Melatonin Alleviates The Bone Senescence And Bone Loss Induced By Type 1 Diabetes Through The MT1/MT2-Sirt1 Pathway

Objective: The prevalence of diabetic osteoporosis(DM-OS)is increasing rapidly with the increase of age,metabolic disorders and other factors.In recent years,it has been found that there is a causal relationship between senescence and osteoporosis.With the increase of age,the proportion of senescent cells in bone microenvironment gradually increases,which leads to the disorder of bone synthesis and metabolism and the decrease of bone formation.Interestingly,senescence is believed to be one of the causes of bone loss in osteoporosis due to diabetes.Melatonin is a product produced in the pineal gland of mammals.It has been found that melatonin has a strong anti-senescence and osteogenic effects in recent years.However,it has not been reported that melatonin can alleviate the bone senescence and bone loss induced by type 1 diabetes mellitus(DM1).Therefore,this study explored whether melatonin can inhibit senescence and bone loss induced by DM1.Method: For in vivo experiments,we established DM1 mouse model with one bullet intraperitoneal injection of STZ in C57 BL / 6 mice(180mg/kg).Experimental animal groups included 10 mice in the normal control group,10 mice in the DM1 group,10 mice in the melatonin feeding group,and 10 mice in the DM1 plus melatonin feeding group.After 2 months of melatonin infusion,the experiment was ended with inhalation ether anesthesia and cervical dislocation.At the same time,the protein in the other side of the femur was extracted with the invest kit and detected by western blot.In vitro experiments,we used glucose concentration gradient to culture cells.Glucose concentration: 5mm,10 mm,15 mm,20 mm,25 mm.Finally,MC3T3-E1 cells were cultured in 25 mm /L high glucose medium(Hg)to establish a high glucose cell model in vitro.Cells were cultured with different concentrations of melatonin: 0,1nm,10 nm,100nm,1um,10 um,100um.Finally,1 um concentration of melatonin was selected for in vitro experiment.Western blot,RT-q PCR,CCK-8,SA-?-gal staining,ROS detection,?-H2 AX fluorescence staining,cell cycle detection,micro-CT detection were used to verify whether DM1 can lead to bone senescence and bone loss in vivo and in vitro,whether melatonin can inhibit DM1 induced bone senescence,alleviate bone loss,and explore the mechanism of its role.Result: 1.High glucose induced senescence of MC3T3-E1.High glucose environment increased the protein expression of ?-H2 AX,p21,p16,p15,p53 and ATM,and promoted the m RNA expression of p16(P = 0.0011),p21(P = 0.0015),IL-1 ?(P = 0.0036)and TNF-?(P = 0.0023).The results of SA-?-gal staining showed that high glucose significantly increased cell senescence,showing a glucose concentration dependent trend.ROS detection showed that high glucose significantly increased the level of ROS in cells,which was glucose concentration dependent.It was found that high glucose significantly increased DNA damage level in cells,which was glucose concentration dependent.2.Melatonin alleviated the senescence of MC3T3-E1 cells induced by high glucose.Melatonin significantly reduced the high expression levels of ?-H2 AX,p21,p16,p15,p53 and ATM induced by Hg,and alleviated the high m RNA expression of p16(P =0.0004),p21(P = 0.0018),IL-1 ?(P = 0.0284)and TNF-?(P = 0.0180)induced by Hg.Melatonin significantly alleviated ROS production,DNA damage and cell senescence induced by Hg.3.Melatonin promoted the proliferation of MC3T3-E1 cells through the MT1 / MT2 pathway,and alleviated the cell cycle arrest induced by HG.The results of cell cycle experiment showed that Hg induced G1 phase arrest(P = 0.0010)and decreased the proportion of S phase cells(P = 0.0079).Melatonin can alleviate the G1 phase arrest induced by Hg(P = 0.0116),and increase the proportion of S phase(P < 0.0001).Melatonin promoted the m RNA and protein expression of CDK2(P = 0.0196),CDK4(P = 0.0266),Cyclin D1(P = 0.0023).These effects disappeared after knockdown of melatonin receptor MT1 or MT2,and Cyclin D1 was the most obvious.These results indicated that the effect of melatonin on cell cycle progression depends on MT1 and MT2 receptors.4.Melatonin promoted the expression of PRC complex through the receptor MT1 /MT2 pathway.Melatonin can significantly improve the m RNA expression levels of Bmi1(P = 0.0008),Rnf2(P < 0.0001),EZH2(P = 0.0039),Suz12(P = 0.0203)and protein expression level of PRC complex,and it has a concentration dependent trend.These effects were weakened after knockdown of MT1 or MT2.It is suggested that melatonin promoted the expression of PRC complex through MT1 / MT2 pathway.5.Melatonin inhibited the senescence of MC3T3-E1 cells induced by HG environment through receptor MT1 and MT2.After transfection of knockdown melatonin receptors MT1 or MT2,the effects of melatonin on Hg induced ROS production,DNA damage and cell senescence was significantly weakened,indicating that melatonin can alleviate Hg induced ROS production,DNA damage and cell aging through MT1 or MT2 receptor.6.Melatonin increased SIRT1 protein level in a dose-dependent manner,while the knockdown of MT1 or MT2 eliminated this effect.In addition,SIRT1 knockdown by si RNA reduced the anti-ROS,anti-DNA damage and anti-senescence effects of melatonin.7.The protein levels of p16,p21,p15 and p53 in the femur of DM1 mice treated with melatonin were significantly decreased.The protein levels of Cyclin D1,Runx2,Bmi1,Rnf2,BCL2 and SIRT1 in the femur of DM1 mice treated with melatonin were significantly increased.Micro CT results showed that melatonin significantly alleviated the trabecular bone loss induced by DM1,among which BV/TV(P <0.0001),TB.N(P = 0.0005),TB.Sp(P = 0.0023)were statistically significant,Tb.Th(P = 0.2477)was not statistically significant.Conclusion: Melatonin alleviated the bone senescence and bone loss induced by type1 diabetes through the MT1/MT2-SIRT1 pathway.Melatonin may prevent bone senescence and bone loss via MT1/MT2-PRC complex pathway.Melatonin may prevent bone senescence and bone loss via MT1/MT2-CDKs pathway.