Research On The Survival Of Relationship Betweenautophagy And Porphyromonas Gingivalis Intracellular KB Cell

ObjectiveTo investigate the relationship between autophagy and Porphyromonas gingivalis surviving in oral epithelial cells,reveal the mechanism of Porphyromonas gingivalis surviving in oral epithelial cells.Methods1.MOI values(number of bacteria: cell number)of 50: 1,100: 1,150:1,200: 1,250: 1 of P.gingivalis co-culture with KB cells for 3h,washed away Pg that not invasived the cells,continue to culture 24 h,Cell viability of Pg-infected KB cells with different MOI values was detected by the CKK-8.2.KB cells were transfected into GFP-LC3 for 24 h,the fluorescence microscope to observe the transfection effect,after successful transfection,incubation for 3h,washed away P.gingivalis that not invasived the cells,and continue cultured,the fluorescence of GFP-LC3 was observed and the expression of autophagy-related proteins LC3 and p62 was detected by Western blotting at 12 h,18 h,24 h,and36h.3.KB cells were transfected into GFP-p62 for 24 h,the fluorescence microscope to observe the transfection effect,after successful transfection,incubation for 3h,washed away P.gingivalis that not invasived the cells,and continue cultured,the expression of GFP-p62 and autophagy were observed by fluorescence microscopy.4.KB cells were transfected into Synthetic p62 si RNA and LC3 si RNA in order to interfere the p62 and LC3 expression,then detecte the expression ofp62 and LC3 by Western blot.5.KB cells were interfere the p62 express,KB cells were transfected into GFP-LC3,KB cells were interfere the LC3 express and normal KB cell co-culture with the Pg of MOI 100 for 3h,washed away P.gingivalis that not invasived the cells,and continue cultured,observe the cell form and detecte the cell viability of KB cells by CCK-8.6.KB cells were interfere the p62 express,KB cells were transfected into GFP-LC3,KB cells were interfere the LC3 express and normal KB cell co-culture with the Pg of MOI 100 at 0h,12 h,24h and 36 h,respectively.the number of viable bacteria in the cells was measured.,The cells were digested and counted,and the number of cells in each group was equal.The cells were lysed and the lysate was applied to BHI blood agar plates for 5 to 6 days.The number of colonies after bacterial culture was observed.Results1.GFP-LC3 was successfully transfected into KB cells.After incubation for 3 hours,the stained P.gingivalis was observed under the microscope.The autophagic fluorescence was observed under fluorescence microscope at 12 h compared with 0h(P <0.01).The expression of LC3 protein was significantly increased by Western blot(P <0.01),and the expression of p62 protein was also increased(P <0.01).The number of autophagy decreased significantly at18 h and 12h(P <0.01),the expression of LC3 was decreased(P<0.05),and the expression of p62 was decreased(P<0.05),and the expression of LC3 and p62 was not significant(P <0.01),the expression of LC3 was also decreased(P<0.01),and the expression of p62 was decreased(P<0.01).There was no significant difference in the number of autophagy and the decrease of LC3 between 30h and 0h(P> 0.05),and the expression of p62 was decreased(P<0.01).2.P.gingivalis infect the KB cells that transfected with GFP-LC3.The stained P.gingivalis were observed in 0 h cells.The stained P.gingivalis werestill observed in the cells after 36 h.There was no significant difference in the number of bacteria at 36h(P> 0.05).3.GFP-p62 cells transfected with were added to the P.gingivalis of MOI value of 100,and the aggregation and fusion of p62 protein were observed at12 h and 0h,).After 24 h,the p62 fluorescent spot was dispersed and decreased compared with 12 h.After 36 h the microscope can hardly see the fluorescent point.4.KB cells were transfected into p62 si RNA and interfere p62 expression,after 24 h Western blot analysis showed that he expression of p62 was lower than control group(P <0.01).5.KB cells were interfere the p62,KB cells were transfected into GFP-p62 and normal KB cell co-culture with the P.gingivalis of MOI 100,there was no significant difference between three grops on svrvival rate of cells((P>0.05),and fewer cell morphology becomes round between three groups at 12 h.After 24 h,the survival rate of p62 overexpressing KB cells was lower than that of KB cells(P<0.05),The survival rate of KB cells interfered p62 expression was higher than that of KB cells(P<0.05),p62 high expression of KB cells and KB cell morphology into a circular cell was significantly increased,interfering p62 expression of KB cells in the cell morphology is relatively good.After 36 h,the survival rate of p62 overexpressing KB cells was significantly lower than that of KB cells(P<0.01),the survival rate of KB cells interfered p62 expression was significantly higher than that of KB cells(P<0.01).KB cells and p62 overexpression of KB cells cell morphology almost all become round,While the p62 expression of the interference of the morphology of KB cells become less round,a large part of the cells to maintain the original cell morphology.The number of colonies in the BHI blood agar plate showed no difference between three groups at 0h(P>0.05).After 12 h,the number of colonies in the p62 high expression group was no significant difference compared with the KB group(P>0.05),the number of colonies in p62 interfered group was lower than that in KB group(P<0.05).After 24 h he number ofcolonies in p62 group was higher than that in KB group(P<0.05).The number of colonies in p62 interfered group was significantly lower than that in KB group(P<0.01).After 36 h the number of colonies in p62 group was significantly higher than that in KB group(P<0.01),the number of colonies in p62 interfered group was decreased by 3 times compared with KB group(P<0.01).6.The P.gingivalis of MOI 100 co-culture with KB cells and KB cells were interfered LC3 express.there was no significant difference between two groups on svrvival rate of cells(P>0.05)and fewer cell morphology becomes round between two groups at 12 h.After 24 h,the survival rate of KB cells interfered LC3 was higher than that of KB cells(P<0.05),KB cells morphologically changed to round cells significantly increased,while interference with LC3 KB cell morphology is relatively good.After 36 h,the morphology of KB cells became almost round,while the cells of interfered LC3 had a large proportion of cells to maintain the original cell morphology.The survival rate of KB cells interference with LC3 was 2.7 times that of KB cells(P<0.01).After 12 h,the number of colonies in the LC3 interference group was less than that in the KB group(P<0.05).After 24 h the colony number of LC3 interference group was decreased by 2 times compared with KB group(P<0.01).After 36 h,the colony number of LC3 interference group was decreased by 4 times compared with KB group(P<0.01).ConclusionsP.gingivalis induced autophagy in KB cells,P.gingivalis can survive in the autophagy of KB cells,autophagy levels were reduced,P.gingivalis survived or proliferated in KB cells,and KB cell survival increased.The effect of p62 on the survival of P.gingivalis in KB cells plays a supporting role.