目的 探讨大黄素对多重耐药金黄色葡萄球菌(多重耐葡菌)的抑菌效果及其作用机制。方法 全自动微生物鉴定药敏分析仪鉴定临床分离耐甲氧西林金黄色葡萄球菌(MRSA)株耐药情况,二倍稀释法检测MRSA标准株和临床分离株的大黄素的最低抑菌浓度(MIC)、最低杀菌浓度(MBC)和半数抑制浓度(IC50)。以MRSA标准株为研究背景,将其分为对照组、大黄素(1×MIC)组和大黄素(2×MIC)组,观察一段时间内(0、0.5、1、2、4、8、16 h)MRSA标准株细菌数量,双色荧光染色观察细胞膜通透性,实时定量PCR(RT-PCR)检测生物膜相关基因表达水平。制备MRSA感染小鼠模型并将其分为感染组、大黄素(2.5 mg/kg)组、大黄素(5 mg/kg)组、大黄素(10 mg/kg)组,另设置不做感染处理的对照组,感染后1 h大黄素各组小鼠给予相应剂量干预,给药12 h后,比较小鼠血液中细菌含量、炎性因子水平和腹腔巨噬细胞吞噬功能。结果 耐药菌结果证实临床分离的4株均为MRSA。MRSA标准株和临床分离株的大黄素MIC分别为4、8、8、8、2 μg/ml,MBC分别为8、16、8、8、4 μg/ml,IC50分别为2.51、4.15、5.36、4.01、1.43 μg/ml。大黄素(1×MIC)组、大黄素(2×MIC)组的MRSA标准株细菌数量、cidA、icaA、agrA、sortaseA和sarA表达低于对照组(P<0.05),且荧光染色图像中有红色荧光。大黄素(2.5 mg/kg)组、大黄素(5 mg/kg)组、大黄素(10 mg/kg)组小鼠血液中耐药菌含量和血清肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-8(IL-8)、干扰素-γ(INF-γ)、降钙素原(PCT)水平低于感染组(P<0.05),腹腔巨噬细胞吞噬率和吞噬指数均高于感染组(P<0.05)。结论 大黄素对多重耐葡菌具有抑菌作用,可提高菌株细胞膜的通透性,调节细胞生物膜的相关基因表达水平,还可降低MRSA感染小鼠体内炎症水平并恢复巨噬细胞吞噬功能。
Abstract
Objective To explore antibacterial effect of emodin on multi-resistant staphylococcus aureus (staphylococcus multidrug-resistant) and its mechanism. Methods The drug resistance of clinically isolated methicillin-resistant staphylococcus aureus (MRSA) was determined by full-automatic microbial identification and drug sensitivity analyzer. The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and the half inhibitory concentration (IC50) of emodin in MRSA standard strains and clinically isolated strains were detected by double dilution method. With MRSA standard strains as the research background, they were divided into control group, emodin (1×MIC) group and emodin (2×MIC) group. The number of bacteria in MRSA standard strains was observed during a period of time (0, 0.5, 1, 2, 4, 8, 16 h). The permeability of cell membrane was observed by double-color fluorescent staining. The levels of biofilm-related genes were detected by real-time quantitative PCR (RT-PCR). The models of mice with MRSA infection were prepared, and they were divided into infection group, emodin (2.5 mg/kg) group, emodin (5 mg/kg) group and emodin (10 mg/kg) group. The mice models without infection treatment were enrolled as control group. At 1h after infection, mice in all emodin groups were given corresponding doses of emodin. At 12h after administration, bacteria content in blood, inflammatory factors and phagocytic function of peritoneal macrophages were compared. Results The results of drug-resistant bacteria confirmed that the 4 clinically isolated strains were MRSA. MIC, MBC and IC50 of emodin for standard and clinically isolated MRSA strains were (4, 8 , 8, 8, 2 μg/ml),(8, 16, 8, 8, 4 μg/ml) and (2.51, 4.15, 5.36, 4.01, 1.43 μg/ml), respectively. The number of standard MRSA strains, expressions of cidA, icaA, agrA, sortaseA and sarA in emodin (1×MIC) group and emodin (2×MIC) group were lower than those in control group (P<0.05), and there was red fluorescence in fluorescence images. The levels of drug-resistant bacteria in blood, serum tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-8 (IL-8), interferon gamma (INF-γ) and procalcitonin (PCT) in emodin (2.5 mg/kg) group, emodin (5 mg/kg) group and emodin (10 mg/kg) group were lower than those in infection group (P<0.05), phagocytic rate and index of peritoneal macrophages were higher than those in infection group (P<0.05). Conclusions Emodin can improve the permeability of cell membrane, regulate the expression of biofilm-related genes, reduce inflammation level and recover the phagocytic function of macrophages.
关键词
多重耐药金黄色葡萄球菌 /
大黄素 /
抑菌效果 /
细胞通透性 /
炎症反应 /
作用机制
Key words
multi-resistant staphylococcus aureus /
emodin /
antibacterial effect /
cell permeability /
inflammation response /
action mechanism
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