The Role Of PI3K In Chronic Apical Periodontitis

Objective:Based on the clinical samples of chronic periapical periodontitis,the osteoclast inflammatory environment,and the osteoblastic inflammatory environment,study the expression of PI3 K in chronic periapical periodontitis,and explore the role of PI3 K in the bone destruction of chronic periapical periodontitis Provide reference for the treatment of chronic apical periodontitis.Methods:Collect periapical tissue samples from patients with chronic periapical periodontitis,establish LPS-mediated osteoclast-level inflammatory environment and osteoblast-level inflammatory environment,and study the expression of PI3 K and related factors.1.Detection of gene expression levels of PI3 K and osteoclast-related factors in clinical samplesReal-Time PCR was used to study the expression of PI3 K,TRAP and NFATc1 in 26 patients with chronic periapical tissues and 10 healthy periapical tissues.2.The expression of PI3 K in osteoclasts(1)Induction of RAW264.7 differentiation into osteoclastsThe osteoclast precursor RAW264.7 cells were cultured to the third generation and induced with 0.1 ?g/ml RANKL induction solution for 5 days.The TRAP gene was detected by light microscopy,TRAP staining,and Real-Time PCR for osteoclast verification.(2)Expression of PI3 K and osteoclast-related factors in osteoclasts under LPS-mediated inflammation100 ng / ml LPS was applied to osteoclasts.Real-Time PCR and Western Blot were used to detect PI3 K,TRAP,and NFATc1 gene and protein expression levels,respectively.(3)Effect of PI3 K inhibitors on osteoclast proliferation,differentiation and PI3 K /Akt signaling pathwayOsteoclasts were treated with PI3 K inhibitor LY294002,and cell proliferation activity was detected by CCK-8 method.Real-Time PCR was used to detect Akt,TRAP,and NFATc1 gene expression levels.Western Blot was used to detect Akt,p-Akt,and TRAP and NFATc1 protein expression levels.3.PI3 K expression in osteoblasts(1)Inducing MC3T3-E1 to differentiate into osteoblastsThe osteoblast precursor MC3T3-E1 cells were cultured to the third generation,and the osteogenic induction fluid was induced for 7 days.The ALP gene was detected by ALP staining and Real-Time PCR for osteoblast verification.The osteoblast induction fluid was induced for 21 days.Pigment red staining was performed for osteoblast verification.(2)Expression of PI3 K and osteogenesis-related factors in osteoblasts under inflammatory environment mediated by LPS1000 ng / ml LPS was applied to osteoblasts.Real-Time PCR and Western Blot were used to detect PI3 K and BMP2,Runx2 gene and protein expression levels,respectively.(3)Effect of PI3 K inhibitors on osteoblast proliferation,differentiation and PI3 K /Akt signaling pathwayThe inhibitor LY294002 acts on osteoblasts,respectively,and the cell proliferation activity is detected by CCK-8 method.Real-Time PCR detected Akt,BMP2,and Runx2 gene expression levels.Western Blot was used to detect Akt,p-Akt,BMP2 and Runx2 protein expression levels.Results:1.Expression of PI3 K,TRAP and NFATc1 in clinical samplesCompared with healthy periodontal ligament tissue,the expression of PI3 K,TRAP and NFATc1 genes in chronic periapical periodontitis sample group increased,and the difference was statistically significant(P <0.05).2.The expression of PI3 K in osteoclasts(1)After 5 days of RANKL induction solution induction,osteoclasts were successfully induced by light microscope,TRAP staining,and Real-Time PCR detection of the TRAP gene.(2)Real-Time PCR results showed that the m RNA levels of PI3 K,TRAP and NFATc1 were increased in the experimental group,and the difference was statistically significant(P <0.05).Western Blot results showed that the protein levels of PI3 K,TRAP,and NFATc1 were consistent with Real-Time PCR results,and the differences were statistically significant(P <0.05).(3)CCK8 results showed that the proliferation activity of PI3 K inhibitors on osteoclasts at 24 h and 48 h was lower than that of the control group,and the difference was statistically significant(P <0.05).Real-Time PCR results showed that the m RNA levels of PI3 K,TRAP,and NFATc1 were decreased in the experimental group,and the differences were statistically significant(P <0.05).Western Blot results showed that the p-Akt / Akt protein ratio and the expression of TRAP and NFATc1 protein levels in the experimental group were consistent with the results of Real-Time PCR,and the differences were statistically significant(P <0.05).3.PI3 K expression in osteoblasts(1)After 7 days and 21 days of induction with osteogenic induction solution,respectively,ALP staining,Real-Time PCR detection of ALP gene,and Alizarin red staining revealed that osteoblasts were successfully induced.(2)Real-Time PCR results showed that the m RNA levels of PI3 K,BMP2 and Runx2 decreased in the experimental group,and the difference was statistically significant(P<0.05).Western Blot results showed that the protein levels of PI3 K,BMP2 and Runx2 were consistent with the results of Real-Time PCR,and the differences were statistically significant(P <0.05).(3)CCK8 results showed that the proliferation activity of PI3 K inhibitors on osteoblasts at 24 h and 48 h was lower than that of the control group,and the difference was statistically significant(P <0.05).Real-Time PCR results showed that the m RNA levels of PI3 K,BMP2,and Runx2 in the experimental group were reduced,and thedifferences were statistically significant(P <0.05).Western Blot results showed that the p-Akt / Akt protein ratio and the expression of BMP2 and Runx2 protein levels in the experimental group were consistent with Real-Time PCR results,and the differences were statistically significant(P <0.05).Conclusion:1.The gene expression level of PI3 K in chronic periapical periodontitis increased.2.In the inflammatory environment mediated by LPS,the expression of PI3 K in osteoclasts increased and the expression of PI3 K decreased in osteoblasts.3.Inhibition of PI3 K affects the proliferation and differentiation of osteoclasts and osteoblasts.PI3 K participates in the development of chronic apical periodontitis by regulating the proliferation and differentiation of osteoclasts and osteoblasts.