Effects of diosgenin dose on alveolar bone loss in rats with experimental periodontitis

Effects of diosgenin dose on alveolar bone loss in rats with experimental periodontitis | 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 Effects of diosgenin dose on alveolar bone loss in rats with experimental periodontitis Alper Kızıldağ, Aysan Lektemür Alpan, Tuğba Köseoğlu Aydın, Melih Özdede, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4142299/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jul, 2024 Read the published version in BMC Oral Health → Version 1 posted 16 You are reading this latest preprint version Abstract Background This study was performed to determine the therapeutic effects of diosgenin (DG) which is a steroidal saponin, administered at different doses on alveolar bone loss (ABL) in rats with experimental periodontitis using immunohistochemical and cone-beam computed tomography (CBCT). Methods Thirty-two male Wistar rats divided into four equal groups: control (non-ligated), periodontitis (P), DG-48, and DG-96. Sutures were placed at the gingival margin of the lower first molars to induce experimental periodontitis. Then, 48 and 96 mg/kg of DG was administered to the study groups by oral gavage for 29 days. At day 30, the animals were sacrificed and ABL was determined via CBCT. Results The expression patterns of osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (Col-1), B cell lymphoma 2 (Bcl 2), Bcl 2-associated X protein (Bax), bone morphogenetic protein 2 (BMP-2), and receptor activator of NF κB ligand (RANKL) were examined immunohistochemically. DG treatment increased the ALP, OCN, Bcl 2, Col-1, and BMP-2 levels in a dose-dependent manner, compared with the P group (p < 0.05); it decreased the receptor RANKL, Bax, and ABL levels in a dose-dependent manner (p < 0.05). Conclusion DG contributes to bone formation by decreasing ABL and apoptosis in rats with periodontitis. Alveolar bone loss apoptosis diosgenin periodontitis Figures Figure 1 Figure 2 Figure 3 Background Periodontitis is an inflammatory disease in which the interactions between periodontal bacteria and the host tissue response lead to tissue destruction [ 1 ]. Increases in oxidative stress, proinflammatory cytokines, and osteoclast cells have major roles in periodontal destruction [ 2 ]. Reactive oxygen species (ROS) can help to eliminate invading pathogenic microorganisms in healthy tissues [ 3 ]. However, an excessive increase in the ROS level causes an imbalance between ROS products and antioxidants, resulting in oxidative stress within tissues. Oxidative stress stimulates the transformation of precursor osteoclast cells into mature osteoclasts, leading to pathological changes, followed by the destruction of affected tissue [ 3 – 5 ]. ROS causes apoptosis by reducing B-cell lymphoma 2 proteins (Bcl-2) and elevating the expression of Bcl-2-associated X protein (BAX).6 In addition, the elevation of ROS levels can damage tissue cells by stimulating proinflammatory cytokine cells and increasing receptor activator of NFκB ligand (RANKL) levels [ 1 , 7 , 8 ]. RANKL is expressed by several types of cells, including osteoblasts, osteocytes, fibroblasts, and lymphocytes[9]. RANKL induces the activation of osteoclast cells and osteoclastogenesis because it stimulates the formation of osteoclast precursor cells. RANKL-mediated osteoclastogenesis has a critical role in periodontal destruction [ 1 , 2 , 10 ]. Growth factors organize cellular activities and improve tissue healing by binding to specific cell receptors. Several studies have used growth factors to enhance periodontal tissue and bone regeneration [ 11 , 12 ]. Bone morphogenetic protein 2 (BMP-2) is a growth factor with roles in tissue regeneration, including the transformation of undifferentiated mesenchymal cells and enhancement of osteoblast differentiation [ 13 ]. Furthermore, it stimulates the secretion of several osteoblastic-specific molecules, such as alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen (Col-1) [ 10 , 14 ]. Diosgenin (DG) is a naturally occurring bioactive steroid saponin. It has been used in several steroidal drugs in the pharmaceutical industry because its chemical structure is similar to the structures of sex hormones [ 15 – 17 ]. DG exhibits various therapeutic effects, including antioxidative, antidiabetic, anti-inflammatory, and antihyperlipidemic activities [ 16 , 18 , 19 ]. Moreover, DG modulates RANKL and OCN levels, stimulates signaling in the BMP pathways, and prevents apoptosis [ 17 , 20 – 22 ]. To our knowledge, no study has evaluated the effects of DG on periodontal destruction. Here, we hypothesized that DG could prevent periodontal tissue destruction by decreasing RANKL levels, inhibiting periodontal inflammation and cell apoptosis, and inducing bone formation. This study was performed to investigate the therapeutic effects of DG on ALP, OCN, Col-1, BAX, Bcl-2, BMP-2, and RANKL levels, as well as alveolar bone loss (ABL), in rats with experimental periodontitis. Methods Animals All experimental procedures in the present study were approved by the University Ethics Committee for Animal Experiments, Denizli (PAUHADYEK-2018/33). The Animal Research: Reporting of In Vivo Experiments guidelines were followed in this study. Thirty-two male Wistar albino rats (4 months old, 350 − 400 g) were used in this study. Before initiation of the experimental procedures, the rats were acclimated to the experimental environment for 10 days; they were housed separately in cages in a room at 21 ± 2°C and with a 12-h light:12-h dark cycle. All animals had free access to water and food. Rats were randomly separated into four groups (n = 8/group): control (non-ligated), periodontitis (P; ligature only), DG-48 (ligature + DG 48 mg/kg/day), and DG-96 (ligature + DG 96 mg/kg/day). The DG (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in distilled water and administered by oral gavage for 29 days, as in previous studies [ 17 , 23 ]. All rats were decapitated under general anesthesia at day 30 [ 10 , 24 ]. Induction of periodontitis model The experimental procedure was performed under general anesthesia by intraperitoneal administration of 50 and 5 mg/kg body weight of ketamine (Eczacibasi Ilac Sanayi, Istanbul, Turkey) and xylazine chloride (Virbaxil®, São Paulo, Brazil), respectively. The cervical regions of the first lower right and left mandibular molars were submarginally ligatured using a 4 − 0 sterile silk suture (Dogsan Ilac Sanayi, Istanbul, Turkey) to stimulate plaque accumulation and periodontal inflammation. The ligatures were checked daily by the same operator (AK). Three-dimensional imaging A supine-position cone-beam computed tomography (CBCT) unit (Newtom 5G-XL; QR, Verona, Italy) was used for three-dimensional imaging. The smallest field-of-view of this device (6 cm × 6 cm) was chosen; the exposure settings were 100 mm voxel, 110 kV, 11.4 mA, 9.0 s exposure time, 26.0 s scanning time, enhanced scan, boosted dose, and high-resolution (HiRes) mode. The unit’s proprietary software (NNT, version 12.1; QR) was used for image analysis. All specimens were exposed in the same position with the same exposure parameters. A dentomaxillofacial radiologist with 9 years of experience was blinded to the specimens (MO); this radiologist performed all tomographic procedures and analyzed the images. Figure 1 shows three-dimensional reconstructed and cross-sectional slice images. Linear bone loss (in mm) was measured from the cementoenamel junction to the alveolar bone crest and averaged across six areas (the mesial, medial, and distal parts of the buccal–lingual surfaces) of the mandibular first molar teeth. Histopathological method After rats had been sacrificed, mandibular samples were obtained and fixed in 10% neutral-buffered formalin for histopathological evaluation. The samples were decalcified in a solution (Osteofast 1; Biognost, Zagreb, Croatia) for 2 weeks, then routinely processed using automatic tissue processor equipment (Leica ASP300S; Leica Microsystems, Wetzlar, Germany) and immersed in paraffin. Subsequently, a rotary microtome (Leica RM 2155; Leica Microsystems) was used to obtained 5µm sections from each sample. Each sample was cut along the long axis of the tooth in the mesiodistal direction and stained with hematoxylin and eosin. Histopathological examinations were performed by a single specialist who was blinded to the samples (ÖÖ). Observations were conducted using a light microscope at ×40 magnification, according to a modified version of histopathological scoring criteria established by Leitao et al [ 25 ]. Inflammatory cell infiltrations, alveolar bone resorption, and degeneration and destruction of the cementum were evaluated with the following scores: 0 for absence of or only discrete cellular infiltration, and preserved alveolar process and cementum; 1 for moderate cellular infiltration and some minor alveolar process resorption, and intact cementum; 2 for accentuated cellular infiltration and accentuated degradation of the alveolar process, and partial destruction of cementum; and 3 for accentuated cellular infiltrate, complete resorption of the alveolar process, and severe destruction of cementum. Immunohistochemical method For immunohistochemical assays, sections were stained with anti-BMP-2 (ab59348; all from Abcam plc, Cambridge, UK), antiRANKL (ab216484), antiALP (ab224335), BAX (ab53154), antiBcl-2 (ab59348), antiCol-1 (ab34710), and antiOCN (ab93876) antibodies using the streptavidin–biotin peroxidase technique, in accordance with the manufacturer’s recommendations. The primary antibodies were used at a dilution of 1:100. A micro-polymer detection kit (Mouse and Rabbit Specific HRP/DAB Detection Kit-Micro-polymer, ab236466; Abcam plc) was used as the secondary antibody with 3,3´diaminobenzidine as the chromogen. The primary antibody was not used in negative controls. All immunohistochemical evaluations were performed by a specialized pathologist who was blinded to the samples (ÖÖ). Seven serial sections were prepared and analyzed for each rat; two areas of each sample were scored semiquantitatively according to the staining intensity, as described previously (0, absence of staining; 1, slight; 2, medium; and 3, marked) [26]. After the classic microscopic analyses, we obtained histomorphometric and immunohistochemical evaluations using an automated image analysis system (Olympus CX41; Olympus Corporation, Tokyo, Japan). The lesioned area was evaluated using proprietary software (cellSens Life Science Imaging Software System; Olympus Corporation). Statistical analysis The Shapiro–Wilk test was used to assess whether data exhibited normal distributions. The post hoc Duncan multiple comparison test and one-way analysis of variance were used to analyze the ABL. Independent variables (ALP, BAX, Bcl-2, BMP-2, Col-1, OCN, RANKL, and histopathological scores) were compared using the Kruskal–Wallis test. All data are reported as means ± standard deviations for each group (p < 0.05). All analyses were conducted using SPSS software (version 23; IBM Corporation, Armonk, NY, USA). Results CBCT findings Periodontitis was induced in all ligated groups, according to the CBCT findings. The results showed that the control group had no ABL. ABL was significantly lower in the DG-48 and DG-96 groups than in the P group (p < 0.05; Fig. 1 , Table 1 ). Table 1 Statistical analysis of histopathological, immunohistochemical, and cone-beam computed tomography results. Variable Group Control Periodontitis Diosgenin 48 mg/kg/day Diosgenin 96 mg/kg/day Alveolar bone loss 0.29 ± 0.012 b,c,d 0.945 ± 0.164 a,c,d 0.835 ± 0.016 a,b,d 0.761 ± 0.011 a,b,c Alkaline phosphatase 1.42 ± 0.53 b,d 0.28 ± 0.18 a,c,d 1.57 ± 0.53 b,d 2.57 ± 0.78 a,b,c Bcell lymphoma 2-associated X protein 0.14 ± 0.03 b,c,d 2.57 ± 0.78 a,c,d 1.57 ± 0.53 a,b 1.28 ± 0.48 a,b Bcell lymphoma 2 1.57 ± 0.53 b,d 0.57 ± 0.29 a,c,d 1.85 ± 0.69 b,d 2.42 ± 0.78 a,b,c Bone morphogenetic protein 2 1.42 ± 0.42 b,d 0.57 ± 0.29 a,c,d 1.71 ± 0.75 b,d 2.57 ± 0.53 a,b,c Receptor activator of NF-κB ligand 0.42 ± 0.20 b,c 2.71 ± 0.48 a,c,d 1.00 ± 0.81 a,b,d 0.42 ± 0.20 b,c Type I collagen 2.42 ± 0.53 b,c,d 0.71 ± 0.28 a,c,d 1.57 ± 0.78 a,b 1.85 ± 0.69 a,b Osteocalcin 2.85 ± 0.37 b,c,d 1.00 ± 0.57 a,c,d 1.85 ± 0.37 a,b 1.85 ± 1.06 a,b a,b,c,d Statistically significant difference (p < 0.05) between the control, periodontitis, or diosgenin 48 or 96 mg/kg/day group, respectively, and the other groups. All values expressed as means ± standard deviations. Histopathological findings Histological examination showed that the control group had normal gingival tissue architecture and gingival epithelium; it showed no pathological findings. Hyperemia, ulcers in the gingival epithelial layer, inflammatory reactions in the gingival tissue and periodontal ligament, partial to severe cement destruction, and alveolar bone degradation were observed in the P group. Microscopic evaluations of the DG-48 and DG-96 groups revealed that the treatments ameliorated the pathological findings, compared with the P group. Furthermore, cellular infiltration, ABL, and cement destruction were reduced in the DG-96 group, compared with the DG-48 group (Fig. 2 ). Immunohistochemical findings The expression patterns of ALP, Bcl-2, BAX, Col-1, BMP-2, OCN, and RANKL in mesenchymal cells in all groups were observed immunohistochemically. Positive immunoexpression was indicated by a brown color. During examinations of the ALP, BAX, Bcl-2, BMP-2, Col-1, OCN, and RANKL immunostained sections, slight to negative immunoexpression findings were observed in the control group. ALP, Bcl-2, BMP-2, Col-1, and OCN expression levels were significantly lower in the P group than in the control group (p < 0.05). Treatment significantly increased the expression levels of ALP, Bcl-2, BMP-2, Col-1, and OCN in the DG groups, compared with the P group (p < 0.05). Additionally, DG-96 was more effective than DG-48 for normalizing immunoexpression (Fig. 3 ). Statistical analysis results of the immunohistochemical scores are shown in Table 1 . BAX and RANKL expression levels increased in the P group, compared with the control group (p < 0.05). Treatment significantly decreased RANKL and BAX levels in the DG groups, compared with the P group (p < 0.05). Finally, DG-96 significantly decreased the expression levels of RANKL and BAX, compared with DG-48 (p < 0.05; Fig. 3 , Table 1 ). Discussion In the present study, we used histomorphometry, immunohistochemistry, and CBCT to evaluate the effects of DG dose on ABL in experimental periodontitis. To our knowledge, this is the first study regarding the effects of DG in rats with experimental periodontitis. The doses of DG were determined on the basis of previous findings [ 17 , 23 , 27 ]. Micro-computerized tomography is regarded as the “gold standard” method for analyzing trabecular bone and tooth microstructure, evaluating the development of the skull bones, and assessing tissue engineering[ 28 ]. However, several studies have evaluated the efficacy of CBCT as an alternative for assessing periodontal defects because micro-computerized tomography involves ultra-high radiation doses and is not routinely used in clinical settings[ 29 ]. Thus, Tayman et al. investigated the use of CBCT to measure periodontal defects; they concluded that it provides useful linear and volumetric measurements of such defects in vitro [ 29 ]. Other studies have suggested that CBCT can be used to evaluate periodontal defects and the structures and trabecular microarchitecture of alveolar bone[ 30 ]. In an experimental study, Lektemur Alpan et al. demonstrated that CBCT measurements of ABL levels were accurate[ 30 ]. Thus, we measured ABL using CBCT in this study. RANKL is the primary regulator of osteoclastogenesis; it has a critical role in osteoclast-associated diseases[ 31 ]. Several studies have demonstrated that ABL is associated with high RANKL levels [ 28 , 32 ]. One study suggested that DG suppresses osteoclastogenesis by reducing NF-κB-regulated gene expression[ 33 ]. Zhang et al. reported that a high dose of DG decreased bone loss by modulating the RANKL and osteoprotegerin levels in an ovariectomized rat model [ 17 ]. In an another study, Zhang et al. indicated that DG inhibited osteogenesis and osteoclastogenesis by regulating the expression levels of important molecules, including RANKL[ 34 ]. In the present study, DG treatments significantly downregulated the RANKL levels and inhibited RANKL-induced osteoclastogenesis in rats in a dose-dependent manner, compared with the untreated periodontitis group. Furthermore, DG significantly decreased ABL in a dose-dependent manner, compared with the untreated group. These results suggested that DG prevents ABL by inhibiting RANKL expression and RANKL-induced osteoclastogenesis, consistent with the findings of earlier reports [ 17 , 33 , 34 ]. Inflammation can increase oxidative stress, thus, worsening DNA damage and tissue apoptosis[ 35 ].. Moreover, periodontal disease reportedly leads to an imbalance between pro- and anti‐apoptotic processes [ 36 ]. Therefore, we evaluated apoptotic marker levels in our study. BAX is a member of the Bcl-2 family; expression levels of BAX and Bcl-2 are considered indicators of apoptosis or survival in cells [ 10 , 37 ]. In the present study, we evaluated BAX and Bcl-2 levels to identify the effects of DG on apoptosis signaling pathways. The results showed that experimental periodontitis upregulated and downregulated the expression levels of BAX and Bcl-2, respectively. In contrast, DG treatment upregulated and downregulated the expression levels of Bcl-2 and BAX, respectively, in our experimental periodontitis model. These results indicate that DG treatment decreases periodontitis–induced apoptosis by suppressing the expression of BAX and inducing the expression of Bcl-2; these results are also consistent with previous findings [ 21 ]. Several biochemical markers have been used to evaluate bone metabolic activity, including ALP, Col-1, OCN, and BMP-2 [ 10 , 37 ]. ALP is released by osteoblast cells; measurements of ALP level are used to evaluate osteoblastic activity. OCN controls mineral deposition; thus, it has critical roles in bone formation and remodeling [ 38 ]. Col-1 is an important factor that stimulates osteoblast differentiation and mineral matrix deposition[ 39 ]. Furthermore, BMP-2 mediates the differentiation of osteoblastic cells and induces the release of ALP, OCN, and Col-1 [ 10 , 13 , 14 ]. Zhao et al. reported that DG significantly promotes bone formation and inhibits bone absorption by regulating bone metabolism and mineralization [ 22 ]. Another study found that DG could enhance the bone formation process through increased Wnt and BMP signaling activity; these pathways regulate the osteogenic differentiation of mesenchymal stem cells and preosteoblasts [ 20 ]. Liao et al. found that the arginyl–DG conjugate stimulates BMP-2-induced osteoblastic differentiation with synergistic effects on ALP activity and mineralization [ 40 ]. In the present study, DG significantly promoted the expression of ALP, OCN, Col-1, and BMP-2. These findings suggest that DG enhances bone formation by increasing new bone activity through enhanced expression of ALP, OCN, Col-1, and BMP-2; this is also consistent with previous findings [ 20 , 22 , 40 ]. Conclusion The present study indicated that both doses of DG—particularly the higher dose—regulate bone activity, prevent RANKL-induced osteoclastogenesis and cell apoptosis, and improve new bone activity and bone formation. However, we did not evaluate the effect of DG on the Wnt pathways or osteoprotegerin levels and did not compare the micro-computerized tomography and CBCT; these were limitations of the present study. Therefore, further studies are needed to investigate the effects of DG on the Wnt pathways and osteoprotegerin expression in periodontal disease. Our results indicate that DG administration can prevent alveolar bone damage in periodontal disease. Abbreviations ROS Reactive oxygen species Bcl‑2 B-cell lymphoma 2 proteins BAX Bcl‑2-associated X protein RANKL NF‑κB ligand BMP‑2 Bone morphogenetic protein 2 ALP Alkaline phosphatase Osteocalcin (OCN) Col‑1 Type I collagen DG Diosgenin ABL Alveolar bone loss P Periodontitis DG‑48 ligature + DG 48 mg/kg/day DG‑96 ligature + DG 96 mg/kg/day CBCT Cone-beam computed tomography Declarations Ethics approval and consent to participate All experimental procedures in the present study were approved by the University Ethics Committee for Animal Experiments, Denizli (PAUHADYEK-2018/33) Consent for Publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors report no conflicts of interest related to this study Funding This study was self-funded by the authors Authors' contributions AK, ALA and TA induced periodontitis, applied experimental procedures. MÖ performed radiographic analyzed and ÖÖ applied the histological examinations. AK and ALA wrote the article. All authors read and approved the final manuscript. Acknowledgements Not applicable References Arabacı T, Kermen E, Özkanlar S, et al. Therapeutic effects of melatonin on alveolar bone resorption after experimental periodontitis in rats: a biochemical and immunohistochemical study. J Periodontol. 2015;86(7):874–81. Kızıldağ A, Arabacı T, Albayrak M, et al. Therapeutic effects of caffeic acid phenethyl ester on alveolar bone loss in rats with endotoxin-induced periodontitis. J Dent Sci. 2019;14(4):39–45. Sczepanik FSC, Grossi ML, Casati M, et al. Periodontitis is an inflammatory disease of oxidative stress: We should treat it that way. 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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-4142299","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283990706,"identity":"a3161c74-5ae9-4abf-99ca-90bf4a80b460","order_by":0,"name":"Alper Kızıldağ","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFElEQVRIiWNgGAWjYDACdhBxQIKBjZ3BgIGh4gBY8MADfFqYYVqYQVrOHGDgAfETCGsBMwwYGNsgWhjwaeFv5j34uOKMhT0fM/PGj1/n3ZGzFzv8EGiLnZxuA3YtEof5kg3P3JBgZmNmK5aW3fbMmEc6zQCoJdnY7AAOaw7zmEk2fJBgY2PmMZCW3HY4sUc6AaTlQOI2HFrkD/OY/wRq4QFqMf4tOQekJf0DXi0GQFsYG25ISAC1mEl+bABpycFviyHQL5INZyQMgH4ps2Y4dtiY53ZOwYEEA9x+kTvee/Bjw7E6e/n25s03f9QclmOfnb75w4cKOzmc3ofEAwQwI9gGuJSjaWH8gU/hKBgFo2AUjFgAAFAZXDBKNaAUAAAAAElFTkSuQmCC","orcid":"","institution":"Pamukkale University","correspondingAuthor":true,"prefix":"","firstName":"Alper","middleName":"","lastName":"Kızıldağ","suffix":""},{"id":283990709,"identity":"9f34265a-0aa8-4111-88e7-f99316e3983d","order_by":1,"name":"Aysan Lektemür Alpan","email":"","orcid":"","institution":"Pamukkale University","correspondingAuthor":false,"prefix":"","firstName":"Aysan","middleName":"Lektemür","lastName":"Alpan","suffix":""},{"id":283990711,"identity":"bffca7f1-514a-4533-a278-a32440fe2d39","order_by":2,"name":"Tuğba Köseoğlu Aydın","email":"","orcid":"","institution":"Atatürk University","correspondingAuthor":false,"prefix":"","firstName":"Tuğba","middleName":"Köseoğlu","lastName":"Aydın","suffix":""},{"id":283990712,"identity":"67971491-a882-4b5c-8ad6-86c55e44f741","order_by":3,"name":"Melih Özdede","email":"","orcid":"","institution":"Dokuz Eylül University","correspondingAuthor":false,"prefix":"","firstName":"Melih","middleName":"","lastName":"Özdede","suffix":""},{"id":283990713,"identity":"5c36057f-7ce4-436e-8c09-e7d4dd53bce9","order_by":4,"name":"Özlem Özmen","email":"","orcid":"","institution":"Burdur Mehmet Akif Ersoy University","correspondingAuthor":false,"prefix":"","firstName":"Özlem","middleName":"","lastName":"Özmen","suffix":""}],"badges":[],"createdAt":"2024-03-21 09:27:00","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4142299/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4142299/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12903-024-04646-3","type":"published","date":"2024-07-28T16:15:35+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53760896,"identity":"c495251e-c921-428e-ac0a-dc52eb716071","added_by":"auto","created_at":"2024-03-29 20:25:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":208655,"visible":true,"origin":"","legend":"\u003cp\u003eCone-beam computed tomography images from all groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure Legends:\u003c/strong\u003e The left and middle columns show three-dimensional reconstruction images of the buccal and lingual areas, respectively; the right column shows cross-sectional images.\u003c/p\u003e","description":"","filename":"Fig.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142299/v1/6cdd12eb1737a8c9d0dbf5d3.jpg"},{"id":53760898,"identity":"17d03163-acdb-4a8d-8b6a-ac2bd7e922d2","added_by":"auto","created_at":"2024-03-29 20:25:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":513045,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological appearance of the groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure Legends:\u003c/strong\u003eNormal gingival histology in the control group (arrow); gingival epithelial loss and severe inflammatory reaction in the periodontitis group; moderate inflammatory reaction in the diosgenin 48 mg/kg/day (DG‑48) group; and decreased inflammatory reaction and periodontal lesions and increased epithelization in the diosgenin 96 mg/kg/day (DG‑96) group. T: teeth; the bars represent 100 µm.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142299/v1/f3723509c20c412a549a8108.jpg"},{"id":53761249,"identity":"4c351664-4e11-4204-8ae3-57fc3c0e4d61","added_by":"auto","created_at":"2024-03-29 20:33:49","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":568338,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical expression results for all groups according to the streptavidin–biotin method.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFigure Legends:\u003c/strong\u003eNegative-to-slight expression in the control group; marked decreases in alkaline phosphatase (ALP), B‑cell lymphoma 2 (Bcl‑2), bone morphogenetic protein 2 (BMP‑2), Col‑1, and osteocalcin (OCN) but increases in Bcl‑2-associated X protein (BAX) and receptor activator of NF-κB ligand (RANKL) in the periodontitis (P) group; and amelioration by diosgenin 48 mg/kg/day (DG‑48) and diosgenin 96 mg/kg/day (DG‑96) in those groups. The arrows indicate cells expressing markers; the bars represent 50 µm.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4142299/v1/b79f5024500272851533fb4d.jpg"},{"id":61596165,"identity":"f293e80b-6b35-4712-b072-712c6dfba778","added_by":"auto","created_at":"2024-08-01 17:25:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1751998,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4142299/v1/645808c9-25d2-4e98-9b41-2330cc4663fd.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of diosgenin dose on alveolar bone loss in rats with experimental periodontitis","fulltext":[{"header":"Background","content":"\u003cp\u003ePeriodontitis is an inflammatory disease in which the interactions between periodontal bacteria and the host tissue response lead to tissue destruction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Increases in oxidative stress, proinflammatory cytokines, and osteoclast cells have major roles in periodontal destruction [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Reactive oxygen species (ROS) can help to eliminate invading pathogenic microorganisms in healthy tissues [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, an excessive increase in the ROS level causes an imbalance between ROS products and antioxidants, resulting in oxidative stress within tissues. Oxidative stress stimulates the transformation of precursor osteoclast cells into mature osteoclasts, leading to pathological changes, followed by the destruction of affected tissue [\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. ROS causes apoptosis by reducing B-cell lymphoma 2 proteins (Bcl-2) and elevating the expression of Bcl-2-associated X protein (BAX).6 In addition, the elevation of ROS levels can damage tissue cells by stimulating proinflammatory cytokine cells and increasing receptor activator of NFκB ligand (RANKL) levels [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. RANKL is expressed by several types of cells, including osteoblasts, osteocytes, fibroblasts, and lymphocytes[9]. RANKL induces the activation of osteoclast cells and osteoclastogenesis because it stimulates the formation of osteoclast precursor cells. RANKL-mediated osteoclastogenesis has a critical role in periodontal destruction [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGrowth factors organize cellular activities and improve tissue healing by binding to specific cell receptors. Several studies have used growth factors to enhance periodontal tissue and bone regeneration [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Bone morphogenetic protein 2 (BMP-2) is a growth factor with roles in tissue regeneration, including the transformation of undifferentiated mesenchymal cells and enhancement of osteoblast differentiation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Furthermore, it stimulates the secretion of several osteoblastic-specific molecules, such as alkaline phosphatase (ALP), osteocalcin (OCN), and type I collagen (Col-1) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDiosgenin (DG) is a naturally occurring bioactive steroid saponin. It has been used in several steroidal drugs in the pharmaceutical industry because its chemical structure is similar to the structures of sex hormones [\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. DG exhibits various therapeutic effects, including antioxidative, antidiabetic, anti-inflammatory, and antihyperlipidemic activities [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Moreover, DG modulates RANKL and OCN levels, stimulates signaling in the BMP pathways, and prevents apoptosis [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo our knowledge, no study has evaluated the effects of DG on periodontal destruction. Here, we hypothesized that DG could prevent periodontal tissue destruction by decreasing RANKL levels, inhibiting periodontal inflammation and cell apoptosis, and inducing bone formation. This study was performed to investigate the therapeutic effects of DG on ALP, OCN, Col-1, BAX, Bcl-2, BMP-2, and RANKL levels, as well as alveolar bone loss (ABL), in rats with experimental periodontitis.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003e All experimental procedures in the present study were approved by the University Ethics Committee for Animal Experiments, Denizli (PAUHADYEK-2018/33). The Animal Research: Reporting of In Vivo Experiments guidelines were followed in this study. Thirty-two male Wistar albino rats (4 months old, 350\u0026thinsp;\u0026minus;\u0026thinsp;400 g) were used in this study. Before initiation of the experimental procedures, the rats were acclimated to the experimental environment for 10 days; they were housed separately in cages in a room at 21\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and with a 12-h light:12-h dark cycle. All animals had free access to water and food. Rats were randomly separated into four groups (n\u0026thinsp;=\u0026thinsp;8/group): control (non-ligated), periodontitis (P; ligature only), DG-48 (ligature\u0026thinsp;+\u0026thinsp;DG 48 mg/kg/day), and DG-96 (ligature\u0026thinsp;+\u0026thinsp;DG 96 mg/kg/day). The DG (Sigma-Aldrich, Saint Louis, MO, USA) was dissolved in distilled water and administered by oral gavage for 29 days, as in previous studies [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. All rats were decapitated under general anesthesia at day 30 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eInduction of periodontitis model\u003c/h2\u003e \u003cp\u003eThe experimental procedure was performed under general anesthesia by intraperitoneal administration of 50 and 5 mg/kg body weight of ketamine (Eczacibasi Ilac Sanayi, Istanbul, Turkey) and xylazine chloride (Virbaxil\u0026reg;, S\u0026atilde;o Paulo, Brazil), respectively. The cervical regions of the first lower right and left mandibular molars were submarginally ligatured using a 4\u0026thinsp;\u0026minus;\u0026thinsp;0 sterile silk suture (Dogsan Ilac Sanayi, Istanbul, Turkey) to stimulate plaque accumulation and periodontal inflammation. The ligatures were checked daily by the same operator (AK).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eThree-dimensional imaging\u003c/h2\u003e \u003cp\u003eA supine-position cone-beam computed tomography (CBCT) unit (Newtom 5G-XL; QR, Verona, Italy) was used for three-dimensional imaging. The smallest field-of-view of this device (6 cm \u0026times; 6 cm) was chosen; the exposure settings were 100 mm voxel, 110 kV, 11.4 mA, 9.0 s exposure time, 26.0 s scanning time, enhanced scan, boosted dose, and high-resolution (HiRes) mode. The unit\u0026rsquo;s proprietary software (NNT, version 12.1; QR) was used for image analysis. All specimens were exposed in the same position with the same exposure parameters. A dentomaxillofacial radiologist with 9 years of experience was blinded to the specimens (MO); this radiologist performed all tomographic procedures and analyzed the images. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows three-dimensional reconstructed and cross-sectional slice images. Linear bone loss (in mm) was measured from the cementoenamel junction to the alveolar bone crest and averaged across six areas (the mesial, medial, and distal parts of the buccal\u0026ndash;lingual surfaces) of the mandibular first molar teeth.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological method\u003c/h2\u003e \u003cp\u003eAfter rats had been sacrificed, mandibular samples were obtained and fixed in 10% neutral-buffered formalin for histopathological evaluation. The samples were decalcified in a solution (Osteofast 1; Biognost, Zagreb, Croatia) for 2 weeks, then routinely processed using automatic tissue processor equipment (Leica ASP300S; Leica Microsystems, Wetzlar, Germany) and immersed in paraffin. Subsequently, a rotary microtome (Leica RM 2155; Leica Microsystems) was used to obtained 5\u0026micro;m sections from each sample. Each sample was cut along the long axis of the tooth in the mesiodistal direction and stained with hematoxylin and eosin. Histopathological examinations were performed by a single specialist who was blinded to the samples (\u0026Ouml;\u0026Ouml;).\u003c/p\u003e \u003cp\u003eObservations were conducted using a light microscope at \u0026times;40 magnification, according to a modified version of histopathological scoring criteria established by Leitao et al [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Inflammatory cell infiltrations, alveolar bone resorption, and degeneration and destruction of the cementum were evaluated with the following scores: 0 for absence of or only discrete cellular infiltration, and preserved alveolar process and cementum; 1 for moderate cellular infiltration and some minor alveolar process resorption, and intact cementum; 2 for accentuated cellular infiltration and accentuated degradation of the alveolar process, and partial destruction of cementum; and 3 for accentuated cellular infiltrate, complete resorption of the alveolar process, and severe destruction of cementum.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical method\u003c/h2\u003e \u003cp\u003eFor immunohistochemical assays, sections were stained with anti-BMP-2 (ab59348; all from Abcam plc, Cambridge, UK), antiRANKL (ab216484), antiALP (ab224335), BAX (ab53154), antiBcl-2 (ab59348), antiCol-1 (ab34710), and antiOCN (ab93876) antibodies using the streptavidin\u0026ndash;biotin peroxidase technique, in accordance with the manufacturer\u0026rsquo;s recommendations. The primary antibodies were used at a dilution of 1:100.\u003c/p\u003e \u003cp\u003eA micro-polymer detection kit (Mouse and Rabbit Specific HRP/DAB Detection Kit-Micro-polymer, ab236466; Abcam plc) was used as the secondary antibody with 3,3\u0026acute;diaminobenzidine as the chromogen. The primary antibody was not used in negative controls. All immunohistochemical evaluations were performed by a specialized pathologist who was blinded to the samples (\u0026Ouml;\u0026Ouml;). Seven serial sections were prepared and analyzed for each rat; two areas of each sample were scored semiquantitatively according to the staining intensity, as described previously (0, absence of staining; 1, slight; 2, medium; and 3, marked) [26].\u003c/p\u003e \u003cp\u003eAfter the classic microscopic analyses, we obtained histomorphometric and immunohistochemical evaluations using an automated image analysis system (Olympus CX41; Olympus Corporation, Tokyo, Japan). The lesioned area was evaluated using proprietary software (cellSens Life Science Imaging Software System; Olympus Corporation).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe Shapiro\u0026ndash;Wilk test was used to assess whether data exhibited normal distributions. The post hoc Duncan multiple comparison test and one-way analysis of variance were used to analyze the ABL. Independent variables (ALP, BAX, Bcl-2, BMP-2, Col-1, OCN, RANKL, and histopathological scores) were compared using the Kruskal\u0026ndash;Wallis test. All data are reported as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations for each group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). All analyses were conducted using SPSS software (version 23; IBM Corporation, Armonk, NY, USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCBCT findings\u003c/h2\u003e \u003cp\u003ePeriodontitis was induced in all ligated groups, according to the CBCT findings. The results showed that the control group had no ABL. ABL was significantly lower in the DG-48 and DG-96 groups than in the P group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical analysis of histopathological, immunohistochemical, and cone-beam computed tomography results.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVariable\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ePeriodontitis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDiosgenin\u003c/p\u003e \u003cp\u003e48 mg/kg/day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eDiosgenin\u003c/p\u003e \u003cp\u003e96 mg/kg/day\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlveolar bone loss\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.012\u003csup\u003eb,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.945\u0026thinsp;\u0026plusmn;\u0026thinsp;0.164\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.835\u0026thinsp;\u0026plusmn;\u0026thinsp;0.016\u003csup\u003ea,b,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.761\u0026thinsp;\u0026plusmn;\u0026thinsp;0.011\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAlkaline phosphatase\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBcell lymphoma 2-associated X protein\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03\u003csup\u003eb,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBcell lymphoma 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBone morphogenetic protein 2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003csup\u003eb,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003ea,b,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReceptor activator of NF-κB ligand\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.48\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81\u003csup\u003ea,b,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e0.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003csup\u003eb,c\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eType I collagen\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.42\u0026thinsp;\u0026plusmn;\u0026thinsp;0.53\u003csup\u003eb,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.71\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.57\u0026thinsp;\u0026plusmn;\u0026thinsp;0.78\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eOsteocalcin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003eb,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.57\u003csup\u003ea,c,d\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.85\u0026thinsp;\u0026plusmn;\u0026thinsp;1.06\u003csup\u003ea,b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003e\u003csup\u003ea,b,c,d\u003c/sup\u003e Statistically significant difference (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) between the control, periodontitis, or diosgenin 48 or 96 mg/kg/day group, respectively, and the other groups.\u003c/td\u003e\u003c/tr\u003e \u003ctr\u003e\u003ctd colspan=\"5\"\u003eAll values expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eHistopathological findings\u003c/h2\u003e \u003cp\u003eHistological examination showed that the control group had normal gingival tissue architecture and gingival epithelium; it showed no pathological findings. Hyperemia, ulcers in the gingival epithelial layer, inflammatory reactions in the gingival tissue and periodontal ligament, partial to severe cement destruction, and alveolar bone degradation were observed in the P group. Microscopic evaluations of the DG-48 and DG-96 groups revealed that the treatments ameliorated the pathological findings, compared with the P group. Furthermore, cellular infiltration, ABL, and cement destruction were reduced in the DG-96 group, compared with the DG-48 group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemical findings\u003c/h2\u003e \u003cp\u003eThe expression patterns of ALP, Bcl-2, BAX, Col-1, BMP-2, OCN, and RANKL in mesenchymal cells in all groups were observed immunohistochemically. Positive immunoexpression was indicated by a brown color. During examinations of the ALP, BAX, Bcl-2, BMP-2, Col-1, OCN, and RANKL immunostained sections, slight to negative immunoexpression findings were observed in the control group.\u003c/p\u003e \u003cp\u003eALP, Bcl-2, BMP-2, Col-1, and OCN expression levels were significantly lower in the P group than in the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Treatment significantly increased the expression levels of ALP, Bcl-2, BMP-2, Col-1, and OCN in the DG groups, compared with the P group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Additionally, DG-96 was more effective than DG-48 for normalizing immunoexpression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Statistical analysis results of the immunohistochemical scores are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBAX and RANKL expression levels increased in the P group, compared with the control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Treatment significantly decreased RANKL and BAX levels in the DG groups, compared with the P group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Finally, DG-96 significantly decreased the expression levels of RANKL and BAX, compared with DG-48 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn the present study, we used histomorphometry, immunohistochemistry, and CBCT to evaluate the effects of DG dose on ABL in experimental periodontitis. To our knowledge, this is the first study regarding the effects of DG in rats with experimental periodontitis. The doses of DG were determined on the basis of previous findings [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMicro-computerized tomography is regarded as the \u0026ldquo;gold standard\u0026rdquo; method for analyzing trabecular bone and tooth microstructure, evaluating the development of the skull bones, and assessing tissue engineering[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. However, several studies have evaluated the efficacy of CBCT as an alternative for assessing periodontal defects because micro-computerized tomography involves ultra-high radiation doses and is not routinely used in clinical settings[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Thus, Tayman et al. investigated the use of CBCT to measure periodontal defects; they concluded that it provides useful linear and volumetric measurements of such defects in vitro [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Other studies have suggested that CBCT can be used to evaluate periodontal defects and the structures and trabecular microarchitecture of alveolar bone[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In an experimental study, Lektemur Alpan et al. demonstrated that CBCT measurements of ABL levels were accurate[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Thus, we measured ABL using CBCT in this study.\u003c/p\u003e \u003cp\u003eRANKL is the primary regulator of osteoclastogenesis; it has a critical role in osteoclast-associated diseases[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Several studies have demonstrated that ABL is associated with high RANKL levels [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. One study suggested that DG suppresses osteoclastogenesis by reducing NF-κB-regulated gene expression[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Zhang et al. reported that a high dose of DG decreased bone loss by modulating the RANKL and osteoprotegerin levels in an ovariectomized rat model [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In an another study, Zhang et al. indicated that DG inhibited osteogenesis and osteoclastogenesis by regulating the expression levels of important molecules, including RANKL[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In the present study, DG treatments significantly downregulated the RANKL levels and inhibited RANKL-induced osteoclastogenesis in rats in a dose-dependent manner, compared with the untreated periodontitis group. Furthermore, DG significantly decreased ABL in a dose-dependent manner, compared with the untreated group. These results suggested that DG prevents ABL by inhibiting RANKL expression and RANKL-induced osteoclastogenesis, consistent with the findings of earlier reports [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eInflammation can increase oxidative stress, thus, worsening DNA damage and tissue apoptosis[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].. Moreover, periodontal disease reportedly leads to an imbalance between pro- and anti‐apoptotic processes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Therefore, we evaluated apoptotic marker levels in our study. BAX is a member of the Bcl-2 family; expression levels of BAX and Bcl-2 are considered indicators of apoptosis or survival in cells [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the present study, we evaluated BAX and Bcl-2 levels to identify the effects of DG on apoptosis signaling pathways. The results showed that experimental periodontitis upregulated and downregulated the expression levels of BAX and Bcl-2, respectively. In contrast, DG treatment upregulated and downregulated the expression levels of Bcl-2 and BAX, respectively, in our experimental periodontitis model. These results indicate that DG treatment decreases periodontitis\u0026ndash;induced apoptosis by suppressing the expression of BAX and inducing the expression of Bcl-2; these results are also consistent with previous findings [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eSeveral biochemical markers have been used to evaluate bone metabolic activity, including ALP, Col-1, OCN, and BMP-2 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. ALP is released by osteoblast cells; measurements of ALP level are used to evaluate osteoblastic activity. OCN controls mineral deposition; thus, it has critical roles in bone formation and remodeling [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Col-1 is an important factor that stimulates osteoblast differentiation and mineral matrix deposition[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Furthermore, BMP-2 mediates the differentiation of osteoblastic cells and induces the release of ALP, OCN, and Col-1 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Zhao et al. reported that DG significantly promotes bone formation and inhibits bone absorption by regulating bone metabolism and mineralization [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Another study found that DG could enhance the bone formation process through increased Wnt and BMP signaling activity; these pathways regulate the osteogenic differentiation of mesenchymal stem cells and preosteoblasts [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Liao et al. found that the arginyl\u0026ndash;DG conjugate stimulates BMP-2-induced osteoblastic differentiation with synergistic effects on ALP activity and mineralization [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In the present study, DG significantly promoted the expression of ALP, OCN, Col-1, and BMP-2. These findings suggest that DG enhances bone formation by increasing new bone activity through enhanced expression of ALP, OCN, Col-1, and BMP-2; this is also consistent with previous findings [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe present study indicated that both doses of DG\u0026mdash;particularly the higher dose\u0026mdash;regulate bone activity, prevent RANKL-induced osteoclastogenesis and cell apoptosis, and improve new bone activity and bone formation. However, we did not evaluate the effect of DG on the Wnt pathways or osteoprotegerin levels and did not compare the micro-computerized tomography and CBCT; these were limitations of the present study. Therefore, further studies are needed to investigate the effects of DG on the Wnt pathways and osteoprotegerin expression in periodontal disease. Our results indicate that DG administration can prevent alveolar bone damage in periodontal disease.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eROS \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Reactive oxygen species\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBcl‑2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; B-cell lymphoma 2 proteins\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eBAX \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bcl‑2-associated X protein\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRANKL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; NF‑\u0026kappa;B ligand\u003c/p\u003e\n\u003cp\u003eBMP‑2 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Bone morphogenetic protein 2\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eALP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Alkaline phosphatase Osteocalcin (OCN)\u003c/p\u003e\n\u003cp\u003eCol‑1 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Type I collagen\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDG \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Diosgenin\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eABL \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Alveolar bone loss\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eP \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Periodontitis\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDG‑48 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;ligature + DG 48 mg/kg/day\u003c/p\u003e\n\u003cp\u003eDG‑96 \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;ligature + DG 96 mg/kg/day\u003c/p\u003e\n\u003cp\u003eCBCT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Cone-beam computed tomography\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures in the present study were approved by the University Ethics Committee for Animal Experiments, Denizli (PAUHADYEK-2018/33)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors report no conflicts of interest related to this study\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was self-funded by the authors\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAK, ALA and TA induced periodontitis, applied experimental procedures. M\u0026Ouml; performed radiographic analyzed and \u0026Ouml;\u0026Ouml; applied the histological examinations. AK and ALA wrote the article. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eArabacı T, Kermen E, \u0026Ouml;zkanlar S, et al. Therapeutic effects of melatonin on alveolar bone resorption after experimental periodontitis in rats: a biochemical and immunohistochemical study. J Periodontol. 2015;86(7):874\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKızıldağ A, Arabacı T, Albayrak M, et al. Therapeutic effects of caffeic acid phenethyl ester on alveolar bone loss in rats with endotoxin-induced periodontitis. 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Chem Biol Drug Des. 2018;91(1):17\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Alveolar bone loss, apoptosis, diosgenin, periodontitis","lastPublishedDoi":"10.21203/rs.3.rs-4142299/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4142299/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThis study was performed to determine the therapeutic effects of diosgenin (DG) which is a steroidal saponin, administered at different doses on alveolar bone loss (ABL) in rats with experimental periodontitis using immunohistochemical and cone-beam computed tomography (CBCT).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThirty-two male Wistar rats divided into four equal groups: control (non-ligated), periodontitis (P), DG-48, and DG-96. Sutures were placed at the gingival margin of the lower first molars to induce experimental periodontitis. Then, 48 and 96 mg/kg of DG was administered to the study groups by oral gavage for 29 days. At day 30, the animals were sacrificed and ABL was determined via CBCT.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe expression patterns of osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (Col-1), B cell lymphoma 2 (Bcl 2), Bcl 2-associated X protein (Bax), bone morphogenetic protein 2 (BMP-2), and receptor activator of NF κB ligand (RANKL) were examined immunohistochemically. DG treatment increased the ALP, OCN, Bcl 2, Col-1, and BMP-2 levels in a dose-dependent manner, compared with the P group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05); it decreased the receptor RANKL, Bax, and ABL levels in a dose-dependent manner (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eDG contributes to bone formation by decreasing ABL and apoptosis in rats with periodontitis.\u003c/p\u003e","manuscriptTitle":"Effects of diosgenin dose on alveolar bone loss in rats with experimental periodontitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-29 20:25:45","doi":"10.21203/rs.3.rs-4142299/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-24T04:30:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-23T14:54:13+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-22T21:54:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102543179274235751622031148693177425913","date":"2024-06-15T00:30:08+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-13T12:02:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"271696872635525392504419603952800632690","date":"2024-06-12T08:38:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"93374143883978008402199680572435477900","date":"2024-06-10T10:05:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-03T08:59:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-28T23:37:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"194451296145987371712054101699180865754","date":"2024-05-27T08:42:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"58462747591570426046582352190082716864","date":"2024-05-25T09:53:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-25T08:21:40+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-05-24T11:45:30+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-03-26T09:19:20+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-26T09:14:02+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Oral Health","date":"2024-03-21T09:23:58+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-oral-health","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ohea","sideBox":"Learn more about [BMC Oral Health](http://bmcoralhealth.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/ohea/default.aspx","title":"BMC Oral Health","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e728b574-5d36-4f92-8647-b6ebad5887b0","owner":[],"postedDate":"March 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-01T17:06:06+00:00","versionOfRecord":{"articleIdentity":"rs-4142299","link":"https://doi.org/10.1186/s12903-024-04646-3","journal":{"identity":"bmc-oral-health","isVorOnly":false,"title":"BMC Oral Health"},"publishedOn":"2024-07-28 16:15:35","publishedOnDateReadable":"July 28th, 2024"},"versionCreatedAt":"2024-03-29 20:25:45","video":"","vorDoi":"10.1186/s12903-024-04646-3","vorDoiUrl":"https://doi.org/10.1186/s12903-024-04646-3","workflowStages":[]},"version":"v1","identity":"rs-4142299","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4142299","identity":"rs-4142299","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}