{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T23:30:27Z","timestamp":1785799827273,"version":"3.56.0"},"reference-count":50,"publisher":"MDPI AG","issue":"13","license":[{"start":{"date-parts":[[2023,6,30]],"date-time":"2023-06-30T00:00:00Z","timestamp":1688083200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Youth Project of Shandong Provincial Natural Science Foundation","award":["ZR2020QH330"],"award-info":[{"award-number":["ZR2020QH330"]}]},{"name":"Youth Project of Shandong Provincial Natural Science Foundation","award":["2020M054"],"award-info":[{"award-number":["2020M054"]}]},{"name":"Traditional Chinese Medicine Science and Technology Project of Shandong Province","award":["ZR2020QH330"],"award-info":[{"award-number":["ZR2020QH330"]}]},{"name":"Traditional Chinese Medicine Science and Technology Project of Shandong Province","award":["2020M054"],"award-info":[{"award-number":["2020M054"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Molecules"],"abstract":"<jats:p>Neurofibromatosis type 1 is a rare autosomal dominant genetic disorder, with up to 50% of patients clinically displaying skeletal defects. Currently, the pathogenesis of bone disorders in NF1 patients is unclear, and there are no effective preventive and treatment measures. In this study, we found that knockout of the NF1 gene reduced cAMP levels and osteogenic differentiation in an osteoblast model, and icariin activated the cAMP\/PKA\/CREB pathway to promote osteoblast differentiation of the NF1 gene knockout cell model by increasing intracellular cAMP levels. The PKA selective inhibitor H89 significantly impaired the stimulatory effect of icariin on osteogenesis in the NF1 cell model. In this study, an osteoblast model of NF1 was successfully constructed, and icariin was applied to the cell model for the first time. The results will help to elucidate the molecular mechanism of NF1 bone disease and provide new ideas for the clinical prevention and treatment of NF1 bone disease and drug development in the future.<\/jats:p>","DOI":"10.3390\/molecules28135128","type":"journal-article","created":{"date-parts":[[2023,7,3]],"date-time":"2023-07-03T00:53:16Z","timestamp":1688345596000},"page":"5128","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":10,"title":["Icariin Promotes Osteogenic Differentiation in a Cell Model with NF1 Gene Knockout by Activating the cAMP\/PKA\/CREB Pathway"],"prefix":"10.3390","volume":"28","author":[{"given":"Meng","family":"Chen","sequence":"first","affiliation":[{"name":"Shandong Center for Disease Control and Prevention, Jinan 250014, China"},{"name":"Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China"},{"name":"Shandong Qidu Pharmaceutical Co., Ltd., Shandong Provincial Key Laboratory of Neuroprotective Drugs, Zibo 255400, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lianhua","family":"Lu","sequence":"additional","affiliation":[{"name":"Shandong Center for Disease Control and Prevention, Jinan 250014, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dong","family":"Cheng","sequence":"additional","affiliation":[{"name":"Shandong Center for Disease Control and Prevention, Jinan 250014, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jing","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shandong Center for Disease Control and Prevention, Jinan 250014, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7302-2214","authenticated-orcid":false,"given":"Xinyong","family":"Liu","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong University, Jinan 250012, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianli","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shandong Qidu Pharmaceutical Co., Ltd., Shandong Provincial Key Laboratory of Neuroprotective Drugs, Zibo 255400, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tianliang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Shandong Center for Disease Control and Prevention, Jinan 250014, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,6,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Ge, L.-L., Xing, M.-Y., Zhang, H.-B., and Wang, Z.-C. (2022). Neurofibroma Development in Neurofibromatosis Type 1: Insights from Cellular Origin and Schwann Cell Lineage Development. Cancers, 14.","DOI":"10.3390\/cancers14184513"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"2327","DOI":"10.1002\/ajmg.a.33045","article-title":"Skeletal abnormalities in neurofibromatosis type 1: Approaches to therapeutic options","volume":"149A","author":"Elefteriou","year":"2009","journal-title":"Am. J. Med. Genet. A"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1002\/jbmr.2316","article-title":"Combined MEK inhibition and BMP2 treatment promotes osteoblast differentiation and bone healing in Nf1Osx -\/- mice","volume":"30","author":"Stevens","year":"2015","journal-title":"J. Bone Miner. Res."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Kaspiris, A., Savvidou, O.D., Vasiliadis, E.S., Hadjimichael, A.C., Melissaridou, D., Iliopoulou-Kosmadaki, S., Iliopoulos, I.D., Papadimitriou, E., and Chronopoulos, E. (2022). Current Aspects on the Pathophysiology of Bone Metabolic Defects during Progression of Scoliosis in Neurofibromatosis Type 1. J. Clin. Med., 11.","DOI":"10.3390\/jcm11020444"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"874","DOI":"10.1093\/hmg\/ddm032","article-title":"Multiple roles for neurofibromin in skeletal development and growth","volume":"16","author":"Kolanczyk","year":"2007","journal-title":"Hum. Mol. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1186\/s13023-021-01773-w","article-title":"Hyperpigmented spots at fundus examination: A new ocular sign in Neurofibromatosis Type I","volume":"16","author":"Moramarco","year":"2021","journal-title":"Orphanet. J. Rare Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"290","DOI":"10.1038\/nrc3911","article-title":"A RASopathy gene commonly mutated in cancer: The neurofibromatosis type 1 tumour suppressor","volume":"15","author":"Ratner","year":"2015","journal-title":"Nat. Rev. Cancer"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"5758","DOI":"10.1038\/s41467-021-26031-2","article-title":"Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling","volume":"12","author":"Stickley","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_9","first-page":"297","article-title":"Neurofibromin in neurofibromatosis type 1\u2014Mutations in NF1gene as a cause of disease","volume":"18","author":"Abramowicz","year":"2014","journal-title":"Dev. Period Med."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"325","DOI":"10.1007\/s00403-011-1191-3","article-title":"Bone mineral metabolism in patients with neurofibromatosis type 1 (von Recklingausen disease)","volume":"304","author":"Petramala","year":"2012","journal-title":"Arch Dermatol. Res."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"116060","DOI":"10.1016\/j.bone.2021.116060","article-title":"RASopathies: The musculoskeletal consequences and their etiology and pathogenesis","volume":"152","author":"Fowlkes","year":"2021","journal-title":"Bone"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"259","DOI":"10.1136\/jmg.2008.061895","article-title":"Bone health and fracture rate in individuals with neurofibromatosis 1 (NF1)","volume":"46","author":"Tucker","year":"2009","journal-title":"J. Med. Genet."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"697","DOI":"10.1203\/PDR.0b013e31816fee45","article-title":"Evidence of increased bone resorption in neurofibromatosis type 1 using urinary pyridinium crosslink analysis","volume":"63","author":"Stevenson","year":"2008","journal-title":"Pediatr. Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"230","DOI":"10.4055\/cios.2011.3.3.230","article-title":"Disturbed osteoblastic differentiation of fibrous hamartoma cell from congenital pseudarthrosis of the tibia associated with neurofibromatosis type I","volume":"3","author":"Lee","year":"2011","journal-title":"Clin. Orthop. Surg."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"983","DOI":"10.1359\/JBMR.040130","article-title":"NF1 tumor suppressor protein and mRNA in skeletal tissues of developing and adult normal mouse and NF1-deficient embryos","volume":"19","author":"Kuorilehto","year":"2004","journal-title":"J. Bone Miner. Res."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"2837","DOI":"10.1093\/hmg\/ddl208","article-title":"Neurofibromin plays a critical role in modulating osteoblast differentiation of mesenchymal stem\/progenitor cells","volume":"15","author":"Wu","year":"2006","journal-title":"Hum. Mol. Genet."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"441","DOI":"10.1016\/j.cmet.2006.10.010","article-title":"ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae","volume":"4","author":"Elefteriou","year":"2006","journal-title":"Cell Metab."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"889","DOI":"10.3892\/ijmm.2012.1079","article-title":"Icariin promotes bone formation via the BMP-2\/Smad4 signal transduction pathway in the hFOB 1.19 human osteoblastic cell line","volume":"30","author":"Liang","year":"2012","journal-title":"Int. J. Mol. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2514","DOI":"10.1016\/j.biochi.2012.06.033","article-title":"Herba epimedii flavonoids suppress osteoclastic differentiation and bone resorption by inducing G2\/M arrest and apoptosis","volume":"94","author":"Zhang","year":"2012","journal-title":"Biochimie"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"20883","DOI":"10.1074\/jbc.M117.809517","article-title":"The flavonol glycoside icariin promotes bone formation in growing rats by activating the cAMP signaling pathway in primary cilia of osteoblasts","volume":"292","author":"Shi","year":"2017","journal-title":"J. Biol. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"928","DOI":"10.1007\/s00198-004-1801-4","article-title":"Decreased bone mineral density and content in neurofibromatosis type 1: Lowest local values are located in the load-carrying parts of the body","volume":"16","author":"Kuorilehto","year":"2005","journal-title":"Osteoporos. Int."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1038\/270725a0","article-title":"Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle","volume":"270","author":"Yaffe","year":"1977","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"591","DOI":"10.1016\/j.bbrc.2019.03.097","article-title":"MST2 kinase regulates osteoblast differentiation by phosphorylating and inhibiting Runx2 in C2C12 cells","volume":"512","author":"Won","year":"2019","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1016\/j.phymed.2018.03.019","article-title":"Limonene promotes osteoblast differentiation and 2-deoxy-d-glucose uptake through p38MAPK and Akt signaling pathways in C2C12 skeletal muscle cells","volume":"45","author":"Soundharrajan","year":"2018","journal-title":"Phytomedicine"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Hidaka, Y., Chiba-Ohkuma, R., Karakida, T., Onuma, K., Yamamoto, R., Fujii-Abe, K., Saito, M.M., Yamakoshi, Y., and Kawahara, H. (2020). Combined Effect of Midazolam and Bone Morphogenetic Protein-2 for Differentiation Induction from C2C12 Myoblast Cells to Osteoblasts. Pharmaceutics, 12.","DOI":"10.3390\/pharmaceutics12030218"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Chen, M., Cui, Y., Li, H., Luan, J., Zhou, X., and Han, J. (2019). Icariin Promotes the Osteogenic Action of BMP2 by Activating the cAMP Signaling Pathway. Molecules, 24.","DOI":"10.3390\/molecules24213875"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"10123","DOI":"10.3390\/molecules161210123","article-title":"The dose-effect of icariin on the proliferation and osteogenic differentiation of human bone mesenchymal stem cells","volume":"16","author":"Fan","year":"2011","journal-title":"Molecules"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"668","DOI":"10.1007\/s00774-017-0889-5","article-title":"Study of the osteogenesis effect of icariside II and icaritin on canine bone marrow mesenchymal stem cells","volume":"36","author":"Luo","year":"2018","journal-title":"J. Bone Miner. Metab."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"444","DOI":"10.1016\/j.bbrc.2008.02.054","article-title":"Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner","volume":"369","author":"Zhao","year":"2008","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1007\/s00223-016-0138-2","article-title":"Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway","volume":"99","author":"Zhang","year":"2016","journal-title":"Calcif. Tissue Int."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.biopha.2017.07.071","article-title":"Icaritin induces MC3T3-E1 subclone14 cell differentiation through estrogen receptor-mediated ERK1\/2 and p38 signaling activation","volume":"94","author":"Wu","year":"2017","journal-title":"Biomed. Pharmacother."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1633","DOI":"10.1016\/j.phymed.2014.07.016","article-title":"Synergistic inhibitory effect of Icariside II with Icaritin from Herba Epimedii on pre-osteoclastic RAW264.7 cell growth","volume":"21","author":"Liu","year":"2014","journal-title":"Phytomedicine"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"153680","DOI":"10.1016\/j.phymed.2021.153680","article-title":"Randomized, double-blind, placebo-controlled trial to examine the safety, pharmacokinetics and effects of Epimedium prenylflavonoids, on bone specific alkaline phosphatase and the osteoclast adaptor protein TRAF6 in post-menopausal women","volume":"91","author":"Yong","year":"2021","journal-title":"Phytomedicine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1385","DOI":"10.1002\/ptr.3733","article-title":"Icariin promotes extracellular matrix synthesis and gene expression of chondrocytes in vitro","volume":"26","author":"Zhang","year":"2012","journal-title":"Phytother. Res."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1647","DOI":"10.1016\/j.joca.2012.08.009","article-title":"Icariin: A potential promoting compound for cartilage tissue engineering","volume":"20","author":"Li","year":"2012","journal-title":"Osteoarthr. Cartil."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Qi, S., He, J., Zheng, H., Chen, C., and Lan, S. (2019). Icariin Prevents Diabetes-Induced Bone Loss in Rats by Reducing Blood Glucose and Suppressing Bone Turnover. Molecules, 24.","DOI":"10.3390\/molecules24101871"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1007","DOI":"10.1097\/GME.0000000000000201","article-title":"Icariin prevents ovariectomy-induced bone loss and lowers marrow adipogenesis","volume":"21","author":"Li","year":"2014","journal-title":"Menopause"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1007\/s12013-013-9533-8","article-title":"Icariin protects against glucocorticoid-induced osteoporosis in vitro and prevents glucocorticoid-induced osteocyte apoptosis in vivo","volume":"67","author":"Feng","year":"2013","journal-title":"Cell Biochem. Biophys."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"652317","DOI":"10.1155\/2013\/652317","article-title":"Icariin Augments Bone Formation and Reverses the Phenotypes of Osteoprotegerin-Deficient Mice through the Activation of Wnt\/\u03b2-Catenin-BMP Signaling","volume":"2013","author":"Li","year":"2013","journal-title":"Evid. Based Complement Alternat. Med."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/0014-5793(96)00137-8","article-title":"Phosphorylation of neurofibromatosis type 1 gene product (neurofibromin) by cAMP-dependent protein kinase","volume":"382","author":"Izawa","year":"1996","journal-title":"FEBS Lett."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"791","DOI":"10.1126\/science.276.5313.791","article-title":"Rescue of a Drosophila NF1 mutant phenotype by protein kinase A","volume":"276","author":"The","year":"1997","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"7281","DOI":"10.1073\/pnas.0711190105","article-title":"cAMP\/PKA pathway activation in human mesenchymal stem cells in vitro results in robust bone formation in vivo","volume":"105","author":"Siddappa","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"2399","DOI":"10.3892\/etm.2016.3177","article-title":"Intermittent parathyroid hormone (1-34) application regulates cAMP-response element binding protein activity to promote the proliferation and osteogenic differentiation of bone mesenchymal stromal cells, via the cAMP\/PKA signaling pathway","volume":"11","author":"Chen","year":"2016","journal-title":"Exp. Ther. Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"872","DOI":"10.1016\/j.bone.2009.01.370","article-title":"Parathyroid hormone enhances bone morphogenetic protein activity by increasing intracellular 3\u2032,5\u2032-cyclic adenosine monophosphate accumulation in osteoblastic MC3T3-E1 cells","volume":"44","author":"Nakao","year":"2009","journal-title":"Bone"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"1048","DOI":"10.1016\/j.cellsig.2016.04.014","article-title":"Communication of cAMP by connexin43 gap junctions regulates osteoblast signaling and gene expression","volume":"28","author":"Gupta","year":"2016","journal-title":"Cell Signal"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"558","DOI":"10.1089\/ten.tea.2011.0312","article-title":"Forskolin enhances in vivo bone formation by human mesenchymal stromal cells","volume":"18","author":"Doorn","year":"2012","journal-title":"Tissue Eng. Part A"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"396","DOI":"10.1016\/S8756-3282(02)00839-6","article-title":"A phosphodiesterase inhibitor, pentoxifylline, enhances the bone morphogenetic protein-4 (BMP-4)-dependent differentiation of osteoprogenitor cells","volume":"31","author":"Tsutsumimoto","year":"2002","journal-title":"Bone"},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"2487","DOI":"10.1002\/glia.23500","article-title":"Polycomb repression regulates Schwann cell proliferation and axon regeneration after nerve injury","volume":"66","author":"Ma","year":"2018","journal-title":"Glia"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"54","DOI":"10.1177\/1073858410365562","article-title":"Ras and Rap signaling in synaptic plasticity and mental disorders","volume":"17","author":"Stornetta","year":"2011","journal-title":"Neuroscientist"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"8949","DOI":"10.1523\/JNEUROSCI.23-26-08949.2003","article-title":"The neurofibromatosis 1 gene product neurofibromin regulates pituitary adenylate cyclase-activating polypeptide-mediated signaling in astrocytes","volume":"23","author":"Dasgupta","year":"2003","journal-title":"J. Neurosci."}],"container-title":["Molecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/13\/5128\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:03:27Z","timestamp":1760126607000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1420-3049\/28\/13\/5128"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,6,30]]},"references-count":50,"journal-issue":{"issue":"13","published-online":{"date-parts":[[2023,7]]}},"alternative-id":["molecules28135128"],"URL":"https:\/\/doi.org\/10.3390\/molecules28135128","relation":{},"ISSN":["1420-3049"],"issn-type":[{"value":"1420-3049","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,6,30]]}}}