Melatonin Restrains Ferroptosis To Restire Osteoblast Differentiation Through Activating Wnt Signaling

Objective: Iron overload has been emerged in degenerative or hematologic diseases in a large population of patients with common outcome such as osteoporosis.The impact of changes in iron homeostasis on osteoblast functions and its underlying mechanisms are poorly defined.We show herein iron overload-induced ferroptosis and its cellular and molecular basis responsible for its dysfunction of osteoblast differentiation in bone marrow stromal cells(BMSCs)at pathological concentration;and propose a working model for melatonin to restore ferroptosis-disrupted osteoblast differentiation by retrieving canonical Wnt signaling.The aim is to provide a new theory and research direction for the treatment of abnormal bone metabolism induced by chronic iron accumulation disease.Methods: Ferric ammonium citrate(FAC)effective simulation of iron overload in patients with plasma the transferrin in combination with iron(NTBI)with BMSCs,concentration of 20μM,5020μM,200 20μM(110 to1100ug/d L,1100ug/d L for the highest concentration of serum in clinical of iron overload disease).q PCR,ALP staining and alizarin red staining detection such as BMSCs osteogenesis ability of differentiation and bone nodule formation.product of lipid peroxidation(LPO)reactive oxygen species(ROS),Intracellular ferroptosis was detected by fluorescence probe and electron microscopy(TEM).Wnt reporter plasmid Topflash and β-catenin were used to detect Wnt signal.Results:(1)Iron overload in a dose-dependent manner inhibited osteogenic differentiation and calcium deposition of BMSCs.(2)Iron overload leads to the accumulation of ROS and lipid peroxidation products(LPO)in BMSCs,and changes in mitochondria induce ferroptosis.Inhibition of ferroptosis can restore the decreased osteolast differentiation of BMSCs induced by iron overload,and apoptosis,pyrotosis and autophagy are not the main causes of decreased osteogenic differentiation.(3)Iron overload dose-dependent inhibition of Wnt signal,activation of Wnt signal can prevent ferroptosis and restore osteolast differentiation.(4)Melatonin inhibits FAC-induced ferroptosis without altering iron overload,thereby resuming osteoblast differentiation of BMSCs through activation of Wnt signaling.Conclusions: Iron overload induced ferroptosis inhibited Wnt signaling to inhibit osteoblast differentiation of BMSCs,and melatonin inhibited ferroptosis to restore osteoblast differentiation by activating Wnt signaling.