伊犁黑蜂蜂胶对不同状态下变形链球菌乳酸脱氢酶及其相关基因影响的实验研究

Effect of Ili black bee propolis on lactate dehydrogenase from Streptococcus mutans and expression of ldh gene in vitro

目的目的通过新疆伊犁黑蜂蜂胶乙醇提取物(Ethanol Extract of Propolis,EEP)对不同状态下变形链球菌乳酸脱氢酶活性及其相关基因表达影响的作用,研究伊犁黑蜂蜂胶抑制变形链球菌产酸的原因并探讨其可能的防龋机制。方法 (1)分别培养浮游状态与生物膜状态下生长的变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、50 mg/L氟化钠的BHI培养基作用18 h,通过还原性辅酶I氧化法测定乳酸脱氢酶活性。(2)分别培养浮游状态与生物膜状态下生长变形链球菌,根据实验分组用含梯度浓度EEP的BHI培养基、含50 mg/L氟化钠的BHI培养基作用18 h,反转录-实时荧光定量PCR(RTq PCR)法测定各组乳酸脱氢酶编码基因ldh表达情况。结果 (1)在浮游状态与生物膜状态下,EEP组和Na F组乳酸脱氢酶活性均有降低,差异具有统计学意义(P<0.05)。(2)浮游状态时,实验组组和阳性对照组ldh表达明显受到抑制(P<0.05);生物膜状态下,实验组在1 MBEC、1/2 MBEC、1/4 MBEC浓度时ldh表达受到抑制(P<0.05),Na F组ldh表达差异没有统计学意义(P>0.05)。结论伊犁黑蜂蜂胶能够抑制浮游状态与生物膜状态下变形链球菌乳酸脱氢酶活性及其编码基因ldh表达,来抑制细菌产酸,伊犁黑蜂蜂胶可能是通过此途径抑制变形链球菌产酸,从而达到防龋的效果。

Objective To explore the possible mechanisms of the inhibion of ethanol extracts of propolis （EEP） from Ili balck bee on acid production of Streptococcus mutans by means of comparing its effects on lactate dehydro- genase （LDH） activity in different states and expression of ldh gene. Methods Streptococcus mutans were cultured in both planktonic and biofilm states, and treated with BHI culture media containing various concentrations of EEP or BHI culture media containing 50 mg/L of NaF respectively for 18 hours. Then the Coenzyme I reduction method was used to assay the LDH activity in each group. The expression of ldh gene was detected by means of RT- qPCR. Results （ 1 ） Significant inhibition of LDH activity was found in both EEP and NaF groups with statistical significance in both planktonic and biofilm states （ P 〈 0. 05 ）. （2） In the planktonic state, significant down-regu- lation of ldh was detected in study groups and positive control group; the ldh down-regulation ratio in study groups was greater than that in positive control group （ P 〈 0. 05 ）. In the state of biofilm, the ldh expression in the study groups with 1 MBEC, 1/2 MBEC and 1/4 MBEC of EEP was significantly down-regulated compared to the negative control （P 〈 0. 05 ）, while the positive control group was not different from negative control group （P 〉 0. 05 ）. Conclusion Propolis from Ili black bee can inhibit the lactate dehvdrogenase acitivitv of Streptococcus mutans and the expression of its ldh gene in both planktonic and biofilm state, thus possibly inhibit the acid production of the bacteria and play a cariostatic role.