The Efficacy And Mechanism Of Rice Bran-Derived Active Peptide In Protecting Against Osteoporosis

With the aging population,osteoporosis(OP)and its associated fractures have become significant public health issues affecting the well-being of people in our country.Existing anti-OP drugs that promote bone regeneration are limited by long treatment periods,high costs,and side effects.Bioactive peptides,short peptides containing2-20 amino acids,are widely sourced,potent,and biocompatible,gaining extensive use in the field of tissue regeneration.Previously,we isolated a short peptide,consisting of 8 amino acids with a molecular weight of1002.6 Da(rice bran-derived active peptide,RBAP),from rice bran through high-resolution mass spectrometry,and reported its antioxidative and anti-aging effects.This study aims to explore the efficacy and mechanism of RBAP against OP.Methods: Functional studies: 1)Menopausal OP mouse models were established using ovariectomy(OVX);OP fracture mouse models were created by mid-femoral fractures;natural aging mouse models were constructed using 16-month-old male C57BL/6 mice;disuse OP mouse models were created by tail suspension.All these models were intervened through gastric feeding and tail vein injection.2)μCT and a biomechanical testing machine were used to detect bone mass and bone mechanical strength.The ability of new bone mineralization was evaluated by calcein-double staining.Enzyme-linked immunosorbent assay(ELISA)was used to measure the serum osteogenic activity marker osteocalcin(OCN)and bone resorption activity marker C-terminal telopeptide of typeI collagen(CTX).OCN,tartrate-resistant acid phosphatase(TRAP),and perilipin staining were performed to assess the formation of osteoblasts,osteoclasts,and adipocytes in vivo.3)Real-time fluorescence quantitative PCR was performed to detect the effect of RBAP on osteogenesis and adipogenesis-related gene expression.Alizarin red,TRAP,and oil red O staining were used to evaluate the effects of RBAP on osteoblast,osteoclast,and adipocyte formation in vitro.4)An aptamer was linked to RBAP to target bone marrow mesenchymal stem cells(BMSCs),termed Apt-RBAP.The bone targeting of Apt-RBAP was assessed by immunofluorescence staining,flow cytometry,and near-infrared in vivo imaging system.The above methods assessed whether the BMSCs targeting aptamer can enhance the pro-osteogenic and anti-adipogenic effects of RBAP to further improve its bone protective function.Mechanism studies: The Tlr4 knockout mouse was used to assess whether RBAP exerts its bone protective function by binding to TLR4.Proteomics and bioinformatics analysis were used to identify proteins in BMSCs that can bind to RBAP and their involved molecular pathways.The candidate target molecules were knocked down by small interfering RNA in BMSCs to evaluate whether the pro-osteogenic and anti-adipogenic effects of RBAP are weakened or disappeared in the absence of target proteins.Western blotting and molecular docking technology were further used to evaluate whether RBAP can directly bind to the target protein.Results: RBAP significantly promoted bone regeneration in OVX mice,tail-suspended mice,naturally aging mice,and OP fracture mice.In vitro and in vivo experiments confirmed that RBAP significantly promoted osteogenesis and inhibited adipogenesis,but had no significant effect on the formation and activity of osteoclasts.RBAP can reach multiple organs in OVX mice,and binding with the BMSCs targeting aptamer significantly increased the enrichment of RBAP in bone tissue.Further,in vitro and in vivo experimental results indicated that the BMSCs targeting aptamer significantly enhanced the bone-protective effect of RBAP by promoting its osteogenic and anti-adipogenic effects.Molecular mechanism experiments revealed that RBAP could bind to eIF3 L,which mediated the pro-osteogenic and anti-adipogenic effects of RBAP.Conclusion: RBAP can bind to eIF3 L in BMSCs to regulate its osteogenic and adipogenic differentiation fate,thereby improving OP.Moreover,binding to the aptamer that targets BMSCs can further ameliorate the bone-fat imbalance in OP,thereby enhancing the bone protective effect of RBAP.