| ObjectiveIn order to provide a theoretical basis for the application of lemon essential oil?LEO?in the prevention and treatment of oral diseases,we investigated the effects of LEO on S.mutans acid production and its regulation mechanism under different glucose concentrations.Methods1.Determination of the minimum inhibitory concentrations?MICs?The MICs of LEO to S.mutans at 0%,0.2%,1%and 5%glucose concentrations were determined by liquid dilution method.2.Acid production assayAccording to the measured MIC,the LEO was diluted in TPY medium containing0%,0.2%,1%,and 5%glucose by the two-fold serial dilutions method,four dilution concentrations under each glucose concentration and three replicates of each concentration.The culture medium?four glucose concentrations?without LEO was used as control group.The pH value was detected in each group of solutions before the addition of S.mutans.After the anaerobic culture of S.mutans for 24h at 37°C,the pH value of each solution was detected again.The effects of LEO on the ability of S.mutans acid production under different glucose concentrations were reflected by the formula(?pH=pH final-pH initial).3.Determination of the activity of lactate dehydrogenaseThe assay was designed as mentioned in the acid production.The S.mutans cultured for 18h were collected and broken by the ultrasonic under ice bath.The protein concentration of each sample was determined by BCA protein quantitative kit and the activity of lactate dehydrogenase in each sample was detected by reductive coenzyme I oxidation method.4.Determination of the transcription and translation of ldh geneThe grouping design was the same as experiment 2,with 3 dilution concentrations under each glucose concentration.S.mutans was added to each group and cultured for18h at 37°C.The total RNA of S.mutans was extracted,and PCR was begin to amplify after the synthesis of cDNA by reverse transcription.The ldh gene expression of S.mutans in each group was determined according to the 2-??Ct??Ct value.LEO?1×MIC?was diluted in TPY medium containing 0%and 1%glucose by the two-fold serial dilutions method,three dilution concentrations under each glucose concentration and three replicates of each concentration.The culture medium?0%and1%glucose?without LEO was used as control group.S.mutans was added into each group,and was incubated for18h at 37?.The protein expression of ldh gene was detected by SDS-PAGE.Results1.The MICs of LEO to S.mutans was 1.125mg/ml at 0%and 5%glucose concentrations and was 2.25mg/ml at 0.2%and 1%glucose concentrations.2.Effects of LEO on the acid production of S.mutans at 0%,0.2%,1%and 5%glucose concentrations?1?Effects of glucose concentration on the acid production of S.mutansWith the increase of glucose concentration?0.2%5%?,the values of?pH increased gradually and were higher than that in the 0%glucose concentration in the control group.The difference was statistically significant?F=1913.266,P<0.05?,indicating that the high concentration of glucose was more beneficial to the acid production of S.mutans.?2?Effects of LEO on the acid production of S.mutans under different glucose concentrationsIn the absence of glucose,the?pH value in 1.125mg/ml?MIC?group was lower than that in control group and the difference was statistically significant?F=12.953,P<0.05?,suggesting that in the glucose free environment,sub-MICs of LEO had no effects on the acid production of S.mutans.In the presence of glucose environment,with the increase of LEO concentration,the?pH value in experimental group decreased gradually,Except for concentrations below 0.563mg/ml?1/4MIC of 0.2%glucose concentration?,the?pH values of all LEO groups were lower than that in the control group of the same glucose concentration,and the difference was statistically significant(F0.2%=197.287,F1%=2229.371,F5%=2603.091,P<0.05),suggesting that sub-MICs of LEO exhibited a dose-dependent inhibition on the acid production of S.mutans under the high glucose,medium glucose and low glucose environment in a certain concentration range.3.Effects of LEO on the activity of lactic dehydrogenase of S.mutans at 0%,0.2%,1%and 5%glucose concentrations?1?Effects of glucose on the activity of lactic dehydrogenase of S.mutansIn the control group,the lactate dehydrogenase activity of S.mutans increased with the growing glucose concentration?0.2%5%?,and were higher than that in 0%glucose concentration.The difference was statistically significant?F=1913.266,P<0.05?,indicating that the high concentration of glucose was more beneficial to improve the activity of S.mutans lactate dehydrogenase.?2?Effects of LEO on the activity of lactic dehydrogenase of S.mutans under different glucose concentrationsThe results showed that the activity of lactic dehydrogenase of S.mutans in 0%,0.2%,1%and 5%glucose groups,except for 0.141mg/ml?1/16MIC of 0.2%glucose concentration?,was lower than that in the control group under the same glucose concentration and the difference was statistically significant(F0%=60.85,F0.2%=78.867,F1%=77.071,F5%=255.594,P<0.05),suggesting that sub-MICs of LEO could inhibit S.mutans lactate dehydrogenase activity under glucose free and glucose environment in a certain concentration range and the inhibitory effect was dose-dependent.4.Effects of LEO on the transcription and translation of ldh gene in S.mutans under the glucose concentration of 0%,0.2%,1%and 5%?1?Determination of transcription of ldh gene in S.mutans in the control groupThe ldh gene transcription of S.mutans in only 1%and 5%glucose concentration groups was higher than that in 0%glucose concentration group,and the difference was statistically significant?F=16.151,P<0.05?.It was suggested that a certain concentration of glucose?1%and 5%glucose concentration?could up-regulate the expression of S.mutans ldh gene.?2?Effects of LEO on the transcription and translation of ldh gene in S.mutans under different glucose concentrations.The transcription of ldh in experimental group was higher than that in the control group?0%glucose concentration?,and the difference was statistically significant compared with the control group?F=14.258,P<0.05?.The translation of ldh gene in the 0.563 mg/ml?1/2MIC?group was lower than that in the control group,and in the0.282mg/ml?1/4MIC?group was higher than that of the control group.Compared with the control group,the difference was statistically significant?F=167.193,P<0.05?.It was suggested that LEO could induce the up regulation of ldh gene transcription but had no regular effects on the translation in in the glucose free environment.In the presence of glucose,except for 0.563mg/ml?1/4MIC of 0.2%glucose concentration?,the transcription of ldh gene in remaining LEO groups was lower than that in the control group,and the differences were statistically significant(F0.2%=18.009,F1%=316.667,F5%=36.528,P<0.05).The ldh gene translation of experimental group was significantly lower than that in the control group,and the difference was statistically significant(F1%=709.001,P<0.05).It was indicated that LEO could inhibit the transcription and translation of ldh gene in the presence of glucose.Conclusions1.LEO could inhibit the acid production of S.mutans under 0%,0.2%,1%and 5%glucose concentrations2.In the presence of glucose,sub-MICs of LEO could also supress the transcription and translation of ldh gene of S.mutans,decreasing the activity of lactic dehydrogenase,reducing the production of acid without necessarily affecting the growth of oral bacteria.3.In the absence of glucose,sub-MICs of LEO may reverse the transcription of ldh gene and has no regular effects on the translation.4.S.mutans could up regulate the expression of ldh gene and increase the activity of lactate dehydrogenase in high glucose environment,which is more conducive to the acid production.5.The inverse regulation of LEO on S.mutans ldh gene transcription in glucose free environment needed further study and confirmation from multiple perspectives. |
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