| The human skeleton is in the process of continuous reconstruction all its life.When stimulated by external force,the skeleton will undergo adaptive changes,and external stimulation can promote the formation of new bone,including clinically common distraction osteogenesis and alveolar bone reconstruction in the process of orthodontic tooth movement.In the process of orthodontic tooth movement,there are two main force surfaces in the tooth,namely pressure surface bone absorption and tension surface bone hyperplasia,so that the tooth moves to the target position.Osteoblasts play an important role in this process.Clinically,many patients with periodontal disease need to focus on the tooth space through orthodontic treatment,so as to prepare for the later restoration treatment.Because periodontal support tissues of patients with periodontal disease are weak,it is particularly important to control the magnitude of the orthodontic force during the orthodontic process.Mechanical stress can promote the remodeling of bone tissue,but the mechanical effects of bone remodeling are more complex under the condition of inflammation.Studies have found that mechanical stretch can regulate osteoblast differentiation through Rho A/ROCK signaling pathway,but the effect of mechanical stretch on osteoblast growth characteristics and intracellular Rho A/ROCK signaling pathway in the inflammatory environment is still not clear.Based on the results of the experiment in the early stage of the experimental study,1ug/ml Pg-LPS(lipopolysaccharide from porphyromonas gingivalis)stimulates mouse osteoblasts MC3T3-E1,the osteoblasts were subjected to 8% stretch simulation using Flexcell-5000.then,we observed the growth of cells,the expression of RhoA,ROCK and osteoblastic differentiation gene Runx-2 were observed byqRT-PCR and Western blot.In addition,by inhibiting RhoA/ROCK signaling pathway,the relationship between Rho A/ROCK signaling and osteoblastic differentiation was explored after stretch force was applied to osteoblasts under Pg-LPS stimulation.Objective: To investigate the effect of Pg-LPS stimulation on the growth characteristics of MC3T3-E1 of osteoblasts and the effect of Pg-LPS stimulation on the intracellular Rho A/ROCK signal.To understand the molecular mechanical mechanism of stretch stress between Rho A/ROCK and osteoblast differentiation under Pg-LPS stimulation.Methods: After 1ug/ml Pg-LPS stimulated mouse osteoblasts MC3T3-E1 for24 h,the cells were subjected to 8% stretch stress for 8h,cell proliferation activity was detected by CCK8 technology,apoptosis was observed by Hoechst 33258 staining,and cell skeleton was observed by Phalloidin-TRITC staining.2)1ug/ml Pg-LPS stimulated MC3T3-E1 cells for 24 h,then 8% stretch force were subjected to MC3T3-E1 cells for 8h,the expression changes of Rho A,ROCK and Runx-2 in the cells were detected by q RT-PCR and Western Blot technology.3)1ug/ml Pg-LPS stimulated MC3T3-E1 cells for 24 h,then inhibiting Rho A/ROCK signaling pathway with 0.5ng/ml C3 transferase,the cells were subjected to 8% stretch for 8h.we detected the expressions of Rho A,ROCK and Runx-2 in the protein and gene levels were by Western blot and q RT-PCR,and the expressions of Rho A,ROCK and Runx-2in the cells were compared with those of Rho A,ROCK and Runx-2 before inhibiting by Rho A/ROCK signaling pathway.Results: 1)After 24 hours of Pg-LPS stimulation,the proliferation rate of osteoblasts significantly decreased,and the number of apoptotic cells significantly increased.When Pg-LPS stimulated cells for 24 h and 8% stretch force was applied for8 h,the cell activity was enhanced and the number of apoptosis was reduced,but it was still lower than that in the group without Pg-LPS stimulation.After stretch force was applied to cells,disordered actin fibers were arranged orderly along the cell axis.2)the protein and gene levels of Rho A,ROCK and Runx-2 were all increased after 8h of 8% stretch.The protein and gene levels of Rho A,ROCK and Runx-2 in the cells were decreased after the addition of C3 transferase and the 8% stretch force was applied for 8h.3)After Pg-LPS incubation for 24 h,the protein and gene levels of Rho A,ROCK and Runx-2 in the cells were all decreased,and the protein and gene levels of Rho A,ROCK and Runx-2 in the cells were increased after 8% stretch forcewas applied to the cells,but were still lower than those in the normal control group.The cells were incubated with Pg-LPS for 24 h.After the treatment with cell inhibitors,the expression of Rho A and ROCK was decreased and the expression of Runx-2 was increased after the 8% stretch force was applied for 8h.Conclusion: Certain mechanical tension has a protective effect on Pg-LPS-induced apoptosis,Rho A/ROCK signaling pathway has a regulatory effect on Pg-LPS-induced inhibition of cell differentiation.and has an effect on osteoblast osteogenesis. |
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