| In orthodontic treatment,root resorption and restoration often happen and when the two are out of balance,"orthodontic induced root resorption(OIIRR)" may occur.In recent years,it has been reported that the decrease of periodontal oxygen pressure under orthodontic action leads to increased apoptosis of cementoblasts,which may also be the mechanism of root resorption.Therefore,this study simulated the hypoxia-induced orthodontic environment in vitro to explore the possible mechanism of inhibiting the apoptosis of cementoblast cells under the hypoxic environment,so as toprovide a possible therapeutic target for reducing the absorption of orthodontic root.Studies have shown that autophagy in hypoxia environment regulation of bone cells,osteoblast and osteoclast apoptosis and mineralization,play an important role in bone metabolism balance,at the same time the autophagy induced by Sirt1 enhancement of osteogenesis sample cell apoptosis induced by fluoride protection of bone cells into similar to osteoblast biology behavior,and low oxygen environment of bone cells into autophagy not seen related research.The purpose of this experiment is to establish the root absorption model of local injections of Sirt1 activator resveratrol and/or autophagy inhibitor chloroquine to further validate the role of "Sirt1-autophagy axis" in root absorption.And simulate orthodontic periodontal low oxygen environment,to research the role of autophagy in cell apoptosis in the hypoxia environment.It is expected that the future research on autophagy of odontoblast can be transformed from the basic to the clinical,providing a new therapeutic scheme for the repair of root resorption in the orthodontic treatment.This experiment is divided into two experiments in vitro and in vivo:Experiment 1:The study of effect of "Sirt1-autophagy axis" on the model of root resorption in rat.Part ?: Changes of proteins related to autophagy and apoptosis in "Sirt1-autophagy axis" in periodontal tissue in rat root resorption model.Methods: A total of 24 healthy 8-week-old SD male SD rats were divided into two groups with 12 rats in each group randomly:the control group and the reinforcement group.The first molar crown of the left maxillary of the rats in the reinforcement group was applied with a force of100 g in the direction of proximal medium,and the rats were killed after 7days.HE staining,immunohistochemical staining against Sirt1,LC3,P62 and TUNEL staining were used to observe autophagy and apoptosis in periodontal tissues.Results: Root resorption fossas increased in the load group compared with the control group.And the expression of LC3 and Sirt1 in the periodontal membrane increased significantly in the load group compared with the control group.The expression of p62 in periodontal membrane was significantly decreased in the load group compared with the control group.The mean optical density of LC3 in periodontal tissues was observed,and the difference was statistically significant(P<0.05).The apoptosis rate of the load group increased compared with the control group,and the difference was statistically significant(P < 0.05).Conclusion: Autophagy related protein quantity changes,autophagy activates,Sirt1 protein level increases,apoptosis increases,and tooth root absorption fossas increase.Part ?: Changes of related proteins and root resorption after local injection of Sirt1 and autophagy regulators in rat root resorption model.Methods:A total of 48 healthy 8-week-old SD male rats were randomly divided into four groups with 12 rats in each group: control group,PBS group,Resveratrol group,Resveratrol+CQ group.In 7days,the specimen was scanned by micro-CT and the volume of the root resorption notch was calculated by using three dimensional convex hull method.The other detection methods are the same as the first part of experiment I.Results: At 7 days after the application of force,the volume of absorption lacuna in PBS group was larger than that in the control group,the difference was not statistically significant.The volume of absorption lacuna in Resveratrol group was significantly smaller than that in PBSgroup,the difference was statistically significant(P < 0.05).The volume of absorption lacuna in Resveratrol +CQ group was significantly larger than that in Resveratrol group,the difference was statistically significant(P <0.05).Compared with PBS group,the expression of LC3 protein in Resveratrol group was significantly increased,p62 protein expression was significantly decreased,autophagy was enhanced;compared with Resveratrol group,the expression of LC3 protein in Resveratrol + CQ group was significantly decreased,p62 protein expression was significantly increased,the difference was statistically significant(P < 0.05).The Sirt1 protein expression in Resveratrol group was significantly higher than that in PBS group(P < 0.05),while that in Resveratrol + CQ group was lower than that in Resveratrol group(P < 0.05).The apoptosis rate of Resveratrol group was lower than that of control group(P < 0.05).The apoptosis rate of Resveratrol + CQ group was significantly higher than that of the control group(P < 0.05).Conclusion: Local application of SIRT1 activator resveratrol can reduce apoptosis and root resorption in periodontal tissues,while chloroquine,an autophagy inhibitor,can block this effect.The results showed that activation of "Sirt1-autophagy axis" could reduce root resorption.Experiment 2: Study on the effect of "Sirt1-autophagy axis" on OCCM-30 cell apoptosis in hypoxia.Part ?: Effects of hypoxic environment on autophagy and apoptosis of OCCM-30 cells.Methods: OCCM-30 cells were collected during the logarithmic growth phase.The cells were divided into control group,hypoxia group and hypoxia + 3-MA group and cultured for 12 h.Changes of autophagylysosomes were observed by MDC staining.;Changes of autophagosomes were detected by LC3 II immunofluorescence.Autophagosomes were observed by transmission electron microscopy.Western Blot was used to detect the expression of Sirt1,LC3 II,p62 and other proteins.Cell apoptosis was detected by PI-Hoechst immunofluorescence.Results: Compared with the control group,the content of autophagy lysosomes in the hypoxia group increased significantly.At the same time,the content of autophagy lysosomes in the hypoxia + 3-MA group was significantly decreased compared with that in the hypoxia group,with statistically significant differences(P<0.05).Compared with the control group,the expression of fluorescence highlights in the hypoxia group increased significantly.Compared with the hypoxia group,the expression of fluorescence highlights was significantly decreased in the hypoxia + 3-MA group.The differences were statistically significant(P<0.05).Compared with the control group,the expression of p62 protein in the hypoxia group was significantly decreased,the expression of LC3 II and Sirt1 protein was significantly increased,and autophagy was enhanced.Compared with the hypoxia group,the expression of protein p62 in the hypoxia + 3-MA group increased significantly,while the expression of protein LC3 II and Sirt1 decreased significantly(P<0.05).The number of apoptotic cells in hypoxia group increased significantly compared with control group.The number of apoptotic cells in the hypoxia + 3-MA group increased further than that in the hypoxic group,with statistically significant differences(P<0.05).Conclusion: Under hypoxia,autophagy of osteoblasts was enhanced,and the level of Sirt1 was increased.Autophagy inhibitor 3-MA was added to reduce autophagy and increase apoptosis of cementoblasts,indicating that autophagy activation is the relevant mechanism to reduce apoptosis ofcementoblasts under hypoxia environment.Part ?: Effect of Sirt1 on autophagy in OCCM-30 cells under hypoxia environmentMethods: the cells were divided into control group,hypoxia group,hypoxia + Resveratrol group and hypoxia+ Res.+ex527 group.The cells were cultured for 12 h,and The detection method is the same as the first part of Experiment 2.Results: Compared with the control group,the content of autophagy lysosomes in the hypoxia group increased significantly.Meanwhile,the content of autophagy lysosomes in the hypoxia + Resveratrol group increased significantly compared with that in the hypoxia group.The content of autophagy lysosomes in the hypoxia+ Res.+ex527 group was significantly decreased compared with the hypoxia+ Resveratrol group.The differences were statistically significant(P<0.05).Compared with the control group,the expression of fluorescence highlights in the hypoxia group increased significantly.Compared with the hypoxia +Resveratrol group,the expression of fluorescence highlights increased significantly.The expression of fluorescence highlights in hypoxia+ Res.+ex527 group was significantly decreased compared with that in hypoxia +Resveratrol.group.The differences were statistically significant(P<0.05).Compared with the control group,the expression of LC3 II protein was significantly increased in the hypoxia group,while the expression of p62 protein was significantly decreased and autophagy was enhanced.The protein expression of LC3 II and Sirt1 in the hypoxia +Resveratrol group was significantly increased compared with the hypoxia group,while the protein expression of p62 was significantly decreased.The expression of LC3 II and Sirt1 in the hypoxia+ Res.+ex527 group was significantly decreased,while the expression of p62 protein wassignificantly increased.The above differences were statistically significant(P<0.05).Conclusion: In hypoxia environment,the Sirt1 activator resveratrol was added to increase the level of Sirt1 and autophagy in osteoblasts.This indicates that resveratrol can further activate autophagy of cementoblasts in hypoxic environment and increase the expression of Sirt1.In addition,the autophagy activated by resveratrol could be reversed by the Sirt1 inhibitor ex527 in the hypoxia environment,indicating that Sirt1 plays an important role in the autophagy process of resveratrol activated cementoblasts.Part ?: Effect of "Sirt1-autophagy axis" on OCCM-30 cell apoptosis in hypoxic environmentMethods: the cells were divided into control group,hypoxia group,hypoxia + Resveratrol group and hypoxia + Res.+ 3-MA group.The detection method is the same as the first part of Experiment 2.Results: The content of autophagy lysosomes in the hypoxia +Resveratrol group increased significantly compared with that in the hypoxia group.The content of autophagy lysosomes in the hypoxia + Res.+3-MA group was significantly decreased compared with that in the hypoxia+ Resveratrol group.The differences were statistically significant(P<0.05).Compared with the hypoxia + Resveratrol group,the expression of fluorescence highlights increased significantly.The expression of fluorescence highlights in hypoxia+ Res.+ 3-MA group was significantly decreased compared with that in hypoxia + Resveratrol group.The differences were statistically significant(P<0.05).The protein expression of LC3 II and Sirt1 in the hypoxia + Resveratrol group was significantly increased compared with the hypoxia group,while the protein expression of p62 was significantly decreased.LC3 II protein expression in the hypoxia+ Res.+ 3-MA group was significantly decreased and p62 protein expression was significantly increased compared with the hypoxia+Resveratrol group(P <0.05),while the Sirt1 protein expression was decreased and the difference was not statistically significant(P=0.146).Compared with the hypoxia + Resveratrol group,the expression of fluorescence highlights was significantly decreased.The expression of fluorescence highlights in hypoxia + Res.+ 3-MA group was significantly increased than that in hypoxia + Resveratrol group.The differences were statistically significant(P<0.05).Conclusion: In the hypoxia environment,the addition of resveratrol,a Sirt1 activator,increased the level of Sirt1,increased autophagy and decreased apoptosis in the osteoblasts.Moreover,the activation of "Sirt1-autophagy axis" in hypoxia environment reduced the apoptosis of cementoblast cells.However,in the hypoxia environment,the autophagy activated by reveratrol can be inhibited by the autophagy inhibitor 3-MA,while the expression of Sirt1 is still increased,suggesting that some autophagy-related molecules may be downstream acting molecules of Sirt1 in the hypoxia environment. |
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