Antibacterial Activity Of Erythritol Onperiodontal Pathogen

ObjectiveTo explore the effect of erythritol on the growth and adhension of Pg,Aa and Av.We also explore how Periodontal Pathogens affected by erythritol influence the mRNA expression level of inflammatory in periodontal cells.Methods1.Pg,Aa,Av was anaerobically cultured（80%N₂,10%CO₂,10%H₂）at 37℃in 2、4、8、16、32、64、128 g/L Erythritol-BHI mixture groups（experimental groups）and BHI groups（control group）.The lowest erythritol concentration without turbidity or precipitation is the minimum inhibitory concentration.2.The experimental solution was added to according to 96-well plates from left to right according to the increasing order of erythritol concentration.Pg,Aa,Av was anaerobically cultured（80%N₂,10%CO₂,10%H₂）at 37℃a certain period of time,then remove the supernatan before dyeing biofilm using crystal violet which was fixed by methanol.Measure the OD of displacement fluid which was exchanged by acetic acid in crystal violet in biofilm using Microplate Reader.3.Pg,Aa,Av was anaerobically cultured（80%N₂,10%CO₂,10%H₂）at 37℃in MIC,1/2,1/4,1/8 MIC Erythritol-BHI mixture groups（experimental groups）and BHI groups（control group）.Every kind bacteria of each concentration group was centrifugalled and cleaned before added into DMEM.The mixed suspension above co-culture with the four generations of the periodontal ligament cells for 24 hours,and then remove the supernatan before extract total RNA in cracking cells,reversing transcription,detecting relative expression of IL-1,IL-6,TNF-a by the real-time fluore-scent quantitative PCR.Results1.The Minimum Inhibitory Concentration of erythritol on three bacteria is as followed:Porphyromonas gingivalis:64 g/LAggregatibacter actinomycetemcomitans:128 g/LActinomices viscosus:128 g/L2.Analysis of the effect of erythritol on the adhension of Pg,Aa and Av.Erythritol can affect the OD value of Pg,and the OD value is related to the concentration of erythritol.Its OD value was similar the control group of 0 g/L,When the concentration of erythritol was 2 g/L and 4 g/L;With the increasing of the concentration of erythritol（≥8 g/L）,the OD value of Pg decreased,and the higher the concentration was,the lower was the OD value.When the concentration reached 128g/L,no biofilm was observed formed with eyes,and there would not be more inhibitory even increasing erythritol concentration.The effect of erythritol on Aa and Av is similar to that of Pg,but it requires a higher concentration of erythritol to play an inhibitory role.Its OD value was similar the 0 g/L of control group,When the concent ration of erythritol wa s2 g/L、4 g/L and 8 g/L;With the increasing of the concentration of erythritol reached 16g/L or more,the OD value of bacteria decreased in a different degree,and the most powerful effect of erythritol on Aa and Av was 128 g/L.To sum up,erythritol had the ability to inhibit Aa and Av adhension in vitro,and the inhibitory effect is concentration depended.3.Analysis of the influence of Pg,Aa and Av which was affected by erythritol on the expression of IL-1β,IL-6 and TNF-αof the periodontal ligament cells.Gene expression of Pg group.The ability of stimulating periodontal ligament cells to produce IL-1β,IL-6 and TNF-αwas different when bacteria was cultured under different concentrations of erythritol.There was no significant difference between 0 g/L of control group and 8 g/L of experimental group.As the concentration reached 16 g/L,the relative expression of IL-1β,IL-6,TNF-αwas reduced,and the higher concentration was,the less inflammatory factors was.However,the inflammatory factors in all the experimental groups were always higher than that in the blank control group.Gene expression of Aa group.There was no obvious effect of erythritol on inducing inflammatory when the concentration was 16 g/L.Aa growed in the 32 g/L of erythritol had began to change its pathogenic of induceing IL-1βdecreased,and the higher the concentration was,the stronger the inhibition ability was;As the concentration reached 16 g/L,the relative expression of TNF-αwas reduced,and the higher concentration was,the less inflammatory factors was.At the concentration of 128g/L,the amount of IL-6 stimulated by Aa had a sharp drop.All the experimental groups had higher inflammatory cytokines than those without bacteria of blank control group.Gene expression of Aa group.It had already begun to inhibit the release of IL-6when the concentration of erythritol was 32 g/L and there had an obvious inhibitory effect on the expression of IL-1βand when the concentration reached 64 g/L。The concentration reached 128 g/L,the amount of TNF-αdroped.All the experimental groups had higher inflammatory cytokines than the blank control group without bacteria.ConclusionErythritol has an inhibitory effect on the growth and adhension of Porphyrom-onas gingivalis,Aggregatibacter actinomycetemcomitans and Actinomyces viscous.In a certain range,higher concentration of erythritol decides better inhibition effect.Erythritol can also reduce the periodontal pathogenicity of pathogenic bacteria in a way inhibiting the virulence of these bacteria,then reducing the production of IL-6,IL-6,and TNF-a in periodontal cells.