| Objective:Type 2 diabetic osteoporosis（T2DOP）is a musculoskeletal system complication caused by type 2 diabetes mellitus（T2DM）characterized by impaired bone microarchitecture,reduced bone mass,reduced bone strength and increased fracture risk,with early an asymptomatic and complex pathogenesis,limited diagnostic methods,and high disability and mortality rates in patients.Current treatment of T2DOP is mainly based on anti-diabetic drugs and anti-osteoporosis drugs and has a large adverse effect.Therefore,it has become imperative to find drugs that promote bone formation and improve type 2 diabetes.Osteoking（OK）is used for fresh and old fractures,femoral head necrosis,osteoarthrosis,and lumbar disc herniation,and recent studies have shown that osteoking has the ability to improve bone formation in postmenopausal osteoporotic rats and tree shrews.The aim of this study was to investigate the mechanism of osteoking action through Wnt signaling pathway in the treatment of type 2 diabetic osteoporosis,and to explore the new molecular mechanism of OK treatment of T2DOP using pharmacometabolomics and osteoblast transcriptomics.Methods:1.To investigate the mechanism of action of osteoking in the treatment of type 2diabetic osteoporosis via Wnt signaling pathway:leptin receptor-deficient（db/db）mice were selected as an animal model of type 2 diabetic osteoporosis（T2DOP）,and db/db mice were treated with osteoking to detect fasting glucose,body weight,insulin resistance,serum glucose metabolic factors（FINS,GHb A1c）and calculate insulin resistance index（HOMA-IR）and insulin sensitivity（HOMA-IS）,detect urinary lipid metabolic factors（HDL-C,LDL-C,TC,TG）,hepatic glucose metabolism（INSR,GLUT4 and PPARγ）,hepatic glycogen staining,tibial bone biomechanics,femoral bone metabolic markers（OCN,OPG and RANKL）,femoral bone trabecular integrity;using CCK8,ELISA and alizarin red methods to detect the effects of osteoking on proliferation,differentiation and mineralization of osteoblasts cultured in high glucose,and using Western blot to detect the Wnt/β-catenin signaling pathway（Wnt1,β-catenin,Osterix,LEF6）and Wnt/Ca2+signaling pathways（Wnt5,CAMKII,NLK）in OK treatment of T2DOP.2.Exploring new molecular mechanisms of osteoking therapy for T2DOP using drug metabolomics and osteoblast transcriptomics.（1）Identification of osteoking compound based on ultra performance liquid chromatography/quadrupole time of flight mass spectrometry（UPLC-Q-TOF/MS）.（2）Analysis of key signaling pathways for OK treatment of T2DOP using osteoblast transcriptomics.（3）Analysis of key active ingredients and targets of osteoking for the treatment of T2DOP using STITCH website.Results:1.Osteoking treatment significantly reduced fasting glucose,serum GHb A1c,urinary LDL-C,TC,TG,hepatic INSR,GLUT4 and PPARγ,increased serum FINS,urinary HDL-C,hepatic glycogen synthesis,reduced insulin resistance and increased insulin sensitivity in db/db mice;increased tibial bone strength,serum bone formation markers OCN,femoral tissue OCN,OPG,femoral trabecular integrity,and reduced bone resorption marker RANKL;OK significantly improved osteoblast proliferation,differentiation（ALP）and mineralization under high glucose.Osteoking significantly increased femoral Wnt/β-catenin signaling pathway（Wnt1,β-catenin,Osterix,LEF6）protein expression in db/db mice.)protein expression,but had no significant effect on Wnt/Ca2+signaling pathway（Wnt5,CAMKII,NLK）;Wnt/β-catenin signaling pathway inhibitors significantly affected the therapeutic effect of osteoking on high glucose cultured osteoblasts（ALP,OCN）,while Wnt/Ca2+signaling pathway inhibitors had no significant effect on it.2.The possible components of osteoking for T2DOP treatment by drug metabolomics and osteoblast transcriptomics analysis were sinapyl alcohol,targeting HPGD and SMPD3,and key pathways were NOD-like receptors and Hippo signaling pathway.Conclusion:Osteoking may improve bone metabolism,bone quality and bone morphology in db/db mice through Wnt/β-catenin signaling pathway,and significantly improve glucolipid metabolism and insulin resistance in db/db mice.The multi-omics approach to explore new targets for osteoking treatment provides new ideas for future exploration of type 2 diabetic osteoporosis treatment,which needs further confirmation by follow-up experiments. |
