Melatonin Induces Mitochondrial Apoptosis In Osteoblasts By Regulating The STIM1/Cytosolic Calcium Elevation/ERK Pathway

Objective:Idiopathic scoliosis is a kind of scoliosis with unclear reasons during growth and development,which is more common in teenagers.Scoliosis can not only cause the collapse,pain and deformity of the spine of patients,but also seriously affect the quality of life and labor ability of patients.At the same time,it causes serious psychological trauma to adolescents.At present,the main treatment of idiopathic scoliosis is surgery,with postoperative complications and obvious trauma.Therefore,new treatment methods need to be developed.It was found that the occurrence of idiopathic scoliosis was related to melatonin deficiency.Application of melatonin antagonists can improve the success rate of treating scoliosis model of bipedal rats,indicating that melatonin deficiency may increase the possibility and severity of scoliosis.A prospective study of adolescent IS found that melatonin supplementation can prevent the progression of scoliosis,especially in mild cases with a curve less than 35 degrees.Interestingly,intraperitoneal injection of melatonin at physiological concentrations did not prevent the occurrence and development of idiopathic scoliosis.However,the concentration of melatonin injected into the abdominal cavity is much higher than the physiological concentration,which can prevent the occurrence and development of idiopathic scoliosis.Therefore,we speculate that high concentration of melatonin may play a therapeutic role in idiopathic scoliosis.In addition,our team's previous study found that high concentrations?1mM or more?of melatonin inhibited osteoblast proliferation.Therefore,we speculate that melatonin can prevent the development of deformity by inhibiting the abnormal proliferation of osteoblasts and restoring normal bone growth.The purpose of this study is to explore the mechanism of melatonin inhibiting the proliferation of osteoblasts.Methods:1.CCK-8 method was used to detect the proliferation of osteoblasts;2.Apoptosis detection kit was used to detect the apoptosis effect of drugs on osteoblasts;3.Alkaline phosphatase kit was used to detect the effect of drugs on the differentiation of osteoblasts;4.RT-qPCR was used to detect the mRNA level expression of the target gene;5.Immunofluorescence was used to detect the expression of the target protein and its intracellular 6.Western blotting was used to detect the expression of the target protein;7.SiRNA was used to down-regulated the expression of the target protein;8.Fluo-4 was used to detect the concentration of Ca²⁺in cells with fluorescence microscope;9.Fluo-4 was used to detect the effect of drugs on Ca²⁺in cells with fluorescence enzyme marker;10.JC-1 method was used to detect the change of mitochondrial membrane potential;11.Statistics Academic analysis:all experiments in this article were repeated at least three times.Statistical analysis was performed using SPSS.Paired t-tests or one-way ANOVA of variance was used to identify statistical significance.p<0.05 was considered statistically significant.Results:1.Melatonin inhibited the proliferation,differentiation and induced apoptosis of osteoblasts in a concentration dependent manner.The osteoblasts were treated with melatonin of 0mM,1mM,2mM and 4mM for 48 hours.The results of CCK-8 showed that the proliferation of osteoblasts was significantly inhibited with the increase of melatonin concentration.Cell death detection ELISA showed that melatonin induced apoptosis of osteoblasts in a concentration dependent manner.The differentiation ability of osteoblasts was detected by alkaline phosphatase kit.The results showed that with the increase of melatonin concentration,the differentiation ability of osteoblasts was significantly inhibited.2.Melatonin inhibited the mRNA expression of Osteopontin?OPN?;Osteoprotegerin?OPG?;Osteocalcin?OCN?in a concentration dependent manner.The osteoblasts were treated with melatonin of 0mM,1mM,2mM and 4mM for 48 hours.The mRNA expression of OPN,OCN,OPG in osteoblasts was detected by RT-qPCR.The mRNA expression of OPN,OCN,OPG decreased significantly.3.Melatonin promoted the mRNA and protein expression of STIM1 and ORAI1 in a concentration dependent manner.The osteoblasts were treated with melatonin of 0mM,1mM,2mM and 4mM for 48 hours.The mRNA expression of STIM1 and ORAI1 in osteoblasts was detected by RT-qPCR.The results showed that melatonin up-regulates mRNA expression of STIM1 and ORAI1 in a concentration-dependent manner.Western blotting was used to detect the protein expression of STIM1and Orai1 in osteoblasts.The results showed that melatonin significantly increased the protein expression of STIM1 and Orai1 in a concentration dependent manner.The expression and distribution of STIM1 in osteoblasts were detected by immunofluorescence microscopy.The results showed that melatonin could significantly enhance the expression and distribution of STIM1.4.Melatonin can significantly increase the intracellular Ca2+content.Fluo-4 was used to detect the Ca2+content of osteoblasts.The results showed that with the increase of melatonin concentration,the Ca2+content of osteoblasts increased significantly.5.SiRNA down-regulated protein expression of STIM1 reversed melatonin-induced increase of cytosolic calcium in osteoblasts.The results showed that melatonin could increase the level of intracellular Ca2+.When the STIM1 protein was down-regulated,the elevated Ca2+was significantly inhibited,which confirmed that melatonin induced the increase of intracellular Ca2+by up regulating STIM1.6.Calcium channel blocker TRPC can reverse the inhibition of melatonin on osteoblast proliferation and the promotion of apoptosis.The results of CCK-8 showed that TRPC reverse the inhibition of proliferation and the promotion of apoptosis of osteoblasts,indicating that the increase of intracellular Ca2+was the key to induce apoptosis of osteoblasts.7.Down-regulated the protein expression of STIM1 by SiRNA could reversed the inhibition of melatonin on osteoblast proliferation and the promotion of apoptosis.The results showed that melatonin could inhibit the proliferation of osteoblasts,but the inhibition was reversed after down-regulation of STIM1.In addition,melatonin can promote the apoptosis of osteoblasts and down-regulate the STIM1 protein,this promotion was significantly reversed,which confirmed that up-regulated STIM1 is the key to induce the apoptosis of osteoblasts.8.Melatonin induced apoptosis of osteoblasts through mitochondrial pathway.The osteoblasts were treated with melatonin of 0mM,1mM,2mM and 4mM for 48 hours.The mitochondrial membrane potential in osteoblasts was detected by JC-1 method.The results showed that melatonin could significantly inhibit the mitochondrial membrane potential in a concentration dependent manner.Western blotting was used to detect the protein expression of Bax;Bcl-2;Cyclochrome C;Cleaved-caspase 3 in osteoblasts.The results showed that melatonin significantly up-regulated the protein expression of Bax;Cyclochrome C;Cleaved-caspase 3 and down-regulated the protein expression of Bcl-2 in a concentration dependent manner.9.Down-regulating the protein expression of STIM1 can reverse the apoptosis of mitochondrial pathway in melatonin-mediated osteoblasts.SiRNA was used to transfect osteoblasts to down-regulated STIM1.JC-1was used to detect mitochondrial membrane potential,the results showed melatonin reduces intracellular mitochondrial membrane potential levels which was reversed significantly.The protein expression of Bax;Bcl-2;Cyclochrome C;Cleaved-caspase 3 was detected by western blotting.The results showed that up-regulation protein expression of Bax;Cyclochrome C;Cleaved-caspase 3 was significantly decreased,while the down-regulation protein expression of Bcl-2 protein expression was significantly increased.10.Melatonin significantly inhibited the activation of ERK and AKT pathway.The osteoblasts were treated with melatonin of 0mM,1mM,2mM and 4mM for 48 hours.The protein expression of ERK and AKT pathway in osteoblasts was detected by western blotting.The results showed that the phosphorylated protein expression of ERK and AKT were significantly inhibited with the increase of melatonin concentration.11.Down-regulating the protein expression of STIM1 can up-regulated the phosphorylation protein expression of ERK mediated by melatonin in osteoblasts,but it has no significant effect on the phosphorylation protein expression of AKT.The results showed that the ERK pathway inhibited by melatonin was activated,but the AKT pathway did not change significantly.It is confirmed that ERK is involved in the apoptosis of osteoblasts induced by melatonin.12.Calcium channel blocker TRPC can reverse the inhibition of melanin on ERK pathway in osteoblasts.With the application of calcium channel blocker TRPC,the ERK pathway inhibited by melatonin was significantly activated,further confirming that SOCE induces osteoblast apoptosis through the ERK pathway.Conclusion:1.Melatonin inhibited the proliferation,differentiation and induced apoptosis of osteoblasts in a concentration dependent manner.2.Melatonin can increase intracellular Ca2+by up-regulating STIM1 and induce osteoblast apoptosis through mitochondrial pathway.3.STIM1 mediated SOCE induces osteoblasts apoptosis through ERK pathway.