Calcitriol Decreases Live Porphyromonas Gingivalis Internalized Into Macrophages By Promoting Autophagy

Objective: Porphyromonas gingivalis(P.gingivalis)is the most important putative bacteria in the chronic periodontitis,and can be widely internalized into various host cells,which is closely associated with the development of systemic diseases.Calcitriol(1?,25-dihydroxyvitamin D 3,1 alpha,25 Dihydroxyvitamin D3,1,25D)has the ability of reducing the number of P.gingivalis in epithelial cells and monocytes by promoting autophagy.This study aims to observe the effects of 1,25 D on internalized P.gingivalis in U937 macrophages,the co-localization of the autophagic marker products and internalized P.gingivalis and the formation of autophagy in U937-derived macrophages to clarify the effect of 1,25 D on P.gingivalis internalized into U937 macrophages and the potential role of autophagy during this process.Methods: 1.U937 monocyte cell lines were induced to macrophages.2.A model of P.gingivalis internalized U937-derived macrophages was established by antibiotic protection method.3.The effect of 1,25 D on the number of live P.gingivalis internalized into U937-derived macrophage were detected by RT-PCR.4.The effect of 1,25 D and P.gingivalis on autophagy of U937-derived macrophages was studied by the treatment with P.gingivalis,1,25 D,and 1,25 D combined with P.gingivalis.The expression level of LC3(Microtubule-associated protein 1 light chain 3)and p62 was detected by Western Blot.5.The co-localization of P.gingivalis and LC3,and the co-localization of P.gingivalis and lysosome marker Lysosomal-Associated Membrane Protein 1(LAMP1)were observed by the confocal microscope.6.The effect of 1,25 D on the number of live P.gingivalis bacteria in U937-derived macrophages was detected with or without autophagy inhibitor 3-MA(3-Methyladenine)by RT-PCR.Results: 1.1,25 D caused a dose-dependent decrease in the number of live P.gingivalis bacteria in U937-derived macrophages(P < 0.05).2.The effect of P.gingivalis and 1,25 D on autophagy of U937-derived macrophages:(1)After establishing P.gingivalis internalization model in U937-derived macrophages for 18 h,the expression level of LC3-II increased with the increase of MOI(P < 0.05),and the expression level of p62 gradually decreased(P < 0.05).(2)1,25 D promoted the expression of LC3-II(P < 0.05)and decreased the expression of p62(P < 0.05)in U937 macrophages in a dose-and time-dependent manner.(3)After 18 hours treatment of combined 1,25 D and P.gingivalis on U937 macrophages,the expression of LC3-II significantly increased(P < 0.01)and the expression of p62 significantly decreased(P < 0.01).3.Comparing with P.gingivalis internalization alone,1,25 D comibined with P.gingivalis significantly increased the number of LC3 puncta in U937 macrophages(P < 0.05),and the co-localization rate of LC3 with P.gingivalis,and LAMP1 with P.gingivalis both significantly increased(P < 0.05).4.After inhibiting autophagy,the inhibition effect of 1,25 D on the number of P.gingivalis in macrophages decreased significantly(P < 0.01).Conclusion: In U937-derived macrophages,calcitriol may promote colocalization of P.gingivalis with autophagosomes and lysosomes,namely autophagy process,to degrade live P.gingivalis.