Verificação da inibição da expressão de genes envolvidos na formação de biofilme cariogênico em Streptococcus mutans

Abstract

Dental caries results from a metabolic imbalance in the biofilm, formed by bacteria, especially streptococci and lactobacilli. One way of preventing caries process would be inhibit cariogenic biofilm formation. This work therefore aims to verify the possible inhibition of biofilm formation by Streptococcus mutans (S. mutans) after the treatment with four substances (epigallocatechin, chlorhexidine, xylitol and resveratrol), and to investigate the action mechanism through the expression of the genes glycosyltrasferase (GTF) and fructosyltransferase (FTF). The test of quantitative polymerase chain reaction (qPCR) was carried out to quantify the expression of these enzymes. Cell viability assays as well as tests for verification of biofilm formation were also carried out after the treatment with the test substances to determine the concentration when the inhibition of biofilm formation occurred under conditions of cell viability.The xylitol, resveratrol and chlorhexidine concentrations that proved effective were 100 mg/ml, 0.1 mg/ml and 0.001 mg/mL, respectively, concentrations in which there was inhibition of biofilm formation without modification of the bacterial viability. The epigallocatechin didn’t demonstrate a concentration that would provide biofilm inhibition without the consequent inhibition of bacterial viability. The treatments were performed with xylitol, resveratrol and chlorhexidine, and after completion of RT-qPCR (reverse transcription real time polymerase chain reaction), they’re obtained the results for enzyme expression of FTF and GTFB. There was a significant decrease (72.8%) in the enzyme FTF expression after treatment with xylitol. The resveratrol didn’t affect the expression of this enzyme and chlorhexidine caused reduced gene expression control (housekeeping gene), precluding to evaluate the FTF gene expression in the assessed concentration. There was no significant change in GTFB expression after treatment with resveratrol and chlorhexidine in defined concentrations. Xylitol reduced housekeeping gene 16S rRNA expression, indicating cell death in the concentration tested. The results led us to conclude that the three substances (resveratrol, xylitol and chlorhexidine) act as potential dental biofilm inhibitors formed by S. mutans. It was found, however, that xylitol inhibits FTF gene expression, indicating that the probable mechanism of action of such inhibition of biofilm formation was by reducing the expression of this S. mutans gene. Chlorhexidine has already recognized bactericidal action, however this work we see a concentration of this substance that was inhibiting biofilm without subsequent cell death. Considering those findings may suggest other ways of applying the xylitol and chlorhexidine in dentistry and also the use of resveratrol as an inhibitory agent of the carious process, since it is capable of inhibiting dental plaque formation. Biofilm inhibition these substances would be interesting to such an extent that there would generate an imbalance in the bacterial flora, thus keeping bacteria that act in a positive manner in the oral cavity. Thus, clinical studies are necessary to demonstrate the efficacy of these substances to prevent or even caries treatment.