| Dental caries,as one of the most common chronic diseases in humans,is a multi-factorial bacterial infectious disease,whose main initiating factor is plaque biofilm.Streptococcus mutans(S.mutans)has been recognized as the main cariogenic bacteria.The intake of sucrose is directly related to the occurrence and development of caries,so the use of sucrose substitutes is of great significance for the control and prevention of caries.Rubusoside is a kind of sweetener with high sweetness extracted from sweet tea,a natural Chinese herbal plant.Previous studies have proved that rubusoside can inhibit the growth,acid production and adhesion of S.mutans in the planktonic state,and it can also inhibit the activity of glucosyltransferase and reduce the production of water-insoluble extracellular polysaccharide.Objective:The purpose of this study was,from the perspective of biofilm,to study the effect of rubusoside as sucrose substitute on cariogenicity of S.mutans biofilms,exploring the role of rubusoside in preventing caries.By comparing the effect of different sweeteners on the expression levels of cariogenic virulence factor-related genes in biofilms,the underlying anti-caries mechanisms were analyzed,which will develop a more favorable theoretical basis for the further development and utilization of rubusoside,and provide a new idea for the prevention of dental caries.Methods:1.The effect of rubusoside on the growth and acid production of S.mutans biofilm.After preparing a BHI solution containing 1%sucrose,rubusoside,xylitol and a blank BHI liquid medium,the initial pH was adjusted to 7.04.S.mutans was inoculated into a 96-well plate in the proportion of 1:10(v:v)to each medium solution,and cultured at 37? for 12,24,36,48,60,72,and 96 hours under anaerobic static culture.Crystal violet staining was used to detect the amount of S.mutans biofilm,and the biofilm growth curve was drawn.At the same time,another 24-well plate was cultured according to the above operation,and the pH value of the culture medium was measured with a pH meter every 24 hours,and the pH of each group was compared.2.The effect of rubusoside on the biomass and viability of S.mutans biofilmBHI solution containing 1%sucrose,rubusoside and xylitol and blank BHI liquid medium were prepared.According to the proportion of 1:10(v:v),S.mutans and medium solution was inoculated into a 24 well plate with a cover slide,and cultured for 5 days under an anaerobic condition at 37?.The biofilm on the cover slide was scraped off to form a bacterial suspension.The dry weight of the biofilm was measured.And the total soluble protein in the bacterial solution was extracted and measured with BCA protein quantitative kit.The bacterial activity of biofilm was measured by dilution coating plate method.The bacterial suspension was inoculated on the solid BHI medium,and cultured for 48h at 37?.The number of colony forming unit(CFU)was calculated,and the result was corrected by dilution factor,expressed by the dry weight of biofilm log10cfu/mg.3.The effect of rubusoside on the morphological structure of S.mutans biofilmResin sheets were prepared with the upper cover of 96-well plate and put into 24-well plate.The BHI solution containing 1%sucrose,rubusoside and xylitol and a blank BHI liquid medium were prepared.According to the proportion of 1:10(v:v),S.mutans and each medium solution was inoculated into a 24 well plate with resin sheets and cultured for 5 days at 37? under an anaerobic condition.The resin sheets with biofilm were taken out,dehydrated with ethanol in gradient and freeze-drying.The biofilm of S.mutans was observed with scanning electron microscope(SEM).4.The effect of rubusoside on the production of polysaccharides in S.mutans biofilmBHI solution containing 1%sucrose,rubusoside and xylitol and blank BHI liquid medium were prepared.According to the proportion of 1:10(v:v),S.mutans and each medium solution was inoculated into a 24 well plate with a cover slide,and cultured for 5 days under an anaerobic condition at 37?.The biofilm on the cover slide was scraped off to form bacterial suspension,and then soluble extracellular polysaccharide(SEPS),insoluble extracellular polysaccharide(IEPS)and intracellular polysaccharide(IPS)were extracted respectively.The content of polysaccharide was determined by phenol sulfuric acid method.5.Effect of rubusoside on cariogenic gene expression of S.mutans biofilmAccording to the proportion of 1:10(v:v),S.mutans and each medium solution was inoculated into a 24 well plate with a cover slide,and cultured for 5 days under an anaerobic condition at 37?.The biofilm on the cover slide was scraped off to form bacterial suspension,and RNA was extracted.After reverse transcription,the expression level of cariogenic virulence factors in the biofilm was determined by real-time quantitative PCR and compared.Results:1.The amount of S.mutans biofilm formed in BHI medium containing sucrose was far more than that of the other three groups(P<0.05).There was no significant difference between the control and xylitol in the first 12 h(P>0.05),but after 12 h,the biofilm amount of xylitol was significantly less than that of the control(P<0.05),and reached the maximum biomass at 48 hours.The growth of S.mutans biofilm reached its peak at 24 h in the rubusoside,and compared with the control,sucrose and xylitol group,the amount of biofilm formed was lower(P<0.05).After exposure to sucrose medium for 24 h,the pH value decreased rapidly to below 5.5(critical pH of enamel demineralization),and kept at the lowest level during the whole experiment(P<0.05),and the pH value gradually decreased with the increase of time(P<0.05).There was no significant difference in pH value between the control and xylitol at 48h,72h and 96h(P>0.05).At 24h,the pH value of xylitol was higher than that of the control,while at 120h,the pH value of xylitol was lower(P<0.05).The pH value of the rubusoside remained at the highest level in the four groups(close to 7),and the pH value did not change significantly with time(P>0.05).2.The dry weight of biofilm in sucrose was higher than that in other treatment groups(P<0.001),and the dry weight of biofilm in xylitol was similar to that in the control(P>0.05),while the dry weight of biofilm in rubusoside was the lowest(P<0.01).The total soluble protein content of S.mutans biofilm in sucrose was significantly higher than other groups(P<0.001),and there was no significant difference among the other three groups(P>0.05).The number of viable bacteria in the biofilm grown in sucrose was significantly higher than that in the control(P<0.05).At the same time,the number of viable bacteria of the biofilm treated by xylitol was slightly lower than that of the control(P<0.05),while the number of viable bacteria in the rubusoside was significantly lower than that of other groups(P<0.05).3.The morphology and structure of biofilm were observed by scanning electron microscopy.In the control,the bacteria had regular morphology and smooth surface.The extracellular matrix produced by bacterial metabolism is very little,and the number of bacteria is low.Bacteria arranged loosely in chains without obvious cluster network distribution.In sucrose,the number of S.mutans increased,the chain became longer and more.Bacteria produced a large number of amorphous extracellular matrix materials,which made bacterial cells wrapped in the three-dimensional structure formed by extracellular matrix.The number of S.mutans in rubusoside and xylitol decreased significantly,and the extracellular matrix on the surface of the bacteria was also less.The bacteria were distributed in short chains,the three-dimensional structure was not obvious,and the difference between them was not obvious.4.The content of SEPS in the biofilm treated with sucrose was higher than that in the other three groups(P<0.001),but there was no significant difference between xylitol and control(P>0.05).The content of SEPS in the biofilm grown in the medium containing rubusoside was the least.The addition of sucrose could significantly increase the content of IEPS in the biofilm(P<0.001),and the content of IEPS was the least in the rubusoside(P<0.05).The production of IEPS in xylitol and control were between sucrose and rubusoside,while xylitol was lower than the control.As for the IPS,the content in sucrose was the highest(P<0.001),but there was no significant difference among the other three groups(P>0.05).5.Genes related to polysaccharide synthesis(gtfB,gtfC,gtfD,ftf)were significantly up-regulated in sucrose,and the level was the highest in all groups(P<0.001).The expression of gtfB and gtfC in rubusoside was lower than xylitol(P<0.05).However,for the expression levels of gtfD,rubusoside,xylitol and control group were not significantly different(P>0.05).And the expression level of ftf in rubusoside was slightly higher than that in xylitol(P<0.01).Among the genes related to adhesion,the expression of spaP in rubusosides and xylitol was not significantly different from that of the control(P>0.05),but both were much lower than sucrose(P<0.001).For gbpB,the expression level in rubusoside was slightly high compared with the control(P<0.05),but much lower than that in the xylitol and sucrose(P<0.05).In the presence of sucrose,the up-regulation of gbpB was the largest(P<0.001).Among genes related to acid production and acid tolerance,the ldh expression levels of biofilms grown in rubusoside were similar to those of the control(P>0.05),and were significantly lower than those of sucrose(P<0.001)and xylitol(P<0.01).The expression of atpF in rubusoside was up-regulated compared with the control and xylitol(P<0.001),but much lower than that of sucrose.In sucrose,the expression levels of ldh and atpF were significantly increased(P<0.001).For genes related to the stress regulation system,vicR expression was down-regulated in rubusoside and xylitol(P<0.05),but there was no difference between the two groups(P>0.05).But in sucrose,vicR gene expression was up-regulated compared to other groups(P<0.001).For comD,the expression in rubusoside was not significantly different from that in the control(P>0.05),but was lower than that in xylitol(P<0.05).Sucrose caused the largest up-regulation of comD(P<0.001).Conclusion:1.Rubusoside can inhibit the growth and acid production of S.mutans biofilm.2.Rubusoside can reduce the biomass and bacterial viability of S.mutans biofilm.3.By scanning electron microscopy,we found that rubusoside can inhibit the adhesion of bacteria and reduce the secretion of extracellular matrix.The biofilm is thin,and the three-dimensional structure is not obvious.4.Rubusoside can reduce the synthesis of water-insoluble exopolysaccharide,water-soluble exopolysaccharide and intracellular polysaccharide in S.mutans biofilm.5.Rubusoside can affect the expression of caries-associated virulence genes of S.mutans biofilms,and make the expression level of spaP,gbpB genes related to adhesion,gtfB,gtfC,gtfD and ftf genes related to polysaccharide synthesis,ldh and atpF genes related to acid production and acid resistance decrease,stress regulation related vicR and comD genes were reduced.This difference in gene expression may lead to the effect of rubusoside on the growth,acid production,biomass,bacterial viability,polysaccharide content of S.mutans biofilm.The research results of this project show that the natural sweetener,rubusoside,has the potential to be used as a sucrose substitute in the field of caries control,that it has the potential to be used as a sucrose substitute in the field of caries prevention and treatment,and has very high development and utilization value and broad market prospects. |
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