The Radiologic Evaluation And Pathological Mechanism Study Of Periodontitis With Diabetes Mellitus

BackgroundPeriodontitis is a common chronic localized infectious disease,and diabetes can affect immune function in vivo.Several studies have proved that diabetes and periodontal diseases have a mutual promoting relationship that induces severe tissue damage and cell death.Numerous studies have shown that periodontitis or diabetes could induce cell apoptosis or autophagy.We want to figure out whether other cell death patterns emerged in pathological process of the periodontitis with diabetes.ObjectiveCompare the periodontal condition of patients with diabetes-associated periodontitis to that of patients with simple chronic periodontitis by oral clinical probing and imaging observation.Compare the differences of CBCT,Clinical Examination and Curved body tomography in the evaluation of alveolar bone defect in periodontitis.To figure out whether other cell death patterns emerged in patients with diabetes-associated periodontitis,and study the relative mechanism through periodontal tissue samples and MC3T3-E1 cell line culture.Materials and methods1.Clinical data was collected,and clinical periodontal examination and CBCT imaging examination were performed to evaluate patients with diabetes-associated periodontitis.The difference between CBCT and clinical exploration and curved tomography in the evaluation of alveolar bone defect in periodontitis was evaluated.2.Gingival tissue samples from different patients were collected for immunohistochemistry,western blot and fluorescence quantitative PCR experiments to determine the expression level of programmed death-related proteins RIP1,RIP3,p-MLKL and ATF4 in periodontal tissues.3.High glucose and Lipopolysaccharide(LPS)were used to stimulate MC3T3-E1 cells,CCK-8 kit were used to detect cell proliferation,and ROS DCFH-DA probe kit was used to determine the release of intracellular ROS levels,cytokine secretion was confirmed by ELISA assay.Gene expression was performed by q RT-PCR,and protein expression was assessed by western blot and immunofluorescence analysis.The expression of RIP1,RIP3,p-MLKL and ATF4 was performed by q RT-PCR,or assessed by western blot and immunofluorescence analysis.4.ROS inhibitor NAC and RIP1 inhibitor Nec-1 were used to pre-stimulate cells,and the expression of RIP1,RIP3,p-MLKL and ATF4 were detected after high glucose and LPS co-stimulation.5.Antagomir-214,an inhibitor of mi R-214,was used to pretreat cells,and the expression of RIP1,RIP3,p-MLKL and ATF4 were determined after high glucose and LPS co-stimulation.6.ATF4 si RNA were utilized to inhibit the expression of ATF4,antagomir-214 was used to pretreat cells,and the expression of RIP1,RIP3,p-MLKL and ATF4 were determined.Results1.CBCT imaging and clinical examination results showed that periodontal conditions such as alveolar bone resorption in patients with diabetes-associated periodontitis were more serious than those in patients with simple chronic periodontitis,and CBCT imaging results could better reflect the alveolar bone defect in patients than curved tomography X-ray.2.In this study,we proved that RIP1/RIP3-dependent programmed necrosis emerged in the swollen gingival tissue of patients with diabetes-associated periodontitis by immunohistochemistry,western blot and fluorescence quantitative PCR assays.3.After MC3T3-E1 cells were co-stimulated with high glucose and LPS,it was found that high glucose and LPS promoted ROS and AGEs accumulation in cells,induced LDH release and increased the expression levels of programmed death-related proteins RIP1,RIP3,p-MLKL and ATF4,resulting in RIP1/ RIP3-dependent programmed necrosis.4.In this experiment,it was found that the expression of mi R-214 in gingival tissues and cells increased with the increase of high glucose and LPS,while the expression of ATF4 decreased.Necroptosisis were inhibited by ROS inhibitor NAC and RIP1 inhibitor Nec-1.The expression of ATF4 increased with ROS clearance,while the expression of ATF4 did not change with only inhibition of RIP1 by Nec-1.The antagomir-214 inhibitor of mi R-214 inhibited necroptosisis and increased the expression of ATF4,but it was found that antagomir-214 did not inhibit necroptosisis after the use of ATF4 si RNA to interfere with the expression of ATF4.These results suggested that mi R-214 played a role in the regulation of ATF4 in necroptosisis.The mi R-214 level,receptor-interacting serine-threonine protein(RIP)1,RIP3 and phospho-mixed lineage kinase domain-like(p-MLKL)protein expression were increased in the inflamed gingival tissues of diabetes-associated periodontitis patients,with ATF4 expression showing the opposite effect.The high glucose(22 m M)couldn’t induce significant increase of RIP1,RIP3 and p-MLKL,however,the high glucose and LPS(500-1000 ng/m L)co-treated resulted in increase of RIP1,RIP3 and p-MLKL in MC3T3-E1 cells.NAC(ROS inhibitor)inhibited RIP1,RIP3 and increased ATF4,however,Nec-1(RIP1 inhibitor)specifically inhibited the protein levels of RIP1 and RIP3 and have no influence on ATF4.The use of antagomir-214 suppressed the expression of mi R-214,RIP1,RIP3 and p-MLKL,but increased ATF4 protein level in glucose and LPS-induced cells.ATF4 knockdown by ATF4 si RNA offset the effect of antagomir-214.RIP1-and RIP3-dependent necroptosis was confirmed in the inflamed gingival tissues of diabetes-associated periodontitis patients and high glucose-and LPS-co-treated cells.ConclusionIt was suggested that mi R-214-targeted ATF4 participated in the regulation of RIP1/ RIP3-dependent necroptosis in vivo and in vitro.It is suggested that mi R-214 play an important role in diabetes-associated periodontitis,which may contribute to the clinical prevention and treatment of periodontitis with diabetes.