亚抑菌浓度痰热清注射液抑制金黄色葡萄球菌α-溶血素溶血活力

Study on Sub-minimal Inhibitory Concentration of Tanreqing Injection in Inhibiting Hemolytic Activity of Staphylococcus aureus α-hemolysin

目的:研究痰热清注射液(TRQ)在亚抑菌浓度下是否抑制金黄色葡萄球菌α-溶血素的产生、释放,进而缓解感染过程中机体损伤,为更好地指导临床用药提供实验依据。方法:微孔板法和时间-生长曲线首先测定TRQ对金黄色葡萄球菌的最小抑菌浓度(MIC)和对细菌生长的影响;而后不同亚抑菌浓度TRQ与细菌或细菌上清共培养,再与脱纤维兔血共孵育,检测TRQ对金黄色葡萄球菌α-溶血素的抑制和中和作用;细胞增殖与活性检测(CCK-8)法检测TRQ对金黄色葡萄球菌感染介导的人肺泡上皮细胞(A549)损伤的保护作用;实时荧光定量聚合酶链式反应(Real-time PCR)检测亚抑菌浓度TRQ对金黄色葡萄球菌α-溶血素调控基因hla,agrA mRNA表达的影响。结果:TRQ对金黄色葡萄球菌的MIC为1/8原液,亚抑菌浓度(1/64MIC~1/16MIC)TRQ不影响细菌生长;1/64MIC~1/16MIC浓度TRQ具有抑制和中和α-溶血素溶血活力的作用,且对金黄色葡萄球菌感染介导的A549细胞损伤具有保护作用;其对α-溶血素的抑制作用与抑制hla,agrA mRNA表达密切相关。结论:亚抑菌浓度TRQ能抑制和中和金黄色葡萄球菌的α-溶血素溶血活力,对金黄色葡萄球菌感染介导的A549细胞损伤具有保护作用,其抑制α-溶血素的机制与干扰agr调控系统密切相关。

Objective: To study whether Tanreqing injection(TRQ)can alleviate the body injury in the process of infection by inhibiting the production and release of α-hemolysin of Staphylococcus aureus under sub-minimal inhibitory concentration, and to provide experimental basis for better guidance of clinical medication. Method:The effects of TRQ on the minimum inhibitory concentration(MIC)and bacterial growth of S. aureus were determined firstly by microplate method and time-growth curve. The different sub-minimal inhibitory concentrations of TRQ were co-cultured with bacteria or bacterial supernatants,and then co-incubated with defibrillated rabbit blood to detect the inhibitory and neutralizing effects of TRQ on S.aureus α-hemolysin.Cell counting kit-8(CCK-8)cell viability assay was used to detect the protective effect of TRQ on S. aureusmediated damage to human alveolar epithelial cells(A549). Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect the effect of sub-minimal inhibitory concentration of TRQ on the mRNA expression of S. aureus α-hemolysin regulatory genes hla and agrA. Result: The MIC of TRQ to S.aureus was 1/8 of the stock solution,and the sub-minimal inhibitory concentration(1/64 MIC-1/16 MIC)TRQ used in this study did not affect the growth of bacteria. 1/64 MIC-1/16 MIC TRQ had the effect of inhibiting and neutralizing the hemolytic activity of α-hemolysin,with a protective effect on S. aureus supernatant-mediated A549 cell damage,and its inhibitory effect on α-hemolysin was closely related to the inhibition of hla and agrA mRNA expression. Conclusion: The sub-minimal inhibitory concentration TRQ can inhibit and neutralize the hemolytic activity of α-hemolysin of S. aureus,with a protective effect on A549 cell damage mediated by S. aureus infection,and its mechanism of inhibiting α-hemolysin is closely related to the interference with agr regulatory system.