Study On The Pathogenesis Of Postmenopausal Osteoporosis Based On Energy Metabolomics And The Intervention Of Melatonin

Objective: Osteoporosis(OP)is a chronic,systemic endocrine and metabolic disorder.Bone loss due to estrogen deficiency is common in elderly women,and the root cause is imbalance of bone remodeling homeostasis.The bone regeneration of osteoblasts and bone resorption of osteoclasts are highly dependent on energy consumption.In recent years,the exploration of the pathogenesis of various diseases based on energy metabolism has gradually become a research hotspot,and the energy metabolism of bone microenvironment is also closely related to bone remodeling.However,the specific metabolic processes and metabolites involved in the occurrence and development of osteoporosis are still unclear.It has been reported that circadian rhythm disturbances can affect cellular energy metabolism,and the rhythmic hormone melatonin secreted by the pineal gland is involved in regulating many metabolic processes,including glucose metabolism,lipid metabolism,and amino acid metabolism.Melatonin corrects metabolic disorders by regulating circadian rhythms.In addition,studies have found that postmenopausal osteoporosis women have lower serum melatonin levels,disrupted circadian rhythms,and that melatonin are positively correlated with bone mass.Our previous studies also found that melatonin can regulate osteoblast activity.However,it remains to be further studied whether menopause can cause energy metabolism disorder in bone microenvironment and whether melatonin can promote bone formation by restoring energy metabolism osteoblasts.Therefore,the aim of this study is to explore the pathogenesis of osteoporosis based on energy metabolism by means of metabonomics,bioinformatics,bibliometrics,network pharmacology and in vitro and in vivo experiments to further reveal the new mechanism of melatonin on osteoporosis and provide a new target for bone metabolism research.Methods: The anmial model of postmenopausal osteoporosis was established by oophorectomy and bone mass was determined by Micro-CT.The UHPLC-MS technique was used to accurately quantify targeted energy metabolomics in the bone tissue of the two groups,and functional annotation,enrichment analysis and target prediction of differential metabolites were performed.Bibliometrics was used to analyze the melatonin related publications in the past 20 years,and figure out the current status,gaps and future hotspot of melatonin research.The potential target of melatonin was predicted by network pharmacology method based on the structure information,and the functional annotation were performed.Ovariectomized mice were treated with melatonin intraperitoneally,bone mass was measured by Micro-CT.The citrate kit was used to detect citrate in unit bone and net secretion by osteoblasts,alkaline phosphatase kit and alizarin red staining were used to detect osteogenic differentiation and matrix mineralization,respectively.PCR,Western blotting and immunofluorescence staining were used to analyze the potential target of melatonin(ZIP-1),and the mechanism was verified by small interfering RNA and plasmid overexpression.Results: The results showed as follows: 1.The targeted energy metabolomics of ovariectomized mice was significantly different from that of the control group.2.There were nine distinct metabolites,adenine was abundant in osteoporotic bone,while the contents of citrate,L-lactate,L-tyrosine,pyruvic acid,phenyllactate,AMP,cyclic-AMP and UDP-Glc NAc were lower.3.Differential metabolites are mainly involved in purine metabolism,secondary metabolite synthesis,amino acid synthesis and carbon metabolism.4.The differential metabolites mainly involved the c AMP signaling pathway,AMPK signaling pathway,CPMP-PKG signaling pathway and HIF-1 signaling pathway.The tricarboxylic acid cycle,glucagon pathway and purine signaling pathway are the key links of energy metabolism disorder in osteoporosis.5.The biological processes of the predicted targets by differential metabolites include: carbon metabolism,circadian rhythm regulation,hypoxia response,and ion transmembrane transport.6.In recent years,the exploration of various biological functions of melatonin has attracted more and more attention from scholars at home and abroad.A total of 8 hotspots of melatonin research have been identified by bibliometrics,including the metabolic regulation.7.The main biological pathways and molecular functions of melatonin targets include: cascade amplification of G protein-coupled receptor signals,regulation of kinase activity to affect protein phosphorylation,neurotransmitter transmission between synapses,receptor of exogenous drugs and ligands of ATP.8.Intraperitoneal injection of melatonin can restore bone loss and increase citrate content in ovariectomized mice.9.Melatonin enhances matrix mineralization by promoting the net secretion of citrate from osteoblasts.10.Melatonin inhibits aconitase by promoting the expression of ZIP-1 in osteoblasts,resulting in a net efflux of mitochondrial citrate.11.The promotion of histone acetylation in osteoblasts by melatonin is partially dependent on the expression of ZIP-1.Conclusion: This study found that the disturbance of energy metabolism in bone microenvironment is closely related to the occurrence and development of osteoporosis.The imbalance of bone remodeling homeostasis is due to the abnormal content of energy metabolism substrates and intermediates.As an endogenous substance,melatonin plays a multi-angle,multi-level and multi-target role in the physiological regulation of the body,and in-depth molecular mechanism research is a hot topic.Melatonin can restore citrate in postmenopausal bones and promote citrate net secretion by up-regulating the expression of ZIP-1 in osteoblasts,thereby enhancing matrix mineralization on the one hand and histone acetylation on the other.