Preliminary Study On The Involvement Of Prevotella.denticola In Dentin Caries By Metabolizing Carbohydrates And Proteolysis

Objectives:In vitro experiments,we explored the acid production and demineralization of P.denticola and its ability to degrade human dentin collagen,revealed its role in oral microecological transformation and caries occurrence,and explored its molecular mechanism of caries.At the same time,the etiology microbiology of caries is improved,which provides new ideas and research basis for the effective and targeted prevention and treatment of caries.Methods:（1）In this part of the experiment,the model strain of P.denticola was used as the experimental strain to observe the culture cycle and physiological characteristics of P.denticola.The morphology of the strain was observed by scanning electron microscopy and its film-forming ability was evaluated.The growth curve(OD₆₀₀)of P.denticola was determined,and the factors affecting its growth and reproduction were explored by changing the p H of the medium,glucose concentration or co-culture with Streptococcus mutans.（2）Firstly,the ability of P.denticola to produce acid was determined by p H meter and high performance liquid chromatography,and then the effects of p H and S.mutans on its sugar consumption and succinic acid production were investigated.Finally,P.denticola and S.mutans were co-incubated with dentin blocks in vitro to establish a dentin caries model.S.mutans was used as a reference bacterium to detect the changes in the hardness of tissue blocks under the action of bacterial acidic products by micro-Vickers hardness tester.Laser confocal microscopy was used to quantify the depth of dentin demineralization.Scanning electron microscopy was used to observe the surface morphology of dentin to evaluate the demineralization ability of P.denticola.（3）In this part of the experiment,a completely demineralized dentin model was first prepared,and then the dentin specimen was incubated with P.denticola.The hydroxyproline content of the collagen hydrolysate in the P.denticola bacterial solution was determined by the hydroxyproline kit to determine the appropriate bacterial inoculation concentration.The effects of two factors on the hydrolysis of collagen by P.denticola were investigated by changing the p H of the medium or co-culturing with S.mutans.Finally,the damage degree of dentin collagen fibers was observed by scanning electron microscopy.（4）Transcriptome data combined with genome-wide mapping were used to analyze the effects of glucose/nitrogen stress conditions on the functions and metabolic pathways of glycogen metabolism and collagen hydrolysis genes in P.denticola,and the related genes were preliminarily screened.Results:（1）In this study,P.denticola was successfully cultured and found to have adhesion ability and acid resistance.P.denticola grew best in a neutral environment containing 0.5%carbon source.The results showed that the bacteria were Gram-negative short bacilli with blunt round and non-flagella at both ends.The length was about 0.4-1.2 um.The growth cycle on the plate was 3-4 days.The colony was gray,round,uplifted,smooth surface,regular edge,1-2 mm in diameter,with hemolysis characteristics.The growth cycle in liquid medium was 1 day.P.denticola can adhere to the surface of smooth cell slides after culture,with adhesion ability and acid resistance.The OD₆₀₀ value of P.denticola increased with the increase of p H and glucose concentration,and decreased after reaching the highest value at p H=7.3 and 0.5%glucose concentration.S.mutans initially promoted the growth of P.denticola,and reached the maximum promotion effect at S.mutans(OD₆₀₀=0.2),and then the OD₆₀₀ value of P.denticola gradually decreased with the increase of S.mutans concentration.（2）The experimental results showed that P.denticola could produce organic acids under p H conditions of 8.3-4.3,and P.denticola had the best glucose metabolism ability at p H 7.3.The presence of S.mutans also affected the glucose metabolism of P.denticola.When the concentration was OD₆₀₀=0.1,0.2,0.3,S.mutans promoted the glucose metabolism of P.denticola（P<0.05）.As the concentration increased,S.mutans showed an inhibitory effect on the metabolism of P.denticola.When OD₆₀₀=0.2,the glucose metabolism activity of P.denticola reached a peak.The results of in vitro dentin demineralization model showed that P.denticola could induce the demineralization of dentin hard tissue,showing a decrease in dentin hardness and an increase in fluorescence staining thickness.However,by comparing the two demineralization indexes of S.mutans and P.denticola groups in the same period,it can be seen that the demineralization effect of P.denticola was weaker than that of S.mutans（P<0.05）.（3）The accumulation of hydroxyproline in P.denticola and the results of scanning electron microscopy showed that P.denticola could hydrolyze collagen and release hydroxyproline.The release of hydroxyproline increased first and then decreased at p H 8.3-4.3,and the amino acid content was the highest at p H 7.3.The presence of S.mutans also affected the glucose metabolism of P.denticola.When the concentration was OD₆₀₀=0.1,0.2,0.3,S.mutans promoted the collagen hydrolysis of P.denticola（P<0.05）,suggesting that the synergistic effect of the two in dentin caries at low concentrations of S.mutans.However,with the increase of concentration(OD₆₀₀=0.4,0.5),there was no significant difference between the S.mutans group and the P.denticola pure culture group without S.mutans（P>0.05）,suggesting that the synergistic effect of the two in dentin caries at low concentrations of S.mutans.Scanning electron microscopy showed that the collagen fibers of the specimens incubated in P.denticola broth were hydrolyzed and broken,the fiber network had obvious structural damage,and the interval between the tubules became thinner,supporting the quantitative results of collagen degradation and release of hydroxyproline.（4）Transcriptome sequencing of P.denticola cultured under different concentrations of sugar and nitrogen sources was carried out to explore the molecular mechanism of acid production and hydrolysis of dentin matrix.The results showed that compared with the control group,68 genes were differentially expressed in the P.denticola high glucose culture group,and the expression levels of 8 glucose metabolism-related genes were significantly higher than those in the control group（P<0.05）.Conclusion:The above experiments showed that this study found that P.denticola has the ability of hemolysis and adhesion.It can ferment glucose in vitro to produce succinic acid,which in turn induces environmental acidification to promote dentin demineralization,and can break the collagen exposed to dentin.Hydrolysis releases hydroxyproline,affirming the role of P.denticola in the development of dentin caries.Furthermore,under the conditions of different concentrations of sugar and nitrogen sources,the prokaryotic specific transcription sequencing was carried out by sequencing technology to explore the molecular mechanism of acid production and hydrolysis of dentin matrix by P.denticola,and the etiology microbiology of caries was further supplemented,so as to lay a foundation for the study of the pathogenic mechanism of P.denticola and the establishment of a future platform for caries prevention and control strategies,such as a vaccine,phage therapy,and antibiotic treatment.