Lactoferrin Preserves Bone Homeostasis Based On Bone-Immune

Lactoferrin(LF)is a trace multifunctional glycoprotein in milk and can act as an active ingredient to regulate bone and immune system.However,the mechanisms involved in the regulation of bone remodeling in ovariectomized mice have not been fully elucidated.The effects of lactoferrin on bone in OVX mice,digestion and absorption of lactoferrin in mice and mechanism of bone remodeling based on boneimmune were investigated in the present study.Firstly,the effect of lactoferrin on bone in mice was studied.The ovariectomy model was successfully established.By using double energy X-ray absorption method,micro-computed tomography and three-point bending method,the results showed as follows: lactoferrin significantly inhibited weight gain and improved bone mineral density by 11.93%,and increased the serum alkaline phosphatase activity by 80.76%,while it decreased the serum tartrate-resistant acid phosphatase activity by 60.38%.Additionally,lactoferrin increased the content of Ca and P,and enhanced the maxload and max-stress values of the femur.Furthermore,lactoferrin improved the microstructure of the femur by elevating the bone volume per tissue volume,trabecular thickness and trabecular number and reducing the structure model index.Lactoferrin preserved bone homeostasis by promoting bone formation and inhibiting bone resorption.Secondly,the effect of lactoferrin on bone-immune in mice was studied.Luminex technology was used to detect cytokines in the serum.The results showed that lactoferrin increased the IL-1α,IL-2,IL-3,IL-5,IL-7,IL-10,IL-12(p70),IL-13,IL-15 and IFN-γ and decreased IL-1α and IL-9.Among these cytokines,IL-5,IL-10,IL-12(p70)and IFN-γ can affect RANKL and OPG.By using enzyme-linked immunosorbent assay,the RANKL/OPG ratio of the serum and T cells was decreased by 57.14% and 21.57% after treatment with lactoferrin.Moreover,lactoferrin significantly promoted the proliferation of macrophage and B cells and affected thymus and spleen indexes.Lactoferrin preserved bone homeostasis by bone-immune pathway.Thirdly,the digestion and absorption of lactoferrin in mice was detected.The peptides in the contents of the stomach,duodenum,jejunum and ileum were identified by electrospray ionization quadrupole time-of-flight mass spectrometry(ESI-Q-TOFMS)coupled with ultra performance liquid chromatography(UPLC)or capillary electrophoresis(CE).A total of 578 unique lactoferrin peptides were identified in the gastrointestinal tract of mice.The peptide LRPVAAEIY(LF-LR)was screened by bioinformatics analysis.The Caco-2 monolayer cell was used as absorption model in vitro and the peptide LF-LR was quantitatively analyzed by mass spectrometry.The peptide LF-LR can be absorbed by intestinal epithelial cells through endocytosis and has a good absorption rate.The LF-LR also can be detected in the serum of mice,indicating that LF-LR may be one of active fragments of lactoferrin.Finally,the effect of LF-LR on bone-immune was studied.Based on the identification result of lactoferrin peptides,five peptides were studied by MTT and molecular docking.Cell experiments showed that lactoferrin peptide LF-LR significantly promoted osteoblast proliferation at 500 ng/mL and bonded to integrin α5β1 to regulate the differentiation.The lactoferrin peptide LF-LR bonded with TRAF6 by the interaction between Leu and Pro of peptide and Glu448,Met450,Phe471 and Tyr473 of TRAF6,respectively,blocking the interaction between TRAF6 and RANK and inhibiting the RANKL/RANK/OPG pathway.The peptides KESPQTHY,ESPQTHYY,ISQPEWFK and KPVTEAQ,stable in the gastrointestinal tract,can also bond to TRAF6 and have the potential activity regulating bone remodeling through the bone-immune,which needs further study.The mechanism of lactoferrin regulating bone remodeling via immunological pathways was investigated in this study,which effectively enriched the biological connotation of lactoferrin and provided a theoretical foundation for the application of lactoferrin in functional foods.