| Objective: The purpose of this study was to first analyze and verify the number and compositional changes of the flora on the surface of the buccal mucosa in OLP patients,and then analyze the possible relationship between the changed bacteria and OLP lesions.Methods: The oral buccal surface swabs of 10 healthy controls and 20 OLP patients were collected,and the 16 S rRNA gene of bacteria was detected by high-throughput technology.The bacteria with the most obvious difference between the healthy control group and OLP patients were screened out.(1)Firstly,real-time PCR was used to detect and verify the relative gene expression of the above different genera in the DNA sample of experiment 1.Second,collect damage and non-damage oral buccal surface swabs of 25 OLP patients,detect the relative gene expression of certain bacteria by real-time PCR.Finally,30 OLP patients and 10 healthy controls of buccal mucosal tissues were collected to detect the expression level of bacterial genes in buccal mucosal epithelial tissues.Meanwhile,in situ hybridization was used to detect the presence of 16 S rRNA gene specific signal in the buccal mucosal epithelial tissues.(2)Infection model of human normal oral keratinocytes infected with F.nucleatum was established in vitro,and cell proliferation(CCK8 assay),cytokine IL-1β,IL-6 were detected at different time points after infection(0h,2h,4h and 6h).Expression of IL-1β、IL-6 and TNF-α(ELISA assay),and expression of cellular TLR4/NF-κB on genes(real-time PCR experiments)and protein levels(Western Blot experiments).Reasults:(1)Results of 16 S rRNA gene sequencing analysis are as follows:(1)At genus level,compared with the healthy control,Fusobacterium,Granulicatella,Peptostreptococcus and Parvimonas were significantly increased in OLP group;(2)At genus level,compared with the healthy control,the abundance of Peptostreptococcus(P=0.0125<0.05),Fusobacterium(P=0.0159<0.05),Filifactor(P=0.0181<0.05)and Exiguobacterium(P=0.0378<0.05)increased significantly on the buccal surface of reticular OLP patients.(3)At genus level,compared with the healthy control,the abundance of Burkholderia(P=0.0445﹤0.05)and Parvimonas(P=0.0356﹤0.05)was significantly increased in erosive OLP group.(4)At genus level,compared with the reticular OLP group,the abundance of Porphyromonas(P=0.0322<0.050)and Corynebacterium(P=0.0332<0.05)was reduced in erosive group.(5)Fusobacterium and Granulicatella play an most important role in the OLP group.(2)The expression level of Fusobacterium and Fusobacterium nucleatum(F.n)was significantly increased on the surface of the OLP patients.Compared with non-injured buccal mucosa in patients with OLP(n=25),F.n significantly increased on the surface of buccal mucosa on the damaged buccal surface(P=0.001<0.01).In the reticular OLP group,the relative expression level of F.n on the buccal mucosa was significantly increased compared with the non-damaged mucosa(P=0.0174<0.05).Compared with healthy controls,there was no significant difference in the expression levels of bacteria and F.n genes in buccal lesions in OLP patients.in situ hybridization revealed no signal of bacteria in the buccal mucosa of OLP patients.(3)When keratinocytes were infected for 2 hours,the expression level of IL-β gene was the highest and then decreased.When F.n infected keratinocytes for 4 hours,the level of cell proliferation and the expression levels of TLR4 and phosphorylated NF-κB p65 protein peaked.The expression of IL-6 and TNF-α genes peaked when keratinocytes infected with F.n for 6 hours.Conclusions: F.n,whose abundance on the buccal mucosa surface of OLP patients,may have association with the formation of lesions.In vitro,F.n can promote the proliferation of oral keratinocytes,the expression of IL-1β,IL-6 and TNF-α,and the activation of TLR4/NF-κB pathway.It is suggested that F.n may activate the TLR4/NF-κB pathway of local epithelial cells,and then promote the immune inflammatory response in the development of OLP. |
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