Study On The Interaction Between Autophagy And Apoptosis Of MC3T3-E1 At Starvation State

When confronted with the orthodontic force which is of right size and appropriate direction, the pressure side of alveolar bone will endure considerable death of bone cells, like osteoblasts, hyperplasia of osteoclasts and alveolar bone loss, while the tension side will undergo the death of osteoclasts, osteoblasts hyperplasia and alveolar bone formation. This process happened in the alveolar bone remodeling is the foundation of orthodontic tooth movement. Present study suggests that the death of osteoblasts in the pressure side of alveolar bone is due to the microenvironment change of alveolar caused by orthodontic force, including compression of periodontal ligament, occlusion and partial disintegration of capillaries as well as the decline of blood supply, which leads to the formation of a hypoxic and nutritional deficiency environment, where the alveolar bone tissue cells of pressure side stay in. Here is the question, whether the starvation induced the alveolar bone loss and the death of alveolar cells in pressure side? It is well known that bone remodeling under orthodontic force is physiological tooth movement, and pathological changes like cell necrosis do not occur in bone tissue. There are three ways of cell death: necrosis and autophagy, apoptosis, between which autophagy and apoptosis are all programmed cell death which led by itself. We want to know what were the role of them in the course of cell death in the pressure side of the bone tissue? What is the relationship between them? In vivo experiments, we establish orthodontic tooth movement models in beagle dogs, By the models, the tooth movement was measured, the absorption of alveolar bone under orthodontic force were observed at different times, and the expression level changes of autophagy and apoptosis in bone tissue at the pressure side also been recorded. Furthermore, in vitro study we simulated death of osteoblasts in starvation environment, detected the expression and relationship of autophagy and apoptosis. This research includes the following two aspects:1. Autophagy and apoptosis levels of the bone tissue during tooth movement in animal studiesObjective: To investigate the morphology changes and expression of autophagy and apoptosis of the alveolar bone tissue in the pressure side in Beagles orthodontic tooth movement model.Methods: 6 adult beagle dogs were randomly divided into control group and orthodontic treatment group. The orthodontic treatment group was subdivided into 7 days orthodontic treatment group, 14 days orthodontic treatment group, 28 days orthodontic treatment group. The control group was subjected to no disposal, in the orthodontic treatment groups, canines were moved distally movement under 150 g force application with implants as anchorage. The effects of tooth movement were observed, while the alveolar bone remodeling at the pressure side was analyzed with histomorphology study under HE stain. The autophagy and apoptosis expression of the alveolar bone tissue in the pressure side were detected by Western blot and immunohistochemical staining.Results: under the orthodontic force, ① In comparison with control group, canines in each orthodontic treatment group were moved significantly(P <0.05), and the distance increased along with time(P <0.05) ② Western blot analysis showed that, compared with the control group, LC3- Ⅱ/ LC3- Ⅰconversion rate in alveolar bone tissue in the pressure side of increased rapidly and peaked at 7 days(P <0.05), while caspase 3 protein levels increased rapidly after 7 days and peaked at 28 days(P <0.05); ③ HE staining study showed that the alveolar bone resorption increased as the orthodontic force going; ④ The immunohistochemical staining showed that, in 7 days and 28 days orthodontic treatment groups, LC3 staining was positive, while caspase 3 staining was positive in 14 days and 28 days orthodontic treatment groups.Conclusion: In the early stage of orthodontic bone remodeling under suitable and continuous orthodontic force, autophagic response of bone cells enhanced and the bone resorption was in a small extent. Then, with the orthodontic force going, the autophagy decreased and apoptosis began to increase, while the bone resorption increased. It was suggested that the autophagy at the early stage was a kind of protection which might postpone the apoptosis.2. The effects of starvation on autophagy and apoptosis of MC3T3-E1Objective: To explore the interaction between autophagy and apoptosis of MC3T3-E1 at starvation state.Methods: MC3T3-E1 of 3-MA group was pretreated with 3-MA for 1 hour before being cultured in earle’s balanced salt solution(EBSS)for another 6 hours, and the samples without 3-MA pretreatment were taken as starvation group. MC3T3-E1 of different groups were sampled hourly. The morphological change of MC3T3-E1 form starvation group was observed by TEM. To determine the expression of LC3 and caspase 3, western blot and flow cytometry were performed.Results: The observation of TEM showed that MC3T3-E1 from starvation group was dominated by autophagy in the first two hours of the 6-hours period cultured in EBSS, whereas apoptosis was the main process after the first two hours and the level increased with time. The observation of western blot showed that, compared with those from starvation group, MC3T3-E1 from 3-MA group presented a reduced LC3 expression level(P<0.05) throughout the 6-hours period, and an enhanced caspase 3 expression level in the first 4 hours(P<0.05) while there was no difference observed between the two groups(P>0.05) in the last 2 hours(P>0.05). The observation of flow cytometry showed that, compared with those from starvation group, MC3T3-E1 from 3-MA group presented an enhanced apoptosis level in the first 4 hours(P<0.05), while there while there was no difference observed between the two groups(P>0.05) in the last 2 hours(P>0.05).Conclusion: Under starvation condition, autophagy occurs in osteoblasts at the early stage, which may have a protective effect in delaying apoptotic.Synthesize the above results, in vivo and vitro experiments, we found that in early period, the response of autophagy appeared in alveolar bone may be a protection mechanism which fight against hypoxia and inadequate nutrition in the pressure side caused by orthodontic force. Once this protection weakened or disappeared, alveolar bone loss happened along with the die out of vast bone cells caused by apoptosis.