Mechanisms Of Isoflavones Isolated From Chickpea Sprouts In Preventing And Treating Osteoporosis By Regulating Bone Remodeling

Objective: Isoflavones Isolated from Chickpea Sprouts(ICS)is the product of chickpea sprouting,and the enrichment rate of isoflavones is significantly increased after chickpea sprouting.The aim of this study was to investigate the mechanism of ICS in preventing and treating osteoporosis by regulating bone remodeling and to further explore the potential medicinal value of ICS for preventing and treating osteoporosis.The main objectives of this study were: 1)to predict the main targets and signaling pathways of isoflavones with drug-like properties in ICS for the prevention and treatment of osteoporosis based on network pharmacology,and to reveal their potential mechanisms of action;2)to establish a rat model of bilateral ovariectomy osteoporosis and explore the preventive and therapeutic effects of ICS on osteoporosis and its mechanism;3)to verify the regulatory mechanism of ICS on bone metabolism by in vitro cell experiments.Method: 1)The first part of the study looked for isoflavones currently found in chickpea sprouts,screened for isoflavones with drug-like properties according to Lipinski’s five principles,and searched for targets of action on the Swiss Targetprediction platform.The Human Gene Database(Gene Cards)database,The Online Mendelian Inheritance in Man(OMIM)database,Therapeutic Target Database(TTD)database were used to search for osteoporosis targets and to screen for ICS and osteoporosis.Protein-protein interaction(PPI)analysis of the intersecting targets was performed using the STRING database,GO and KEGG pathway analysis of the intersecting targets was performed using the Metascape platform,molecular docking of core components and core targets was performed,binding energies were calculated,and the results were visualized;2)the study of The second part established a rat osteoporosis model by removing the bilateral ovaries of female Sprague-Dawley(SD)rats,which were divided into five groups according to the experimental protocol: sham-operated group(Sham),ovariectomy group(OVX),ICS low-dose group(ICS(L)),ICS high-dose group(ICS(H))and raloxifene group(Raloxifene,RLX).Bone Mineral Density(BMD)and trabecular structural parameters of the distal femur and lumbar quadratus were measured by Micro-CT,the biomechanical characteristics of the femur by three-point bending,bone conversion markers in serum by ELISA,and osteogenic differentiation of the lower femoral epiphysis by immunohistochemical staining.(3)The third part of the study firstly examined the effects of ICS on the proliferation and toxicity of MC3T3-E1,Bone marrow mesenchymal stem cells(BMSCs)and RAW264.7.proliferation and toxic effects.Osteogenic induction of differentiation of M3T3-E1,BMSCs,while intervening with different concentrations of ICS,and the effect of ICS on osteogenic differentiation was examined using alkaline phosphatase staining,alizarin red staining,RT-q PCR and protein immunoblotting(Western Blot,WB),while whether ICS promotes osteogenic differentiation through estrogen receptors after intervention with ICI 182,780.osteogenic differentiation.In RAW264.7 cells,the effect of ICS on osteoclast bone resorption was examined by measuring TRAP enzyme activity,TRAP staining,RT-q PCR and WB using different concentrations of ICS under RANKL-mediated intervention.In addition,the activation of NF-κB signalling pathway was examined at different time points under ICS intervention to investigate the mechanism of ICS inhibition of osteoclast bone resorption.Results:1)The first part of the results showed that 14 isoflavone components with drug-like properties were screened among all reported isoflavones contained in chickpea sprouts,and 255 targets were queried using the Swiss Targetprediction database.A total of 5008 osteoporosis disease targets were obtained from the Gene Card,OMIM and TTD databases,and a total of 151 ICS and osteoporosis intersection targets were identified through screening.PPI analysis of the interactions between the intersecting targets resulted in the screening of 25 core targets.GO and KEGG analyses of the intersecting targets yielded1395 biological processes,85 cellular components and 128 molecular functions,enriching a total of 19 groups(160)of signalling pathways.Molecular docking of representative core components and core targets showed binding energies in the range of-7.4 to-10.4kcal/mol;2)The second part of the results showed that the BMD of the femur and lumbar4 vertebrae of rats in the OVX group was significantly reduced and the trabecular structure was destroyed,while the bone mass and trabecular structure of the ICS(L),ICS(H)and RLX groups were restored to varying degrees and the biomechanical properties were improved The ICS(H)group showed comparable improvement to the RLX group.Serum bone turnover markers showed that ICS reduced the high bone metabolic status of OVX rats,ICS increased the expression of proteins related to osteogenic differentiation of the distal femur and inhibited the expression of enzymes and proteins related to bone resorption,and ICS increased the ratio of OPG/RANKL expression in serum and femur;3)The third part of the results showed that ICS promoted the osteogenic differentiation of MC3T3-E1 and BMSCs by promoting the osteogenic differentiation of BMSCs,alkaline phosphatase activity,extracellular calcium salt deposition and expression of genes and proteins related to osteogenic differentiation in BMSCs,while ICI 182,780 was able to inhibit these promoting effects.For bone resorption,ICS inhibits the enzymatic activity of osteoclasts and the expression of genes and proteins related to bone resorption,and ICS inhibits the activation of NF-κB signalling pathway under RANKL-mediated conditions.Conclusion:1)Through network pharmacological analysis,ICS has multi-component,multi-target and multi-pathway characteristics in the treatment of osteoporosis and regulation of bone metabolism,among which MAKP,NF-κB and ER-related signaling pathways may be the key pathways of ICS in the prevention and treatment of osteoporosis;2)ICS has the effects of promoting osteogenic differentiation and inhibiting bone resorption in OVX rats,decreasing bone turnover and improving bone mass and bone strength.ICS may regulate bone metabolism through the OPG/RANKL signaling pathway;3)In vitro experiments demonstrated that ICS regulates bone metabolism by promoting osteogenic differentiation and regulating the ratio of OPG/RANKL through ER,and inhibits bone resorption by osteoclasts through NF-κB signaling pathway.ICS has a dual role in regulating bone metabolism.