| Background and Objectives:Refractory apical periodontitis(RAP)is an inflammatory disease that occurs in periapical tissue.In recent years,the prevalence has gradually risen,which poses a serious threat to public’s health,particularly the elderly and immunocompromised people.Enterococcus faecalis(E.faecalis)is considered to be the main pathogen causing intractable and secondary root canal infections,with a detection rate of 77%in refractory periapical infections.E.faecalis mainly exists in the form of biofilm in the root canal.It can tolerate high alkaline and low nutrient environment,invade the dentin tubules,and easily escape the phagocytosis of host cells.Besides,it has strong resistance and resistance to conventional mechanical and chemical preparation measures as well as a variety of bactericidal drugs.Therefore,it is difficult to completely remove during root canal treatment.In recent years,plant compounds have attracted extensive attention due to their excellent biosafety and diverse pharmacological actions,providing new approaches for the treatment of resistant root canal infections.Ginsenosides(Ginsenosides or panaxosides),as the most significant active ingredients in ginseng,has a variety of biological effects,such as delaying aging,anti-tumor,improving immunity,enhancing memory,protecting nerves,anti-inflammatory and so on.In recent years,the antibacterial effect of ginsenosides has attracted the attention of researchers to a certain extent.At present,studies have proved that it has inhibitory effects on Streptococcus mutans,Porphyromonas gingivalis and other oral pathogens,and can intervene and reverse microbial resistance by inhibiting bacterial respiratory metabolism and protein synthesis,but its antibacterial effect on E.faecalis is still unclear.Therefore,in this study,Ginsenoside Rh2(G-Rh2)with strong antibacterial activity against E.faecalis was screened through previous literature review and preliminary experiment.Through crystal violet staining,Methyl thiazolyl tetrazolium(MTT),Scanning electron microscope(SEM),the inhibitory effect of G-Rh2 on the growth of E.faecalis plankton and biofilm formation was observed in vitro,which provided a new hypothesis for further research on E.faecalis scavenging drugs in the root canal,and provided laboratory basis for clinical improvement of the success rate of refractory periapical inflammation.Methods:1.Ginsenosides(G-Rh2,G-Rg3,and G-Rd)solutions with different concentrations and E.faecalis solutions with a concentration of 1×106 CFU/ml were co-cultured in 96-well cell culture plates.The turbidity of the medium in each well was observed 24 hours later.2.Through preliminary experiment,ginsenoside monomeric G-Rh2 with antibacterial effect on E.faecalis was successfully screened.Different concentrations of G-Rh2 solution and E.faecalis bacterial solution with concentration of 1×106CFU/mL were prepared and co-cultured in 96-well cell culture plates.After 24h,the turbidity of the medium in each well was observed.The minimum drug concentration corresponding to the clarified well of the medium was the MIC of ginsenoside against E.faecalis.100 μL bacterial solution was taken from the clarifying hole and coated on the surface of BHI solid medium.The results were observed after 24 h constant temperature culture at 37℃.MBC was the minimum drug concentration corresponding to no colony growth on the medium.3.G-Rh2 solution with different concentration and E.faecalis solution with concentration of 1×106 CFU/mL were prepared in centrifuge tube for co-culture,and control group was set up.OD600 of each group was measured by enzymelabeled instrument every 1h.The growth curve of E.faecalis within 10 h was drawn to evaluate the bacteriostatic effect of G-Rh2 on E.faecalis.4.Different concentrations of G-Rh2 solution and 1×106 CFU/mL of E.faecalis bacterial solution were prepared and co-cultured on 24-well cell culture plates,and control group was set up.The effect of G-Rh2 on the amount of E.faecalis biofilm was evaluated by crystal violet staining after 24h.5.Different concentrations of G-Rh2 solution and 1×106 CFU/mL of E.faecalis bacterial solution were prepared for co-culture in 96-well cell culture plates,and control group was set up.After 24h,the effects of G-Rh2 on the metabolic activity of E.faecalis biofilm were evaluated by MTT method.6.Different concentrations of G-Rh2 solution and 1×106 CFU/mL of E.faecalis bacterial solution were prepared and co-cultured on 24-well cell culture plates,and control group was set up.After 24h,the samples were fixed with 2.5%glutaraldehyde solution overnight.After gradient dehydration with ethanol,sample drying and gold spraying,the morphology and structure of E.faecalis and its biofilm were observed under SEM.7.G-Rh2 solution with different concentration and E.faecalis solution with concentration of 1×106 CFU/mL were prepared and co-cultured in CLSM culture dishes,and control group was set up.After 24 h,E.faecalis biofilm was stained with a lived-dead cell staining kit.The composition and distribution of live and dead bacteria were observed by Confocal Laser confocal canning microscopy(CLSM).Results:1.The bacterial liquid in the orifice plate is turbid due to bacterial growth,and the bacterial liquid in the orifice plate is clarified due to sterile growth.The results of the 96-well plate showed that the bacterial liquid in the hole became clear when the concentration of G-Rh2 started from 3.125 mg/L.When the concentration of G-Rd and G-Rg3 was from 3.125 mg/L to 1000 mg/L,the bacterial liquid in the pore was still cloudy.2.When the concentration of G-Rh2 was more than 3.125 mg/L,the bacterial liquid in the 96-well plate became clear,that is,the minimum inhibitory concentration(MIC)of G-Rh2 against E.faecalis was 3.125 mg/L.The AGAR plate counting results showed that there was no bacterial growth on the solid plate when the concentration of G-Rh2 was 6.250 mg/L,that is,the MBC of G-Rh2 to E.faecalis was 6.250 mg/L.3.The growth curve of E.faecalis within 10h showed that,compared with the control group,the growth of E.faecalis was inhibited when the concentration of G-Rh2 was MIC and 1/2 MIC;When the concentration of G-Rh2 was 1/4 MIC and 1/8 MIC,there was no inhibitory effect.4.Crystal violet staining showed that,compared with the control group,the formation of E.faecalis biofilm was inhibited when the concentration of G-Rh2 was MIC and 1/2 MIC,and the difference was statistically significant.When the concentration of G-Rh2 was 1/4 MIC and 1/8 MIC,there was no inhibitory effect,and the difference was statistically significant.5.The MTT results showed that when the concentration of G-Rh2 was MIC and 1/2 MIC,the metabolic activity of bacteria in the biofilm could be inhibited,and the difference was statistically significant when compared to the control group.When the concentration of G-Rh2 was 1/4 MIC and 1/8 MIC,there was no inhibitory effect,and the difference was not statistically significant.6.SEM image results showed that,compared with the control group,when the concentration of G-Rh2 was 1/4 MIC,the number of bacteria in the E.faecalis biofilm increased,but some bacteria’s morphology changed.When the concentration of G-Rh2 was between 1/2 and MIC,the structure of the E.faecalis biofilm disappeared,the number of bacteria decreased significantly,and the morphology of almost all bacteria changed.7.CLSM image results showed that,compared with the control group,when the concentration of G-Rh2 was MIC and 1/2 MIC,the number of viable bacteria in the E.faecalis biofilm was reduced,the biofilm formation was inhibited,and the biofilm thickness was reduced.When the concentration of G-Rh2 is 1/4 MIC,it has no significant effect on the thickness of the biofilm but can reduce the number of viable bacteria.Conclusions:1.G-Rh2 had an inhibitory effect on E.faecalis.When the concentration was less than 1000 mg/L,G-Rd and G-Rg3 might have no inhibitory effect on E.faecalis.2.When the concentration was greater than 1/2 MIC,G-Rh2 had an inhibitory effect on the growth and biofilm formationof E.faecalis.When the concentration was less than 1/4 of the MIC,G-Rh2 had no inhibitory effect on the growth and biofilm formation of E.faecalis.3.When the concentration was greater than 1/2 MIC,G-Rh2 may play an antibacterial role by destroying bacterial cell membrane and reducing bacterial adhesion. |
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