鸡蛋清溶菌酶对头孢他啶诱导的痢疾杆菌溶解和致炎活性物质释放的抑制作用

Inhibitory effect of egg white lysozyme on ceftazidimeinduced release of inflammatory factor from Shigella dysenteriae

目的 :研究鸡蛋清溶菌酶 (L ZM)与头孢他啶 (CFT)联合应用对痢疾杆菌的生长、形态及其致炎活性物质释放的影响。方法 :在营养肉汤或稀释血液培养的痢疾杆菌液中加入 L ZM或 CFT作用一定时间后 ,采用营养琼脂平板接种 ,计数各培养管中存活细菌数。将细菌悬液离心 ,收集培养上清液 ,将上清液加入到培养的巨噬细胞中 ,测定其对巨噬细胞炎性因子如肿瘤坏死因子 α(TNFα)、一氧化氮 (NO)的诱生能力 ;同时用光学显微镜观察各培养管中细菌的形态。结果 :营养肉汤培养中的痢疾杆菌在 CFT(16× MIC、32× MIC)作用下 ,可造成细菌迅速溶解 ,同时伴有大量致炎活性物质释放 ,其培养上清在体外培养的巨噬细胞上可诱导大量的TNFα、NO产生 ;L ZM与 CFT联合使用既能阻止细菌溶解 ,又能保持与 CFT单独用药相当的杀菌作用 ;镜下观察 ,死亡的细菌相互粘连 ,形成聚集体 ;联合用药后可降低巨噬细胞的激活 ,减少 TNFα、NO的产生。在血液培养系统中 ,CFT(16× MIC、32× MIC)对痢疾杆菌有较强的杀菌作用 ,但较在肉汤培养体系中弱 ;L ZM与 CFT联合用药较 CFT(16× MIC、32× MIC)单独用药有更强的杀菌作用 ,并能显著降低 NO的释放。结论 :L ZM与 CFT联合应用能阻止痢疾杆菌溶解 ,降低细菌致炎活性物质 (主要为内毒素 )

Objective:To investigate the effects of egg white lysozyme(LZM) in combination with Ceftazidime(CFT) on the growth,the morphological changes and the release of inflammatory substances of Shigella dysenteriae.Methods:Shigella dysenteriae ATCC 51067 which was cultured in the nutrition broth or the diluted rabbit blood was treated with CFT alone or in combination with LZM.The viability of the bacteria in the various cultures was determined by inoculating the suspensions from the treated cultures onto the nutrition agar plates.The supernatants from the bacterial cultures were collected by centrifuge and added into RAW 264 7 cells,a macrophage cell line.Tumor necrosis factorα(TNFα) productivity of the supernatants from the cell cultures was assayed with enzymelinked immunosorbent assay(ELISA) methods.Nitric oxide(NO) productivity of the supernatants from the cell cultures was assayed with greiss kits.In addition,the morphological changes of the bacteria were observed with a microscopy.Results:Shigella dysenteriae ATCC 51067 could be rapidly lysed causing a large amount of inflammatory factor releasing from bacteria into the culture supernatant after the bacteria were treated with CFT(16×MIC,32×MIC) when it was cultured in the nutrition broth.Those supernatants induced a plenty of TNFα production from RAW 264 7 cells.LZM in combination with CFT blocked the lysis of bacteria without disturbance of bactericidal effect of CFT.The obituary bacteria conglutinated each other and forming a lot of large congeries.As a result,the combination LZM with CFT reduced release of inflammatory substances and depressed the following activation of macrophages assessed by TNFα and NO production.A study by using diluted rabbit blood culture showed that more bacteria were killed and less amount of inflammatory factor was released from bacteria into the culture supernatants after the conbination treatment of LZM with CFT than the treatment with CFT(16×MIC,32×MIC) alone.The study also showed that the combinative LZM with CFT also depressed the following activation of macrophages assessed by NO production.Conclusions:The combination of LZM with CFT can prevent bacteriolysis and decrease the release of inflammatory factors (mostly including endotoxin),and consequently abate endotoxemia.