Abstract Osteogenic differentiation is a crucial biological process for maintaining bone remodelling. Aerobic glycolysis is the main source of energy for osteogenic differentiation. Alpha-enolase (Eno1), a glycolytic enzyme, is a therapeutic target for numerous diseases. Icariin, a principal active component of the traditional Chinese medicine Epimedium grandiflorum, can stimulate osteogenic differentiation. Here, we aimed to determine if icariin promotes osteogenic differentiation via Eno1. Icariin (1 μM) significantly promoted osteogenic differentiation of MC3T3-E1 cells. Icariin upregulated Eno1 protein and gene expressions during osteogenic differentiation. Moreover, ENOblock, a specific inhibitor of Eno1, markedly inhibited icariin-induced osteogenic differentiation. Futhermore, western blot assay showed that Eno1 might mediate osteogenic differentiation through the BMP/Smad4 signalling pathway. Collectively, Eno1 could be a promising drug target for icariin to regulate osteogenic differentiation. Keywords: Eno1, Aerobic glycolysis, Osteogenic differentiation, Icariin, BMP/Smad4 signalling pathway Highlights * • Icariin remarkedly promotes the osteoblast differentiation of MC3T3-E1 cells. * • Alpha-enolase (Eno1) is a potential target to regulate the osteoblast differentiation. * • Icariin promotes the osteoblast differentiation via the upregulation of Eno1. * • BMP/Smad4 signalling pathway plays a vital role on Eno1-mediated osteoblast differentiation. 1. Introduction Bone remodelling is a dynamic process resulting from both bone formation and bone resorption [[39]1]. The orchestrated interplay between osteoblasts and osteoclasts is crucial for bone remodelling [[40]2]. Abnormal osteogenesis jeopardises bone homeostasis by inducing an imbalance between bone formation and bone resorption, leading to disorders, such as osteoporosis and osteoarthritis [[41]3,[42]4]. Energy metabolism plays a crucial role in maintaining healthy tissues [[43]5]. Skeletal metabolic energy disorders disrupt bone metabolism balance, leading to bone-related diseases [[44]6]. Osteoblast formation is an energy-consuming process, and the energy demands are met by active energy metabolism [[45]7]. Aerobic glycolysis is the main source of energy during osteogenic differentiation [[46]8]. Augmented aerobic glycolysis also promotes osteoblast differentiation [[47]9]. Several glycolytic enzymes and metabolites, such as glucose-6-phosphate isomerase 1 and pyruvate, were reported to be involved in regulating osteoblast differentiation [[48]10,[49]11]. Alpha-enolase (Eno1), which catalyses the conversion between 2-phosphoglyceric acid and phosphoenolpyruvic acid, is involved in many biological functions, including cellular stress, cancer metastasis and autoantigen activities [[50]12]. The differential expression of Eno1 leads to several diseases, including cancer, Alzheimer's disease and rheumatoid arthritis [[51]13]. However, the role of Eno1 in the regulation of bone metabolism is unclear. Icariin is the main ingredient of Epimedium grandiflorum, which is a traditional Chinese medicine. Icariin was proved to modulate bone remodelling through promoting osteogenic differentiation and inhibit osteoclast formation [[52][14], [53][15], [54][16]]. BMP/Smad signalling pathway was confirmed to be a key target for icariin efficacy both in vivo and in vitro [[55]17,[56]18]. When the glucose metabolism disorder occurs under the condition where the bone remodelling is destructive, such as diabetes-induced osteoporosis, BMP/Smad is closely involved in the regulation of osteoblast differentiation [[57]19]. Given that Eno1 expression markedly changes in ovariectomised and icariin-treated ovariectomised rats (unpublished data), the relationship between Eno1, BMP/Smad signalling pathway and icariin-regulated bone metabolism is of great interest. In the present study, we confirmed the efficacy of icariin on osteogenic differentiation using Alizarin red staining, osteogenic gene detection and alkaline phosphatase (ALP) activity measurements. Eno1 gene and protein levels were determined in icariin-induced osteogenic differentiation. Further, ENOblock, an inhibitor of Eno1, was used to explore the function of Eno1 in osteogenic differentiation and icariin efficacy. Finally, the role of BMP/Smad4 signalling in Eno1-regulated osteogenic differentiation was determined. 2. Materials and methods 2.1. Cell culture The MC3T3-E1 cell line was purchased from China Procell Biotechnology Co., Ltd. Cells were cultured in α-MEM medium containing 10% foetal bovine serum (Gibco), 100 U/ml penicillin and 100 U/ml streptomycin at 37 °C in a 5% CO[2]-saturated humidified incubator. Osteogenic differentiation was induced when cells reached more than 80% confluence. Fifth passage of MC3T3-E1 cells were induced with osteogenic differentiation culture medium (HyCyte^TM, EOMX-D101, China). The experiment was divided into two steps. To determine the optimal concentration of icariin to induce osteogenic differentiation of MC3T3-E1 cells, the cells were treated with osteogenic differentiation culture medium containing four concentrations of icariin: 0(blank control group), 0.01, 0.1 and 1 μM. To determine the role of Eno1 in osteogenic differentiation and icariin efficacy, osteogenic differentiation culture medium containing 5 μM ENOblock (MedChemExpress) was used. The cells were divided into blank control, ENOblock, icariin and icariin + ENOblock groups. 2.2. Cell viability assessment MC3T3-E1 cells in the logarithmic growth phase were seeded into a 96-well plate at a density of 10^3 cells/well. The experiment was conducted in two steps. First, to determine the effect of ICA on cell viability, cells were cultured with or without different concentrations of icariin (0.01, 0.1 and 1 μM). Second, to determine the effect of ENOblock on cell viability, cells were fed with or without different concentrations of ENOblock (0.05, 0.5 and 5 μM). Cells were cultured at 37 °C and 5% CO[2] for 24, 48 and 72 h after treatment. 100 μL CCK8 (APExBIO, USA)was added and the cells were incubated at 37 °C for an additional 2h. Absorbance was measured at 450 nm with a microplate reader. 2.3. Alizarin red S staining MC3T3-E1 cells were seeded into 6-well plates. After reaching 80% confluence, the cells were treated with icariin and ENOblock, as described above. After culturing the treated cells for 21 days, Alizarin red S staining was performed according to the manufacturer’s instructions (Solarbio, China). Alizarin red S was destained with 10% cetylpyridinium chloride for 30 min and the absorbance at 562 nm was measured to determine the calcium deposits. 2.4. ALP activity assay MC3T3-E1 cells were seeded into 6-well plates. The cells were cultured in osteoblast differentiation induction medium with or without different concentrations of icariin for 7 days. ALP activity was measured according to the ALP detection kit instructions (Nanjing Jiancheng Biotechnology Co., Ltd.) The enzymatic activity unit was defined as 1 mg of phenol produced per mg of lytic protein at 37 °C for 15 min as a Guinness unit. 2.5. Western blot A standard western-blot analysis was used for detecting the protein levels of Eno1, BMP2, BMP4, Smad4 and p-Smad1/5/9. Briefly, cells were induced for 7 days and then lysed with RAPI (Beyotime, China) and PMSF (Beyotime, China) (R:P = 100:1) at a ratio of 10:1. Proteins were quantified using a bicinchoninic acid protein assay kit (Beyotime, China), following the manufacturer’s instructions. Protein lysates (20 μg per lane) were separated with 10% SDS-PAGE and transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked (Beyotime, China) for 20 min. The membranes were then incubated with primary antibodies overnight at 4 °C. The primary antibodies were as follows: Eno1 (Abcam, USA); BMP2 (Abcam, USA); BMP4(Abcam, USA); Smad4 (Abcam, USA); Smad1/5/9 (Abcam, USA); p-Smad1/5/9 (Cell Signaling Technology, USA); Vinculin (Affinity Biosciences, China). The membranes were incubated with corresponding HRP-conjugated secondary antibodies (Goat Anti-Rabbit IgG, Bioss, China) for 1 h at room temperature. The immunoreactive proteins were visualised using an enhanced chemiluminescence western detection kit (Abbkine, China). Image Lab software was used to quantify the density of each band. 2.6. Real-time quantitative PCR Cells were induced for 7 days and RNA was extracted using a RNA extraction kit (Aidlab Biotech, China). The cDNA was synthesised on a Veriti-96 Well Thermal Cycler (Applied Biosystems, USA) using a PrimeScript^TM RT Reagent Kit (TaKaRa, Dalian, China). Real-time quantitative PCR (qPCR) was performed using a CFX Opus 96 (Bio-Rad, USA) with TB Green® Premix Ex Taq^TM (TaKaRa, Dalian, China). The qPCR conditions were as follows: denaturation at 95 °C for 30 s, 40 cycles of PCR reaction at 95 °C for 5 s and 60 °C for 30 s. The melting curves at the end of amplification were analysed. β-actin was used as a control, and the data were calculated using the comparative Ct (2^−ΔΔCT) method and normalized against β-actin. All the primer sequences are shown in [58]Supplementary Table S1. 2.7. Bioinformatics analysis Genes related to postmenopausal osteoporosis and diabetes mellitus were obtained from the GeneCards, Online Mendelian Inheritance in Man, PharmGKB, Drugbank and TTD databases. The common target genes were imported into the String database to construct a protein-protein interaction network. A Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway enrichment analysis of common targets was performed using R language software. 2.8. Statistical analyses Statistical analyses were performed using the Prism software (GraphPad Software Inc., La Jolla, CA, USA). T-tests and one-way ANOVA were used for two-group and multiple group comparisons, respectively. The results are presented as mean ± standard deviation (SD) and P < 0.05 was considered statistically significant. 3. Results 3.1. Icariin promoted osteogenic differentiation of MC3T3-E1 cells To determine the effects of icariin on the proliferation of MC3T3-E1 cells, cell viability was measured in basal medium with different concentrations of icariin (0, 0.01, 0.1 and 1 μM) for 24, 48 and 72 h. As shown in [59]Fig. 1A, MC3T3-E1 cell viability did not change significantly after icariin treatment. Fig. 1. [60]Fig. 1 [61]Open in a new tab Icariin promoted osteogenic differentiation of MC3T3-E1. (A) Cell viability of MC3T3-E1 under different concentration of icariin were measured by CCK8 assay for 24, 48, 72h. (B) ALP enzyme activity of 7-days different concentrations icariin-induced osteogenic differentiation were tested. (C) The mineralization of 21-days induction under different concentrations of icariin were performed by Alizarin red S staining, and calcium deposits were quantified by measuring OD[562] after destaining (D). (E–G) mRNA levels of Alp (E), Bgp (F) and Runx2 (G), were tested as osteogenic markers by qPCR. All the data are shown as mean [MATH: ± :MATH] SD (n = 3) in three independent experiments. *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001 versus the 0 μM icariin group. (For interpretation of the references to colour in this figure legend, the