Study On The Mechanism Of Egg Yolk Glycoprotein In Improving Osteoporosis

Osteoporosis is a metabolic bone disease characterized by low bone mass and microstructural changes in bone tissue,leading to increased bone fragility and increased fracture risk.With the development of the aging population,osteoporosis has become an increasingly and seriously health problem all over the world.Eggs are rich in a variety of biologically active ingredients,including polyunsaturated fatty acids,immunoglobulins,and lecithin.The egg yolk protein is a by-product of the extraction of lecithin.Therefore,in this study,the basic componets of egg yolk glycoprotein,which was prepared form egg yolk,were analyzed.Osteoclasts differentiated from RAW264.7 induced by RANKL and preosteogenic MC3T3-E1 cells were used to evaluate the inhibiting mechanism of osteoclastogenesis and the promoting mechanism of osteogenesis.Ovariectomized osteoporosis rats were used to assess the effect of EYG on osteoporosis.The main research results are as follows:(1)Preparation and identification of EYGUse egg yolk as raw material to prepare crude EYG.Separate and purify DEAE-52 and Sephadex G-50 to obtain EYG with a purity of 94.57% and a relative molecular mass of 28045.61 Da.It consists of three kinds of Gal,Man and Glc-AC Monosaccharide composition,contains 17 kinds of amino acids,with the highest content of aspartic acid and glutamic acid.(2)The mechanism of EYG inhibiting osteoclast differentiation and maturationThe osteoclast model was established by inducing RAW264.7 macrophages with RANKL,and the effects of EYG on osteoclast differentiation and maturation were evaluated by MTT,tartrate-resistant acid phosphatase staining,ELISA,q RT-PCR and Western blot.The results showed that EYG significantly inhibited the formation of osteoclasts,inhibited the secretion of cathepsin k and matrix metalloproteinase 9 by osteoclasts.It also significantly inhibited the gene expression of key regulators ERK2 and JNK of the mitogenactivated protein kinase(MAPK)signaling pathway,and the key regulators of c-Fos and NFATc1 in the nucleus,and significantly suppressed the protein expression of NFATc1.EYG can inhibit osteoclast differentiation and maturation and reduce bone resorption by inhibiting the activation of MAPK signaling pathway.(3)Action mechanism of EYG to promote osteoblast differentiation and maturationIn the previous osteoblast MC3T3-E1 cell model,MTT,ELISA,q RT-PCR,transcriptomics and other methods were used to analyze the effect of EYG on the proliferation and differentiation of osteoblasts.The results showed that EYG significantly promoted the proliferation of osteoblasts and the synthesis and secretion of alkaline phosphatase,type I collagen and osteocalcin,and promoted the formation of mineralized nodules.EYG can activate the expression of the marker genes COL Ⅰ,Lrp5,β-catenin,Runx2 and BMP-2 genes during osteoblast differentiation,and inhibit the expression of RANKL gene.Transcriptomics studies showed that the expression of EYG group significantly upregulated 45 genes and significantly downregulated 78 genes.Among them,EYG can upregulate GF and ECM-mediated PI3K-Akt,Wnt and MAPK signals in signaling pathways related to bone metabolism path.The results of q RT-PCR showed that EY-SGP can regulate the differentiation and maturation of osteoblasts and promote bone formation by activating the gene expression of key regulatory factors Lrp5,β-catenin,Runx2 and BMP-2 in Wnt signaling pathway.(4)EYG protects ovariectomized rats from osteoporosis.To investigate the effect of EYG on osteoporosis in ovariectomized rats,the rats were addministred with EYG.The results showed that EYG significantly increased bone volume fraction,cortical bone density and trabecular bone number,and considerably reduced trabecular bone separation,thereby improving bone microstructure in osteoporotic rats.EYG can reduce the levels of bone resorption markers serum TRAP,type I collagen C-terminal peptide,Cath-k and MMP-9(P<0.05),and reduce the levels of deoxypyridinoline,urine calcium and urine phosphorus in urine(P<0.05).EYG substantially increased the levels of bone-derived markers bone-derived alkaline phosphatase,type I collagen carboxy-terminal propeptide,osteocalcin and Runx2 in rat serum.This indicated that EYG can significantly improve the symptoms of osteoporosis in ovariectomized rats.