Pyk2 Regulated Porphyromonas Gingivalis Internalization And Autophagy Of Gingival Epithelial Cells

Objectives: Porphyromonas gingivalis,which is the main pathogen of chronic periodontitis,can be internalized in and chronically persist in gingival epithelial cells on the surface of periodontal tissue.Protein-rich tyrosine kinase 2(Pyk2)was found to get involved in the infection in previous study.In this study,the expression of autophagy-related molecules of gingival epithelial cells in the process of P.gingivalis infection was investigated and the effect of Pyk2 on P.gingivalis internalization and the autophagy of gingival epithelial cells induced was further explored.The aims of this study are to elucidate the role Pyk2 in the pathogenesis of P.gingivalis and to provide new perspectives and targets for the treatment and prevention of periodontitis.Materials and methods: Immortalized gingival epithelial cells,epi4 cells were infected by P.gingivalis with MOI 100:1.Autophagy vesicles were detected by transmission electron microscopy(TEM).Autophagosomes were observed with immunofluorescence.LC3Ⅱ m RNA expression was detected by Real-time PCR.Protein expression of LC3Ⅱ and P62 was analyzed by Western blot.After epi4 cells were pretreated with TAE226(Pyk2 Inhibitor)and then infected by P.gingivalis,LC3Ⅱ protein expression was analyzed and P.gingivalis internalization into epi4 cells was detected by antibiotic protection assay.Results: 1.After P.gingivalis infection,double-membrane structure of autophagy vesicles can be detected in cells and P.gingivalis can be found in vesicles with TEM;2.Increased autophagosomes were observed with immunofluorescence after P.gingivalis infection;3.The m RNA expression of LC3Ⅱ increased time-dependently,and the peak was reached at 6 h(P<0.05);4.The protein expression of LC3Ⅱ increased time-dependently,and P62 decreased time-dependently(P<0.05);5.The protein expression of LC3Ⅱ decreased after cells were pretreated with TAE226(P<0.05);6.P.gingivalis internalization into epi4 cells decreased after cells were pretreated with TAE226(P<0.05).Conclusions: 1.Autophagy of gingival epithelial cells and P.gingivalis internalization can be induced by P.gingivalis infection;2.Pyk2 plays an important role in the P.gingivalis internalization;3.Pyk2 may regulate the process of autophagy.