The Molecular Mechanism Of Milk Derived MFG-E8 On Repair And Regeneration Of Aging Sarcopenic Rats

Sarcopenia is defined as a disease associated with ageing process in human,leading to the muscle dysfunction and action disorder.It is caused by an imbalance of protein degradation and synthesis.At present,drugs,resistance exercises and nutritional interventions are effective ways to prevent and treat sarcopenia.Milk fat globule membrane?MFGM?has been demonstrated to be an effector in skeletal muscles.However,there are hundreds of proteins in MFGM.The molecular mechanism of promoting cell proliferation was unclear.In this thesis,milk fat globule epidermal growth factor-8?MFG-E8?,a high-abundance protein was isolated and purified from milk fat globule membrane protein.The structure,properties and biological characteristics of MFG-E8 were systematically studied.The mechanism of MFG-E8 in C₂C₁₂ cell proliferation and differentiation was studied.Furthermore,the mechanism of MFG-E8 in skeletal muscle repair and regeneration was further clarified by aging sarcopenic rats.The MFGM protein fractions were separated by DEAE-52 cellulose ion exchange chromatography.The components of MFGM protein fraction were identified by SDS-PAGE,MALDI-TOF/TOF and LC-MS/MS.The major component of MFGM protein,MFG-E8,is accounted for 98.33%.The data of LC-MS/MS,FT-IR,Circular dichroism and Bioinformatics showed that the MFG-E8 consist of 431 amino acids with a molecular weight of 47.82 k Da.Secondary structure of MFG-E8 consist of 5% helix,70% sheet and 25% random coil.The tertiary structure of MFG-E8 was globulin.Moreover,MFG-E8 is a hydrophilic globulin with a hydrophobic cavity,two transmembrane regions,three phosphorylation sites and two N-terminal glycosylation sites.The analysis of protein-protein interactions and KEGG Pathway found that MFG-E8 can regulate the biological processes,such as angiogenesis,apoptotic cell clearance,cell adhesion and protein metabolism,mainly related to ILK/Akt,?v?6-ERK and?v?6/PKC/PKD/HDAC5 signaling pathway.MFG-E8 can promote C₂C₁₂ cell proliferation in a concentration and time-dependent manner.The proliferation rate reached a maximum?35.8%?at 200?g/m L at 48 h.MFG-E8 can decrease the cell population of G0/G1 and S phase,increase the cell population of G2/M phase,and increase the number of mitochondria which could promote C₂C₁₂ cell proliferation;MFG-E8 can also up-regulate the expression of Myo D,Myo G and My HC that further promote the differentiation and fusion of C₂C₁₂ cells to form multinucleated myotubes.8high-expressed proteins were screened,which could regulate proliferation andapoptosis of C₂C₁₂ cell via PI3 K,MAPK,oxidative phosphorylation and AMPK signaling pathways.Results of q RT-PCR and western blot verified that MFG-E8 promote C₂C₁₂ cell proliferation mainly via PI3 K and ERK signaling pathway.Meanwhile,MFG-E8 could activate PI3 K signaling pathway and increase the expression of p-Akt which further increase mitochondria number and repair damaged cells.The results of q RT-PCR and western blot demonstrated that MFG-E8 could promote C₂C₁₂ cells proliferation by activating PI3K/Akt/m TOR/P70S6 K signaling pathway.In addition,Activated Akt could up-regulate the expression of Myo D,Myo G and My HC,which promote the differentiation of C₂C₁₂ cells into multinucleated myotubes.The rats were interfered by whey protein concentrate?WPC?and MFG-E8 for4 weeks.The levels of MDA,lipofuscin,TG,NEFA and ketone body in serum were reduced,and SOD activity and IGF-1 level were increased.Gastrocnemius intercellular edema and waxy degeneration disappeared.Muscle fiber cross-sectional area was increased.The numbers of nuclei were increased and gastrocnemius muscle was repaired and regenerated.q RT-PCR and western blot demonstrated that both MFG-E8 and WPC could activate PI3K/Akt/m TOR signaling pathway to regulate protein metabolism process.MFG-E8 promoted protein anabolism via PI3K/Akt/m TOR/P70S6 K signaling pathway.WPC inhibitied catabolism protein via PI3K/Akt/m TOR/4E-BP signaling pathway.Synergistic effect of WPC and MFG-E8 could enhance the inhibition of MFG-E8 on protein catabolism,which achieved by the down-regulation of negative regulatory factors expression including 4E-BP,Atrogin-1 and MURF.