Studies On The Regulation Of L-arginine In Apoptosis Of Immortalized Murine Cementoblast Cells And Root Resorption During Orthodontic Tooth Movement

Nowadays,with the improvement of the quality of life,orthodontic treatment has become more and more popular.However,during the treatment,absorption of dental root could often appear,and when the balance of absorption and restoration is broken,the negative effect“Orthodontically Induced Inflammatory Root Resorption?OIIRR?”may happen.The effective methods for prevention have not been found becouse of the lack of understanding of mechanism.Recently,it reported that the reduce of oxygen caused by orthodontic force may lead to the decrease of proliferation and the increase of apoptosis of cementoblast,and that can be another mechanism of root resorption.L-Arginine?L-arg?,as a donor of nitric oxide,has been reported to increase the hypoxia tolerance of tissue cells,inhibit apoptosis,and promote the proliferation and functional activities of a variety of cells,including osteoblast.Therefore,in the periodontal anoxic environment during orthodontic treatment,whether l-arginine has the same effect on cementoblast cells,so as to reduce root resorption caused by orthodontic treatment.It has not been studied.The purpose of this experiment is to simulate periodontal local hypoxic environment,and to study the effect of l-arginine on the proliferation and apoptosis of cementoblasts in hypoxic environment.Moreover,we research the effect of l-arg on root resorption during orthodontic treatment in vivo by establishing of the orthodontic tooth movement model in rats,in order to provide evidence for the new direction of root resorption mechanism research and further guide clinical treatment.The experiment is divided into two parts:in vitro and in vivo1:Effect of l-arginine on the proliferation and apoptosis of immortalized murine cementoblast cells in hypoxia.Objective:To study the effects of different concentrations of l-arg solution on the proliferation level and apoptosis of OCCM-30 in a hypoxic environment.Methods:OCCM-30 was cultured in hypoxic environment(2%O_2,5%CO₂,93%N₂)with different concentrations of L-arg?0?mol/l,30?mol/l,60?mol/l,120?mol/l,240?mol/l,360?mol/l?.Cell proliferation was detected by CCK-8 and the apoptosis level was detected by flow cytometer at 24h and 48h,respectively.Then,OCCM-30 was cultured in hypoxic environment containing L-arg at 0?mol/l and 120?mol/l respectively.The gene expressions of Caspase-3,Caspase-9,Bax and Bcl-2were detected by real-time PCR and the protein expression levels of Caspase-3,Caspase-9,Bax and Bcl-2 were detected by Western-blot at 48hours.Results:At 24 hours,the viability of OCCM-30 was significantly decreased when the L-arg concentration was 0?mol/l in the hypoxic environment,and the difference was statistically significant?P<0.05?.The apoptosis level of OCCM-30 cells increased significantly when the L-arg concentration was 0?mol/l in hypoxia for 24 hours compared with the control group in the normal oxygen environment,and the difference was statistically significant?P<0.05?.The cell viability was significantly increased and the apoptosis level was significantly decreased when the concentration was 30?mol/l,and the difference was statistically significant compared with the 0?mol/l concentration.Until the concentration became120?mol/l,the cell activity showed a rise to the peak and the apoptosis level decreased to the lowest level,then,with the concentration increased,the activity decreased and the apoptosis increased.Similar results were obtained at the 48-hour test.The gene expressions and protein expressions of caspase-3,caspase-9and Bax in OCCM-30 cells were increased when the L-arg concentration was 0?mol/l after 48 hours compared with that in the control group.However,when the L-arg concentration was 120 mol/l,the expression was significantly decreased compared with that in the 0?mol/l group.Bcl-2 was contrary to the above three.Conclusion:Hypoxic stimulation can inhibit the proliferation and promote the apoptosis of OCCM-30,and L-arg can effectively promote the proliferation and inhibit the apoptosis of OCCM-30 in hypoxic environment.The proliferation level reaches the peak and the apoptosis level can be minimized at the concentration of 120?mol/l.The mechanism may be to inhibit the expression of Caspase-3,Caspase-9,Bax gene and protein,and promote the expression of Bcl-2 gene and protein.2:Effects of local application of L-arg on root resorption during orthodontic tooth movement in rats.Objective:To study the effect of L-arg on root resorption during orthodontic tooth movement in rats.Methods:36 male SD rats aged 8 weeks were randomly divided into 3groups,the blank group,the control?PBS?group and experimental?L-arg?group,then added 50 g force to the left maxillary first molars of rats in each group and injected the corresponding drug every other day.After 7and 14 days,the first molar alveolar bone specimen was isolated for Micro–Computed Tomography?Micro-CT?scanning,Hematoxylin-eosin staining,TRAP staining,PCNA immunohistochemical staining and TUNEL staining were used to detect the morphological changes.Results:After 7 and 14 days,there was no significant difference between the control group and the blank group.The volume of root resorption fossa in the experimental group was smaller than that in the control group,and the difference was statistically significant?P<0.05?.Compared with the control group,the osteoclast count of the experimental group did not change significantly.In the experimental group,the number of periodontal tissue cells in proliferationin increased and the number of apoptotic cells decreased compared with the control group.Conclusion:Local application of l-arg can reduce root resorption during orthodontic tooth movement in rats.