Effects Of Advanced Glycation End Products(AGEs) On Osteoclasts Of Differentiation

Background:In the process of oral clinical diagnosis and treatment,it is found that patients with inferior control of blood glucose may accompany with high probability of tooth loosening,long healing time of extraction wound,low rate of new bone formation after grafting,weak stability of implant teeth in alveolar bone,poor effect of orthodontic tooth movement and so on.Advanced glycation end products(AGEs)are a group of highly oxidized complex products with certain pathogenicity to diabetes and other chronic diseases,which are produced and accumuLated with age in normal physiological state.While in the case of inferior condition of blood glucose control in patients with diabetes,the production rate of AGEs can be accelerated,which may directly or indirectly affect the balance of bone metabolism.The normal and stable bone metabolism depends on the dynamic balance of bone formation and resorption.The effective tooth movement of clinical orthodontic patients is affected by the remodeling of periodontal tissue and the functional activity ofosteoblasts and osteoclasts.Most of the studies on the effects of AGEs on bone metabolism are focused on osteoblasts,but the effects of which on bone resorption are still controversial.Osteoclasts,derived from hematopoietic stem cells,are highly differentiated muLtinucleated giant cells,which are the main functional cells for bone resorption on the pressure side of alveolar bone during orthodontic tooth movement.Notch signaling pathway,involved in the reguLation of tissue balance,has a specific reguLation on the differentiation and activity of osteoclasts and may affect the polarization of macrophages.Macrophages and monocytes can both be used as progenitor cells to differentiate into mature osteoclasts.Macrophages play an important role in the function of immune defense and they can be differentiated into different phenotypes in different microenvironments in vivo and in vitro,including classical activated M1-type macrophages and alternative activated M2-type macrophages.More and more scholars have proposed that the polarization of macrophages has a certain effect on the differentiation ability of osteoclasts.It can be seen that a large number of AGEs accumuLated in the host can affect the process of bone metabolism,in which AGEs play a crucial role in reguLating the differentiation of downstream osteoclasts by affecting the polarization of macrophages.In-depth study of the effects of AGEs on osteoclast differentiationand reveal the related mechanism is of great significance to improve the control of tooth movement in orthodontic patients with diabetes.Objective:The purpose of this experiment is to explore the effects of AGEs on the polarization of macrophages as well as the differentiation ability of osteoclasts,and to preliminarily explore the relevant mechanisms,so as to provide new ideas for the control of tooth movement in clinical diabetic patients during orthodontic process.Methods:Part ?: LPS and IL-4 were used to induce the polarization of macrophages to M1-type and M2-type respectively,and the polarization state of macrophages was identified by qRT-PCR and Griess detections.Osteoclastic induction was carried out on M1-type macrophages and M2-type macrophages respectively,and the differentiation ability of osteoclasts on macrophages in different states of polarization was detected by qRT-PCR.Part ?: The different stages of osteoclast differentiation were observed by TRAP staining.The effects of AGEs on osteoclast differentiation of macrophages at different stages were detected by TRAP staining,qRT-PCR and Western blot.The trend of polarization of AGEs on macrophages was detected by qRT-PCR.And then the effects of AGEs on osteoclast differentiation of polarizedmacrophages were detected by TRAP staining,qRT-PCR and Western blot.Part ?: DAPT was used to inhibit the Notch signal pathway,and the relative mRNA expression of osteoclast-related factors as well as polarization-related factors were detected by qRT-PCR in the experiment of osteoclast differentiation treated with AGEs.ResuLts:Part ?: After inducing the polarization of macrophages,the content of NO and the relative mRNA expression of M1-type-related markers in LPS group were higher than those in control group,and the relative mRNA expression of M2-type-related markers in IL-4 group was higher than that in control group,of which the difference is statistically significant.After inducing the formation of osteoclasts,the relative mRNA expression of osteoclast-related factors in M1-type macrophages group was higher than that in M2-type macrophages group,and the experimental resuLts of BMMs and Raw264.7 were consistent.Part ?: There were no muLtinucleated cells in the first 3 days of osteoclastic induction of macrophages,but the muLtinucleated cells began to appear 3 days later.The number of TRAP positive cells in the early stage intervention group and in the later stage intervention group were both more than that in the control group,but significant effect was found only in early stage intervention group.Compared with the control group,the relativemRNA expression of osteoclast-related factors was higher in the early stage intervention group and the difference is statistically significant.The trend of protein expression was consistent with that of mRNA expression.The relative mRNA expression of related markers of M1-type macrophages was higher than that of M2-type macrophages under the action of AGEs.The resuLts of TRAP staining,qRT-PCR and Western blot of the effects of AGEs on the differentiation ability of osteoclasts induced by M1-type macrophages was consistent with those on Raw264.7 and BMMs.Part ?: Compared with RANKL group,the relative mRNA expression of related markers of M1-type macrophages was higher in RANKL+AGEs group,and the expression of related markers of M2-type macrophage was lower in RANKL+AGEs group.After adding DAPT,the relative mRNA expression of related markers was opposite.Compared with RANKL group,the relative mRNA expression of osteoclast-related factors in RANKL+AGEs group was higher,which was decreased under the action of DAPT.Conclusion:Part ?: LPS and IL-4 can induce the polarization of macrophages to M1-type and M2-type respectively.M1-type macrophages possess stronger ability to differentiate into osteoclasts than M2-type macrophages.Part ?: It is a dynamic process of inducing macrophages to differentiate into osteoclasts,in which the first three days are considered asthe early stage of differentiation,and then the later stage of differentiation.AGEs can facilitate the differentiation of osteoclasts more obviously in the early stage of differentiation than in the later stage.AGEs may promote the differentiation ability of osteoclasts by mediating the polarization of macrophages to M1-type.Part ?: AGEs may promote the ability of osteoclast differentiation by mediating the polarization of macrophages to M1-type through Notch signaling pathway.