人参皂苷Rh2型联合氟化钠对变异链球菌生长及生物膜形成的抑制作用

doi:10.13481/j.1671-587X.20220218

摘要/Abstract

摘要： 目的

探讨人参皂苷Rh2型（G-Rh2）联合氟化钠（NaF）对体外变异链球菌生长及生物膜形成的抑制作用，为其将来在口腔临床上的应用提供理论依据。

方法

采用微量稀释法分别测量G-Rh2与NaF对变异链球菌的半数最小抑制浓度（MIC₅₀）；采用棋盘微量稀释法测量G-Rh2与NaF联合应用于变异链球菌的MIC₅₀值，并计算分级抑菌浓度（FIC）指数来判断两者的联合效应，当FIC<0.5时证明2种药物间具有协同抑制变异链球菌的作用；采用结晶紫染色实验分别测量G-Rh2与NaF对变异链球菌的半数最小生物膜抑制浓度（MBIC₅₀）值；实验分为空白对照组、MIC₅₀G-Rh2组、MIC₅₀NaF组和MIC₅₀G-Rh2+MIC₅₀NaF联合组，采用生长曲线及产酸实验评估各组变异链球菌浮游菌生长速度及产酸抑制率；实验分为空白对照组、MBIC₅₀G-Rh2组、MBIC₅₀NaF组及MBIC₅₀G-Rh2+MBIC₅₀NaF联合组，采用结晶紫染色实验及扫描电镜观察各组变异链球菌生物膜形成量及结构的变化；空白对照组内不含任何药物。

结果

G-Rh2与NaF作用于变异链球菌的MIC₅₀分别为25.000和125.000 mg·L^－1；G-Rh2与NaF联合应用时MIC₅₀分别为5.00和31.25 mg·L^－1，FIC指数为0.45，小于0.50，证明2种药物间具有协同抑制变异链球菌的作用；G-Rh2与NaF作用于变异链球菌的MBIC₅₀分别为22.5和62.5 mg·L^－1。与空白对照组、MIC₅₀G-Rh2组和MIC₅₀NaF组比较，MIC₅₀G-Rh2+MIC₅₀NaF联合组变异链球菌的生长速度明显降低（P<0.05）。与空白对照组比较，MIC₅₀G-Rh2组、MIC₅₀NaF组和MIC₅₀G-Rh2+MIC₅₀NaF联合组ΔpH值均降低，且MIC₅₀G-Rh2+MIC₅₀NaF联合组ΔpH值低于MIC₅₀G-Rh2组和MIC₅₀NaF组（P<0.05）；MIC₅₀G-Rh2+MIC₅₀NaF联合组的产酸抑制率高于MIC₅₀G-Rh2组和MIC₅₀NaF组（P<0.05）。MBIC₅₀G-Rh2+MBIC₅₀NaF联合组的生物膜形成量明显少于空白对照组、MBIC₅₀G-Rh2组和MBIC₅₀NaF组（P<0.01）。扫描电镜观察，MBIC₅₀G-Rh2组和MBIC₅₀NaF组生物膜厚度降低，胞外基质量减少，细菌之间排列疏松，而MBIC₅₀G-Rh2+MBIC₅₀NaF联合组生物膜结构消失，细菌数量减少且细菌形态发生变化。

结论

G-Rh2与NaF联合应用对体外变异链球菌生长及生物膜形成具有协同抑制作用。

关键词: 变异链球菌, 人参皂苷Rh2型, 氟化钠, 联合药敏实验

Abstract: Objective

To explore the inhibitory effects of ginsenoside Rh2 （G-Rh2） combined with sodium fluoride （NaF） on the growth of Streptococcus mutans and biofilm formation in vitro， and to provide theoretical basis for their application in stomatological clinic.

Methods

The half minimum inhibitory concentrations （MIC₅₀） of G-Rh2 and NaF against Streptococcus mutans were measured by microdilution method；the MIC₅₀ value of G-Rh2 combined with NaF on Streptococcus mutans was measured by chessboard microdilution method，and the fractional inhibitory concentration （FIC） value was calculated to judge the combined effect of these two drugs. When FIC<0.5， it was proved that the two drugs had synergistic inhibitory effect on Streptococcus mutans. The half minimum biofilm inhibition concentrations（MBIC₅₀） of G-Rh2 and NaF against Streptococcus mutans were measured by crystal violet staining test.The experiment was divided into blank control group， MIC₅₀G-Rh2 group， MIC₅₀NaF group and MIC₅₀G-Rh2+MIC₅₀NaF combined group， the growth curve and acid production test were used to evaluate the growth speeds and inhibitory rates of acid production of Streptococcus mutans in each group； the experiment was divided into blank control group， MBIC₅₀G-Rh2 group， MBIC₅₀NaF group and MBIC₅₀G-Rh2+MBIC₅₀NaF combined group，the changes of biofilm formation of Streptococcus mutans and structures were observed by crystal violet staining test and scanning electron microscope in each group.The blank control group did not contain any drugs.

Results

The MIC₅₀ of G-Rh2 and NaF acting on Streptococcus mutans alone were 25.000 and 125.000 mg·L^－1，respectively；the MIC₅₀ of G-Rh2 combined with NaF on Streptococcus mutans were 5.00 and 31.25 mg·L^－1，the FIC index was 0.45 and less than 0.5， which proved that the two drugs had synergistic inhibitory effect on Streptococcus mutans.The MBIC₅₀ of G-Rh2 and NaF were 22.5 and 62.5 mg·L^－1.The growth speed of Streptococcus mutans in MIC₅₀G-Rh2+MIC₅₀NaF combined group was significantly lower than those in blank control group， MIC₅₀G-Rh2 group and MIC₅₀NaF group（P<0.05）.The ΔpH values in MIC₅₀G-Rh2 group，MIC₅₀NaF group and MIC₅₀G-Rh2+MIC₅₀NaF combined group were lower than that in blank control group， and the ΔpH value in MIC₅₀G-Rh2+MIC₅₀NaF combined group was lower than those in MIC₅₀G-Rh2 group and MIC₅₀NaF group（P<0.05）.The inhibition rate of acid production in MIC₅₀G-Rh2+MIC₅₀NaF combined group was higher than those in MIC₅₀G-Rh2 group and MIC₅₀NaF group（P<0.05），and the amount of biofilm formation in MBIC₅₀G-Rh2+MBIC₅₀NaF combined group was significantly lower than those in blank control group，MBIC₅₀G-Rh2 group and MBIC₅₀NaF group （P<0.01）.The scanning electron microscope results showed that the thickness of biofilm， and the mass of extracellular base in MBIC₅₀G-Rh2 group and MBIC₅₀NaF group were decreased， the arrangement of bacteria was loose； while in MBIC₅₀G-Rh2+MBIC₅₀NaF combined group， the biofilm structure was disappeared， the number of bacteria was decreased，and the morphology of bacteria was changed.

Conclusion

The combination of G-Rh2 and NaF has the synergistic inhibitory effect on the growth and biofilm formation of Streptococcus mutans in vitro.

Key words: Streptococcus mutans, Ginsenoside Rh2, Sodium fluoride, Combined drug susceptibility experiments

中图分类号:

R781.1

方圆,刘晓璇,王瑞. 人参皂苷Rh2型联合氟化钠对变异链球菌生长及生物膜形成的抑制作用[J]. 吉林大学学报(医学版), 2022, 48(2): 407-413.

Yuan FANG,Xiaoxuan LIU,Rui WANG. Inhibitory effects of ginsenoside Rh2 combined with sodium fluoride on growth and biofilm formation of Streptococcus mutans[J]. Journal of Jilin University(Medicine Edition), 2022, 48(2): 407-413.

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