The Effects Of Vitamin K2 On Bone Metabolism And Mitochondrial Recovery

Osteoporosis(OP)is a systemic bone metabolic disease featuring the deteriorated microarchitecture,reduced density and decreased mass of bones.Osteoporosis is prone to bone fracture,which has a great impact on the life quality of individuals.Bone remodeling imbalance is one of the important pathogenesis of OP.The process of bone remodeling is mainly composed of osteoclast-mediated bone resorption and osteoblast-mediated bone formation,and both increased bone resorption and decreased bone formation can lead to disruption of the balance of bone remodeling,resulting in bone hyperplasia or OP.Vitamin K2 has the effect of inhibiting bone resorption and promoting bone formation and has been clinically used to prevent and treat osteoporosis,but the principle for vitamin K2 to affect bone metabolism is complicated.In particular,the effect difference of the vitamin K2 with different sidechain lengths(MK4 and MK7)on bone metabolism and its functional mechanism are not clear.Moreover,As the energy metabolism center of cells,mitochondria have dysfunction that promotes the occurrence and development of OP.Vitamin K2 is an electron carrier in mitochondrial oxidative respiratory chain and has antioxidant activity,however,the physiological mechanism for vitamin K2 to resist the dysfunction caused by oxidative stress in mitochondria is not clear.In order to apply vitamin K2 to the treatment of OP better,the function and mechanism of vitamin K2 in bone metabolism and mitochondrial recovery were studied,and the main activities and results of this dissertation are summarized as follows:1.The mediated bone metabolism by vitamin K2 is systematically investigated.Based on the proposed osteoblast and osteoclast differentiation model,it is found that the vitamin K2 molecules with the sidechains of different lengths(MK4,MK7)can both promote osteoblast differentiation and inhibit osteoclast differentiation,where the MK4 have a stronger ability to inhibit osteoclast differentiation than the MK7,but the MK7 molecules have a much better capability to promote osteoblast differentiation.In the dexamethasone(Dex)-induced osteoporosis model,MK4 and MK7 both significantly increase the number,thickness and bone density of trabeculae,but reduce the trabecular separation and bone damage.Moreover,MK7 mainly promotes osteoblast differentiation and bone formation,while MK4 mainly inhibits osteoclast differentiation and bone resorption.The action mechanism of MK4 and MK7 in bone metabolism was studied,and it was found that:(1)Transcriptome sequencing results showed that IL-6 played a key role during inhibiting osteoclast differentiation of MK4;in vitro and in vivo experiments showed that MK4 significantly inhibits the expression of IL-6 and interferes with the interaction between RANK and TRAF6.It is suggested that the mechanism of MK4 inhibiting osteoclast differentiation may be to weaken the interaction between RANK and TRAF6 by inhibiting the expression of IL-6,and then inhibit the expression of NFATc1;(2)MK7 promotes the transformation of LC3A I to LC3A Ⅱ,inhibits the expression of p62 and increases the level of autophagy.The experimental results of adding autophagy inhibitor(3-MA)confirm that a decreased autophagy level can inhibit the promoting effect of MK7 on osteoblast differentiation and mineralization,indicating that the process of MK7 promoting osteoblast differentiation and bone formation is strongly dependent on autophagy.Transcriptome sequencing results showed that MK7 was involved in regulating the PI3K/AKT signaling pathway.In vitro and in vitro experimental results showed that MK7 inhibited the phosphorylation of AKT and mTOR,suggesting that the promotion effect of MK7 on autophagy may be related to the inhibition of mTOR signaling tranduction.2.The mechanism for MK7 to mitochondrial recovery is investigated.MK7 was used in vitro to repair mitochondrial damage caused by 6-hydroxydopamine(6-OHDA).It was found that MK7 is capable to effectively remove the ROS induced by 6-OHDA and restore mitochondrial membrane potential,leading to the inhibition of cell apoptosis.Mechanistic studies show that MK7 is able to repair mitochondrial damage by maintaining the operation of mitochondrial quality control ring.On the one hand,MK7 promotes the dynamic balance of mitochondrial morphology and quantity by inhibiting abnormal mitochondrial fusion and division caused by 6-OHDA.On the other hand,MK7 accelerates the clearance of damaged and dysfunctional mitochondria by promoting mitophagy and biogenesis and maintains the stable operation and energy supply of mitochondria.3.The effect of fungal hydrophobic protein on enhancing K7 performance is studied.Hydrophilic HGFI-MK7 nanoparticles are obtained by modifying hydrophobic MK7 with HGFI,and it is revealed that HGFI molecules are self-assembled on the surface of MK7 nanoparticles by physical adsorption.In vitro cell experiments show that HGFI-MK7 nanoparticles can significantly improve the activity of alkaline phosphatase and the relative content of osteoprotegerin in MC3T3-E1 cells,which enhance the differentiation ability of osteoblasts promoted by MK7.