Osteoking ameliorates type 2 diabetes osteoporosis by promoting osteoblasts proliferation via PI3K/AKT/GSK-3β pathway activation

Osteoking ameliorates type 2 diabetes osteoporosis by promoting osteoblasts proliferation via PI3K/AKT/GSK-3β pathway activation | 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 Article Osteoking ameliorates type 2 diabetes osteoporosis by promoting osteoblasts proliferation via PI3K/AKT/GSK-3β pathway activation Rong Li, Jiangli Lu, Peijin Wang, Yulan Zhao, Yi Yang, Jianlin Jiao, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3833910/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Osteoking (OK) is a Yi folk Chinese herb from the Yunnan province, which exerts bone formation-promoting effects on menopausal osteoporosis and osteoporotic fractures. However, it remains to be determined whether OK ameliorates type 2 diabetic osteoporosis (T2DOP). Thus, T2DOP animal model was established in db/db mice in this study. Micro-computed tomography (micro-CT) analysis revealed that OK significantly increased bone strength, improved bone metabolism, and promoted bone formation. GS and p-GSK-3β expression levels were increased in OK group as compared with db/db group by Western blot analysis. IL-6, IL-17A, IFN-γ, TNF-α, and IL-1β were lower levels in the OK group compared to the db/db group, nevertheless, the IL-10 level was significantly higher. Furthermore, an In vitro cells model was constructed by stimulating with high glucose (HG, 30 mM). ALP protein was significantly elevated in OK treatment group. Administration of OK at 1.44 mg/mL significantly increased p-AKT/AKT expression, while, combined with LY294002, an inhibitor of PI3K, OK significantly reduced the expression levels of p-PI3K/PI3K, p-AKT/AKT and p-GSK-3β/GSK-3β. In conclusion, to our knowledge, this study is the first to reveal OK exhibits efficacy against T2DOP in db/db mice by promoting osteogenesis of preosteoblast MC3T3-E1 cells through PI3K/AKT/GSK-3β pathway regulation. Biological sciences/Drug discovery Biological sciences/Molecular biology Health sciences/Diseases Health sciences/Medical research Health sciences/Molecular medicine Osteoking Type 2 diabetes osteoporosis preosteoblast osteoblasts PI3K/AKT/GSK-3β pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. Introduction Type 2 diabetes mellitus (T2DM) is a widespread chronic metabolic disease. Epidemiological studies have shown that about 90% of the nearly 500 million diabetic patients worldwide have T2DM [ 1 ]. Diabetic osteoporosis (DO), a severe complication of T2DM, is increasing annually, causing an increased healthcare burden on patients [ 2 – 4 ]. Studies have shown that the high glucose environment inhibits osteoblasts and disrupts the dynamic balance of bone metabolism [ 5 , 6 ], eventually leading to osteoporosis (OP). Additionally, hyperglycemia stimulates the production of advanced glycation end products (AGEs), and AGEs product receptors inhibit PI3K, which can prevent osteoblast proliferation and reduce osteosynthesis, resulting in OP [ 7 ]. Currently, no available drug can simultaneously decrease blood glucose and improve type 2 diabetes osteoporosis T2DOP [ 2 ]. Treatment strategies for the disease have primarily been glucose lowering and calcium supplementation. Unfortunately, insulin and thiazolidinedione hypoglycemic agents can affect bone health [ 8 , 9 ]. Additionally, bisphosphonate usage against OP can cause severe gastrointestinal reactions and medication adherence [ 10 ]. Therefore, it is necessary to develop harmless and affordable drugs to treat T2DOP. Osteoking (OK), a compound preparation from the Yi nationality in the Yunnan province of China, is composed of Carthamus tinctorius L. , Panax notoginseng (Burkill) F.H.Chen , Panax ginseng C.A.Mey. , Astragalus mongholicus Bunge , Eucommia ulmoides Oliv , Trionycis carapace, Datura metel L. , Schizophragma integrifolium Oliv , and Citrus reticulata Blanco. Each plant name has been verified in “The Plant List” ( www.theplantlist.org ) and MPNS ( http://mpns.kew.org ). OK was approved by the Chinese State Food and Drug Administration (Z20025103) for clinical use in 2002 [ 11 ], after which it was clinically confirmed to treat fractures while preventing deep venous thrombosis [ 12 ]. OK reduces osteocalcin levels, improves bone density and strength, and prevents osteoporosis in ovariectomized rabbits [ 13 ]. OK increase bone ALP levels in OP model rats by enhancing HSP90-β expression and protecting MC3T3-E1 cells from oxidative stress to improve OP [ 11 , 14 ]. Meanwhile, the potential of various natural medicines in reducing blood sugar in the prescription, including the active components of Carthamus tinctorius L , Panax ginseng C.A.Mey. , Panax notoginseng (Burkill) F.H.Chen , Astragalus mongholicus Bunge and Citrus reticulata Blanco . These natural medicines may exert anti-diabetic effects by regulating the PI3K/AKT pathway to reduce insulin resistance and blood glucose levels in db/db mice [ 15 – 19 ]. Additionally, extracts of Panax ginseng C.A.Mey. and Panax notoginseng (Burkill) FH.Chen can reduce the level of inflammatory factors, including tumor necrosis factor-α (TNF-α), IL-6, and IL-1β [ 20 ]. Collectively, we predict a potential hypoglycemic effect of OK and explore its role in treating T2DOP. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway is involved in cell growth, proliferation, differentiation, and metabolism [ 21 ]. The PI3K/AKT pathway is critical in glucose metabolism and bone homeostasis. Its activation can promote osteoblast proliferation and differentiation [ 22 ]. Moreover, the PI3K/AKT pathway negatively regulates glycogen synthase kinase (GSK-3β) by promoting glycogen synthase (GS) activation, thus increasing glycogen synthesis and lowering blood glucose levels [ 23 – 25 ]. The PI3K/ AKT pathway may be a key mechanism in T2DOP treatment. T2DOP has gained widespread attention, but whether OK benefits this condition is unclear. Our study aimed to explore the role and mechanism of OK in improving T2DOP. Using in vivo and in vitro models, we investigated whether OK protects osteoblasts and regulates glucose and bone homeostasis by regulating the PI3K/AKT pathway. These findings will provide new strategies for the clinical treatment of T2DOP. 2. Methods 2.1 Osteoking preparation The OK used in this experiment (batch No. 20191111) was prepared according to the Chinese Pharmacopoeia (version 2015) purchased from Yunnan Crystal Natural Pharmaceutical Co., Ltd. (Kunming, China) (batch No. Z20025103). Briefly, the following natural plants were used: Carthamus tinctorius L. (15 g), Panax notoginseng (Burkill) FH.Chen (30 g), Panax ginseng C.A.Mey . (20 g), Astragalus mongholicus Bunge (40 g), Eucommia ulmoides Oliv (30 g), Trionycis carapace (10 g), Datura metel L. (6 g), Schizophragma integrifolium Oliv . (l0 g), and Citrus reticulata Blanco (10 g). A previously published method was used for OK (0.36 g/mL) preparation by boiling, filtering, centrifuging, and adjusting the pH value [ 26 ]. All natural plants used to prepare the OK were identified following the Chinese Pharmacopoeia (version 2015) by Yunnan Crystal Natural Pharmaceutical Co., Ltd. The test report was consistent with previous studies on OK [ 27 ]. According to [ 28 ], the drug active ingredients were determined using UltraPerformance Liquid Chromatography according to the Chinese Pharmacopeia (version 2015), and the quality of the drug from the manufacturer was confirmed to be of good quality. 2.2. Animal experiments and ethics statement Seven-weeks-old male diabetic (SPF) db/db mice on the C57BKS background (BKS-Lepr em2Cd479 /Gpt) and their homozygous littermate wild-type (WT) control mice purchased from GemPharmatech Co., Ltd. (SCXK (SU) 2018-0008, China, Nanjing) were used in the current study. All animals were housed under controlled temperature (22–24°C), 12 h light/dark cycles, and 50% – 60% humidity. Six WT mice and 12 db/db mice were randomly assigned to the WT group, db/db group, and db/db treatment group receiving OK. All the mice were administered intragastric (0.1 mL/10 g/d) doses of OK. The WT and db/db group mice were given 0.9% normal saline, while the OK group was given 0.72 g/mL of OK for 10 weeks. Dosages administered were determined according to a human-mouse conversion algorithm of body surface area. Blood glucose levels were measured before the start of treatment and at the end of the study. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The blood samples were collected from the eyelids of mice using ambrosia. The collected serum was stored in heparinized tubes at 4°C overnight (at 3500 rpm for 15 min). After the animals were humanely sacrificed by intraperitoneal injection of excessive pentobarbital sodium, the liver, femurs, tibia, and lumbar spine were dissected for measurement. Animal welfare were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Research Council Institute for Laboratory Animals, R. (1996), Washington (DC), National Academy Press (US)). All procedures in this study were conducted in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Kunming Medical University (Approval No. : KMMU2021652). All the authors complied with the ethical standards and practices. 2.3. Periodic Acid-Schiff Stain (PAS) and Histopathology PAS staining Liver tissues were fixed, embedded, and sectioned. After dehydration, the sections were placed sequentially in periodic acid and Schiff’s reagent for 10 min, then stained with hematoxylin for 3 min. Next, routine dehydration and transparency procedures were followed, and pathological changes were observed. Histopathology The right femur specimens were first immersed in 4% paraformaldehyde for 48 h at 4°C, then transferred into 10% EDTA for 20 days. The specimens were subsequently dehydrated in ethanol, defatted in xylene, and embedded in paraffin wax. Paraffin sections with 5 µm thickness were stained with hematoxylin and eosin (H&E, Solar bio, G1100, Beijing, China) and Masson (Solar bio, G1340, Beijing, China). The sections were then examined for pathological changes using a Leica Fluorescence Microscope (Leica, Heidelberg, Germany) and Adobe Photoshop (version 2018) software (Adobe Systems, USA). 2.4. Cytokine Analysis Liver supernatants were extracted from db/db mice livers. A mouse cytokine Bio-Plex Pro™ Th17 Panel A 6-Plex (BIO-RAD, M6000007NY, California, USA) was used to measure inflammatory cytokine concentration. The kit was used to measure TNF-α, interferon-γ (IFN-γ), IL-6, IL-17A, IL-1β, and IL-10 levels in liver supernatants, following the manufacturer’s protocol. A Luminex X-200 IS System (Luminex, TX, USA) was used to read out the samples, compute standard curves, and estimate cytokine concentrations. 2.5. Biomechanical 3-point bending test The right tibias were subjected to 3-point bending tests using a bone strength tester machine (MUROMACHI KIKAI CO., LTD, MODEL TK-252D, No. 170417). Each right tibia was wrapped in gauze soaked in normal saline and stored overnight in a refrigerator at 4°C. The tibia specimens were taken out and placed at room temperature for 1 h. The specific methods used were as follows: the midpoint of the tibia was determined using a vernier caliper; the tibia was then placed on a self-made 3-point bending platform with a bracket span of 10 mm and at a 5 mm distance between the two ends of the bracket and the midpoint of the tibia; a horizontal downward force was applied at 20 mm/min speed at the midpoint of the tibia until the tibia was broken. The maximum load, maximum displacement, stiffness, and maximum energy absorption were obtained from the load-displacement curve. 2.6. Enzyme-linked immunosorbent assay (ELISA) The concentration of osteoprotegerin (OPG) (Mouse OPG ELISA kit, QY-M30277, QIYI Biological Technology, Shanghai, China), Bone Gla protein (BGP) (Mouse BGP ELISA kit, QIY-01670, QIYI Biological Technology, Shanghai, China), and C- terminal telopeptide of type I collagen (CTX- 1) (Mouse CTX- 1 ELISA kit, QY-M30118, QIYI Biological Technology, Shanghai, China) was measured in the mice plasma after OK administration for 10 weeks, according to the manufacturer's protocol. 2.7. Micro-computed tomography (Micro-CT) scanning The right femur was scanned using a Brucker Skyscan1172 (Skyscan, Aartselaar, Belgium). The µCT scanning parameters chosen for each specimen included 50 kV voltage, 180 µA current, 950 ms exposure time, 180° total rotation angle, and 0.4° rotation angle of increment. After scanning, 2-dimensional image sequences were reconstructed to the 3-dimensional image with an isotropic voxel size of 15.9 µm. To quantify femoral trabecular bone microarchitecture, a volume of interest (VOI) with 1.0 mm height, which started at 0.5 mm distance from the lowest end of the growth plate of the distal femur and extended to the proximal end with 1.0 mm distance, was selected, containing the second spongiosa. All 3D image manipulations and analyses were performed using the system software (CTAn, Skyscan). The following trabecular bone architectural parameters were determined, including trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structure model index (SMI). 2.8. Western blotting Relevant proteins were extracted from the liver, femur, and tibia using RIPA buffer containing protease inhibitor cocktails (R0020, Solar bio, China). The proteins were separated by SDS-PAGE and detected using the following primary antibodies: Phospho-glycogen synthase kinase-3β (p-GSK-3β; 1:1000, 9323T, CST), Glycogen synthase (GS; ab40810, 1:10000, Abcam), GSK-3β (ab75814, 1:10000, Abcam), Phospho-glycogen synthase (p-GS; ab81230, 1:5000, Abcam), Alkaline phosphatase (ALP; ab108337, 1:2000, Abcam), Tartrate-resistant acid phosphatase (TRACP; ab191406, 1:5000, Abcam), Phosphatidylinositol-3 kinase (p-PI3K; 1:1000, AF3241, Affinity), P13K (ab191606, 1:1000, Abcam), Phosphorylation protein kinase (p-AKT; 4060T, 1:500, CST), AKT (ab179463, 1:10000, Abcam), and β-actin (E-AB-20058, 1:1000, Elabscience). The primary antibodies were incubated with the protein samples at 4°C overnight, followed by incubation with fluorescent secondary antibodies (incubated with horseradish peroxidase (HRP, ZB2301, 1:5000, ZSGB-BIO) at 37°C for 1 h). Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) system, and the intensity ratio of each band to the β-actin band was calculated for each sample using densitometry. 2.9. Cell culture MC3T3-E1, a mouse pre-osteoblastic cell line used in experiments, was purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were maintained in α-MEM (Biological Industries, 01-042-1ACS, Israel) medium supplemented with 10% fetal bovine serum (FBS, Every Green, 11011 − 8611, Zhejiang, China) and 0.1% penicillin-streptomycin (Hyclone, SV30010, UT, USA). 2.10. Cell counting kit-8 (CCK-8) assays Cell viability was assessed using the CCK-8 (Dojindo, Tokyo, Japan) assay. Briefly, cells were cultured at a density of 1 × 10 4 cells/well in 96-well plates at 37°C. Then, 5 mM glucose, 30 mM glucose, 30 mM glucose + 7.2 mg/mL OK, 30 mM glucose + 1.44 mg/mL OK, 30 mM glucose + 0.288 mg/mL OK, and 30 mM glucose + 0.0576 mg/mL OK were added for 12, 24, and 36 h. A 10 µL of CCK-8 was added to each well, and the cells were cultured at 37°C for 3 h. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA). Cell viability% = (OD sample - OD blank) / (OD control - OD blank) × 100% 2.11. ALP activity tests ALP activity was detected using an Alkaline Phosphatase Detection Kit, following the manufacturer’s instructions. Briefly, MC3T3-E1 cells were seeded into 6-well plates at a density of 5 × 10 3 cells/well for 24 h. Afterward, the cells were exposed to different conditions for 48 h: 5 mM glucose, 30 mM glucose, 30 mM glucose + 1.44 mg/mL OK, and 30 mM glucose + 0.288 mg/mL OK. Cells were treated with an osteogenesis induction medium containing penicillin-streptomycin (0.1%), FBS (10%), L-Ascorbic acid (50 µg/mL), β-glycerophosphate (10 mM), and dexamethasone (10 8 mM) for 7 and 14 days, with the medium being replaced every 3 days. Cells were washed thrice with PBS before being lysed with 0.05% Triton X-100 on ice for 30 min. Cells were incubated with p-nitrophenol phosphate for 1 h at 37°C, and the reaction was stopped with 2 M NaOH. Finally, the absorbance at 405 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA). 2.12. Alizarin Red S(ARS) staining The MC3T3-E1 cells were seeded into 6-well plates at the density of 5 × 10 3 cells/well for 24 h. Afterward, the cells were exposed to different conditions for 48 h: 5 mM glucose, 30 mM glucose, 30 mM glucose + 1.44 mg/mL OK, and 30 mM glucose + 0.288 mg/mL OK. Cells were treated with an osteogenesis induction medium for 21 days. After induction for a specified time, cells were fixed in 95% ethanol for 30 min and stained with 0.2% ARS (Solar bio, G1450, Beijing, China) at pH 8.3 for 30 min at room temperature. The 6-well plates were observed under an optical microscope (Olympus Corporation, Tokyo, Japan), and the total area of red calcified nodules was measured using ImageJ 2.0 software. 2.13. Statistical analysis All data were presented as mean ± SD (Standard Deviation), except for Fig. 2 , Fig. 6 A, and Fig. 7C, which were analyzed using two-way ANOVA followed by Tukey's post hoc test. The remaining figures were analyzed using one-way ANOVA with Tukey’s multiple comparison test. We performed all statistical analyses using GraphPad Prism 7 (GraphPad, San Diego, CA, USA) and considered P < 0.05 statistically significant. 2.14. Ethics statement All the methods were carried out according to ARRIVE guidelines and regulations in this study. 3. Result 3.1. OK decreased blood glucose and increased glycogen synthesis in db/db mice Body weights of db/db and OK group mice were significantly higher than in the WT group, but there was an insignificant difference between db/db and OK groups (Fig. 1 A). Blood glucose level of the db/db group before administration was significantly higher than that of the WT group (P < 0.01). After the OK intervention, the blood glucose level of the OK group was significantly lower than that of the db/db group (P < 0.01) (Fig. 1 B). Upon observing PAS-reactive substances in liver tissues, the db/db group showed impaired cell morphology, a lighter purplish-red color of cytoplasm, reduced glycogen content, and significantly lower mean optical density (P < 0.01) compared to the WT group. Contrarily, the OK group, which showed cells returning to normal, exhibited a deepened purplish-red color, higher glycogen content, and significantly higher mean optical density (P < 0.05) compared to the db/db group (Fig. 1 C, D). To assess the expression levels of four proteins: GS, p-GS, GSK-3β, and p-GSK-3β by Western blot analysis on db/db mice liver tissues. GS and p-GSK-3β expressions were significant decreases (P < 0.01), along with a significant increase in p-GS expression (P < 0.01), GSK-3β expression was not significantly higher in the db/db group compared with the WT group. In the OK group, GS and p-GSK-3β expression levels were significant increases (P < 0.01, P < 0.05), along with a significant decrease in p-GS expression level (P < 0.01). However, GSK-3β expression level was not significant decrease in the OK group compared with the db/db group (Fig. 1 E, F). 3.2. OK reduces inflammatory cytokines in the livers of the db/db mice The inflammatory cytokines levels TNF-α, IFN-γ, IL-6, IL-17A (P < 0.01), and IL-1β (P < 0.05) were increased in the db/db group compared to the WT group. The IL-10 level was not significantly decreased. Furthermore, in the OK group, IL-6, IL-17A (P < 0.01), IFN-γ, TNF-α, and IL-1β (P < 0.05) were significantly decreased compared to the db/db group. Additionally, the IL-10 level was significantly higher (P < 0.05) in the OK group (Fig. 2 ). 3.3. OK enhances bone strength and improves bone metabolism in the db/db mice The maximum load, energy, maximum displacement, and stiffness were significantly declined in the db/db group than in the WT group (P < 0.01). Nevertheless, OK significantly enhanced the maximum load (P < 0.01), energy (P < 0.05), maximum displacement (P < 0.01), and stiffness (P < 0.01) levels of the tibias in the OK group than in the db/db group (Fig. 3 A, B, C, D). Elisa and Western blotting were performed to measure the levels of bone formation (OPG, BGP, and ALP) and bone resorption markers (CTX1 and TRACP) to evaluate the effect of OK on bone metabolism in db/db mice. Elisa assay revealed significantly lower OPG and BGP levels (P < 0.01) and significantly higher CTX1 levels (P < 0.05) in the serum of the db/db group compared to the WT group, with the same result in the db/db group compared to the OK group. Therefore, OK intervention could significantly increase the OPG and BGP levels (P < 0.01, P < 0.05, P < 0.05, P < 0.05, and P < 0.05) and significantly decrease the CTX1 level (P < 0.01) in the serum and lumbar spine of the db/db mice (Fig. 3 E, F, G, H, I, J). Western blotting showed that the ALP expression was significantly lower (P < 0.01) and TRACP expression was significantly higher (P < 0.01) in the db/db group compared to the WT group. After the OK intervention, ALP expression was significantly higher (P < 0.05), and TRACP expression was significantly lower (P < 0.01) in db/db mice (Fig. 3 K, L). 3.4. OK improves bone microstructure in db/db mice To further investigate the effects of OK on bone formation, the changes in bone microstructure were analyzed in the femur of db/db mice. Compared to the WT group, the db/db group exhibited significantly lower levels of BV/TV (P < 0.01), Tb.N (P < 0.01), and Tb.Th (P < 0.05), and significantly higher levels of Tb.Sp and SMI (P < 0.01). However, after the OK intervention, the levels of BV/TV (P < 0.01), Tb.N (P < 0.01), Tb.Th (P < 0.05), Tb.Sp and SMI (P < 0.01) were significantly improved in the OK group compared to the db/db group (Fig. 4 A). Additionally, the WT group had dense trabecular bone structure and normal microstructure, while the trabecular bone structure in the db/db group was sparse in number, morphologically impaired, and microstructurally deteriorated. Furthermore, the OK intervention significantly improved the trabecular bone and microstructure (Fig. 4 B, C). Pathological section staining showed that the db/db group had decreased collagen fibers and new bone area, broken bone trabeculae, and structural incompleteness compared to the WT group. Nevertheless, the collagen fibers and new bone area increased, and bone trabeculae restored connections in the OK group compared to the db/db group (Fig. 4 D, E). 3.5. OK increased MC3T3-E1 cells viability and improved the balance of intracellular bone metabolic factor The effect of different concentrations of OK on the proliferation of MC3T3-E1 cells were assessed using CCK8. Cell viability was significantly inhibited in the high glucose (HG) environment compared to the normal glucose (NG) environment. However, administration of different concentrations of OK (1.44 mg/mL, 0.288 mg/mL, and 0.0576 mg/mL) for 12, 24, and 36 h significantly increased cell viability (P < 0.01, P < 0.05, and P < 0.01). The effect of administering 7.2 mg/mL OK to improve cell viability was insignificant (Fig. 5 A). In the HG environment, ALP expression was significantly decreased (P < 0.01) compared to that in the NG environment. However, after administering different concentrations of OK (7.2 mg/mL, 1.44 mg/mL, 0.288 mg/mL, and 0.0576 mg/mL), the ALP protein was significantly elevated (P < 0.05, P < 0.01, P < 0.01, and P < 0.01) (Fig. 5 B, C). 3.6. OK promotes bone differentiation and mineralization of MC3T3-E1 cells in HG environment by regulating PI3K/AKT/GSK-3β pathway to enhance bone formation Based on the results of the preceding tests, concentrations of 1.44 and 0.288 mg/mL of OK was chosen for the subsequent experiments to explore the optimal concentration and avoid cytotoxicity. The expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-GSK-3β/GSK-3β proteins were significantly lower in the HG environment compared to NG (P < 0.01). Administration of OK at 0.288 mg/mL significantly increased the expression levels of these proteins (P < 0.01), while 1.44 mg/mL of OK significantly increased p-AKT/AKT expression (P < 0.05). However, when combined with LY294002, an inhibitor of PI3K, 1.44 mg/mL of OK significantly reduced the expression levels of p-PI3K/PI3K, p-AKT/AKT and p-GSK-3β/GSK-3β (P < 0.01), whereas 0.288 mg/mL of OK showed no statistically significant reduction (Fig. 6 A, B). In vitro , differentiation assays were performed using the ALP detection kit to examine MC3T3-E1 cells. ALP expression level was significantly lower in the HG environment compared to the NG environment (P < 0.01). The addition of OK at different concentrations significantly increased ALP expression in the HG environment (P < 0.01), and longer treatment time further enhanced this effect (P < 0.01). However, the addition of LY294002 significantly inhibited ALP expression (P < 0.01) (Fig. 6 C). Furthermore, a significant decrease in the mineralized area of MC3T3-E1 cells in the HG environment compared to the NG environment (P < 0.01). Contrarily, the mineralized area of osteoblasts in the HG environment significantly increased after OK administration (P < 0.01). However, this phenomenon was significantly inhibited by the addition of LY294002 (P < 0.01) (Fig. 6 D, E). 4. Discussion T2DM is associated with abnormal bone metabolism, and OK has a potential therapeutic effect on T2DOP. This study, OK decreased glucose values and improved T2DOP in db/db mice. GSK-3β is essential in regulating blood glucose [ 29 ]. In high glucose levels, the p-GSK-3β was decreased, which increased p-GS, leading to abnormal glucose metabolism [ 30 ]. In db/db mice experiments, treatment with OK significantly increased liver tissue glycogen content. The expressions of GS and p-GSK-3β proteins decreased considerably in liver tissues of db/db mice, while p-GS protein expression increased significantly. However, OK treatment reversed the expressions of these proteins. These findings are consistent with a previous study, which showed that Sea Buckthorn Fruit Oil Extract effectively alleviated insulin resistance in rats with high-fat diet-induced diabetes through the PI3K/AKT pathway [ 31 ]. Therefore, OK can regulate the expression of critical proteins that convert glucose to hepatic glycogen and achieve glycemic control in the db/db mice. The liver plays a crucial role in maintaining blood glucose homeostasis, and persistent hyperglycemia in T2DM can lead to low-grade inflammation [ 32 ]. Chronic liver disease patients are also at increased risk of fractures [ 33 ]. Studies have shown that patients with Type 2 diabetes have significantly increased levels of pro-inflammatory factors, including IL-1β, IL-6, TNF-α, and IFN-γ [ 34 ]. In our study, we observed that db/db mice developed an inflammatory response, but the level of anti-inflammatory factors increased, and the levels of pro-inflammatory factors were reduced after the OK intervention. Our results suggest that OK could reduce liver inflammation to control blood glucose and improve bone health in the db/db mice. Reduced biomechanical parameters and damage to trabecular bone structures in db/db mice, as determined by three-point mechanical bending and Micro-CT [ 35 ]. The expression levels of bone formation markers (OPG, BGP, and ALP) were decreased, while bone resorption markers (CTX1, TRACP) were increased in osteoporotic rats [ 11 , 36 ]. Interestingly, the present study observed deterioration in trabecular bone microarchitecture, reduced collagen fibers, and impaired new bone formation in the db/db mice. However, after treatment with OK, the tibial strength was significant improved, expression levels of bone formation markers were increased, bone resorption markers were decreased compared with the db/db group. A related study found that OK treatment improved trabecular bone microstructure, restored collagen fiber levels, promoted new bone regeneration, and enhanced the expression of osteogenic-related proteins in ovariectomy-induced osteoporosis rats by reducing oxidative stress [ 11 ]. These observations suggest that OK stimulation can regulate bone metabolism disorders, restore collagen fibers, and promote the formation of new bone at the tissue level in the db/db mice. It is well known that osteoblasts are responsible for bone formation and remodeling. ALP is an early osteoblast differentiation marker involved in osteoblasts differentiation and mineralization [ 37 ]. MC3T3-E1 cell proliferation was inhibited in a hyperglycemic environment, where the ALP level was decreased, and the bone formation was disturbed [ 38 ]. Consequently, MC3T3-E1 cells were cultured under high glucose conditions. Our findings demonstrated inhibition of MC3T3-E1 cell viability and decreased ALP levels under high glucose. Furthermore, optimal concentrations of OK increased MC3T3-E1 cell viability and reversed the ALP expression. Osteogenesis involves numerous cytokines and signaling pathways, including parathyroid hormone, PI3K/AKT/GSK, and Wnt [ 39 , 40 ]. Activated PI3K/AKT not only increases the expression of osteogenic differentiation marker genes (ALP) but also activates downstream GSK-3β and promotes bone differentiation [ 22 , 41 ]. Previous studies have shown that plus LY294002 inhibited MC3T3-E1 cell mineralization under high glucose conditions and attenuated the expression of ALP mRNA [ 42 ]. Consistent with the above results, our experiments showed that the levels of p-PI3K/PI3K, p-AKT/AKT, p-GSK-3β/GSK-3β, and ALP were significantly decreased, and the mineralized area was decreased in hyperglycemia-injured MC3T3-E1 cells and that these indicators were significantly reversed by OK treatment. Contrarily, the addition of LY294002 to MC3T3-E1 cells attenuated OK-induced osteogenesis. These results suggest that OK can improve high glucose injury by regulating the PI3K/AKT/GSK-3β pathway, which is involved in the differentiation and mineralization of MC3T3-E1 cells. In conclusion, our study demonstrated that OK had beneficial effects both in vivo and in vitro . In vivo , OK decreased blood glucose levels, regulated bone metabolism disorders, and improved bone biomechanical properties in db/db mice. In vitro , OK promoted osteoblastic differentiation and mineralization of MC3T3-E1 cells under high glucose conditions through the PI3K/AKT/GSK-3β pathway. These findings provide new insights into the clinical treatment of T2DOP and suggest that OK could be a promising therapeutic agent for this disease. Declarations Acknowledgements Not applicable. Funding The research was supported by Applied Basic Research Key Project of Yunnan Province (No.202201AY070001-032); Applied Basic Research Foundation of Yunnan Province (No. 2019FE001 (-026), 202201AT070296). Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Authors' contributions R. L. and J. L. 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Liu, Concentrate Growth Factors Regulate Osteogenic Dysfunction of MC3T3-E1 Cells Induced by High Glucose Through PI3K/Akt Signaling Pathway, Implant Dentistry 28(5) (2019) 478–483. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-3833910","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":267157893,"identity":"556b1598-b5cb-4248-8a90-e779f97d7f9f","order_by":0,"name":"Rong Li","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Li","suffix":""},{"id":267157894,"identity":"af701c90-d82a-44a0-bf6a-38922df51ce3","order_by":1,"name":"Jiangli Lu","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jiangli","middleName":"","lastName":"Lu","suffix":""},{"id":267157895,"identity":"43c3a2b5-684f-4e16-9112-6c0ff058c372","order_by":2,"name":"Peijin Wang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peijin","middleName":"","lastName":"Wang","suffix":""},{"id":267157896,"identity":"aadde25e-7f75-42c7-a5a0-986bde5cbb46","order_by":3,"name":"Yulan Zhao","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yulan","middleName":"","lastName":"Zhao","suffix":""},{"id":267157897,"identity":"fd94c7aa-6c2b-4f04-9ead-1d8620ec77c2","order_by":4,"name":"Yi Yang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Yang","suffix":""},{"id":267157898,"identity":"65aaff5d-3fcb-43a2-8b5c-d91431ec2ad1","order_by":5,"name":"Jianlin Jiao","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jianlin","middleName":"","lastName":"Jiao","suffix":""},{"id":267157899,"identity":"d106441d-70c8-444e-abac-23088ea1b716","order_by":6,"name":"Zhongyi Qian","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhongyi","middleName":"","lastName":"Qian","suffix":""},{"id":267157900,"identity":"3b2c6aef-69d0-476e-ad8e-8f6562127780","order_by":7,"name":"Limei Wang","email":"","orcid":"","institution":"Kunming Medical University","correspondingAuthor":false,"prefix":"","firstName":"Limei","middleName":"","lastName":"Wang","suffix":""},{"id":267157901,"identity":"a0d40eed-caf9-4e5f-99b8-0d68dbdeb5b0","order_by":8,"name":"Hong Zheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5UlEQVRIiWNgGAWjYDACCQjFuAFEfjCwkSNNC+OMgjRj0rQw83w4nEhQh/zs5mMPv9Tckd3O3nv4tY0BcwID++GjG/BpYZxzLN1Y5tgz450959KscwzY8hh40tJu4NPCLJFjJi3Bdjhxw40cM+McA55iBgkeM7xa2CTyv0lL/INqsTCQSGwgpIVHIodN8mMbWIvxYwYDA8JaJCTSzKQZ+w4bbzhzxoyxxyDBmI2QX+RnJD+T/PHtsOyG4z3GH378+S/Hz374GF4tIMDMA/MXmCSkHAQYf0C1fiBG9SgYBaNgFIw8AADD7ExSZGRC0QAAAABJRU5ErkJggg==","orcid":"","institution":"Kunming Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hong","middleName":"","lastName":"Zheng","suffix":""}],"badges":[],"createdAt":"2024-01-04 07:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3833910/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3833910/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":49727977,"identity":"21bb10ca-fc01-4c01-b3c0-a9a80c209a67","added_by":"auto","created_at":"2024-01-17 04:57:53","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":878629,"visible":true,"origin":"","legend":"\u003cp\u003eOK decreased blood glucose and increased glycogen synthesis in the db/db mice. OK had an insignificant effect on (A) body weight, but (B) reduced fasting glucose values in db/db mice. (C, D) Images of different groups of liver tissues stained with PAS (200X) and (400X), the integrated optical density (IOD) and total area (Area) of PAS positive sites in the sample sections were determined by ImageJ 2.0 software and the mean optical density (MOD = IOD/Area) was calculated, the results showed an increase in glycogen content after OK intervention. （E, F）Western blot analysis showed that OK decreased p-GS and increased GS, P-GSK-3β protein expression in liver tissue. The gray value was determined using the ImageJ software strip based on immunoblotting. All data were shown as the means ± SD, n = 6. \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 versus WT mice. \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 versus db/db mice. \u003csup\u003e^^\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 means the OK group at week 10 versus week 0.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/d6440bf928b9bc9dc0840a88.png"},{"id":49728414,"identity":"44012f74-4c18-47db-9a1e-490d96af0103","added_by":"auto","created_at":"2024-01-17 05:05:53","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96129,"visible":true,"origin":"","legend":"\u003cp\u003eOK improves liver inflammation. OK reduces the levels of (A)TNF-α, (B) IFN-γ, (C) IL-1β, (D) IL-6, and (E) IL-17A and increases the levels of (F) IL-10 in the liver. All data were shown as the means ± SD, n = 6. *P \u0026lt; 0.05, **P \u0026lt; 0.01 versus WT mice. \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 versus db/db mice.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/d716bb9b2e32ffea0acd3ea8.png"},{"id":49727980,"identity":"b7a9976b-2e3b-45c7-8867-205ff6e26a0c","added_by":"auto","created_at":"2024-01-17 04:57:53","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":292823,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of OK irritation on the biomechanical structure and bone metabolism of bone tissue in db/db mice. OK treatment improves (A) maximum load; (B) energy; (C) maximum displacement; and (D) stiffness in tibias. OK increases (E, F) OPG, (G, H) BGP and decreases (I, J) CTX1 in serum and lumbar, respectively. (K, L) Western blot analysis demonstrated significantly induced ALP levels and significantly decreased TRACP levels following OK treatment in liver tissue. The grayscale value was determined by ImageJ software based on immunoblots bands. All data were shown as means ± SD, n = 6. \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 versus WT mice. \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 versus db/db mice.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/7d5f381cc0edc6244ca55b7d.png"},{"id":49728415,"identity":"082113ac-9af2-4bed-9376-42a31fd3eedb","added_by":"auto","created_at":"2024-01-17 05:05:53","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2592706,"visible":true,"origin":"","legend":"\u003cp\u003eOK improved db/db mice trabecular bone structure via µCT and histological analysis. (A) Statistical quantification of trabecular microarchitecture parameters, OK enhanced BV/TV, Tb.Th, and Tb.N, and reduced Tb.Sp and SMI. (B, C) Representative µCT images showing obvious deterioration of cancellous bone microstructure in db/db mice were ameliorated after 10 weeks of OK administration. (D, E) Representative histological analysis by Masson staining and H\u0026amp;E. All data were shown as means ± SD, (n = 6).\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05,\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e**\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 versus WT mice.\u003csup\u003e \u003c/sup\u003e\u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP \u003c/em\u003e\u0026lt; 0.01 versus db/db mice.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/79d5f708ef11215ed8adeacc.png"},{"id":49727981,"identity":"892c77d4-a93f-4ded-b2e3-51358146a7b8","added_by":"auto","created_at":"2024-01-17 04:57:53","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165628,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of OK given at different concentrations on the proliferation of MC3T3-E1 in the HG environment. (A) Cell viability was measured by CCK-8 assay for 12, 24, and 36 h. (B) Different concentrations of OK increased ALP protein expression in MC3T3-E1 cells with HG environment. (C) The data for the protein expression level of the MC3T3-E1. The grayscale value was determined by ImageJ software based on immunoblots bands. All data were shown as means ± SD. \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 versus NG environment. \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 versus HG environment.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/30ee8fb954e5208603bfa471.png"},{"id":49727982,"identity":"a20419dc-0d51-43b9-b29c-dadda185968d","added_by":"auto","created_at":"2024-01-17 04:57:53","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":962592,"visible":true,"origin":"","legend":"\u003cp\u003eOK (1.44 mg/mL, 0.288 mg/ml) regulated the PI3K/AKT/GSK-3β signaling pathway and promoted bone mineralization in MC3T3-E1 cells in the HG environment. (A) Western blot showed that OK regulated PI3K/AKT signaling pathway-related protein; (B) Grayscale value was determined by ImageJ software based on immunoblots bands; (C) OK effectively increased ALP activity, but was eliminated by LY294002 on days 7 and 14. (D) Osteogenic differentiation was determined by ARS staining. demonstrating that OK increased mineralization area but was reversed by LY294002. (E) Total area (Area) was determined by ImageJ 2.0 software. All data were shown as means ± SD. \u003csup\u003e\u003cem\u003e*\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01 versus HG environment; \u003csup\u003e\u003cem\u003e#\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, \u003csup\u003e\u003cem\u003e##\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 versus OK (1.44 mg/mL, 0.288 mg/mL) group. \u003cem\u003e^^P\u003c/em\u003e \u0026lt; 0.01 means that 14 days were compared with 7 days.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/6eeb7fa2ff535f41fcf2d07f.png"},{"id":55974665,"identity":"3055d6f9-6f0c-4fe0-a24a-04b08d031cc6","added_by":"auto","created_at":"2024-05-07 04:57:30","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4174630,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3833910/v1/c66b89c6-6ee3-412b-ab64-9b279fb2e175.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Osteoking ameliorates type 2 diabetes osteoporosis by promoting osteoblasts proliferation via PI3K/AKT/GSK-3β pathway activation","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eType 2 diabetes mellitus (T2DM) is a widespread chronic metabolic disease. Epidemiological studies have shown that about 90% of the nearly 500\u0026nbsp;million diabetic patients worldwide have T2DM [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Diabetic osteoporosis (DO), a severe complication of T2DM, is increasing annually, causing an increased healthcare burden on patients [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Studies have shown that the high glucose environment inhibits osteoblasts and disrupts the dynamic balance of bone metabolism [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], eventually leading to osteoporosis (OP). Additionally, hyperglycemia stimulates the production of advanced glycation end products (AGEs), and AGEs product receptors inhibit PI3K, which can prevent osteoblast proliferation and reduce osteosynthesis, resulting in OP [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Currently, no available drug can simultaneously decrease blood glucose and improve type 2 diabetes osteoporosis T2DOP [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Treatment strategies for the disease have primarily been glucose lowering and calcium supplementation. Unfortunately, insulin and thiazolidinedione hypoglycemic agents can affect bone health [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, bisphosphonate usage against OP can cause severe gastrointestinal reactions and medication adherence [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, it is necessary to develop harmless and affordable drugs to treat T2DOP.\u003c/p\u003e \u003cp\u003eOsteoking (OK), a compound preparation from the Yi nationality in the Yunnan province of China, is composed of \u003cem\u003eCarthamus tinctorius L.\u003c/em\u003e, \u003cem\u003ePanax notoginseng (Burkill) F.H.Chen\u003c/em\u003e, \u003cem\u003ePanax ginseng C.A.Mey.\u003c/em\u003e, \u003cem\u003eAstragalus mongholicus Bunge\u003c/em\u003e, \u003cem\u003eEucommia ulmoides Oliv\u003c/em\u003e, \u003cem\u003eTrionycis carapace, Datura metel L.\u003c/em\u003e, \u003cem\u003eSchizophragma integrifolium Oliv\u003c/em\u003e, and \u003cem\u003eCitrus reticulata Blanco.\u003c/em\u003e Each plant name has been verified in \u0026ldquo;The Plant List\u0026rdquo; (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.theplantlist.org\" target=\"_blank\"\u003ewww.theplantlist.org\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.theplantlist.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) and MPNS (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://mpns.kew.org\u003c/span\u003e\u003cspan address=\"http://mpns.kew.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). OK was approved by the Chinese State Food and Drug Administration (Z20025103) for clinical use in 2002 [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], after which it was clinically confirmed to treat fractures while preventing deep venous thrombosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. OK reduces osteocalcin levels, improves bone density and strength, and prevents osteoporosis in ovariectomized rabbits [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. OK increase bone ALP levels in OP model rats by enhancing HSP90-β expression and protecting MC3T3-E1 cells from oxidative stress to improve OP [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Meanwhile, the potential of various natural medicines in reducing blood sugar in the prescription, including the active components of \u003cem\u003eCarthamus tinctorius L\u003c/em\u003e, \u003cem\u003ePanax ginseng C.A.Mey.\u003c/em\u003e, \u003cem\u003ePanax notoginseng (Burkill) F.H.Chen\u003c/em\u003e, \u003cem\u003eAstragalus mongholicus Bunge\u003c/em\u003e and \u003cem\u003eCitrus reticulata Blanco\u003c/em\u003e. These natural medicines may exert anti-diabetic effects by regulating the PI3K/AKT pathway to reduce insulin resistance and blood glucose levels in db/db mice [\u003cspan additionalcitationids=\"CR16 CR17 CR18\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Additionally, extracts of \u003cem\u003ePanax ginseng C.A.Mey.\u003c/em\u003e and \u003cem\u003ePanax notoginseng (Burkill) FH.Chen\u003c/em\u003e can reduce the level of inflammatory factors, including tumor necrosis factor-α (TNF-α), IL-6, and IL-1β [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Collectively, we predict a potential hypoglycemic effect of OK and explore its role in treating T2DOP.\u003c/p\u003e \u003cp\u003eThe phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway is involved in cell growth, proliferation, differentiation, and metabolism [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The PI3K/AKT pathway is critical in glucose metabolism and bone homeostasis. Its activation can promote osteoblast proliferation and differentiation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Moreover, the PI3K/AKT pathway negatively regulates glycogen synthase kinase (GSK-3β) by promoting glycogen synthase (GS) activation, thus increasing glycogen synthesis and lowering blood glucose levels [\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The PI3K/ AKT pathway may be a key mechanism in T2DOP treatment.\u003c/p\u003e \u003cp\u003eT2DOP has gained widespread attention, but whether OK benefits this condition is unclear. Our study aimed to explore the role and mechanism of OK in improving T2DOP. Using in vivo and in vitro models, we investigated whether OK protects osteoblasts and regulates glucose and bone homeostasis by regulating the PI3K/AKT pathway. These findings will provide new strategies for the clinical treatment of T2DOP.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Osteoking preparation\u003c/h2\u003e \u003cp\u003eThe OK used in this experiment (batch No. 20191111) was prepared according to the Chinese Pharmacopoeia (version 2015) purchased from Yunnan Crystal Natural Pharmaceutical Co., Ltd. (Kunming, China) (batch No. Z20025103). Briefly, the following natural plants were used: \u003cem\u003eCarthamus tinctorius L.\u003c/em\u003e (15 g), \u003cem\u003ePanax notoginseng (Burkill) FH.Chen\u003c/em\u003e (30 g), \u003cem\u003ePanax ginseng C.A.Mey\u003c/em\u003e. (20 g), \u003cem\u003eAstragalus mongholicus Bunge\u003c/em\u003e (40 g), \u003cem\u003eEucommia ulmoides Oliv\u003c/em\u003e (30 g), \u003cem\u003eTrionycis carapace\u003c/em\u003e (10 g), \u003cem\u003eDatura metel L.\u003c/em\u003e (6 g), \u003cem\u003eSchizophragma integrifolium Oliv\u003c/em\u003e. (l0 g), and \u003cem\u003eCitrus reticulata Blanco\u003c/em\u003e (10 g). A previously published method was used for OK (0.36 g/mL) preparation by boiling, filtering, centrifuging, and adjusting the pH value [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. All natural plants used to prepare the OK were identified following the Chinese Pharmacopoeia (version 2015) by Yunnan Crystal Natural Pharmaceutical Co., Ltd. The test report was consistent with previous studies on OK [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. According to [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], the drug active ingredients were determined using UltraPerformance Liquid Chromatography according to the Chinese Pharmacopeia (version 2015), and the quality of the drug from the manufacturer was confirmed to be of good quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Animal experiments and ethics statement\u003c/h2\u003e \u003cp\u003eSeven-weeks-old male diabetic (SPF) db/db mice on the C57BKS background (BKS-Lepr\u003csup\u003eem2Cd479\u003c/sup\u003e/Gpt) and their homozygous littermate wild-type (WT) control mice purchased from GemPharmatech Co., Ltd. (SCXK (SU) 2018-0008, China, Nanjing) were used in the current study. All animals were housed under controlled temperature (22\u0026ndash;24\u0026deg;C), 12 h light/dark cycles, and 50% \u0026ndash; 60% humidity.\u003c/p\u003e \u003cp\u003eSix WT mice and 12 db/db mice were randomly assigned to the WT group, db/db group, and db/db treatment group receiving OK. All the mice were administered intragastric (0.1 mL/10 g/d) doses of OK. The WT and db/db group mice were given 0.9% normal saline, while the OK group was given 0.72 g/mL of OK for 10 weeks. Dosages administered were determined according to a human-mouse conversion algorithm of body surface area. Blood glucose levels were measured before the start of treatment and at the end of the study. The mice were anesthetized by intraperitoneal injection of sodium pentobarbital (50 mg/kg). The blood samples were collected from the eyelids of mice using ambrosia. The collected serum was stored in heparinized tubes at 4\u0026deg;C overnight (at 3500 rpm for 15 min). After the animals were humanely sacrificed by intraperitoneal injection of excessive pentobarbital sodium, the liver, femurs, tibia, and lumbar spine were dissected for measurement.\u003c/p\u003e \u003cp\u003e Animal welfare were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals (National Research Council Institute for Laboratory Animals, R. (1996), Washington (DC), National Academy Press (US)). All procedures in this study were conducted in accordance with ARRIVE guidelines and approved by the Institutional Animal Care and Use Committee of Kunming Medical University (Approval No. : KMMU2021652). All the authors complied with the ethical standards and practices.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Periodic Acid-Schiff Stain (PAS) and Histopathology\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003ePAS staining\u003c/strong\u003e \u003cp\u003eLiver tissues were fixed, embedded, and sectioned. After dehydration, the sections were placed sequentially in periodic acid and Schiff\u0026rsquo;s reagent for 10 min, then stained with hematoxylin for 3 min. Next, routine dehydration and transparency procedures were followed, and pathological changes were observed.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eHistopathology\u003c/strong\u003e \u003cp\u003eThe right femur specimens were first immersed in 4% paraformaldehyde for 48 h at 4\u0026deg;C, then transferred into 10% EDTA for 20 days. The specimens were subsequently dehydrated in ethanol, defatted in xylene, and embedded in paraffin wax. Paraffin sections with 5 \u0026micro;m thickness were stained with hematoxylin and eosin (H\u0026amp;E, Solar bio, G1100, Beijing, China) and Masson (Solar bio, G1340, Beijing, China). The sections were then examined for pathological changes using a Leica Fluorescence Microscope (Leica, Heidelberg, Germany) and Adobe Photoshop (version 2018) software (Adobe Systems, USA).\u003c/p\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cytokine Analysis\u003c/h2\u003e \u003cp\u003eLiver supernatants were extracted from db/db mice livers. A mouse cytokine Bio-Plex Pro\u0026trade; Th17 Panel A 6-Plex (BIO-RAD, M6000007NY, California, USA) was used to measure inflammatory cytokine concentration. The kit was used to measure TNF-α, interferon-γ (IFN-γ), IL-6, IL-17A, IL-1β, and IL-10 levels in liver supernatants, following the manufacturer\u0026rsquo;s protocol. A Luminex X-200 IS System (Luminex, TX, USA) was used to read out the samples, compute standard curves, and estimate cytokine concentrations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Biomechanical 3-point bending test\u003c/h2\u003e \u003cp\u003eThe right tibias were subjected to 3-point bending tests using a bone strength tester machine (MUROMACHI KIKAI CO., LTD, MODEL TK-252D, No. 170417). Each right tibia was wrapped in gauze soaked in normal saline and stored overnight in a refrigerator at 4\u0026deg;C. The tibia specimens were taken out and placed at room temperature for 1 h. The specific methods used were as follows: the midpoint of the tibia was determined using a vernier caliper; the tibia was then placed on a self-made 3-point bending platform with a bracket span of 10 mm and at a 5 mm distance between the two ends of the bracket and the midpoint of the tibia; a horizontal downward force was applied at 20 mm/min speed at the midpoint of the tibia until the tibia was broken. The maximum load, maximum displacement, stiffness, and maximum energy absorption were obtained from the load-displacement curve.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Enzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eThe concentration of osteoprotegerin (OPG) (Mouse OPG ELISA kit, QY-M30277, QIYI Biological Technology, Shanghai, China), Bone Gla protein (BGP) (Mouse BGP ELISA kit, QIY-01670, QIYI Biological Technology, Shanghai, China), and C- terminal telopeptide of type I collagen (CTX- 1) (Mouse CTX- 1 ELISA kit, QY-M30118, QIYI Biological Technology, Shanghai, China) was measured in the mice plasma after OK administration for 10 weeks, according to the manufacturer's protocol.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Micro-computed tomography (Micro-CT) scanning\u003c/h2\u003e \u003cp\u003eThe right femur was scanned using a Brucker Skyscan1172 (Skyscan, Aartselaar, Belgium). The \u0026micro;CT scanning parameters chosen for each specimen included 50 kV voltage, 180 \u0026micro;A current, 950 ms exposure time, 180\u0026deg; total rotation angle, and 0.4\u0026deg; rotation angle of increment. After scanning, 2-dimensional image sequences were reconstructed to the 3-dimensional image with an isotropic voxel size of 15.9 \u0026micro;m. To quantify femoral trabecular bone microarchitecture, a volume of interest (VOI) with 1.0 mm height, which started at 0.5 mm distance from the lowest end of the growth plate of the distal femur and extended to the proximal end with 1.0 mm distance, was selected, containing the second spongiosa. All 3D image manipulations and analyses were performed using the system software (CTAn, Skyscan). The following trabecular bone architectural parameters were determined, including trabecular bone volume per tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and structure model index (SMI).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Western blotting\u003c/h2\u003e \u003cp\u003eRelevant proteins were extracted from the liver, femur, and tibia using RIPA buffer containing protease inhibitor cocktails (R0020, Solar bio, China). The proteins were separated by SDS-PAGE and detected using the following primary antibodies: Phospho-glycogen synthase kinase-3β (p-GSK-3β; 1:1000, 9323T, CST), Glycogen synthase (GS; ab40810, 1:10000, Abcam), GSK-3β (ab75814, 1:10000, Abcam), Phospho-glycogen synthase (p-GS; ab81230, 1:5000, Abcam), Alkaline phosphatase (ALP; ab108337, 1:2000, Abcam), Tartrate-resistant acid phosphatase (TRACP; ab191406, 1:5000, Abcam), Phosphatidylinositol-3 kinase (p-PI3K; 1:1000, AF3241, Affinity), P13K (ab191606, 1:1000, Abcam), Phosphorylation protein kinase (p-AKT; 4060T, 1:500, CST), AKT (ab179463, 1:10000, Abcam), and β-actin (E-AB-20058, 1:1000, Elabscience). The primary antibodies were incubated with the protein samples at 4\u0026deg;C overnight, followed by incubation with fluorescent secondary antibodies (incubated with horseradish peroxidase (HRP, ZB2301, 1:5000, ZSGB-BIO) at 37\u0026deg;C for 1 h). Finally, the protein bands were visualized using an enhanced chemiluminescence (ECL) system, and the intensity ratio of each band to the β-actin band was calculated for each sample using densitometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.9. Cell culture\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eMC3T3-E1, a mouse pre-osteoblastic cell line used in experiments, was purchased from the Cell Bank of the Chinese Academy of Sciences. The cells were maintained in α-MEM (Biological Industries, 01-042-1ACS, Israel) medium supplemented with 10% fetal bovine serum (FBS, Every Green, 11011\u0026thinsp;\u0026minus;\u0026thinsp;8611, Zhejiang, China) and 0.1% penicillin-streptomycin (Hyclone, SV30010, UT, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Cell counting kit-8 (CCK-8) assays\u003c/h2\u003e \u003cp\u003eCell viability was assessed using the CCK-8 (Dojindo, Tokyo, Japan) assay. Briefly, cells were cultured at a density of 1 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells/well in 96-well plates at 37\u0026deg;C. Then, 5 mM glucose, 30 mM glucose, 30 mM glucose\u0026thinsp;+\u0026thinsp;7.2 mg/mL OK, 30 mM glucose\u0026thinsp;+\u0026thinsp;1.44 mg/mL OK, 30 mM glucose\u0026thinsp;+\u0026thinsp;0.288 mg/mL OK, and 30 mM glucose\u0026thinsp;+\u0026thinsp;0.0576 mg/mL OK were added for 12, 24, and 36 h. A 10 \u0026micro;L of CCK-8 was added to each well, and the cells were cultured at 37\u0026deg;C for 3 h. The absorbance at 450 nm was measured using a microplate reader (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003cp\u003eCell viability% = (OD sample - OD blank) / (OD control - OD blank) \u0026times; 100%\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.11. ALP activity tests\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eALP activity was detected using an Alkaline Phosphatase Detection Kit, following the manufacturer\u0026rsquo;s instructions. Briefly, MC3T3-E1 cells were seeded into 6-well plates at a density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well for 24 h. Afterward, the cells were exposed to different conditions for 48 h: 5 mM glucose, 30 mM glucose, 30 mM glucose\u0026thinsp;+\u0026thinsp;1.44 mg/mL OK, and 30 mM glucose\u0026thinsp;+\u0026thinsp;0.288 mg/mL OK. Cells were treated with an osteogenesis induction medium containing penicillin-streptomycin (0.1%), FBS (10%), L-Ascorbic acid (50 \u0026micro;g/mL), β-glycerophosphate (10 mM), and dexamethasone (10\u003csup\u003e8\u003c/sup\u003e mM) for 7 and 14 days, with the medium being replaced every 3 days. Cells were washed thrice with PBS before being lysed with 0.05% Triton X-100 on ice for 30 min. Cells were incubated with p-nitrophenol phosphate for 1 h at 37\u0026deg;C, and the reaction was stopped with 2 M NaOH. Finally, the absorbance at 405 nm was detected using a microplate reader (Bio-Rad, Hercules, CA, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12. Alizarin Red S(ARS) staining\u003c/h2\u003e \u003cp\u003eThe MC3T3-E1 cells were seeded into 6-well plates at the density of 5 \u0026times; 10\u003csup\u003e3\u003c/sup\u003e cells/well for 24 h. Afterward, the cells were exposed to different conditions for 48 h: 5 mM glucose, 30 mM glucose, 30 mM glucose\u0026thinsp;+\u0026thinsp;1.44 mg/mL OK, and 30 mM glucose\u0026thinsp;+\u0026thinsp;0.288 mg/mL OK. Cells were treated with an osteogenesis induction medium for 21 days. After induction for a specified time, cells were fixed in 95% ethanol for 30 min and stained with 0.2% ARS (Solar bio, G1450, Beijing, China) at pH 8.3 for 30 min at room temperature. The 6-well plates were observed under an optical microscope (Olympus Corporation, Tokyo, Japan), and the total area of red calcified nodules was measured using ImageJ 2.0 software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13. Statistical analysis\u003c/h2\u003e \u003cp\u003eAll data were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD (Standard Deviation), except for Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, and Fig.\u0026nbsp;7C, which were analyzed using two-way ANOVA followed by Tukey's post hoc test. The remaining figures were analyzed using one-way ANOVA with Tukey\u0026rsquo;s multiple comparison test. We performed all statistical analyses using GraphPad Prism 7 (GraphPad, San Diego, CA, USA) and considered P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 statistically significant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14. Ethics statement\u003c/h2\u003e \u003cp\u003eAll the methods were carried out according to ARRIVE guidelines and regulations in this study.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Result","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1. OK decreased blood glucose and increased glycogen synthesis in db/db mice\u003c/h2\u003e \u003cp\u003eBody weights of db/db and OK group mice were significantly higher than in the WT group, but there was an insignificant difference between db/db and OK groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Blood glucose level of the db/db group before administration was significantly higher than that of the WT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). After the OK intervention, the blood glucose level of the OK group was significantly lower than that of the db/db group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Upon observing PAS-reactive substances in liver tissues, the db/db group showed impaired cell morphology, a lighter purplish-red color of cytoplasm, reduced glycogen content, and significantly lower mean optical density (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to the WT group. Contrarily, the OK group, which showed cells returning to normal, exhibited a deepened purplish-red color, higher glycogen content, and significantly higher mean optical density (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) compared to the db/db group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, D).\u003c/p\u003e \u003cp\u003eTo assess the expression levels of four proteins: GS, p-GS, GSK-3β, and p-GSK-3β by Western blot analysis on db/db mice liver tissues. GS and p-GSK-3β expressions were significant decreases (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), along with a significant increase in p-GS expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), GSK-3β expression was not significantly higher in the db/db group compared with the WT group. In the OK group, GS and p-GSK-3β expression levels were significant increases (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), along with a significant decrease in p-GS expression level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, GSK-3β expression level was not significant decrease in the OK group compared with the db/db group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE, F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2. OK reduces inflammatory cytokines in the livers of the db/db mice\u003c/h2\u003e \u003cp\u003eThe inflammatory cytokines levels TNF-α, IFN-γ, IL-6, IL-17A (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and IL-1β (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were increased in the db/db group compared to the WT group. The IL-10 level was not significantly decreased. Furthermore, in the OK group, IL-6, IL-17A (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), IFN-γ, TNF-α, and IL-1β (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were significantly decreased compared to the db/db group. Additionally, the IL-10 level was significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the OK 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=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3. OK enhances bone strength and improves bone metabolism in the db/db mice\u003c/h2\u003e \u003cp\u003eThe maximum load, energy, maximum displacement, and stiffness were significantly declined in the db/db group than in the WT group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Nevertheless, OK significantly enhanced the maximum load (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), energy (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), maximum displacement (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and stiffness (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) levels of the tibias in the OK group than in the db/db group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B, C, D). Elisa and Western blotting were performed to measure the levels of bone formation (OPG, BGP, and ALP) and bone resorption markers (CTX1 and TRACP) to evaluate the effect of OK on bone metabolism in db/db mice. Elisa assay revealed significantly lower OPG and BGP levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and significantly higher CTX1 levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the serum of the db/db group compared to the WT group, with the same result in the db/db group compared to the OK group. Therefore, OK intervention could significantly increase the OPG and BGP levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and significantly decrease the CTX1 level (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the serum and lumbar spine of the db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE, F, G, H, I, J). Western blotting showed that the ALP expression was significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) and TRACP expression was significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in the db/db group compared to the WT group. After the OK intervention, ALP expression was significantly higher (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and TRACP expression was significantly lower (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) in db/db mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eK, L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4. OK improves bone microstructure in db/db mice\u003c/h2\u003e \u003cp\u003eTo further investigate the effects of OK on bone formation, the changes in bone microstructure were analyzed in the femur of db/db mice. Compared to the WT group, the db/db group exhibited significantly lower levels of BV/TV (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Tb.N (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and Tb.Th (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), and significantly higher levels of Tb.Sp and SMI (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, after the OK intervention, the levels of BV/TV (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Tb.N (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), Tb.Th (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), Tb.Sp and SMI (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) were significantly improved in the OK group compared to the db/db group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Additionally, the WT group had dense trabecular bone structure and normal microstructure, while the trabecular bone structure in the db/db group was sparse in number, morphologically impaired, and microstructurally deteriorated. Furthermore, the OK intervention significantly improved the trabecular bone and microstructure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C). Pathological section staining showed that the db/db group had decreased collagen fibers and new bone area, broken bone trabeculae, and structural incompleteness compared to the WT group. Nevertheless, the collagen fibers and new bone area increased, and bone trabeculae restored connections in the OK group compared to the db/db group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5. OK increased MC3T3-E1 cells viability and improved the balance of intracellular bone metabolic factor\u003c/h2\u003e \u003cp\u003eThe effect of different concentrations of OK on the proliferation of MC3T3-E1 cells were assessed using CCK8. Cell viability was significantly inhibited in the high glucose (HG) environment compared to the normal glucose (NG) environment. However, administration of different concentrations of OK (1.44 mg/mL, 0.288 mg/mL, and 0.0576 mg/mL) for 12, 24, and 36 h significantly increased cell viability (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The effect of administering 7.2 mg/mL OK to improve cell viability was insignificant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). In the HG environment, ALP expression was significantly decreased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) compared to that in the NG environment. However, after administering different concentrations of OK (7.2 mg/mL, 1.44 mg/mL, 0.288 mg/mL, and 0.0576 mg/mL), the ALP protein was significantly elevated (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB, C).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003e3.6. OK promotes bone differentiation and mineralization of MC3T3-E1 cells in HG environment by regulating PI3K/AKT/GSK-3β pathway to enhance bone formation\u003c/b\u003e \u003c/p\u003e \u003cp\u003eBased on the results of the preceding tests, concentrations of 1.44 and 0.288 mg/mL of OK was chosen for the subsequent experiments to explore the optimal concentration and avoid cytotoxicity. The expression levels of p-PI3K/PI3K, p-AKT/AKT, and p-GSK-3β/GSK-3β proteins were significantly lower in the HG environment compared to NG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Administration of OK at 0.288 mg/mL significantly increased the expression levels of these proteins (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), while 1.44 mg/mL of OK significantly increased p-AKT/AKT expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, when combined with LY294002, an inhibitor of PI3K, 1.44 mg/mL of OK significantly reduced the expression levels of p-PI3K/PI3K, p-AKT/AKT and p-GSK-3β/GSK-3β (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), whereas 0.288 mg/mL of OK showed no statistically significant reduction (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA, B).\u003c/p\u003e \u003cp\u003e \u003cem\u003eIn vitro\u003c/em\u003e, differentiation assays were performed using the ALP detection kit to examine MC3T3-E1 cells. ALP expression level was significantly lower in the HG environment compared to the NG environment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The addition of OK at different concentrations significantly increased ALP expression in the HG environment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and longer treatment time further enhanced this effect (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, the addition of LY294002 significantly inhibited ALP expression (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eFurthermore, a significant decrease in the mineralized area of MC3T3-E1 cells in the HG environment compared to the NG environment (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Contrarily, the mineralized area of osteoblasts in the HG environment significantly increased after OK administration (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). However, this phenomenon was significantly inhibited by the addition of LY294002 (P\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD, E).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eT2DM is associated with abnormal bone metabolism, and OK has a potential therapeutic effect on T2DOP. This study, OK decreased glucose values and improved T2DOP in db/db mice. GSK-3β is essential in regulating blood glucose [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In high glucose levels, the p-GSK-3β was decreased, which increased p-GS, leading to abnormal glucose metabolism [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In db/db mice experiments, treatment with OK significantly increased liver tissue glycogen content. The expressions of GS and p-GSK-3β proteins decreased considerably in liver tissues of db/db mice, while p-GS protein expression increased significantly. However, OK treatment reversed the expressions of these proteins. These findings are consistent with a previous study, which showed that Sea Buckthorn Fruit Oil Extract effectively alleviated insulin resistance in rats with high-fat diet-induced diabetes through the PI3K/AKT pathway [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Therefore, OK can regulate the expression of critical proteins that convert glucose to hepatic glycogen and achieve glycemic control in the db/db mice.\u003c/p\u003e \u003cp\u003eThe liver plays a crucial role in maintaining blood glucose homeostasis, and persistent hyperglycemia in T2DM can lead to low-grade inflammation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Chronic liver disease patients are also at increased risk of fractures [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Studies have shown that patients with Type 2 diabetes have significantly increased levels of pro-inflammatory factors, including IL-1β, IL-6, TNF-α, and IFN-γ [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. In our study, we observed that db/db mice developed an inflammatory response, but the level of anti-inflammatory factors increased, and the levels of pro-inflammatory factors were reduced after the OK intervention. Our results suggest that OK could reduce liver inflammation to control blood glucose and improve bone health in the db/db mice.\u003c/p\u003e \u003cp\u003eReduced biomechanical parameters and damage to trabecular bone structures in db/db mice, as determined by three-point mechanical bending and Micro-CT [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The expression levels of bone formation markers (OPG, BGP, and ALP) were decreased, while bone resorption markers (CTX1, TRACP) were increased in osteoporotic rats [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Interestingly, the present study observed deterioration in trabecular bone microarchitecture, reduced collagen fibers, and impaired new bone formation in the db/db mice. However, after treatment with OK, the tibial strength was significant improved, expression levels of bone formation markers were increased, bone resorption markers were decreased compared with the db/db group. A related study found that OK treatment improved trabecular bone microstructure, restored collagen fiber levels, promoted new bone regeneration, and enhanced the expression of osteogenic-related proteins in ovariectomy-induced osteoporosis rats by reducing oxidative stress [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. These observations suggest that OK stimulation can regulate bone metabolism disorders, restore collagen fibers, and promote the formation of new bone at the tissue level in the db/db mice.\u003c/p\u003e \u003cp\u003eIt is well known that osteoblasts are responsible for bone formation and remodeling. ALP is an early osteoblast differentiation marker involved in osteoblasts differentiation and mineralization [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. MC3T3-E1 cell proliferation was inhibited in a hyperglycemic environment, where the ALP level was decreased, and the bone formation was disturbed [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Consequently, MC3T3-E1 cells were cultured under high glucose conditions. Our findings demonstrated inhibition of MC3T3-E1 cell viability and decreased ALP levels under high glucose. Furthermore, optimal concentrations of OK increased MC3T3-E1 cell viability and reversed the ALP expression. Osteogenesis involves numerous cytokines and signaling pathways, including parathyroid hormone, PI3K/AKT/GSK, and Wnt [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Activated PI3K/AKT not only increases the expression of osteogenic differentiation marker genes (ALP) but also activates downstream GSK-3β and promotes bone differentiation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. Previous studies have shown that plus LY294002 inhibited MC3T3-E1 cell mineralization under high glucose conditions and attenuated the expression of ALP mRNA [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Consistent with the above results, our experiments showed that the levels of p-PI3K/PI3K, p-AKT/AKT, p-GSK-3β/GSK-3β, and ALP were significantly decreased, and the mineralized area was decreased in hyperglycemia-injured MC3T3-E1 cells and that these indicators were significantly reversed by OK treatment. Contrarily, the addition of LY294002 to MC3T3-E1 cells attenuated OK-induced osteogenesis. These results suggest that OK can improve high glucose injury by regulating the PI3K/AKT/GSK-3β pathway, which is involved in the differentiation and mineralization of MC3T3-E1 cells.\u003c/p\u003e \u003cp\u003eIn conclusion, our study demonstrated that OK had beneficial effects both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, OK decreased blood glucose levels, regulated bone metabolism disorders, and improved bone biomechanical properties in db/db mice. \u003cem\u003eIn vitro\u003c/em\u003e, OK promoted osteoblastic differentiation and mineralization of MC3T3-E1 cells under high glucose conditions through the PI3K/AKT/GSK-3β pathway. These findings provide new insights into the clinical treatment of T2DOP and suggest that OK could be a promising therapeutic agent for this disease.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was supported by Applied Basic Research Key Project of Yunnan Province (No.202201AY070001-032); Applied Basic Research Foundation of Yunnan Province (No. 2019FE001 (-026), 202201AT070296).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eR. L. and J. L.\u0026nbsp;Wrote original manuscript.\u0026nbsp;P. W.\u0026nbsp;performed the experiments.\u0026nbsp;Y. Z.\u0026nbsp;and\u0026nbsp;Y. Y.\u0026nbsp;analyzed data.\u0026nbsp;Z. Q. and J. J.\u0026nbsp;prepared\u0026nbsp;figures 1- 6.\u0026nbsp;L. W.\u0026nbsp;and\u0026nbsp;H. Z.\u0026nbsp;designed the study, revised manuscript. All authors reviewed\u0026nbsp;the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatient consent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eJ.I. Shin, Second-line Glucose-Lowering Therapy in Type 2 Diabetes Mellitus, Current Diabetes Reports 19(8) (2019) 54.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eI. Kanazawa, M. Inaba, D. Inoue, K. 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Liu, Concentrate Growth Factors Regulate Osteogenic Dysfunction of MC3T3-E1 Cells Induced by High Glucose Through PI3K/Akt Signaling Pathway, Implant Dentistry 28(5) (2019) 478\u0026ndash;483.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoking, Type 2 diabetes osteoporosis, preosteoblast, osteoblasts, PI3K/AKT/GSK-3β pathway","lastPublishedDoi":"10.21203/rs.3.rs-3833910/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3833910/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eOsteoking (OK) is a Yi folk Chinese herb from the Yunnan province, which exerts bone formation-promoting effects on menopausal osteoporosis and osteoporotic fractures. However, it remains to be determined whether OK ameliorates type 2 diabetic osteoporosis (T2DOP). Thus, T2DOP animal model was established in db/db mice in this study. Micro-computed tomography (micro-CT) analysis revealed that OK significantly increased bone strength, improved bone metabolism, and promoted bone formation. GS and p-GSK-3β expression levels were increased in OK group as compared with db/db group by Western blot analysis. IL-6, IL-17A, IFN-γ, TNF-α, and IL-1β were lower levels in the OK group compared to the db/db group, nevertheless, the IL-10 level was significantly higher. Furthermore, an \u003cem\u003eIn vitro\u003c/em\u003e cells model was constructed by stimulating with high glucose (HG, 30 mM). ALP protein was significantly elevated in OK treatment group. Administration of OK at 1.44 mg/mL significantly increased p-AKT/AKT expression, while, combined with LY294002, an inhibitor of PI3K, OK significantly reduced the expression levels of p-PI3K/PI3K, p-AKT/AKT and p-GSK-3β/GSK-3β. In conclusion, to our knowledge, this study is the first to reveal OK exhibits efficacy against T2DOP in db/db mice by promoting osteogenesis of preosteoblast MC3T3-E1 cells through PI3K/AKT/GSK-3β pathway regulation.\u003c/p\u003e","manuscriptTitle":"Osteoking ameliorates type 2 diabetes osteoporosis by promoting osteoblasts proliferation via PI3K/AKT/GSK-3β pathway activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-17 04:57:48","doi":"10.21203/rs.3.rs-3833910/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0ed79fab-cfeb-4637-9a46-e06f85e1719b","owner":[],"postedDate":"January 17th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":28142062,"name":"Biological sciences/Drug discovery"},{"id":28142063,"name":"Biological sciences/Molecular biology"},{"id":28142064,"name":"Health sciences/Diseases"},{"id":28142065,"name":"Health sciences/Medical research"},{"id":28142066,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2024-05-07T04:56:30+00:00","versionOfRecord":[],"versionCreatedAt":"2024-01-17 04:57:48","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3833910","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3833910","identity":"rs-3833910","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}