Study On The Therapeutic Mechanism Of Melatonin On Postmenopausal Osteoporosis Based On Metabonomics And Network Pharmacology

Objective: Osteoporosis(OP)is a common systemic metabolic disease in the elderly,leading to changes in bone microstructure and bone mass reduction,which can easily lead to fragility fractures.Most of them are postmenopausal women,known as postmenopausal osteoporosis(PMOP).After menopause,female ovarian function fails and declines,resulting in abnormal bone metabolism,increased activity of osteoclasts,decreased bone density,increased bone turnover,massive bone loss,decreased bone mass,changes in bone structure,and increased bone fragility,and show a series of symptoms such as low back pain,hunchback,fracture,and even disability,which is one of the main factors affecting the quality of life of middle-aged and elderly women,and has become a global public health problem.However,most of the existing anti-osteoporosis drugs start from the perspective of inhibiting bone resorption,inhibit osteoclasts,reduce bone destruction in the human body,and improve bone metabolism disorders,but they cannot reverse bone loss,nor can they greatly promote bone formation.At the same time,existing drugs also have certain side effects.Even new monoclonal antibody drugs have problems such as immunosuppression or tumor-causing,which leads to poor compliance of patients,which also affects the progress of osteoporosis treatment.Therefore,the development of new anti-osteoporosis drugs that can promote bone growth in patients with osteoporosis,while overcoming the shortcomings of traditional drugs with low selectivity and high side effects,has important clinical application value.Melatonin is a hormone secreted by the pineal gland,which can regulate various physiological functions of the human body,including regulating the rhythm of the biological clock,eliminating free radicals,delaying aging,inhibiting tumor growth,and regulating immunity.Melatonin is gaining more and more importance in pharmaceutical research as a drug or supplement due to its limited side effects.Changes in serum melatonin were found to be significantly associated with developed markers of bone metabolism.At the same time,a large number of studies have proved that melatonin can regulate bone metabolism from multiple angles,including promoting bone anabolism and inhibiting bone resorption,with extensive and comprehensive effects,which have been confirmed in cell experiments and animal experiments.Therefore,melatonin has the potential and prospect to become a new drug for the treatment of osteoporosis.It is of great significance to elucidate the role of melatonin in resisting the pathological changes of osteoporosis caused by estrogen deficiency and reveal the mechanism of melatonin improving bone metabolism.Therefore,this study explored the pathogenesis of postmenopausal osteoporosis and the network of melatonin in the treatment of postmenopausal osteoporosis by integrating serum non-targeted metabolomics,network pharmacology,and various animal experiments and cell experiments.The metabolic mechanism provides a new therapeutic idea for the treatment of postmenopausal osteoporosis,and also provides a pharmacological experimental basis for the further drug development of melatonin.Methods: 1.We established a postmenopausal osteoporosis mouse model,divided it into sham operation group,model group,and melatonin group,collected femur and serum samples,and used Micro-CT to evaluate the effect of melatonin on postmenopausal osteoporosis,and calculated the corresponding bone index parameters.2.We used non-targeted metabolomics to detect the levels of serum species metabolites in each group to determine the changes in metabolic profiles.3.We used network pharmacology to analyze the targets of melatonin in treating osteoporosis,and found the metabolic pathway of melatonin through GO and KEGG enrichment analysis.4.We combined the results of metabolomics analysis and network pharmacology analysis to obtain the targets and metabolic pathways of melatonin in treating postmenopausal osteoporosis.5.Through animal experiments,the ELISA method was used to verify the changes of key metabolites in serum.6.We used RT-q PCR and western blot to detect the changes of melatonin targets in mouse femur.7.We used RT-q PCR and western blot methods in cell lines to detect the effect of melatonin on osteogenic differentiation indicators.8.We used RT-q PCR and western blot methods in cell lines to detect the effect of melatonin on predicted targets.Results: 1.According to the results of Micro-CT,we found that after undergoing ovariectomy,the femurs of mice in PMOP group showed significant deterioration in trabecular bone microstructure,while the change in Melatonin group was opposite.2.Compared with the Sham group,the main enrichment pathway of differential metabolites in the PMOP group was Steroid hormone biosynthesis(hsa00140),and 5metabolites were enriched in this pathway,including 21-Deoxycortisol,Aldosterone,Corticosterone,Cortisol and Progesterone.The corrected final p value was 0.00785.3.Compared with the Sham group,the differential metabolites in the PMOP group were also enriched in the Pentose phosphate pathway(hsa00030)pathway,and the metabolites involved were D-Ribulose 5-phosphate and 6-Phosphogluconic acid,The p value was 0.00822.4.The differential metabolites between the Melatonin group and the PMOP group were not enriched to a statistically significant pathway.5.In the results of PMOP group vs.Sham group and Melatonin group vs.PMOP group,there are 12 key metabolites with opposite trends in comparison between the two groups.6.The pathways involved in key differential metabolites include Pentose phosphate pathway(hsa00030)and Steroid hormone biosynthesis(hsa00140).7.According to the network pharmacology analysis,there are 18 potential targets for melatonin in the treatment of osteoporosis.The highest score is MTOR,followed by ESR1,IGF1 R,AR,PIK3 CA and CCNE1,and the scores of other proteins are much lower than the above six protein.8.After integrating the results of metabolomics and network pharmacology,the pathway C21-steroid hormone biosynthesis and metabolism of melatonin in the treatment of postmenopausal osteoporosis was obtained,and the target HSD11B1 was obtained.9.The melatonin molecule was combined with the obtained The molecular docking of the target protein HSD11B1 found that the two can be combined through a hydrogen bond,and the docking affinity was-6.8kcal/mol,and the docking result was good.10.The serum cortisol levels of mice in each group were detected by ELISA,which showed that the serum melatonin level of the mice in the Sham group was 23.38±5.49 ng/ml,and that in the PMOP group was 43.28±6.53ng/ml,while after melatonin treatment,the serum cortisol level decreased significantly,which was 26.97±5.79 ng/ml.11.Melatonin can reduce the expression level of HSD11B1 in mouse femur.12.Melatonin alleviates the inhibitory effect of cortisone on osteoblast differentiation and the decrease of ALP activity.13.Melatonin can inhibit the increase of HSD11B1 expression induced by cortisone.Conclusion: Melatonin can reduce the generation of cortisol in the microenvironment of bone tissue by inhibiting the expression level of HSD11B1 and the metabolic pathway of steroid hormone synthesis,thereby reducing the side effects of cortisol on osteoblast differentiation and affecting postmenopausal bone quality.Loosing plays a therapeutic role.