| Objective:Osteoporosis,a chronic bone degenerative disease caused by an imbalance in bone remodeling,has become a global public health problem characterized by decreased bone formation and increased bone resorption,resulting in low bone mass,bone loss and tissue microstructure degeneration,manifested as decreased bone strength,increased fragility,destruction of bone tissue structure,and increased susceptibility to fracture.Among the many primary osteoporosis,the most common is postmenopausal osteoporosis,which greatly affects women’s daily body functions and has a huge negative impact on women’s quality of life.With the aging of our country’s population,the number of patients with postmenopausal osteoporosis continues to increase.If it is not intervened and treated in time,postmenopausal osteoporosis will inevitably bring a heavy economic burden to the whole society.Although the recurrent fracture rate is higher in patients with osteoporosis,new fractures mainly occur in the osteopenic population,based on the principle of secondary prevention of disease,starting intervention at an early stage of bone loss rather than after diagnosis of osteoporosis may provide some protection against postmenopausal osteoporosis fractures.Therefore,it is very important to develop corresponding new early osteoporosis prediction models,however,due to the lack of sensitive biomarkers,the ability to early diagnose osteoporosis is limited.In addition,timely treatment of osteoporosis after early diagnosis is also very important.However,most traditional medicines mainly work by preventing bone loss,but cannot promote new bone formation in a large amount,which has certain limitations.In addition,some drugs also have certain side effects,resulting in poor patient compliance.Therefore,it is of great significance to explore drugs with low side effects,high economic benefits and the ability to promote new bone formation for the treatment of postmenopausal osteoporosis.As a natural endogenous hormone from the pineal gland,melatonin can regulate circadian rhythm and participate in a wide range of physiological processes,and it has been proven to be closely related to postmenopausal osteoporosis.Animal experiments and clinical trials have proved that melatonin can participate in bone remodeling and has a positive effect on maintaining bone metabolism homeostasis.Melatonin can play a role by promoting bone anabolic effect and anti-bone resorption effect at the same time.Therefore,this study explored the correlation between melatonin and postmenopausal osteoporosis through clinical data collection and analysis,cell transcriptomics sequencing analysis,bioinformatics analysis,cell experiments and animal experiments,to explore the predictive effect,therapeutic effect and therapeutic mechanism of melatonin on postmenopausal osteoporosis.Our study can provide a research basis and theoretical basis for melatonin as a candidate drug for postmenopausal osteoporosis in the future,and also provide a new target for the treatment of postmenopausal osteoporosis.Methods:1.Postmenopausal subjects with normal bone mass,osteopenia,and osteoporosis were recruited through the community,and the level of serum melatonin,serotonin,fat-soluble vitamins and estrogen was used to construct the prediction model of postmenopausal bone loss.2.The prediction effect of the bone loss prediction model were evaluated through ROC curve,goodness of fit test,clinical decision curve and calibration curve.3.Ovariectomized mice were constructed to evaluate the therapeutic effect of melatonin on postmenopausal osteoporosis by Micro-CT.4.The effect of melatonin on the expression of osteogenesis-related proteins in mouse femur was determined by western blot experiment.5.CCK-8,RT-q PCR and western blot experiments were used to screen the optimal concentration of melatonin.6.The effect of melatonin on osteoblast differentiation was discovered through transcriptome sequencing,the target of melatonin was found and preliminary verified.7.In order to propose a hypothetical mechanism,the downstream pathway of the target was predicted through bioinformatics analysis.8.Cell biology and molecular biology experiments were applied to verify the downstream pathway of the target.8.The existence of melatonin therapeutic mechanism was conformed in mice and humans by RT-q PCR and western blot.Results:1.BMI,melatonin,serotonin,vitamin K,vitamin D3 and estrogen E2 levels were significantly correlated with the occurrence of bone loss in postmenopausal women.2.Low levels of melatonin,serotonin,VD3 and E2 were independent risk factors for postmenopausal bone loss.The OR values of the four indicators were all less than 1,among which the OR value of VD3 was the smallest,and the OR value of serotonin was the largest.Melatonin had a lower OR value than serotonin,which indicated that melatonin had a stronger protective effect on postmenopausal bone loss than serotonin.3.The results of multicollinearity diagnosis showed that the tolerance of all the included indicators was between 0.9 and 1,and the VIF was between 1 and 2.There was no multicollinearity problem among the indicators.4.The AUC of the model was 0.816,the 95%confidence interval was 0.738 to 0.894,and the best diagnostic cut-off value was 0.659,at which time,the sensitivity was 0.747 and the specificity was0.783.5.Theχ2 of H-L goodness of fit test was 3.773,and the p value was 0.877,suggesting that the model fit was good.6.The clinical decision curve suggested that the model had great clinical application value.7.The results of Micro-CT showed that melatonin can alleviate the bone loss caused by OVX in mice.8.Melatonin can promote the expression of bone formation-related proteins in the femur of OVX mice.9.Melatonin can promote the transcription and protein expression of osteogenesis-related genes in osteoblasts in vitro,and melatonin at a concentration of 10-6 M has the best effect on promoting osteogenic differentiation.10.The results of transcriptome sequencing showed that the gene most affected by melatonin was Mir145a,which could be verified by q PCR.11.Transfection of mi R-145 mimic can reduce the expression of osteogenesis-related proteins,and can also inhibit the promoting effect of melatonin on osteoblast differentiation.12.The bioinformatics database analysis indicated that the target of mi R-145 is the 3’UTR region of Cbfb,with two conserved binding sites.13.In the dual-luciferase experiment,the fluorescence intensity in the experimental group co-transfected with the wild-type Cbfb-3’UTR plasmid of mi R-145 decreased significantly.14.Transfection of mi R-145 mimic can reduce the level of Cbfb m RNA.15.Co-IP result suggested that there is an interaction between Runx2 and Cbfb protein.16.Transfection of mi R-145 mimic can inhibit the protein levels of Cbfb and Runx2 in osteoblasts,while mi R-145 inhibitor does the opposite.17.Melatonin can alleviate the decrease of Runx2 protein level caused by si RNA-Cbfb transfection.18.The level of mi R-145 in the serum of OVX mice was significantly increased,while oral administration of melatonin can reduce the level of mi R-145 in the serum of OVX mice,and the same trend was also observed in the femur.19.The serum mi R-145 level of subjects in osteoporosis and osteopenia group was significantly higher than that in normal bone mass group.20.Human serum mi R-145 and melatonin showed a low negative correlation trend,the correlation coefficient was-0.3855395,and the p value was 0.03618.Conclusions:1.Low levels of melatonin,serotonin,vitamin D3 and estrogen E2 are risk factors for postmenopausal bone loss,and have predictive significance for postmenopausal bone loss.2.Melatonin can treat postmenopausal osteoporosis by promoting osteoblast differentiation.3.High concentration of melatonin（up to 1 m M）can inhibit the activity of MC3T3-E1 preosteoblasts.4.Melatonin can promote osteoblast differentiation,and 1μM is the optimal concentration of melatonin to promote osteoblast differentiation in vitro.5.The promoting effect of melatonin on osteoblast differentiation is mediated through the mi R-145/Cbfb/Runx2 axis.6.The level of mi R-145 in vivo was negatively correlated with bone mass and serum melatonin level. |
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