| Objective: As an antibacterial peptide,histatins 5(Hst5)only exists in saliva of human and senior primates,participating in the defense of oral pathogenic microorganisms.It is an important component of host’s non-immune defense system.The study found that Hst5 has inhibition or killing effects on many microorganisms,such as Candida albicans,Streptococcus mutans,Enterococcus faecalis,Staphylococcus aureus,Escherichia coli,etc.However,its effect on Porphyromonas gingivalis(P.gingivalis) is rarely reported.As one of important periodontal pathogens,P.gingivalis plays a key role in plaque biofilm formation.The purpose of this experiment is to study the effect of Hst5 on P.gingivalis biofilm formation,explore possible mechanism and its role in the occurrence and development of periodontitis via rat periodontitis model.Methods:1.Effect of Hst5 on P.gingivalis biofilm formation and metabolic activity.The effects of Hst5 at different concentrations on planktonic P.gingivalis were observed by drawing bacterial growth curve.Crystal violet staining was used to evaluate the effect of Hst5 on P.gingivalis biofilm biomass.MTT assay was used to study its effect on biofilm metabolic activity.Quantitative PCR was used to analyze the amount of P.gingivalis in biofilm.Scanning electron microscope(SEM)and confocal laser scanning microscope(CLSM)were used to observe the morphology and structure of P.gingivalis biofilm.2.The mechanism of Hst5 inhibiting P.gingivalis biofilm formationAfter P.gingivalis incubated with Hst5(25 μg/m L)for 48 hours,the effect of Hst5 on the expression level of P.gingivalis gene and protein was analyzed by combination of transcriptomics and proteomics,and GO(gene ontology)and KEGG(kyoto encyclopedia of gene and genes)function enrichment analysis were performed.Some differentially expressed genes were screened out,and q RT-PCR was used to evaluate the reliability of transcriptome results.3.The role of Hst5 in rat experimental periodontitisIn vivo,rat periodontitis model was established by ligating maxillary first molar plus P.gingivalis in intragingival sulcus.The concentration of Hst5 was 100 μg/m L.16 rats were divided randomly into 4 groups: blank group,ligation+P.gingivalis group,ligation+P.gingivalis+Hst5 group,ligation+Hst5 group.After 6 weeks,rats were euthanized.Quantitative PCR was used to analyze the amount of P.gingivalis in the perigingival plaque of first molar in each group.Hematoxylin-eosin staining was used to detect inflammatory level in periodontal tissue.Immunohistochemical staining was used to detect the expression level of TNF-α and IL-1β.Microcomputer tomography(Micro-CT)was used to detect alveolar bone resorption of rats.Results:1.Hst5 could inhibit the formation of P.gingivalis biofilm.Bacterial growth curve showed that 12.5-50 μg/m L Hst5 did not affect the growth of P.gingivalis in planktonic state(P > 0.05)and minimum inhibitory concentration was 100μg/m L.The results of crystal violet staining and MTT assay showed that the effect of12.5 μg/m L Hst5 on P.gingivalis biofilm was not significantly different from that of control group(P > 0.05),and Hst5 at ≥ 25 μg/m L could significantly inhibit the formation and activity of P.gingivalis biofilm in a concentration dependent manner(P< 0.05).Quantitative PCR results indicated that the amount of P.gingivalis in biofilm treated with Hst5 was significantly lower(P < 0.05).SEM and CLSM observed the inhibition of Hst5 on P.gingivalis biofilm formation and destruction of biofilm structure.2.Hst5 inhibited biofilm formation by regulating P.gingivalis membrane function and metabolic process.Transcriptomic sequencing analysis showed that compared with control group,there were 877 differentially expressed genes in Hst5 treatment group,including 386 upregulated genes and 491 down-regulated genes.Some transcriptome results were verified by q RT-PCR,and its consistency rate was 78%,indicating that transcriptome results were highly reliable.Proteomic analysis revealed 95 differentially expressed proteins,of which 49 were up-regulated and 46 were down-regulated.GO and KEGG functional enrichment analysis showed that Hst5 mainly regulated cell membrane function and metabolic process of P.gingivalis.The correlation between transcriptomics and proteomics data showed 18 differentially expressed genes/proteins with the same regulation types,among which Rpo D was up-regulated and Feo B was down-regulated.3.Hst5 inhibited the occurrence and development of rat periodontitis by antibacterial and anti-inflammatory effects.Quantitative PCR results showed that the amount of P.gingivalis in plaque of ligation+P.gingivalis+Hst5 group was significantly lower than that of ligation+P.gingivalis group(P < 0.05).The results of hematoxylin-eosin staining and immunohistochemical staining showed that the inflammatory degree and expression level of TNF-α and IL-1β of ligation+P.gingivalis+Hst5 group was significantly lower than that of ligation+P.gingivalis group(P < 0.05).Micro-CT showed that alveolar bone resorption in ligation+P.gingivalis+Hst5 group was significantly lower than that in ligation+P.gingivalis group(P < 0.05).Conclusion: 25 μg/m L Hst5 can inhibit P.gingivalis biofilm formation by regulating membrane function and metabolic process,in which Rpo D and Feo B protein may play an important role.100 μg/m L Hst5 can decrease inflammation level of rat periodontal tissue and alveolar bone absorption through antibacterial and anti-inflammatory effects.Therefore,we speculate that salivary Hst5 can prevent the occurrence and development of chronic periodontitis to a certain extent. |
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