| Within biofilms,microbes are well protected by extracellular polysaccharide-rich matrix which surrounds them and prevents penetration by antimicrobial agents.Thus compared with planktonic cells,these biofilm cells of the same species are constantly more obstinate,leading to persistent infection.In oral cavity,the well-known dental plaque is an example of biofilm.Dental plaque gives rise to dental caries,which is the oral infectious disease with the highest prevalence.Governments need to spend a lot of finance for preventing and treating dental caries every single year.Current methods for eradicating dental plaques include mechanical and chemical methods.Although people can maintain oral hygiene through brushing tooth surface and flossing interdental space,there are still many individuals such as disabled or unskilled persons who cannot take these measures daily in routine.For chemical methods,antibiotics are commonly utilized in clinic and home.Chlorhexidine(CHX)is an effective antibiotic with broad spectrum effects and it has been commonly used in clinics and homes for years.However,there are some inevitable side-effects including the teeth or tongue staining,mucosal desquamation as well as taste perception abnormal.It has been proved that fluoride can prevent dental caries through inhibiting acidogenicity and reducing acid tolerance in the dental plaque.However,fluoride will cause severe health problems such as dental fluorosis and skeletal fluorosis if it is excessively consumed.Moreover,antibiotics cannot inhibit bacterial growth in biofilm directly and antibiotic therapy frequently challenged with the rise in multidrug resistance.Therefore,there is a sense of urgency for developing new methods to control dental plaques without adverse effects.For example,the development of the anti-caries vaccines has aroused numerous attentions among scientists and the effectiveness has been well proven.However,some safety issues and ethical dilemma are the majoy obstables for the clinical application among human beings.Nowadays,more and more researchers point to the plant kingdom in today's fight against biofilm-related diseases.Although natural phytochemicals have arisen increasing interest,to date,there are still almost 300,000 herbs which have not been evaluated for their antimicrobial ability yet.As we know,Streptococcus mutans(S.mutans),Streptococcus sobrinus(S.sobrinus)and Streptococcus sanguinis(S.sanguinis)are major cariogenic bacteria.Although numerous papers have reported that nature's medicinal herb components may be effective against only one of the cariogenic bacteria above,in vitro,ex vivo,and in situ,these results cannot reflect the reality in oral community.Because it is estimated that over 700 bacterial species have been detected in oral biofilms.In addition,different species of microbes may interact with each other during mixed biofilm development.These antimicrobial and antibiofilm agents targeted only one bacterium cannot always have effective inhibition in multispecies dental biofilm.The treasured Chinese herb ginseng is a traditional medicine that has been accepted into use since ancient times.It has shown various outstanding functions including the abilities of antitumor,immune response enhancement,antifatigue and antimicrobial effects.More and more researchers recently focus on its antibacteria effects in infectious diseases.To the best of our knowledge,until now,the report regarding the application of ginseng extracts against multispecies oral biofilms is not available.In order to find out what role the ginsenosides possibly play on cariogenic bacteria,we observed a series of ginsenosides about their effects on S.mutans UA159,S.sobrinus ATCC 6715,and S.sanguinis SK36 due to their high prevalence in number and contribution in oral biofilms.Then the most effective ginsenoside Rh2 was selected for further study.Therefore,in the present research,we investigated whether ginsenoside Rh2 can inhibit monospecies and multispecies cariogenic dental plaques in vitro and in vivo.Meanwhile,we sought to figure out the underlying mechanism about its antimicrobial effects on cariogenic biofilms.Three parts are included in the study:Part One Antimicrobial effects of ginsenosides on monospecies and multispecies cariogenic biofllms In vitroIn this study,we firstly screened out 3 kinds of gisenosides named Rh1 Rh2 and Rg2 which have outstanding biofilm formation inhibition effects on cariogenic bacteria.We then investigated what effects ginsenoside Rh2 had on monospecies and multispecies biofilm formed by cariogenic bacteria S.mutans,S.sobrinus and S.sanguinis in vitro by conducting CLSM observation and polysaccharide measurement.Cytotoxicity of Rh2 was examined by using cell counting kit-8(CCK-8).Rh2's effects on bacterial membranes and biofilm structures were also observed through transmission electron microscopy(TEM)and scanning electron microscopy(SEM).Results:In general,Rh2 application to cariogenic oral biofilms not only significantly decreased biomass accumulation,but also prevented the formed biofilm from developing by inhibiting bacterial growth and extracellular polysaccharide synthesis.We found the Live/Dead confocal photos repeatedly confirmed that Rh2 could significantly inhibit the viability of these bacteria in their biofilm.Nevertheless,no significant change was detected in the multispecies biofilm constitution from the FISH assay.Moreover,Rh2 was essentially non-cytotoxic to periodontal ligament cells at 50%biofilm inhibitory concentration(IC50).TEM and SEM imaging showed that Rh2 could disrupt cell membrane function and disturb the physiology of these bacteria and their biofilms.Part Two Caries protective effects of ginsenoside Rh2 on Rats model in vivoA classical rat caries model with slight modification was built to evaluate the effects of ginsenoside Rh2 on caries protection.18 SPF Sprague-Dawley rats(SD rats)aged 18d were adopted,inoculated with S.mutans,S.sobrinus and S.sanguinis after the antibacterial treatment.Rh2 was orally given in the consective 4 days.And the rats were fed a cariogenic diet(Keyes 2000#).Micro-CT was used for observing the tooth and calculated the volume of the defect.Thereafter,Keyes'caries diagnosis and scoring technique was applied as a secondary evaluation on the molar teeth.Moreover,some safety issues were analyzed and evaluated during the Rh2 application.Results:Both Micro-CT and Keyes' scoring indicated that the Rh2 group showed the least caries defect compared with the controlled group.So far,no observable toxity or safety issues was found during the animal experiment.In sum,Rh2 showed an anti-caries effect in vivo which made it a potential drug in careis prevention.Part Three The underlying mechanism of Rh2'effects on cariogenic bacteriaTo explore the mechanism of Rh2 in inhibiting cariogenic bacteria biofilm formation,isobaric tags for relative and absolute quantification(iTRAQ)were used to profile the common proteins with differential expression.The gene expressions on transcriptional level were analyzed by reverse transcription and quantification polymerase chain reaction(RT-QPCR).Results:Totally there were 2769 proteins identified with different expression in the database,among these,156,84,138 proteins were up-regulated while 62,88,70 proteins were down-regulated in S.mutans,S.sobrinus and S.sanguinis respectively.The common pathways with significantly differences were ten pathways including phosphotransferase system(PTS),butanoate metabolism,serine biosynthesis,alanine,aspartate and glutamate metabolism,lysine biosynthesis,vitamin B6 metabolism,biotin metabolism,pantothenate and Co A biosynthesis,pyrimidine metabolism,and naphthalene degradation.We chose the interest common proteins ManY and ManZ of PTS and acetaldehyde/alcohol dehydrogenase of butanoate metabolism.Their expression levels were down-regulated dramatically.To provide additional transcriptional levels,we identified the corresponding genes for the proteins in PTS and butanoate metabolism pathways,these were mannose-specific IIC/D component and acetaldehyde/alcohol dehydrogenase.Specifically,gene expressions involved the mannose-specific ?C and?D were increased,except that of the mannose-specific ?D in 16 h cultured samples of S.sobrinus.Of note,we detected the gene expressions of bifunctional acetaldehyde-CoA/alcohol dehydrogenase of S.mutans and S.sobrinus were significantly reduced in the Rh2 treated group when compared with the control group,except that of bifunctional acetaldehyde-CoA/alcohol dehydrogenase and aldehyde dehydrogenase in 16 h cultured samples of S.sanguinis. |
PDF Full Text Request