Regulation Of Lysosome-associated Membrane Protein LAMP1 In Keratinocytes By Porphyromonas Gingivalis Lipopolysaccharide

Objective: Porphyromonas gingivalis(P.gingivalis)is the important pathogen of periodontitis,can be internalized into non-phagocytes and survive,thereby evading the host immune defense.Lysosomes are closely related to intracellular bacterial clearance,and lysosome-associate membrane protein 1(LAMP1),as a lysosome marker molecule,is located in the lysosome membrane and is involved in the transport of intracellular substances to the lysosomes.The study is aimed to explore the regulatory effects of p.gingivalis and its Lipopolysaccharide(LPS)on the expression level of LAMP1 in human keratinocyte line HaCaT cells and provide clues to the mechanisms involved in the inhibition of autophagic lysosome formation by P.gingivalis.Methods: 1.A model of the P.gingivalis internalized HaCaT cells was established by antibiotic protection method.2.Transmission electron microscope was used to observe the state of live P.gingivalis internalized into HaCaT cells.3.Brain-Heart Infusion(BHI)solid medium culture method was used to observe the state of living P.gingivalis after internalization.4.The q RT-PCR detected the expression level of LAMP1 m RNA in HaCaT cells.5.The Western Blot detected the protein expression level of LC3(Microtubuleassociated protein 1 light chain 3)and LAMP1.Results:1.BHI solid medium culture method showed that P.gingivalis survived inside HaCaT cells;transmission electron microscope showed that intracellular P.gingivalis was structurally intact and partially surrounded by double-layered membrane structures similar to the autophagosomes.2.P.gingivalis can promote the formation of autophagosomes:(1)After different MOI P.gingivalis was internalized into HaCaT cells for 24 h,the expression of LC3-Ⅱ protein was increased significantly with the increase of MOI compared to the control group(P<0.001);(2)After P.gingivalis(MOI 100)was internalized into HaCaT cells for different times,the expression of LC3-II protein was significantly increased in a timedependent manner compared to the control group(P<0.001).3.The expression of LAMP1 protein decreased in P.gingivalis internalized HaCaT cells:(1)After different MOI P.gingivalis was internalized into HaCaT cells for 24 h,the expression of LAMP1 m RNA significantly increased(P<0.05),while the expression of LAMP1 protein significantly decreased compared to the control group(P<0.01);(2)After P.gingivalis(MOI 100)was internalized into HaCaT cells for different times,the expression of LAMP1 m RNA levels significantly increased(P<0.001),and the expression of LAMP1 protein significantly decreased in a time-dependent manner(P<0.01)compared to the control group。4.Heat-inactivated P.gingivalis reduced the LAMP1 protein levels in HaCaT cells: after treating HaCaT cells with heat-inactivated P.gingivalis at different MOI for 24 h,there was no significant change in the expression of LAMP1 m RNA(P>0.05),while a significant decrease in the expression of LAMP1 protein(P<0.001)compared to the control group.5.P.gingivalis-LPS reduced the LAMP1 protein levels in HaCaT cells:(1)After treating HaCaT cells with different concentrations of P.gingivalis-LPS for 24 h,there was no significant change in the expression of LAMP1 m RNA(P>0.05),while a significant decrease in the expression of LAMP1 protein(P<0.001)compared to the control group.(2)After treating HaCaT cells with 1 μg/m L P.gingivalis-LPS for different times,the expression of LAMP1 m RNA did not significantly change(P>0.05),and the expression of LAMP1 protein significantly decreased(P<0.001)and decreased to a minimum at 24 h(P<0.001)compared to the control group.6.The regulation of LAMP1 protein levels in HaCaT cells by P.gingivalis-LPS and E.coli-LPS is different: after treating HaCaT cells with 1 μg/m L P.gingivalis-LPS and 1 μg/m L E.coli-LPS respectively for 24 h,there was no significant change in the expression of LAMP1 m RNA(P>0.05),but the expression of LAMP1 protein in the former group significantly decreased(P<0.05),while the latter significantly increased(P < 0.05)compared to the control group.Conclusions: P.gingivalis can survive in HaCaT cells,promote the formation of autophagosomes,reduce the expression of LAMP1 protein,and then may inhibit the formation of autolysosome.Compared to E.coli-LPS,P.gingivalis-LPS can decrease the expression of LAMP1 protein in HaCaT cells specifically,which may be an important virulence factor of P.gingivalis affecting the formation of functional autophagic lysosomes.