| Objective:To investigate the antibacterial activities of docosahexaenoic acid(DHA)and eicosapntemacnioc acid(EPA)against Porphyromonas gingivalis(P.gingivalis)and Fusobacterium nucleatum(F.nucleatum),and the anti-inflammatory effects on human gingival fibroblasts(h GFs).Methods:Part ? : 1.The minimum inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of DHA and EPA against P.gingivalis and F.nucleatum were determined through broth microdilution method;2.The effects of DHA and EPA on the planktonic growth and biofilm formation of P.gingivalis and F.nucleatum were evaluated by growth curve determination and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,respectively;3.Colony forming unit(CFU)counting,scanning electron microscopy(SEM)and polymerase chain reaction(real-time PCR)were performed to further investigate the effects of DHA and EPA on the viability,morphology and virulent factor expression of exponential phase P.gingivalis and F.nucleatum;4.Confocal laser scanning microscopy(CLSM)was used to detect the activities of DHA and EPA on the viability and thickness of mature biofilms.Part ?: 1.MTT assay and live-dead cell staining were used to investigate the effects of DHA and EPA on the viability of h GFs;2.Light microscopy and cytoskeleton immunofluorescent staining were used to investigate the effects of DHA and EPA on the morphology of h GFs;3.Flow cytometry was used to investigate the effects of DHA and EPA on the cell cycle of h GFs;4.h GFs were pretreated with DHA and EPA before stimulated by P.gingivalis lipopolysaccharides(LPS)and heat-killed P.gingivalis,then real-time PCR and enzyme linked immunosorbent assay(ELISA)were performed to detect the gene and protein expression of interleukin-6(IL-6),IL-8 and IL-1?,respectively;5.real-time PCR and western blot were used to investigate the effects of DHA and EPA on the gene and protein expression of heme oxygenase-1(HO-1),respectively.Results:Part ?: 1.Against P.gingivalis,the MIC and MBC values were the same 12.5 ?M for DHA,meanwhile,the respective 12.5 ?M and 25 ?M for EPA;however,against F.nucleatum,the MIC and MBC values were both greater than 100 ?M for DHA and EPA;2.DHA and EPA displayed complete inhibition on the planktonic growth and biofilm formation(p ? 0.05)of P.gingivalis from the lowest concentration of 12.5 ?M;the planktonic growth of F.nucleatum was slightly but not completely inhibited by DHA and EPA even at the concentration of 100 ?M,however,the biofilm formation at 24 h was significantly restrained by 100 ?M EPA(p?0.05);3.For exponential-phase bacteria,100 ?M DHA and EPA completely killed P.gingivalis and significantly decreased the viable counts of F.nucleatum(p?0.05);the morphology of P.gingivalis was also apparently damaged;besides,the virulence factor gene expression of P.gingivalis and F.nucleatum was strongly downregulated(p?0.05);4.The viability and thickness of mature P.gingivalis biofilm,together with the viability of mature F.nucleatum biofilm were both significantly decreased in the presence of 100 ?M DHA and EPA(p?0.05).Part ?: 1.DHA and EPA exhibited significant inhibitory effect on the viability of h GFs until the concentration came to 200 ?M;2.100 ?M DHA and EPA had no effects on the morphology of h GFs;3.100 ?M DHA and EPA exert no influence on the cell cycle of h GFs;4.Pretreatment with DHA and EPA could significantly downregulate the gene expression of IL-6 and IL-1?,and the protein expression of IL-6 induced by P.gingivalis LPS and heat-killed P.gingivalis(p?0.05),in a dose-dependent manner;the gene and protein expression of IL8 was also decreased by DHA(p?0.05);5.DHA and EPA significantly increased the gene(p?0.05)and protein expression of HO-1 in a dose dependent manner.Conclusion:DHA and EPA possess both antibacterial activities against periodontal pathogens,and anti-inflammatory actions on periodontal tissues cells,which suggest that they may be potentially supplementary therapeutic agents for prevention and treatment of periodontitis. |
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