Effects Of Lipophagy To MC3T3-E1 Osteogenic Activity In Hyperlipidemia

Background and objectivesThe situation is worrying that hyperlipidemia’s incidence is increasing yearly as people’s living standards improve.There is growing evidence that lipid metabolism disorders are associated with bone dysfunction,which brings increased focus on their relevance.Published results showed that increasing osteoblast apoptosis and inhibiting the activity of osteoblasts were caused by lipids.However,the mechanisms of lipotoxicity in bone are incompletely understood.Autophagy is a highly conservative process to degrade and recycle damaged cellular contents.It has been observed that autophagy was activated in osteoblasts in a high-fat environment concerned with cell death.But it was contradictory that some studies found inhibition of autophagy with inhibitors such as 3-Methyladenine(3-MA)could reduce apoptosis,whereas others have shown that promoting autophagy with rapamycin(RAP)alleviated lipotoxicity.These inconsistent findings demonstrate the urgent need for more comprehensive and systematic studies on the effects of autophagy in hyperlipidemia.Lipophagy is one of the main lipid metabolism processes that degrades triglycerides(TGs)and cholesterol in lipid droplets(LDs)via the autophagy-lysosome system.Therefore,lipophagy is indispensable for regulating lipid content,protecting the cells from potentially toxic lipid molecules,and maintaining the cellular energy homeostasis of eukaryote cells.Impaired lipophagy causes excessive tissue lipid accumulation.However,whether and how lipophagy is involved in the hyperlipidemia-induced poor osteogenesis of osteoblasts needs further exploration.In conclusion,the current study is intended to determine the influence of high-fat-foodinduced hyperlipidemia models on bone regeneration and lipophagy and the impact of lipophagy on the bio function of osteoblasts in the high-fat medium.Overall,we want to clarify the relationship between lipophagy and osteogenesis in a hyperlipidemia environment,thus providing a potential therapeutic approach to lipotoxicity in bone.Materials and methods1.Effect of high-fat environment on the level of lipophagy in osteoblastsThe mouse osteoblast cell line MC3T3-E1 was cultured.Low concentrations of high-fat medium(containing 100 μM oleic acid and 100 μM palmitic acid)and high concentrations of high-fat medium(containing 300 μM oleic acid and 300 μM palmitic acid)were applied to simulate different levels of high-fat environments in vitro.Interventions were performed using the autophagy activator RAP or the autophagy inhibitor 3-MA.The accumulation of lipid droplets in osteoblasts was observed by Bodipy493/503 staining and Oil Red O staining;the internal microstructure of osteoblasts was observed by transmission electron microscope(TEM);the internal microstructure of osteoblasts was observed by monodansyl cadaverine(MDC)staining and immunofluorescence staining.MDC staining,immunofluorescence staining,and Western blot were used to assess the level of lipophagy in osteoblasts.2.Effects of altered lipophagy levels on osteogenic activity in a high-fat environmentOsteoblasts were cultured by applying different concentrations of the high-fat medium in groups and intervened with RAP or 3-MA to observe the alteration of their biological functions.The osteogenic activity of the cells was detected by alkaline phosphatase(ALP)staining,alizarin red staining,and Western blot.3.Effects of altered lipophagy levels on bone regeneration in hyperlipidemic mice with bone defectsThe bone regeneration ability of hyperlipidemic mice was evaluated by micro computed tomography(Micro-CT),hematoxylin and eosin(HE)staining,modified-Masson staining,and immunohistochemical staining after two weeks of surgery.Immunohistochemical staining was used to evaluate bone regeneration and autophagy levels.Results1.High-fat environment activates lipophagy in osteoblastsBoth different concentrations of the high-lipid medium led to further increases in intracellular lipid droplet accumulation,and the level of lipophagy increased under high-lipid stimulation.The results of the drug intervention showed that RAP increased the level of lipophagy in all groups and reduced the intracellular lipid content in the low-concentration lipid medium,but the change of lipid content in the high-concentration high lipid medium was not significant;3-MA attenuated the lipophagy in cells and led to the accumulation of intracellular lipids in all groups.2.The altered level of lipophagy in a high-fat environment had different effects on osteoblast function in different concentrations of high-fat mediumRAP-activated lipophagy promoted the proliferation and restored the function of osteoblasts in low concentrations of the high-fat medium but further inhibited the mineralization of cells in high concentrations of the high-fat medium;3-MA inhibited the proliferation and mineralization of osteoblasts in low concentrations of high-fat medium but promoted osteogenesis in high concentrations of high-fat medium to a small extent.The osteogenic effect was broadcast to a small area in the high-fat medium.3.Altered levels of lipophagy at bone defects in hyperlipidemic mice can affect their bone regeneration capacityMicro-CT,HE staining,and modified-Masson staining showed that the bone volume at the femoral defects in the hyperlipidemic mice was significantly reduced;the immunohistochemical results of autophagy index protein showed that autophagy was activated at the bone defects in the hyperlipidemic mice.RAP increased the level of autophagy and the bone regeneration ability at the bone defects in the hyperlipidemic group;3-MA inhibited autophagy at the bone defects,resulting in a significant decrease in bone volume.Conclusions1.Different high-fat medium concentrations led to lipid droplets accumulation in mouse osteoblasts and activated lipophagy in mouse osteocytes.Autophagy activators or inhibitors could activate or inhibit lipophagy under different levels of high lipid conditions.2.Different hyperlipidemia concentrations inhibited mouse osteoblasts’ proliferation and mineralization.At lower levels of hyperlipidemia,activation of lipophagy had a pro-osteogenic function,and inhibiting lipophagy inhibited osteoblast mineralization.However,the opposite was true at higher levels of hyperlipidemia.3.In this study,we successfully constructed a mouse model of diet-induced hyperlipidemia and clarified that hyperlipidemia impairs bone regeneration at femoral defects in mice.Activation of lipophagy ameliorated the hyperlipidemia-induced bone dysfunction to some extent while inhibiting lipophagy inhibited bone regeneration.