| Objective: Strontium(Sr)is an alkaline earth metal that is homologous to the calcium element in the periodic table.Similar as calcium,Sr has a dual action of improving bone formation and suppressing bone resorption,resulting in increased bone mineral density.The use of Sr drug and the application of Sr-related biomaterials in bone regeneration has become a hot spot of research for svevral years.Although the beneficial effect of Sr on osteogenesis in different models has been corroborated with plenty researches and studies,its molecular mechanism on bone reconstruction still remains to be evaluated to support its clinical application.Autophagy(referred to as macroautophagy)is known as a highly conserved intracellular self-degradation procedure by which eukaryotic cells maintain their homeostasis.Autophagy plays an important role in cell growth,differentiation and homeostasis.In recent years,it has been reported that autophagy participates in the osteogenesis process of human stem cells.However,it is not clear whether autophagy plays a role in the process of Sr promoting osteogenic differentiation.In this study,we examined the osteogenic differentiation and autophagy level of mouse pre-osteoblast MC3T3-E1 subclone 14 induced by Sr,and investigated the related molecular mechanisms.Methods: 1.Cytotoxitity of Sr : MC3T3-E1 cells were treated with different concentrations of Sr(0,3,6,12,24,48,96 mM)for 72 h.MTT assay was performed for cell proliferation to investigate the toxicity of Sr onMC3T3-E1.2.Sr-induced osteogenesis: The expression of RunX-2 and OCN was detected by realtime PCR.Western blot assay was used to detected the expression of OCN.For mineralization analysis,the alkaline phosphatase activity was determined by BCIP/NBT ALP dyeing fluid stain after 7 days-treatment.Mineralized nodules were stained with Alizarin Red solution after 21 days-treatment.Combining with the above results,we determined the optimal Sr stimulation concentration for subsequent experiments.3.Autophagy and Sr-induced osteogenesis: The autophagy related protein SQSTM1/p62 and LC-3 ?/? was measured by Western blot assay.The cells were stained with MDC and measured with 512 nm emission in fluorescence microscope after incubated for 72 h.The acidic vesicles in green fluorescence indicating the activated autophagosome.MC3T3-E1 cells were pre-incubated with 10 mM 3-MA for 12 h,and then treated with strontium for 72 h.The markers of osteogenic differentiation and autophagy were detected to verify the role of autophagy in Sr-induced osteogenesis..4.Role of AMPK/mTOR involved in Sr-induced osteogenesis: We used Western blot assay to measure the phosphorylation of AMPK and mTOR in cells treated with 3 mM Sr.Cells were pre-incubated with 10 ?M compound C to inhibit the activation of AMPK/mTOR pathway.Then the expression level of LC-3 ?/? and OCN was detected by Western blot assay.Results: 1.No significant influence of cell viability was observed when the cells were treated with low concentration of Sr(?12 mM)for three days(p>0.05).However,the exposure of MC3T3-E1 to high concentration Sr(>12mM)markedly inhibited the viability of cells(p<0.001).2.3 mM Sr significantly increased the expression of both RunX-2 and OCN(p<0.01).And it also increased the OCN protein expression(p<0.05).In addition,ALP staining and alizarin red staining respectively represent the early stage and late stage of osteogensis.3 mM Sr treatment group had a high intensity of stain in these two methods.On the other hand,the reatled bone marker of cells with 6 or 12 mM Sr were not increased compared with 3 mM group's.In all,3 mM was slected as the appropriate concentration in the following experiments.3.In Western blot,the expression of SQSTM1/p62 was significantly decreased(p<0.05)and the expression of LC-3 ? was significantly increased(p<0.01).The conversion rates of LC3-? to LC3-I indicated the activated autophagy.MDC staining results showed that the Sr-treated cells exhibited more obvious fluorescent punctate.However,the autophagy parameters had no significantly change(p>0.05)which indicate the inhibition of autophagy by 3-MA in MC3T3-E1.The expression of RunX-2,OCN and the activity of ALP had no significant change in 3-MA+Sr group compared with the control group.Therefore,autophagy participates in the process of Sr-induced osteogenesis.4.Compared with the control,the phosphorylation level of AMPK was significantly increased in the cells exposed to 3 mM Sr(p<0.05).In addition,low phosphorylation level of downstream molecular mTOR was observed in the 3mM Sr-treatment group(p<0.01).We couldn't observe the conversion of LC3-I to LC3-? which represent the inhibition of Sr-induced autophagy when the phosphorylation of AMPK was inhibited by Compound C.Moreover,the expression of OCN was relatively decreased which indicated the inhibition of osteogenic differentiation.In all,these data indicate that the AMPK/mTOR mediated autophagy is involved in Sr-induced osteogenic differentiation of MC3T3-E1.Conclusion: 1.Low concentration of Sr(?12 mM)has no obviously toxic effect on MC3T3-E1 cells.In addition,3 mM is the appropriate concentration of Sr to induce the osteogenic differentiation in vitro.2.The AMPK/mTOR mediated autophagy was involved in Sr-induced osteogenic differentiation of MC3T3-E1. |
PDF Full Text Request