.J.; Ye, Y.; Zhang, F.Y.; Guo, Z.W.; Wang, Q.; Huang, F. Transcriptomics and metabolomics revealed the pulmonary protective mechanism of Xixin-Ganjiang Herb Pair for warming the lungs to dissolve phlegm in COPD rats. J. Chromatogr. B. 2023, 1224, 123665.
[16]	Wang, Z.Y.; Cai, J.F.; Fu, Q.; Cheng, L.P.; Wu, L.H.; Zhang, W.Y.; Zhang, Y.; Jin, Y.; Zhang, C.Z. Anti-inflammatory activities of compounds isolated from the rhizome of Anemarrhena asphodeloides. Molecules. 2018, 23, 2631.
[17]	Shan, L.L.; Wu, Y.Y.; Yuan, L.; Zhang, Y.N.; Xu, Y.Y.; Li, Y.B. Rapid screening of chemical constituents in Rhizoma anemarrhenae by UPLC-Q-TOF/MS combined with data postprocessing techniques. Evid. Based Complement. Alternat. Med. 2017, 2017, 4032820.
[18]	Sun, Y. Ph.D. 000034, Shenyang Pharmaceutical University. 2015.
[19]	Cao, X.T.; Shang, Y.; Kong, W.G.; Jiang, S.Q.; Liao, J.; Dai, R.H. Flavonoids derived from Anemarrhenae Rhizoma ameliorate inflammation of benign prostatic hyperplasia via modulating COX/LOX pathways. J. Ethnopharmacol. 2022, 284, 114740.
[20]	Selvaraj, V.; Sekaran, S.; Dhanasekaran, A.; Warrier, S. Type 1 collagen: Synthesis, structure and key functions in bone mineralization. Differentiation. 2024, 136, 100757.
[21]	Bhattoa, H.P.; Cavalier, E.; Eastell, R.; Heijboer, A.C.; Jørgensen, N.R.; Makris, K.; Ulmer, C.Z.; Kanis, J.A.; Cooper, C.; Silverman, S.L.; Vasikaran, S.D. Analytical considerations and plans to standardize or harmonize assays for the reference bone turnover markers PINP and β-CTX in blood. Clin. Chim. Acta. 2021, 515, 16–20.
[22]	Eastell, R.; Szulc, P. Use of bone turnover markers in postmenopausal osteoporosis. Lancet Diabetes Endocrinol. 2017, 5, 908–923.
[23]	Ensrud, K.E.; Crandall, C.J. Osteoporosis. Ann. Intern. Med. 2018, 168, 306–307.
[24]	Palumbo, C.; Ferretti, M. The osteocyte: from "prisoner" to "orchestrator". J. Funct. Morphol. Kinesiol. 2021, 6, 28.
[25]	Zhou, X.C.; Cao, H.; Guo, J.M.; Yuan, Y.; Ni, G.X. Effects of BMSC-derived EVs on bone metabolism. Pharmaceutics. 2022, 14, 1012.
[26]	Yousefzadeh, N.; Kashfi, K.; Jeddi, S.; Ghasemi, A. Ovariectomized rat model of osteoporosis: a practical guide. EXCLI J. 2020, 19, 89–107.
[27]	Huang, Y.; Bo, Y.H.; Wu, X.; Wang, Q.Y.; Qin, F.; Zhao, L.S.; Xiong, Z.L. An intergated serum and urinary metabonomic research based on UPLC-MS and therapeutic effects of Gushudan on prednisolone-induced osteoporosis rats. J. Chromatogr. B. 2016, 1027, 119–130.
[28]	Ma, B.; Liu, J.N.; Zhang, Q.; Ying, H.J.; Jiye, A.; Sun, J.G.; Wu, D.; Wang, Y.L.; Li, J.; Liu, Y.H. Metabolomic profiles delineate signature metabolic shifts during estrogen deficiency-induced bone loss in rat by GC-TOF/MS. PLoS One. 2013, 8, e54965.
[29]	Lin, G.L.; Wang, H.; Dai, J.; Li, X.; Guan, M.; Gao, S.T.; Ding, Q.; Wang, H.X.; Fang, H. Conjugated linoleic acid prevents age-induced bone loss in mice by regulating both osteoblastogenesis and adipogenesis. Biochem. Biophys. Res. Commun. 2017, 490, 813–820.
[30]	Rahman, M.M.; Fernandes, G.; Williams, P. Conjugated linoleic Acid prevents ovariectomy-induced bone loss in mice by modulating both osteoclastogenesis and osteoblastogenesis. Lipids. 2014, 49, 211–224.
[31]	Gougis, P.; Hilmi, M.; Geraud, A.; Mir, O.; Funck-Brentano, C. Potential cytochrome P450-mediated pharmacokinetic interactions between herbs, food, and dietary supplements and cancer treatments. Crit. Rev. Oncol. Hematol. 2021, 166, 103342.
[32]	Baldwin, W.S. Phase 0 of the xenobiotic response: nuclear receptors and other transcription factors as a first step in protection from xenobiotics. Nucl. Receptor Res. 2019, 6, 101447.
[33]	Bui, P.; Imaizumi, S.; Beedanagari, S.R.; Reddy, S.T.; Hankinson, O. Human CYP2S1 metabolizes cyclooxygenase- and lipoxygenase-derived eicosanoids. Drug Metab. Dispos. 2011, 39, 180–190.
[34]	Lecka-Czernik, B.; Moerman, E.J.; Grant, D.F.; Lehmann, J.M.; Manolagas, S.C.; Jilka, R.L. Divergent effects of selective peroxisome proliferator-activated receptor-gamma 2 ligands on adipocyte versus osteoblast differentiation. Endocrinology. 2002, 143, 2376–2384.