The Role Of Autophagy In The Calcitriol-induced Decrease Of The Living Porphyromonas Gingivalis Internalized Into KB Cells

Objective:Porphyromonas gingivalis?P.gingivalis?,one of the most important putative pathogens of periodontitis,can be widely internalized into a variety of host cells,which is closely related to the development of systemic diseases and cancer.Previous studies suggest that P.gingivalis may promote HIV-1 entry into Hela epithelial cells.The aim of this study is to observe the presence of Porphyromonas gingivalis?P.gingivalis?ATCC33277 in KB cells and its effect on autophagy.And we researched the effects of calcitriol?1,25-Dihydroxyvitamin D3,1,25D?and P.gingivalis on the autophagy of KB cells to analyze the possible mechanism in the process of 1,25-Dihydroxyvitamin D3-induced decrease of the amount of living Porphyromonas gingivalis internalized into KB cells.Methods:1.Transmission electron microscope was used to observe the P.gingivalis organisms internalized into cells?MOI 300?treated with 10^-88 M 1,25D or 0.1%ethanol?control?for 18 h.The experiment was divided into 5 groups:blank control group,0.1%ethanol group,1,25D group,P.gingivalis group,1,25D+P.gingivalis group.After 18 h,we observe the presence of P.gingivalis in KB cells and its relationship with autophagy structure.2.Western blot analysis was performed to observe the LC3 and P62 protein expressions in KB cells to explore the effects of1,25D and P.gingivalis on the autophagy.?1?P.gingivalis treatment:?1?KB cells were co-cultured with P.gingivalis?MOI 10 to 500?for 18 h.?2?P.gingivalis?MOI100?and KB cells were co-cultured for 0-48 h.?2?1,25D treatment:?1?10^-12mol/L,10^-1010 mol/L,10^-88 mol/L 1,25D,or 0.1%ethanol treated KB cells for 18h.?2?10^-8mol/L 1,25D or 0.1%ethanol treated KB cells for 0-48 h.?3?10^-88 mol/L 1,25D combined with P.gingivalis?MOI 100?treated KB cells for 18 h,cells treated with P.gingivalis alone were used as the control.3.To explore the role of autophagy in1,25D-induced decrease of P.gingivalis organisms in KB cells,cells were treated with P.gingivalis+0.1%ethanol,P.gingivalis+1,25D,P.gingivalis+3-MA and P.gingivalis+3-MA+1,25D.The concentration of 1,25D was 10^-88 mol/L,and MOI of P.gingivalis was 100.The number of P.gingivalis in KB cells was detected by qRT-PCRand agar culture.Results:1.In the control group,some P.gingivalis organisms that internalized into KB cells were without vacuoles surrounding them.Some were undergoing division.Some organisms were surrounded by double-layered membrane vacuoles.We found incomplete membrane surrounding some of the organisms.After the 1,25D treatment,incomplete membranes and dividing organisms were not found.Within the double-layered membrane vacuoles,the outer membranes of some P.gingivalis organisms were destructed.2.Both 1,25D and P.gingivalis increased the LC3?/?ratio and reduced P62 expression in KB cells in a dose-and time-dependent manner.The expression of LC3 protein increased in a dose-dependent manner with P.gingivalis?MOI 10 to 500?for 18 h.The expression of P62 was significantly decreased at 24h.10^-1212 mol/L,10^-1010 mol/L,10^-88 mol/L 1,25D treated KB cells for24 h,the expression of LC3 protein in each group was higher than that of 0.1%ethanol group and showed concentration dependency.The ratio of LC3 II/I increased from 2 h at 0,2,6,18,24 and 48 h after treatment with 10^-88 mol/L 1,25D.1,25D promoted the P.gingivalis-induced increase of LC3?/?ratio and reduction of P62protein expression.3.Compared with P.gingivalis+1,25D group,the number of P.gingivalis organisms in P.gingivalis+3-MA+1,25D group was significantly increased to 1.89 times detected by qRT-PCR.Agar culture method and qRT-PCR method have the same trend.Conclusions:P.gingivalis can survive and proliferate in KB cells.1,25D can inhibit the survival of P.gingivalis in the cells by promoting the autophagy process.