Melatonin Suppresses Osteoclast Genesis By Reducing Intracellular ROS Via Activating Nrf2 Pathway

Objective:Osteoporosis is the most common chronic metabolic osteopathy,which often results in fracture due to decrease of bone formation or(and)bone resorption transits.It is prone to fracture.At present,some studies have shown that melatonin can promote osteoblastic differentiation and decreases osteoclasts differentiation,which is related to the reduction of intracellular reactive oxygen species(ROS).However,it is unclear whether melatonin has a definite anti-osteoporosis effect in vivo and through which pathway to suppress osteoclasts genesis from bone marrow mononuclear cells(BMMs).Therefore,we performed experiments in the study to clarify the anti-osteoporosis effect of melatonin.And as if,what is the molecular mechanism.Methods:In vitro:The femurs and tibias of C57BL/6 mice were aseptically removed to extract bone marrow cells.BMMs were isolated from bone marrow cells and induced BMMs to differentiate into multinucleated osteoclasts.Melatonin at different concentrations(20?M/ml?100?M/ml?500?M/ml)is then added during the induction of differentiation.Tartrate resistant acid phosphatase(TRAP)staining and Fibrous Actin(F-Actin)staining is used to label the number and size of TRAP-positive multinucleated osteoclasts.Detecting the expression of osteoclast specific genes and genes related ROS Pathway by PCR,so as to further explore the molecular mechanisms of melatonin.In vivo:8-week-old female C57BL/6 mice induced osteoporosis model by ovariectomy and was intraperitoneal injected with melatonin(0.5 mg/kg?5 mg/kg)twice a week into for 4 weeks.Micro-computed tomography and histological study were performed to assay the effect of different concentration of melatonin.Results:We found a significant reduction in the number of markers of osteoclast-associated specific gene expression in TRAP-positive osteoclasts produced by BMMs in the presence of high concentrations(500uM/ml)of melatonin.Melatonin of medium and low concentration(20 and 100 mu M/ml)had no obvious inhibitory effect on osteoclasts.Through the detection of ROS-related pathway by PCR,we found that the gene expression levels of nuclear factor(erythroid-derived 2)-like 2(Nrf2)and its downstream genes of catalase(CAT)gradually decreased with the maturation of osteoclasts,while high concentration of melatonin could reverse the expression levels of Nrf2 and CAT and increase it.The mice osteoporosis model was successfully established by bilateral ovariectomy.Micro-CT showed that high concentration(5 mg/kg)of melatonin could increase bone mineral density and reduce bone loss in osteoporotic mice,but low concentration(0.5 mg/kg)of melatonin had no obvious effect.Histological study showed that the number of osteoclasts decreased significantly with high concentration of melatonin.conclusion:Melatonin of high concentration can play a definite anti-osteoporosis effect in mouse osteoporosis model.Melatonin inhibits osteoclast genesis of BMMs by reducing intracellular ROS.This effect may be related to the increase of expression of CAT by activating Nrf2 pathway in cells.