苦参总碱抗表皮葡萄球菌的作用机制研究

Study on mechanism of total alkaloids of Sophora flavescens against Staphylococcus epidermidis

目的考察苦参总碱对表皮葡萄球菌体外培养生物膜的抑制作用,观察苦参总碱是否会影响表皮葡萄球菌对乳酸和过氧化氢的耐受性,并探索其可能的作用机制。方法体外培养表皮葡萄球菌产膜菌株,使用半定量黏附实验、结晶紫染色法及刚果红实验评估苦参总碱对表皮葡萄球菌生物膜的抑制作用。采用扫描电镜观测表皮金黄色葡萄球菌生物膜形成。苦参总碱处理后,检测表皮葡萄球菌对氧化胁迫的耐受力的变化,利用实时荧光定量PCR法检测氧化应激响应基因serp2195和gpxA-2表达水平变化。结果结晶紫染色法显示5.0 mg/mL的苦参总碱对表皮葡萄球菌生物膜的形成具有显著的抑制作用(P<0.001),且与刚果红实验结果保持一致。苦参总碱作用后表皮葡萄球菌对氧化胁迫耐受性明显减弱,对乳酸的敏感性增强;实时荧光定量PCR显示氧化应激响应基因serp2195和gpxA-2转录水平明显下降。结论苦参总碱对表皮葡萄球菌生物膜形成具有显著抑制作用,减弱细菌对氧化胁迫耐受性,其作用机制可能与下调表皮葡萄球菌氧化应激响应基因serp2195和gpxA-2表达相关;同时苦参总碱可以减弱表皮葡萄球菌对乳酸耐受性。

Objective To detect the effect of alkaloids of Sophora flavescens on biofilm formation in Staphylococcus epidermidis in vitro,and to observe whether total alkaloids of S.flavescens can affect the tolerance of Staphylococcus epidermidis to lactic acid and hydrogen peroxide,then explore the possible anti-microbial mechanism of alkaloids in Sophora flavescens.Methods The biofilm of Staphylococcus epidermidis was prepared in vitro.We evaluated the effect of alkaloids in S.flavescens on the biofilm-forming ability of Staphylococcus epidermidis via 96-well cell culture microtiter plates with crystal violet staining as well as Congo red experiment.The biofilm formation of Staphylococcus aureus was observed using scanning electron microscope.Then,we measured the change of tolerance to oxidative stress and sensibility to antibiotics for Staphylococcus epidermidis being treated by alkaloids in S.flavescens Ait.qRT-PCR analysis was performed to show the relative transcript level of serp2195 and gpxA-2 upon Staphylococcus epidermidis strain exposed to alkaloids in S.flavescens at different concentrations.Results Alkaloids in S.flavescens significantly prevented biofilm formation in Staphylococcus epidermidis(P<0.001)at the concentration of 5.0 mg/mL during crystal violet staining and Congo red experiment,and thus weakened tolerance to oxidative stress and enhanced sensibility to lactate for Staphylococcus epidermidis.Alkaloids in S.flavescens could downregulate the transcript level of serp2195 and gpxA-2.Conclusion Alkaloids in S.flavescens has a significant inhibitory effect on biofilm formation of Staphylococcus epidermidis,which weakens the tolerance of bacteria to oxidative stress.The mechanism may be realized by downregulating the transcript level of serp2195 and gpxA-2,and alkaloids in S.flavescens could reduce Staphylococcus epidermidis tolerance to lactic acid.To sum up,Alkaloids in S.flavescens can be applied to the gynecological infection caused by biofilm-producing bacteria.