Drynachromoside A from Drynaria roosii induces proliferation and differentiation of MC3T3-E1 osteoblasts involving Wnt/β-catenin signaling pathway

Drynachromoside A from Drynaria roosii induces proliferation and differentiation of MC3T3-E1 osteoblasts involving Wnt/β-catenin signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Drynachromoside A from Drynaria roosii induces proliferation and differentiation of MC3T3-E1 osteoblasts involving Wnt/β-catenin signaling pathway Yafeng Li, Mengya Wang, Siyu Li, Luyue Zhang, Feng Yang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7849846/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Alveolar bone loss belongs to jaw bone defect and is the determinant for the overall restorative outcome and long-term stability of various denture restorations including implant-supported dentures. After tooth extraction or periodontitis, alveolar bone loss often appears. In clinical practice, there are no ideal therapeutic drugs for alveolar bone formation since drugs for osteogenesis usually present side effects. Therefore, discovery of novel therapy is essential. Drynaria roosii Nakaike is a medicinal plant used for osteogenesis in Chinese folk. To search novel phytochemicals targeting alveolar bone loss, we have explored phytochemicals in D. roosii and assessed the effects using MC3T3-E1 cells, which results in the identification of drynachromoside A. Further investigations showed drynachromoside A induced proliferation, osteogenesis and adhesion of MC3T3-E1 cells. and these effects were associated with activation of Wnt/β-catenin signaling pathway. These results could provide evidences for the discovery of novel therapy targeting alveolar bone loss and application of D. roosii in practice. Chromone glycoside Alveolar bone repair Pre-osteoblastic cells Proliferation Osteogenesis Adhesion Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Jaw bone defect is an oral and maxillofacial disorder which results from dentoalveolar trauma, benign and malignant neoplasms, caries, or periodontal disease 1 . As the key bone surrounding the teeth in the jaw, alveolar bone is also an important periodontal tissue component 2 . Alveolar bone is formed by intramembranous bone formation during the formation of the mandible and maxilla and supports the teeth. Following tooth loss, alveolar bone will undergo inevitable resorption. Therefore, the integrity of alveolar bone is the prerequisite for stable osseointegration of dental implants, and the foundation for various denture restorations 3 . However, in clinical practice there are no effective pharmacotherapeutic approached to attenuate alveolar bone resorption. So it is imperative to discover novel therapy to improve that condition. Drynaria roosii Nakaike (syn. Drynaria fortunei (Kunze ex Mett.) J. Sm.) (Polypodiaceae) is a medicinal fern distributed in China, Japan, and Korea 4 . In China, this plant was used for the treatment of osteoporosis and related diseases 5 . Previous pharmacological investigations have shown it can attenuate acute renal failure 6 , cervical spondylitis 7 , osteoporosis 8 and so on. Phytochemical studies on D. roosii revealed there were chromone derivatives as the major phytochemicals 9 , 10 . In our research interests to discover bioactive phytochemicals for alveolar bone repair, we have explored effects of drynachromoside A from D. roosii on the pro-osteoblastic MC3T3-E1 cells and underlying mechanisms. Materials and methods Chemicals and reagents Cell counting kit-8 (CCK-8) assay kit was provided by Meilun Biotechlogy (Dalian, China). Bromodeoxyuridine (BrdU) cell proliferation assay kit was obtained from Boyun Biotechnology (Shanghai, China). Alizarin red staining solution, 4',6-diamidino-2-phenylindole (DAPI) staining solution, secondary antibody and enhanced chemiluminescence (ECL) assay kit were supplied by Beyotime Biotechnology Institute (Shanghai, China). Alkaline phosphatase assay kit was the product of Jiancheng Biotechnology (Nanjing, China). Phalloidin staining solution was obtained from Yeasen Biotechnology (Shanghai, China). All the primary antibodies were products of Abcam (Cambridge, UK). Lipofectamine 3000 was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Negative control siRNA (NC-siRNA) and β-catenin-siRNA were provided by Santa Cruz Biotechnology (Dallas, TX, USA). Titanium plates were supplied by Jinyuan Advanced Materials Technology (Beijing, China). Phytochemical investigations The rhizomes of D. roosii were collected from Yulin (110°17′E, 22°40′N), Guangxi of China in June 2024 and identified by Prof. Yonglin Huang at Guangxi Institute of Botany, Chinese Academy of Sciences. The voucher specimen (No. 20240607) was deposited at Herbarium of Guangxi Institute of Botany (IBK). The air-dried rhizomes of D. roosii (10.0 kg) was ground and extracted with 90% ethanol (10.0 L × 3) under reflux for 2 h each time. After removing the solvent under reduced pressure, the residue (95.0 g) was suspended in water (1.0 L) and partitioned with petroleum ether (1.0 L × 3) and acetyl acetate (1.0 L × 3) successively. Then acetyl acetate part (43.0 g) was subject to column chromatography (30 × 10 cm) on silica gel eluted with gradient dichloromethane/methanol (from 100:0 to 0:100, v/v) (7000 ml) to give 8 fractions. Faction 6 (2.8 g) was separated on Sephadex LH-20 column chromatography (30 × 1.5 cm) eluted with methanol/water (85:15, v/v) (2000 ml) to afford white amorphous powder (26.0 mg). Then NMR spectra of the compound were recorded on a JEOL JNM-ECZ400S NMR spectrometer ( 1 H NMR 400 MHz, 13 C NMR 100 MHz, DMSO- d 6 ). Cell culture and treatment Mouse calvaria pre-osteoblastic MC3T3-E1 cells were provided by Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in minimum essential medium (MEM) including 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37.0 °C at a humid atmosphere with 5.0% CO 2 and 95.0% air. CCK-8 assay The MC3T3-E1 cells were adjusted to 5 × 10 7 /L and seeded in 96-well microplates. After cultured at 60%-80% confluence, the cells were divided into control and experimental groups. The experimental groups were exposed to certain compound obtained above. After 72 h, 10 μl CCK-8 solution was added. After incubation for 1 h at 37.0 °C, the absorbance was measured on a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm. The cell viability was expressed as the relative percentage of absorbance in the experimental groups against that in control group. BrdU incorporation assay To reveal the proliferation of MC3T3-E1 cells, BrdU incorporation assay was performed according to the supplier’s protocol. Briefly, the cells were seeded in 96-well microplates and treated as above. Then the cells were exposed to BrdU solution at 37.0 °C for 3 h. After fixed using FixDenat solution in the kit, the cells were incubated with anti-BrdU-POD working solution at room temperature for 90 min. After washing with PBS, substrate solution was added and incubation was carried out for 30 min. the absorbance was recorded on a microplate reader at 370 nm. Alkaline phosphatase activity To reveal the effects of the compound obtained, alkaline phosphatase activity was determined herein. The cells were cultured in osteogenic induction medium with or without the compound obtained for 72 h, and then washed with PBS twice. The cells were lysed using RIPA lysis buffer. After quantified using BCA assay, the lysates were incubated with the working solution in the assay kit according to the supplier’s protocol. After incubation for 15 min, chromogenic agent was added. Then the absorbance was read on a microplate reader at 520 nm. Alkaline phosphatase activity was calculated from the absorbance versus that of the standard solution. Alizarin red staining The MC3T3-E1 cells were adjusted to 1.5 × 10 7 /L and seeded in 24-well microplates. After cultured for 24 h, the medium was discarded and the cells were washed with PBS for three times. Then osteogenic induction medium with or without certain compound obtained was added. After incubation for two weeks, the cells were washed with PBS twice and then fixed with 4% paraformaldehyde for 15 min at room temperature. After washed with PBS twice, the cells were exposed to 2% alizarin red solution for 15 min. After washed with water, the cells were observed under a microscope (Olympus, Tokyo, Japan) and the images were captured. Phalloidin staining The cells were adjusted to 2×10 7 /L and seeded in 15 mm dishes. After incubation for 24 h, the cells were treated certain compound obtained and another incubation was performed for 24 h. After washed with PBS three times, the cells were fixed using 4% paraformaldehyde for 10 min at room temperature. Then the cells were permeabilized using 0.5% Triton X-100 for 10 min. After washed with PBS again, the cells were exposed to FITC-labeled phalloidin staining solution in darkness for 30 min at room temperature. Then the cells were washed with PBS and incubated with DAPI staining solution. The cells were observed under a laser confocal microscopy (Leica, Wetzlar, Germany) and the images were recorded. Titanium surface culture assay The titanium plates (1.0 cm × 1.0 cm) were sanded and placed in 24-well microplates after sterilized. The cells were seeded in 24-well microplates at 1 × 10 4 per well and treated with or without certain compound obtained above. After incubation for 72 h, CCK-8 assay was implemented. Meanwhile, for SEM observation, the cells were fixed using 4% paraformaldehyde for 4 h at 4 °C. After washed with PBS, the samples were dehydrated with gradient ethanol. Then the samples were dried in a critical point dryer (Leica, Wetzlar, Germany), and observed under a SEM (FEI, Hillsboro, USA). Western blot analysis The cells were treated as above and lysed on ice using RIPA lysis solution. After quantification with BCA kit, the proteins were separated on 10% SDS-PAGE transferred to PVDF membranes. Then the membranes were blocked with 5% defatted milk at room temperature and incubated with primary antibodies against Wnt1 (1:5000), β-catenin (1:5000), Runx2 (1:5000) and GAPDH (1:5000) at 4 °C overnight. After washing with TBS containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) at room temperature for 2 h. Finally, the bands were visualised using ECL substrate and densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD). siRNA interference assay To elucidate the role of Wnt/β-catenin pathway, siRNA interference assay was implemented. The MC3T3-E1 cells were transfected with NC-siRNA or β-catenin-siRNA using lipofectamine 3000 according to the supplier’s protocol. After validating the successful transfection using western blot, the cells were treated as above and CCK-8 was performed. Statistical analysis All the data were expressed as mean ± standard error and analyzed using GraphPad Prism 8.0 (San Diego, CA, USA). One-way analysis of variance (one-way ANOVA) was performed followed by Tukey’s test for multiple comparisons and Student’s t-test for single comparisons. Statistical significance was set at p < 0.05. Results Structure elucidation The structure of the compound obtained was elucidated as drynachromoside A ( 1 ) by analysis of 1 H and 13 C NMR spectra, as well as by comparison of the data with literature 9 . The 1 H and 13 C NMR data were assigned as follows: Drynachromoside A ( 1 ): white amorphous power; 1 H NMR (400 MHz, DMSO- d 6 ) δ: 6.67 (1H, d, J = 2.0 Hz, H-8), 6.44 (1H, d, J = 2.0 Hz, H-6), 6.24 (1H, s, H-3), 5.55 (1H, s, H-1′), 4.45 (1H, d, J = 7.8 Hz, H-1′′), 3.90 (1H, m, H-2′), 3.89 (1H, m, H-4′′), 3.68 (1H, m, H-6′′a), 3.58 (1H, m, H-4′), 3.52 (1H, m, H-5′), 3.45 (1H, m, H-6′′b), 3.18 (1H, m, H-5′′), 3.12 (1H, m, H-3′′), 3.07 (1H, m, H-3′), 3.01 (1H, m, H-2′′), 2.38 (3H, s, H-11), 1.22 (3H, d, J = 6.0 Hz, H-6′); 13 C NMR (100 MHz, DMSO- d 6 ) δ: 182.5 (C-4), 169.0 (C-2), 162.0 (C-7), 161.8 (C-5), 157.9 (C-9), 108.9 (C-3), 105.7 (C-10), 104.9 (C-1′′), 100.1 (C-6), 98.6 (C-1′), 95.1 (C-8), 81.9 (C-4′), 77.6 (C-3′′), 77.2 (C-5′′), 75.0 (C-2′′), 70.8 (C-3′), 70.6 (C-4′′), 70.1 (C-2′), 68.9 (C-5′), 61.7 (C-6′′), 20.6 (C-11), 18.4 (C-6′). Effects of drynachromoside A on the proliferation of MC3T3-E1 cells As shown in Fig. 1A, compared with control group, drynachromoside A significantly increased the viability of MC3T3-E1 cells from 2.5 μM to 10 μM. However, form 20 μM the effect of drynachromoside A on cell viability was decreased. To further clarify the increased cell viability by drynachromoside A was associated with proliferation, BrdU incorporation assay was performed herein. As a result, drynachromoside A could enhance proliferation of MC3T3-E1 cells at both 5 and 10 μM (Fig. 1B). Drynachromoside A promotes the osteogenesis of MC3T3-E1 cells To further elucidate the osteogenic induction of drynachromoside A, alizarin red staining was implemented herein. As shown in Fig. 2A, after incubation for 14 days, more plaque calcified extracellular matrices were observed in the cells following the treatment with 5 and 10 μM drynachromoside A. Even in the cells exposed to 10 μM DSCA, large purple mineralized nodules were found and the cells showed overlapping and cross aggregation. ALP activity confirmed drynachromoside A induced osteogenesis of MC3T3-E1 cells quantitatively (Fig. 2B). Drynachromoside A enhances the adhesion of MC3T3-E1 cells To reveal the adhesion for potential dental implants, we have performed phalloidin staining. As a result, the cells in control group showed slender rod-shape. Where exposed to drynachromoside A at 5 or 10 μM, the cells extended and appeared as filamentous cytoskeleton (Fig. 3A). The cells in control group and drynachromoside A 5 μM group adhered to the surface of titanium plate with some dot-like pseudopods. And in the presence of drynachromoside A at 10 μM the cells extended their filamentous pseudopods, which were much longer than that in control group (Fig. 3B). Drynachromoside A activates Wnt/β-catenin signaling pathway To disclose the mechanisms of drynachromoside A on MC3T3-E1 cells, Wnt/β-catenin signaling pathway was checked herein. Western blot analysis showed one isoform of Wnt family proteins, Wnt1 together with its downstream proteins β-catenin and Rnux2 was up-regulated by drynachromoside A at 5 and 10 μM (Fig. 4A). Densitometric analysis also confirmed the results quantitatively (Fig. 4B-D). These results indicated DCSA could activate Wnt/β-catenin signaling pathway. Wnt/β-catenin pathway is involved in the effects of drynachromoside A To unravel the role of Wnt/β-catenin pathway in the effects of drynachromoside A, siRNA interference was implemented. The results when β-catenin was knocked down (Fig. 5A), the effects of drynachromoside A was reduced sharply (Fig. 5B). However, in the cells transfected with NC-siRNA, the effects of drynachromoside A were also retained. These results implied activation of Wnt/β-catenin pathway is closely associated with the effects of drynachromoside A. Discussion Alveolar bone loss inevitably occurs secondary to the conditions such as tooth extraction and periodontitis, which may potentially give a critical impact on the outcomes of implant osseointegration and other denture restorations 11 , 12 . However, there are no ideal therapeutic drugs to promote alveolar bone formation. Though there are some drugs for osteogenesis in clinic, but the side effects have affected the application in dental sciences 13 . D. roosii is a medicinal plant used for osteogenesis in Chinese medicine. Herein, we have identified drynachromoside A from D. roosii and evaluated its potential on osteogenesis. The results showed drynachromoside A could induce proliferation of MC3T3-E1 cells, promote osteogenesis and enhance adhesion. In the process of osteogenesis, Wnt/β-catenin pathway plays a crucial role 14 . Wnt activates β-catenin-mediated pathway to regulate bone metabolism 15 . Activation of Wnt/β-catenin pathway induces the expression of Runx2. As the master transcription factor for bone development, Rnux2 enhances proliferation of osteoblasts and induces their differentiation 16 . In present study, we have found drynachromoside A activated Wnt/β-catenin signaling. One isoform of Wnt family, Wnt together with downstream β-catenin and Runx2 was up-regulated. In addition, when β-catenin was knocked down, the effects of drynachromoside A was reduced, which indicated drynachromoside A promoted proliferation of MC3T3-E1 cells via activating Wnt/β-catenin pathway. Conclusion In summary, we have identified DCSA from D. roosii and assessed its effects on MC3T3-E1 cells. DCSA induces proliferation, osteogenesis and adhesion of MC3T3-E1 cells via activating Wnt/β-catenin signaling pathway. These results could give evidences to discover novel therapy for alveolar bone loss in dental medicine and application of D. roosii in clinic practice. Declarations Author Contribution: YL: Investigation, Data curation, Formal analysis, Writing -original draft. MW: Investigation, Data curation, Formal analysis. SL: Data curation, Formal analysis. LZ: Formal analysis, Methodology. FY: Conceptualization, Supervision, Funding acquisition, Writing-review & editing. Competing Interests: The authors declare there are no competing interests. Acknowledgments: This work was partly supported by Paired Assistance Scientific Research Project by the Affiliated Hospital of Xuzhou Medical University (Grant No. FXJDBF2024214), and the authors give sincere gratitude to that program. References Calderipe CB, Soares AC, Dos Santos Giorgis R, Fogaça ACM, Torriani MA, Grave LQ, Schuch LF, Vasconcelos ACU (2024) What is the effect of lactoferrin on oral and jawbone tissue repair? A systematic review. 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Int J Mol Sci 23:5776. https://doi.org/10.3390/ijms23105776 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 01 Dec, 2025 Reviewers invited by journal 01 Dec, 2025 Editor invited by journal 27 Nov, 2025 Editor assigned by journal 11 Nov, 2025 First submitted to journal 10 Nov, 2025 Editorial decision: Major revisions 05 Nov, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7849846","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":553352284,"identity":"a7462831-9f47-4626-944d-82d375a7285c","order_by":0,"name":"Yafeng Li","email":"","orcid":"","institution":"Fengxian People's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yafeng","middleName":"","lastName":"Li","suffix":""},{"id":553352285,"identity":"87f1bd9f-cc03-4d55-a1ef-d34c4547b9af","order_by":1,"name":"Mengya Wang","email":"","orcid":"","institution":"Xuzhou Medical 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11:05:44","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":72140,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/4febbb51a1e5a69cdd79c748.html"},{"id":97435298,"identity":"49372d24-e68f-4428-a2c4-df08d7608a22","added_by":"auto","created_at":"2025-12-04 11:05:43","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":852854,"visible":true,"origin":"","legend":"\u003cp\u003eDrynachromoside A promotes proliferation of MC3T3-E1 cells. (A) CCK-8 assay. (B) BrdU incorporation assay. n = 3, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/5aa3441a625bb936bd1b76e4.jpg"},{"id":97435293,"identity":"9a78f1ea-3fc6-4a68-85fb-da289ad99cc5","added_by":"auto","created_at":"2025-12-04 11:05:43","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1083497,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of drynachromoside A on the osteogenesis of MC3T3-E1 cells. (A) Alizarin red staining. (B) ALP activity. n = 3, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 and \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/62350b5c9e92d56e5cab0755.jpg"},{"id":97435300,"identity":"68b91225-ad0a-48bf-bc25-f6620e1de969","added_by":"auto","created_at":"2025-12-04 11:05:43","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4411544,"visible":true,"origin":"","legend":"\u003cp\u003eDrynachromoside A enhances adhesion of MC3T3-E2 cells. (A) Phalloidin staining. (B) SEM analysis.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/4f14e6eb864ed022dc3ee100.jpg"},{"id":97435294,"identity":"06910eae-9d0f-4d63-bf40-95621bff8f57","added_by":"auto","created_at":"2025-12-04 11:05:43","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":807749,"visible":true,"origin":"","legend":"\u003cp\u003eDrynachromoside A activates Wnt/β-catenin pathway in MC3T3-E1 cells. (A) Western blot analysis for Wnt1, β-catenin and Runx2. (B)-(D) Densitometric analysis for Wnt1, β-catenin and Runx2. n = 3, \u003csup\u003e*\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001 \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/6a2c5d8a5ca56c66a4bffd64.jpg"},{"id":97667287,"identity":"431bfb51-95e6-433b-b3c4-944b696a0079","added_by":"auto","created_at":"2025-12-08 09:23:12","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":882147,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of drynachromoside A is associated with activation of Wnt/β-catenin pathway. (A) Western blot analysis for the transfection with NC-siRNA or β-catenin-siRNA. (B) Cell viability of MC3T3-E1 cells transfected with NC-siRNA or β-catenin-siRNA. n = 3, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e NC-siRNA group, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs\u003c/em\u003e control group.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/b05c0ffc941aeadfa2619b02.jpg"},{"id":98421147,"identity":"eb306ce8-da6b-4cb9-94fa-35889cbac544","added_by":"auto","created_at":"2025-12-17 16:24:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8783436,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7849846/v1/eb0db78c-e798-4e76-b5a6-1d3d7d409cd7.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eDrynachromoside A from \u003cem\u003eDrynaria roosii\u003c/em\u003e induces proliferation and differentiation of MC3T3-E1 osteoblasts involving Wnt/β-catenin signaling pathway\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eJaw bone defect is an oral and maxillofacial disorder which results from dentoalveolar trauma, benign and malignant neoplasms, caries, or periodontal disease\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. As the key bone surrounding the teeth in the jaw, alveolar bone is also an important periodontal tissue component\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Alveolar bone is formed by intramembranous bone formation during the formation of the mandible and maxilla and supports the teeth. Following tooth loss, alveolar bone will undergo inevitable resorption. Therefore, the integrity of alveolar bone is the prerequisite for stable osseointegration of dental implants, and the foundation for various denture restorations\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. However, in clinical practice there are no effective pharmacotherapeutic approached to attenuate alveolar bone resorption. So it is imperative to discover novel therapy to improve that condition.\u003c/p\u003e\u003cp\u003e\u003cem\u003eDrynaria roosii\u003c/em\u003e Nakaike (syn. \u003cem\u003eDrynaria fortunei\u003c/em\u003e (Kunze ex Mett.) J. Sm.) (Polypodiaceae) is a medicinal fern distributed in China, Japan, and Korea\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In China, this plant was used for the treatment of osteoporosis and related diseases\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Previous pharmacological investigations have shown it can attenuate acute renal failure\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, cervical spondylitis\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e, osteoporosis\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e and so on. Phytochemical studies on \u003cem\u003eD. roosii\u003c/em\u003e revealed there were chromone derivatives as the major phytochemicals\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. In our research interests to discover bioactive phytochemicals for alveolar bone repair, we have explored effects of drynachromoside A from \u003cem\u003eD. roosii\u003c/em\u003e on the pro-osteoblastic MC3T3-E1 cells and underlying mechanisms.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003ch2\u003eChemicals and reagents\u003c/h2\u003e\n\u003cp\u003eCell counting kit-8 (CCK-8) assay kit was provided by Meilun Biotechlogy (Dalian, China). Bromodeoxyuridine (BrdU) cell proliferation assay kit was obtained from Boyun Biotechnology (Shanghai, China). Alizarin red staining solution, 4\u0026apos;,6-diamidino-2-phenylindole (DAPI) staining solution, secondary antibody and enhanced chemiluminescence (ECL) assay kit were supplied by Beyotime Biotechnology Institute (Shanghai, China). Alkaline phosphatase assay kit was the product of Jiancheng Biotechnology (Nanjing, China). Phalloidin staining solution was obtained from Yeasen Biotechnology (Shanghai, China). All the primary antibodies were products of Abcam (Cambridge, UK). Lipofectamine 3000 was obtained from Thermo Fisher Scientific (Waltham, MA, USA). Negative control siRNA (NC-siRNA) and\u0026nbsp;\u0026beta;-catenin-siRNA were provided by Santa Cruz Biotechnology (Dallas, TX, USA). Titanium plates were supplied by Jinyuan Advanced Materials Technology (Beijing, China).\u003c/p\u003e\n\u003ch2\u003ePhytochemical investigations\u003c/h2\u003e\n\u003cp\u003eThe rhizomes of \u003cem\u003eD. roosii\u0026nbsp;\u003c/em\u003ewere collected from Yulin (110\u0026deg;17\u0026prime;E, 22\u0026deg;40\u0026prime;N), Guangxi of China in June 2024 and identified by Prof. Yonglin Huang at Guangxi Institute of Botany, Chinese Academy of Sciences. The voucher specimen (No. 20240607) was deposited at Herbarium of Guangxi Institute of Botany (IBK). The air-dried rhizomes of \u003cem\u003eD. roosii\u003c/em\u003e (10.0 kg) was ground and extracted with 90% ethanol (10.0 L \u0026times; 3) under reflux for 2 h each time. After removing the solvent under reduced pressure, the residue (95.0 g) was suspended in water (1.0 L) and partitioned with petroleum ether (1.0 L \u0026times; 3) and acetyl acetate (1.0 L \u0026times; 3) successively. Then acetyl acetate part (43.0 g) was subject to column chromatography (30 \u0026times; 10 cm) on silica gel eluted with gradient dichloromethane/methanol (from 100:0 to 0:100, v/v) (7000 ml) to give 8 fractions. Faction 6 (2.8 g) was separated on Sephadex LH-20 column chromatography (30 \u0026times; 1.5 cm) eluted with methanol/water (85:15, v/v) (2000 ml) to afford white amorphous powder (26.0 mg). Then NMR spectra of the compound were recorded on a JEOL JNM-ECZ400S NMR spectrometer (\u003csup\u003e1\u003c/sup\u003eH NMR 400 MHz, \u003csup\u003e13\u003c/sup\u003eC NMR 100 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e).\u003c/p\u003e\n\u003ch2\u003eCell culture and treatment\u003c/h2\u003e\n\u003cp\u003eMouse calvaria pre-osteoblastic MC3T3-E1 cells were provided by Cell Bank of Shanghai Institute of Biological Sciences, Chinese Academy of Sciences (Shanghai, China) and maintained in minimum essential medium (MEM) including 10% FBS, 100 U/ml penicillin, and 100 \u0026mu;g/ml streptomycin at 37.0 \u0026deg;C at a humid atmosphere with 5.0% CO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003eand 95.0% air.\u003c/p\u003e\n\u003ch2\u003eCCK-8 assay\u003c/h2\u003e\n\u003cp\u003eThe MC3T3-E1 cells were adjusted to 5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/L and seeded in 96-well microplates. After cultured at 60%-80% confluence, the cells were divided into control and experimental groups. The experimental groups were exposed to certain compound obtained above. After 72 h, 10 \u0026mu;l CCK-8 solution was added. After incubation for 1 h at 37.0 \u0026deg;C, the absorbance was measured on a microplate reader (Bio-Rad, Hercules, CA, USA) at 450 nm. The cell viability was expressed as the relative percentage of absorbance in the experimental groups against that in control group.\u003c/p\u003e\n\u003ch2\u003eBrdU incorporation assay\u003c/h2\u003e\n\u003cp\u003eTo reveal the proliferation of MC3T3-E1 cells, BrdU incorporation assay was performed according to the supplier\u0026rsquo;s protocol. Briefly, the cells were seeded in 96-well microplates and treated as above. Then the cells were exposed to BrdU solution at 37.0 \u0026deg;C for 3 h. After fixed using FixDenat solution in the kit, the cells were incubated with anti-BrdU-POD working solution at room temperature for 90 min. After washing with PBS, substrate solution was added and incubation was carried out for 30 min. the absorbance was recorded on a microplate reader at 370 nm.\u003c/p\u003e\n\u003ch2\u003eAlkaline phosphatase activity\u003c/h2\u003e\n\u003cp\u003eTo reveal the effects of the compound obtained, alkaline phosphatase activity was determined herein. The cells were cultured in osteogenic induction medium with or without the compound obtained for 72 h, and then washed with PBS twice. The cells were lysed using RIPA lysis buffer. After quantified using BCA assay, the lysates were incubated with the working solution in the assay kit according to the supplier\u0026rsquo;s protocol. After incubation for 15 min, chromogenic agent was added. Then the absorbance was read on a microplate reader at 520 nm. Alkaline phosphatase activity was calculated from the absorbance versus that of the standard solution.\u003c/p\u003e\n\u003ch2\u003eAlizarin red staining\u003c/h2\u003e\n\u003cp\u003eThe MC3T3-E1 cells were adjusted to 1.5 \u0026times; 10\u003csup\u003e7\u003c/sup\u003e/L and seeded in 24-well microplates. After cultured for 24 h, the medium was discarded and the cells were washed with PBS for three times. Then osteogenic induction medium with or without certain compound obtained was added. After incubation for two weeks, the cells were washed with PBS twice and then fixed with 4% paraformaldehyde for 15 min at room temperature. After washed with PBS twice, the cells were exposed to 2% alizarin red solution for 15 min. After washed with water, the cells were observed under a microscope (Olympus, Tokyo, Japan) and the images were captured.\u003c/p\u003e\n\u003ch2\u003ePhalloidin\u0026nbsp;staining\u003c/h2\u003e\n\u003cp\u003eThe cells were adjusted to 2\u0026times;10\u003csup\u003e7\u003c/sup\u003e/L and seeded in 15 mm dishes. After incubation for 24 h, the cells were treated certain compound obtained and another incubation was performed for 24 h. After washed with PBS three times, the cells were fixed using 4% paraformaldehyde for 10 min at room temperature. Then the cells were permeabilized using 0.5% Triton X-100 for 10 min. After washed with PBS again, the cells were exposed to FITC-labeled phalloidin staining solution in darkness for 30 min at room temperature. Then the cells were washed with PBS and incubated with DAPI staining solution. The cells were observed under a laser confocal microscopy (Leica, Wetzlar, Germany) and the images were recorded.\u003c/p\u003e\n\u003ch2\u003eTitanium surface culture assay\u003c/h2\u003e\n\u003cp\u003eThe titanium plates (1.0 cm \u0026times; 1.0 cm) were sanded and placed in 24-well microplates after sterilized. The cells were seeded in 24-well microplates at 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e per well and treated with or without certain compound obtained above. After incubation for 72 h, CCK-8 assay was implemented. Meanwhile, for SEM observation, the cells were fixed using 4% paraformaldehyde for 4 h at 4 \u0026deg;C. After washed with PBS, the samples were dehydrated with gradient ethanol. Then the samples were dried in a critical point dryer (Leica, Wetzlar, Germany), and observed under a SEM (FEI, Hillsboro, USA).\u003c/p\u003e\n\u003ch2\u003eWestern blot analysis\u003c/h2\u003e\n\u003cp\u003eThe cells were treated as above and lysed on ice using RIPA lysis solution. After quantification with BCA kit, the proteins were separated on 10% SDS-PAGE transferred to PVDF membranes. Then the membranes were blocked with 5% defatted milk at room temperature and incubated with primary antibodies against Wnt1 (1:5000), \u0026beta;-catenin (1:5000), Runx2 (1:5000) and GAPDH (1:5000) at 4 \u0026deg;C overnight. After washing with TBS containing 0.1% Tween-20, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:2000) at room temperature for 2 h. Finally, the bands were visualised using ECL substrate and densitometric analysis was performed using ImageJ software (NIH, Bethesda, MD).\u003c/p\u003e\n\u003ch2\u003esiRNA interference assay\u003c/h2\u003e\n\u003cp\u003eTo elucidate the role of Wnt/\u0026beta;-catenin pathway, siRNA interference assay was implemented. The MC3T3-E1 cells were transfected with NC-siRNA or \u0026beta;-catenin-siRNA using lipofectamine 3000 according to the supplier\u0026rsquo;s protocol. After validating the successful transfection using western blot, the cells were treated as above and CCK-8 was performed.\u003c/p\u003e\n\u003ch2\u003eStatistical analysis\u003c/h2\u003e\n\u003cp\u003eAll the data were expressed as mean \u0026plusmn; standard error and analyzed using GraphPad Prism 8.0 (San Diego, CA, USA). One-way analysis of variance (one-way ANOVA) was performed followed by Tukey\u0026rsquo;s test for multiple comparisons and Student\u0026rsquo;s t-test for single comparisons. Statistical significance was set at p \u0026lt; 0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eStructure elucidation\u003c/h2\u003e\n\u003cp\u003eThe structure of the compound obtained was elucidated as drynachromoside A (\u003cstrong\u003e1\u003c/strong\u003e) by analysis of \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR spectra, as well as by comparison of the data with literature \u003csup\u003e9\u003c/sup\u003e. The \u003csup\u003e1\u003c/sup\u003eH and \u003csup\u003e13\u003c/sup\u003eC NMR data were assigned as follows:\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"https://myfiles.space/user_files/58895_8739fc6c57c1c19a/58895_custom_files/img1764846099.png\" width=\"636\" height=\"357\"\u003e\u003c/p\u003e\n\u003cp\u003eDrynachromoside A (\u003cstrong\u003e1\u003c/strong\u003e): white amorphous power; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta;: 6.67 (1H, d, \u003cem\u003eJ\u003c/em\u003e = 2.0 Hz, H-8), 6.44 (1H, d, \u003cem\u003eJ\u003c/em\u003e = 2.0 Hz, H-6), 6.24 (1H, s, H-3), 5.55 (1H, s, H-1\u0026prime;), 4.45 (1H, d, \u003cem\u003eJ\u003c/em\u003e = 7.8 Hz, H-1\u0026prime;\u0026prime;), 3.90 (1H, m, H-2\u0026prime;), 3.89 (1H, m, H-4\u0026prime;\u0026prime;), 3.68 (1H, m, H-6\u0026prime;\u0026prime;a), 3.58 (1H, m, H-4\u0026prime;), 3.52 (1H, m, H-5\u0026prime;), 3.45 (1H, m, H-6\u0026prime;\u0026prime;b), 3.18 (1H, m, H-5\u0026prime;\u0026prime;), 3.12 (1H, m, H-3\u0026prime;\u0026prime;), 3.07 (1H, m, H-3\u0026prime;), 3.01 (1H, m, H-2\u0026prime;\u0026prime;), 2.38 (3H, s, H-11), 1.22 (3H, d, \u003cem\u003eJ\u003c/em\u003e = 6.0 Hz, H-6\u0026prime;); \u003csup\u003e13\u003c/sup\u003eC NMR (100 MHz, DMSO-\u003cem\u003ed\u003csub\u003e6\u003c/sub\u003e\u003c/em\u003e) \u0026delta;: 182.5 (C-4), 169.0 (C-2), 162.0 (C-7), 161.8 (C-5), 157.9 (C-9), 108.9 (C-3), 105.7 (C-10), 104.9 (C-1\u0026prime;\u0026prime;), 100.1 (C-6), 98.6 (C-1\u0026prime;), 95.1 (C-8), 81.9 (C-4\u0026prime;), 77.6 (C-3\u0026prime;\u0026prime;), 77.2 (C-5\u0026prime;\u0026prime;), 75.0 (C-2\u0026prime;\u0026prime;), 70.8 (C-3\u0026prime;), 70.6 (C-4\u0026prime;\u0026prime;), 70.1 (C-2\u0026prime;), 68.9 (C-5\u0026prime;), 61.7 (C-6\u0026prime;\u0026prime;), 20.6 (C-11), 18.4 (C-6\u0026prime;).\u003c/p\u003e\n\u003ch2\u003eEffects of drynachromoside A on the proliferation of MC3T3-E1 cells\u003c/h2\u003e\n\u003cp\u003eAs shown in Fig. 1A, compared with control group, drynachromoside A significantly increased the viability of MC3T3-E1 cells from 2.5 \u0026mu;M to 10 \u0026mu;M. However, form 20 \u0026mu;M the effect of drynachromoside A on cell viability was decreased. To further clarify the increased cell viability by drynachromoside A was associated with proliferation, BrdU incorporation assay was performed herein. As a result, drynachromoside A could enhance proliferation of MC3T3-E1 cells at both 5 and 10 \u0026mu;M (Fig. 1B).\u003c/p\u003e\n\u003ch2\u003eDrynachromoside A promotes the osteogenesis of MC3T3-E1 cells\u003c/h2\u003e\n\u003cp\u003eTo further elucidate the osteogenic induction of drynachromoside A, alizarin red staining was implemented herein. As shown in Fig. 2A, after incubation for 14 days, more plaque calcified extracellular matrices were observed in the cells following the treatment with 5 and 10 \u0026mu;M drynachromoside A. Even in the cells exposed to 10 \u0026mu;M DSCA, large purple mineralized nodules were found and the cells showed overlapping and cross aggregation. ALP activity confirmed drynachromoside A induced osteogenesis of MC3T3-E1 cells quantitatively (Fig. 2B).\u003c/p\u003e\n\u003ch2\u003eDrynachromoside A enhances the adhesion of MC3T3-E1 cells\u003c/h2\u003e\n\u003cp\u003eTo reveal the adhesion for potential dental implants, we have performed phalloidin staining. As a result, the cells in control group showed slender rod-shape. Where exposed to drynachromoside A at 5 or 10 \u0026mu;M, the cells extended and appeared as filamentous cytoskeleton (Fig. 3A). The cells in control group and drynachromoside A 5 \u0026mu;M group adhered to the surface of titanium plate with some dot-like pseudopods. And in the presence of drynachromoside A at 10 \u0026mu;M the cells extended their filamentous pseudopods, which were much longer than that in control group (Fig. 3B).\u003c/p\u003e\n\u003ch2\u003eDrynachromoside A activates Wnt/\u0026beta;-catenin signaling pathway\u003c/h2\u003e\n\u003cp\u003eTo disclose the mechanisms of drynachromoside A on MC3T3-E1 cells, Wnt/\u0026beta;-catenin signaling pathway was checked herein. Western blot analysis showed one isoform of Wnt family proteins, Wnt1 together with its downstream proteins \u0026beta;-catenin and Rnux2 was up-regulated by drynachromoside A at 5 and 10 \u0026mu;M (Fig. 4A). Densitometric analysis also confirmed the results quantitatively (Fig. 4B-D). These results indicated DCSA could activate Wnt/\u0026beta;-catenin signaling pathway.\u003c/p\u003e\n\u003ch2\u003eWnt/\u0026beta;-catenin pathway is involved in the effects of drynachromoside A\u003c/h2\u003e\n\u003cp\u003eTo unravel the role of Wnt/\u0026beta;-catenin pathway in the effects of drynachromoside A, siRNA interference was implemented. The results when \u0026beta;-catenin was knocked down (Fig. 5A), the effects of drynachromoside A was reduced sharply (Fig. 5B). However, in the cells transfected with NC-siRNA, the effects of drynachromoside A were also retained. These results implied activation of Wnt/\u0026beta;-catenin pathway is closely associated with the effects of drynachromoside A.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAlveolar bone loss inevitably occurs secondary to the conditions such as tooth extraction and periodontitis, which may potentially give a critical impact on the outcomes of implant osseointegration and other denture restorations\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e,\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. However, there are no ideal therapeutic drugs to promote alveolar bone formation. Though there are some drugs for osteogenesis in clinic, but the side effects have affected the application in dental sciences\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. \u003cem\u003eD. roosii\u003c/em\u003e is a medicinal plant used for osteogenesis in Chinese medicine. Herein, we have identified drynachromoside A from \u003cem\u003eD. roosii\u003c/em\u003e and evaluated its potential on osteogenesis. The results showed drynachromoside A could induce proliferation of MC3T3-E1 cells, promote osteogenesis and enhance adhesion.\u003c/p\u003e\u003cp\u003eIn the process of osteogenesis, Wnt/β-catenin pathway plays a crucial role\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. Wnt activates β-catenin-mediated pathway to regulate bone metabolism\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Activation of Wnt/β-catenin pathway induces the expression of Runx2. As the master transcription factor for bone development, Rnux2 enhances proliferation of osteoblasts and induces their differentiation\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In present study, we have found drynachromoside A activated Wnt/β-catenin signaling. One isoform of Wnt family, Wnt together with downstream β-catenin and Runx2 was up-regulated. In addition, when β-catenin was knocked down, the effects of drynachromoside A was reduced, which indicated drynachromoside A promoted proliferation of MC3T3-E1 cells via activating Wnt/β-catenin pathway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have identified DCSA from \u003cem\u003eD. roosii\u003c/em\u003e and assessed its effects on MC3T3-E1 cells. DCSA induces proliferation, osteogenesis and adhesion of MC3T3-E1 cells via activating Wnt/β-catenin signaling pathway. These results could give evidences to discover novel therapy for alveolar bone loss in dental medicine and application of \u003cem\u003eD. roosii\u003c/em\u003e in clinic practice.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution:\u003c/h2\u003e\n\u003cp\u003eYL: Investigation, Data curation, Formal analysis, Writing -original draft. MW: Investigation, Data curation, Formal analysis. SL: Data curation, Formal analysis. LZ: Formal analysis, Methodology. FY: Conceptualization, Supervision, Funding acquisition, Writing-review \u0026amp; editing.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eThe authors declare there are no competing interests.\u003c/p\u003e\n\u003ch2\u003eAcknowledgments:\u003c/h2\u003e\n\u003cp\u003eThis work was partly supported by Paired Assistance Scientific Research Project by the Affiliated Hospital of Xuzhou Medical University (Grant No. FXJDBF2024214), and the authors give sincere gratitude to that program.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eCalderipe CB, Soares AC, Dos Santos Giorgis R, Foga\u0026ccedil;a ACM, Torriani MA, Grave LQ, Schuch LF, Vasconcelos ACU (2024) What is the effect of lactoferrin on oral and jawbone tissue repair? A systematic review. 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Bone Res 12:39. https://doi.org/10.1038/s41413-024-00342-8\u003c/li\u003e\n \u003cli\u003eKomori T (2022) Whole Aspect of Runx2 Functions in Skeletal Development. Int J Mol Sci 23:5776. https://doi.org/10.3390/ijms23105776\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Chromone glycoside, Alveolar bone repair, Pre-osteoblastic cells, Proliferation, Osteogenesis, Adhesion","lastPublishedDoi":"10.21203/rs.3.rs-7849846/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7849846/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAlveolar bone loss belongs to jaw bone defect and is the determinant for the overall restorative outcome and long-term stability of various denture restorations including implant-supported dentures. After tooth extraction or periodontitis, alveolar bone loss often appears. In clinical practice, there are no ideal therapeutic drugs for alveolar bone formation since drugs for osteogenesis usually present side effects. Therefore, discovery of novel therapy is essential. \u003cem\u003eDrynaria roosii\u003c/em\u003e Nakaike is a medicinal plant used for osteogenesis in Chinese folk. To search novel phytochemicals targeting alveolar bone loss, we have explored phytochemicals in \u003cem\u003eD. roosii\u003c/em\u003e and assessed the effects using MC3T3-E1 cells, which results in the identification of drynachromoside A. Further investigations showed drynachromoside A induced proliferation, osteogenesis and adhesion of MC3T3-E1 cells. and these effects were associated with activation of Wnt/β-catenin signaling pathway. These results could provide evidences for the discovery of novel therapy targeting alveolar bone loss and application of \u003cem\u003eD. roosii\u003c/em\u003e in practice.\u003c/p\u003e","manuscriptTitle":"Drynachromoside A from Drynaria roosii induces proliferation and differentiation of MC3T3-E1 osteoblasts involving Wnt/β-catenin signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 11:05:38","doi":"10.21203/rs.3.rs-7849846/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-12-01T11:38:27+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-12-01T11:01:04+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Revista Brasileira de Farmacognosia","date":"2025-11-27T13:50:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-11T08:11:39+00:00","index":"","fulltext":""},{"type":"submitted","content":"Revista Brasileira de Farmacognosia","date":"2025-11-11T03:11:31+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2025-11-05T17:59:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"revista-brasileira-de-farmacognosia","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rbfa","sideBox":"Learn more about [Revista Brasileira de Farmacognosia](https://www.springer.com/journal/43450)","snPcode":"43450","submissionUrl":"https://www.editorialmanager.com/rbfa/default2.aspx","title":"Revista Brasileira de Farmacognosia","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"9fa22a6f-795e-409e-a156-fe652512817a","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T21:51:01+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 11:05:38","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7849846","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7849846","identity":"rs-7849846","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}