Study On The Mechanism Of Glaucocalyxin A For Adjuvant Treatment Of Periodontitis

Objective:This study was to obsever the antibacterial activity of glaucocalyxin A(GLA)to Porphyromonas gingivalis(P.gingivalis)in vitro.On the other hand,to investigate the effect of GLA on the expression of inflammatory cytokines in human periodontal ligament cells(hPDLCs)induced by P.gingivalis lipopolysaccharide(LPS)through NF-κB and MAPK signal pathway,and to explore the possibility and possible mechanism of GLA as an adjuvant therapy for periodontitis.Methods:1.The minimal inhibitory concentration(MIC)of GLA against P.gingivalis was determined by broth microdilution assay,and the minimum bactericidal concentration(MBC)was determined by plate culture method.2.Crystal violet staining was used to determine the effect of GLA on the biofilm formation of P.gingivalis.3.MTT assay was used to determine the effect of GLA on the biofilm metabolic activity of P.gingivalis.4.The primary hPDLCs were cultured by tissue explants method.5.The hPDLCs were identified by immunocytochemistry method.6.CCK-8 method was used to determine the effect of GLA on the proliferation of hPDLCs.7.qRT-PCR were used to detect the effect of GLA on the gene expression of IL-1 β,IL-6,IL-8 in hPDLCs treated withP.gingivalis LPS.8.ELISA methods were used to detect the effect of GLA on the protein expression of IL-1 β,IL-6,IL-8 in hPDLCs treated withP.gingivalis LPS.9.Western blot was used to detect the effect of GLA on MAPK and NF-κB signaling pathway after hPDLCs were treated withP.gingivalis LPS.Results:1.GLA has good antibacterial effect on P.gingivalis,the MIC was 5μM and the MBC was 20μM.2.Starting from 1.25μM,GLA could inhibit the formation of P.gingivalis biofilm.With the increase of GLA concentration,the inhibition of P.gingivalis biofilm formation was better.3.Starting from 1.25μM,GLA could inhibit the metabolic activity of P.gingivalis biofilm.The higher the concentration of GLA,the better the inhibitory effect on the metabolic activity on P.gingivalis biofilm.4.hPDLCs were successfully cultured.Under the microscope,the cells were long spindle or polygonal,with long and thin protrusion,abundant cytoplasm and large oval nucleus.The cell has radial or swirling arrangement.5.Immunocytochemical staining showed that the cultured cells were positive for vimentin staining and negative for keratin staining.6.Under the concentration of 2.5μM,GLA did not affect the proliferation of hPDLCs in 24 hours.With the increase of experimental time,the biological activity of hPDLCs began to be inhibited,and the inhibition will be further strengthened with the increase of GLA concentration.7.Compared with the normal group,P.gingivalis LPS could promote the expression of IL-6 and IL-8 m RNA in hPDLCs(p< 0.05);pretreated with 2.5μM and5μM GLA could inhibit the effect of P.gingivalis LPS on the expression of IL-6 and IL-8 m RNA(p<0.05).8.Compared with the normal group,P.gingivalis LPS could promote the secretion of IL-6 and IL-8(p<0.05);pretreated with 2.5μM and 5μM GLA could inhibit the secretion of IL-6 and IL-8(p<0.05).9.Compared with the normal group,P.gingivalis LPS could promote the phosphorylation of ERK,JNK and p38(p<0.05);pretreated with 2.5μM and 5μM GLA could reduce the expression of p-ERK,p-JNK and p-p38(p<0.05).10.Compared with the normal group,P.gingivalis LPS could promote the expression of NF-κB(p<0.05).Conclusion:GLA has good antibacterial activity against Gram-negative bacteria P.gingivalis in vitro,and couled inhibit the growth of its bacterial suspension and the plate colony;in addition,GLA could also inhibit the formation and metabolic activity of P.gingivalis bacterial biofilm.For hPDLCs,GLA can down regulate the expression of inflammatory cytokines induced by P.gingivalis LPS by inhibiting NF-κB and MAPK signaling pathways,so as to play a better anti-inflammatory effect.