外源性一氧化碳释放分子2对大肠杆菌活力及毒力的作用

Effects of exogenous carbon monoxide-releasing molecules 2 on the vitality and toxicity of E. coli

目的 探讨外源性一氧化碳释放分子2（CORM-2）对大肠杆菌ATCC 25922菌株活力及毒力的作用及可能机制。方法 （1）体外实验1。将菌株按照随机数字表法分为细菌组、细菌＋1.2 mmol/L CORM-2组、细菌＋1.6 mmol/L CORM-2组、细菌＋1.2 mmol/L无活性CORM-2（iCORM-2）组、细菌＋1.6 mmol/L iCORM-2组,每组样本数为6。细菌组不添加任何物质，其余4组添加相应浓度的CORM-2或iCORM-2。于培养0、3、5、8、10、12、16、20、24、27、30、48 h，测定各组菌液的增殖活性，结果以吸光度值（波长为600 nm）表示；同时进行菌落计数。（2）体外实验2。另取菌株，将其分为细菌组和细菌＋0.8 mmol/L CORM-2组，采用基因芯片筛查出大肠杆菌的4个相关基因fliA、dnaK、marA和waaQ进行实时定量PCR（qRT-PCR），检测各基因表达量。（3）在体研究。另取菌株同体外实验1分组及处理，培养至细菌组菌液的吸光度值（波长600 nm）为0.4时，各组收集0.5 mL菌液。将72只C57BL/6小鼠按照随机数字表法分为空白对照组、细菌组、细菌＋1.2 mmol/L CORM-2组、细菌＋1.6 mmol/L CORM-2组、细菌＋1.2 mmol/L iCORM-2组、细菌＋1.6 mmol/L iCORM-2组，每组12只。空白对照组小鼠不行任何处理，其余5组小鼠取对应有或无添加物的0.5 mL菌液进行腹腔注射。注射后对后5组小鼠进行大体观察。注射后6、12 h检测后5组小鼠血清中TNF-α、IL-6水平，注射后12 h收集小鼠肝、肺组织标本检测髓过氧化物酶（MPO）活性。空白对照组小鼠行相同检测。对数据进行重复测量设计方差分析、析因设计方差分析、单因素方差分析和t检验。结果 （1）体外实验1。与细菌组和细菌＋1.2 mmol/L iCORM-2组比较，细菌＋1.2 mmol/L CORM-2组多数时相点细菌增殖活性明显受抑，菌落明显数量减少（F值分别为1170.80、217.52，P值均小于0.01）；与细菌组和细菌＋1.6 mmol/L iCORM-2组比较，细菌＋1.6 mmol/L CORM-2组多数时相点细菌增殖活性亦明显受抑，菌落数量亦明显减少（F值分别为7948.34、14 432.85，P值均小于0.01）。（2）体外实验2。qRT-PCR检测结果显示：与细菌组比较，细菌＋0.8 mmol/L CORM-2组fliA基因表达下调，dnaK、marA和waaQ基因表达上调（t值分别为30.28、-165.54、-168.88、-187.28，P值均小于0.01）。（3）在体研究。细菌组和细菌＋1.2 mmol/L iCORM-2组、细菌＋1.6 mmol/L iCORM-2组小鼠出现诸如精神萎靡、呼吸急促等症状，细菌＋1.2 mmol/L CORM-2组、细菌＋1.6 mmol/L CORM-2组小鼠上述症状轻微或不明显。注射后6 h，细菌组、细菌＋1.2 mmol/L iCORM-2组小鼠血清TNF-α、IL-6水平分别为（647.3±3.8）pg/mL、（3.44±0.22）ng/mL以及（639.3±0.8）pg/mL、（2.47±0.32）ng/mL，明显高于细菌＋1.2 mmol/L CORM-2组［（124.6±10.7）pg/mL、（1.03±0.16）ng/mL，t值为15.22~84.03，P值均小于0.01］。与细菌组和细菌＋1.6 mmol/L iCORM-2组比较，注射后6、12 h细菌＋1.6 mmol/L CORM-2组小鼠血清TNF-α、IL-6水平亦均明显降低（t值为19.27~245.34，P值均小于0.01）。注射后12 h，细菌＋1-2 mmol/L CORM-2组小鼠肝、肺组织中MPO活性明显低于细菌组、细菌＋1.2 mmol/L iCORM-2组，细菌＋1.6 mmol/L CORM-2组小鼠肝、肺组织中MPO活性亦明显低于细菌组、细菌＋1.6 mmol/L iCORM-2组（t值分别为17.36~18.92、2.35~3.61，P值均小于0.01）。结论 CORM-2能够明显抑制大肠杆菌的活力和毒力，其抑制作用可能与其调节大肠杆菌部分重要的靶基因（fliA、dnaK、marA和waaQ）有关。

ObjectiveTo explore the effects of exogenous carbon monoxide-releasing molecules 2 （CORM-2） on the vitality and toxicity of E.coli ATCC 25922, and to analyze the potential mechanism. Methods （1） In vitro experiments. Standard strains of E.coli ATCC 25922 were divided into groups A （without addition）, B, C, D, and E according to the random number table, and then the latter 4 groups were respectively cultured with 1.2 mmol/L CORM-2, 1.6 mmol/L CORM-2, 1.2 mmol/L inactive CORM-2 （iCORM-2）, 1.6 mmol/L iCORM-2, with six samples in each group. After being cultured for 0, 3, 5, 8, 10, 12, 16, 20, 24, 27, 30, 48 hours, proliferative vitality of E.coli was examined （denoted as absorption value under 600 nm wavelength）, and bacteria number was counted. Other standard strains of E.coli ATCC 25922 were divided into groups F （without addition） and G （cultured with 0.8 mmol/L CORM-2）, the expressions of genes fliA, dnaK, marA, and waaQ related to E.coli were detected by quantitative real-time （qRT）-PCR. （2） In vivo experiments. Other standard strains of E.coli ATCC 25922 were grouped as A′, B′, C′, D′, and E′ and treated with the same method as that in groups A, B, C, D, and E, and 0.5 mL bacterial liquid of each group were collected when the absorption value of bacterial liquid in group A′ was equal to 0.4 （under 600 nm wavelength）. Seventy-two C57BL/6 mice were divided into groups, namely blank control （without treatment）, H, I, J, K, and L according to the random number table, with 12 mice in each group. The mice in the latter 5 groups were intraperitoneally injected with 0.5 mL bacterial suspension of groups A′, B′, C′, D′, and E′ respectively. After injection, general condition of mice in groups H, I, J, K, and L was observed. The serum levels of TNF-α and IL-6 were determined at post injection hour （PIH） 6, 12. The liver and lung samples were harvested for determination of myeloperoxidase （MPO） activity at PIH 12. The same process was carried out in blank control group. Data were processed with repeated measure analysis of variance （ANOVA）, factorial design ANOVA, oneway ANOVA, and t test. Results （1） In vitro experiments. Compared with those of groups A and D, the proliferative vitality and bacteria number of E.coli in group B were all decreased （with F values respectively 1170.80, 217.52, P values all below 0.01）. Compared with those of groups A and E, the proliferative vitality and bacteria number of E.coli in group C were also obviously decreased （with F values respectively 7948.34, 14 432.85, P values all below 0.01）. Compared with those in group F, the expression of fliA was downregulated, while the expressions of dnaK, marA, and waaQ were upregulated in group G （with t values 30.28, -165.54, -168.88, -187.28, P values all below 0.01）. （2） In vivo experiments. Symptoms including listlessness and tachypnea were observed in mice in groups H, K, and L, and they were ameliorated or not obvious in groups I and J. At PIH 6, the serum levels of TNF-α and IL-6 in groups H and K were respectively （647.3±3.8） pg/mL, （3.44±0.22） ng/mL and （639.3±0.8） pg/mL, （2.47±0.32） ng/mL, which were obviously higher than those in group I [（124.6±10.7） pg/mL, （1.03±0.16） ng/mL, with t values from 15.22 to 84.03, P values all below 0.01]. The serum levels of TNF-α and IL-6 in group J at PIH 6, 12 were also obviously decreased as compared with those in groups H and L （with t values from 19.27 to 245.34, P values all below 0.01）. MPO activity of liver and lung tissues were significantly attenuated in group I at PIH 12 as compared with those in groups H and K, and it was also attenuated in group J when compared with those in groups H and L （with t values respectively from 17.36 to 18.92 and 2.35 to 3.61, P values all below 0.01）. Conclusions CORM-2 can obviously inhibit the vitality and toxicity of E.coli, which might be attributable to regulation of expressions of genes fliA, dnaK, marA, and waaQ of E.coli.