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6h,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.
目的 探討外源性一氧化碳釋放分子2(CORM-2)對大腸桿菌ATCC 25922菌株活力及毒力的作用及可能機製. 方法 (1)體外實驗1.將菌株按照隨機數字錶法分為細菌組、細菌+1.2 mmol/L CORM-2組、細菌+1.6 mmol/L CORM-2組、細菌+1.2 mmol/L無活性CORM-2(iCORM-2)組、細菌+ 1.6 mmol/L iCORM-2組,每組樣本數為6.細菌組不添加任何物質,其餘4組添加相應濃度的CORM-2或iCORM-2.于培養0、3、5、8、10、12、16、20、24、27、30、48 h,測定各組菌液的增殖活性,結果以吸光度值(波長為600 nm)錶示；同時進行菌落計數.(2)體外實驗2.另取菌株,將其分為細菌組和細菌+0.8 mmol/L CORM-2組,採用基因芯片篩查齣大腸桿菌的4箇相關基因fliA、dnaK、marA和waaQ進行實時定量PCR(qRT-PCR),檢測各基因錶達量.(3)在體研究.另取菌株同體外實驗1分組及處理,培養至細菌組菌液的吸光度值(波長600 nm)為0.4時,各組收集0.5 mL菌液.將72隻C57BL/6小鼠按照隨機數字錶法分為空白對照組、細菌組、細菌+1.2 mmol/L CORM-2組、細菌+1.6 mmol/L CORM-2組、細菌+1.2 mmol/L iCORM-2組、細菌+1.6 mmol/L iCORM-2組,每組12隻.空白對照組小鼠不行任何處理,其餘5組小鼠取對應有或無添加物的0.5 mL菌液進行腹腔註射.註射後對後5組小鼠進行大體觀察.註射後6、12 h檢測後5組小鼠血清中TNF-α、IL-6水平,註射後12 h收集小鼠肝、肺組織標本檢測髓過氧化物酶(MPO)活性.空白對照組小鼠行相同檢測.對數據進行重複測量設計方差分析、析因設計方差分析、單因素方差分析和t檢驗. 結果 (1)體外實驗1.與細菌組和細菌+ 1.2 mmol/L iCORM-2組比較,細菌+1.2 mmol/L CORM-2組多數時相點細菌增殖活性明顯受抑,菌落明顯數量減少(F值分彆為1170.80、217.52,P值均小于0.01)；與細菌組和細菌+ 1.6 mmol/L iCORM-2組比較,細菌+1.6 mmol/L CORM-2組多數時相點細菌增殖活性亦明顯受抑,菌落數量亦明顯減少(F值分彆為7948.34、14 432.85,P值均小于0.01).(2)體外實驗2.qRT-PCR檢測結果顯示:與細菌組比較,細菌+0.8 mmol/L CORM-2組fliA基因錶達下調,dnaK、marA和waaQ基因錶達上調(t值分彆為30.28、-165.54、-168.88、-187.28,P值均小于0.01).(3)在體研究.細菌組和細菌+ 1.2 mmol/L iCORM-2組、細菌+1.6 mmol/L iCORM-2組小鼠齣現諸如精神萎靡、呼吸急促等癥狀,細菌+ 1.2 mmol/L CORM-2組、細菌+1.6 mmol/L CORM-2組小鼠上述癥狀輕微或不明顯.註射後6h,細菌組、細菌+1.2 mmol/L iCORM-2組小鼠血清TNF-α、IL-6水平分彆為(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,明顯高于細菌+ 1.2 mmol/L CORM-2組[(124.6±10.7) pg/mL、(1.03 ±0.16)ng/mL,t值為15.22～84.03,P值均小于0.01].與細菌組和細菌+1.6 mmol/L iCORM-2組比較,註射後6、12 h細菌+1.6 mmol/L CORM-2組小鼠血清TNF-α、IL-6水平亦均明顯降低(t值為19.27～245.34,P值均小于0.01).註射後12 h,細菌+1.2 mmol/L CORM-2組小鼠肝、肺組織中MPO活性明顯低于細菌組、細菌+ 1.2 mmol/L iCORM-2組,細菌+1.6 mmol/L CORM-2組小鼠肝、肺組織中MPO活性亦明顯低于細菌組、細菌+ 1.6 mmol/L iCORM-2組(t值分彆為17.36～18.92、2.35 ～3.61,P值均小于0.01).結論 CORM-2能夠明顯抑製大腸桿菌的活力和毒力,其抑製作用可能與其調節大腸桿菌部分重要的靶基因(fliA、dnaK、marA和waaQ)有關.
목적 탐토외원성일양화탄석방분자2(CORM-2)대대장간균ATCC 25922균주활력급독력적작용급가능궤제. 방법 (1)체외실험1.장균주안조수궤수자표법분위세균조、세균+1.2 mmol/L CORM-2조、세균+1.6 mmol/L CORM-2조、세균+1.2 mmol/L무활성CORM-2(iCORM-2)조、세균+ 1.6 mmol/L iCORM-2조,매조양본수위6.세균조불첨가임하물질,기여4조첨가상응농도적CORM-2혹iCORM-2.우배양0、3、5、8、10、12、16、20、24、27、30、48 h,측정각조균액적증식활성,결과이흡광도치(파장위600 nm)표시；동시진행균락계수.(2)체외실험2.령취균주,장기분위세균조화세균+0.8 mmol/L CORM-2조,채용기인심편사사출대장간균적4개상관기인fliA、dnaK、marA화waaQ진행실시정량PCR(qRT-PCR),검측각기인표체량.(3)재체연구.령취균주동체외실험1분조급처리,배양지세균조균액적흡광도치(파장600 nm)위0.4시,각조수집0.5 mL균액.장72지C57BL/6소서안조수궤수자표법분위공백대조조、세균조、세균+1.2 mmol/L CORM-2조、세균+1.6 mmol/L CORM-2조、세균+1.2 mmol/L iCORM-2조、세균+1.6 mmol/L iCORM-2조,매조12지.공백대조조소서불행임하처리,기여5조소서취대응유혹무첨가물적0.5 mL균액진행복강주사.주사후대후5조소서진행대체관찰.주사후6、12 h검측후5조소서혈청중TNF-α、IL-6수평,주사후12 h수집소서간、폐조직표본검측수과양화물매(MPO)활성.공백대조조소서행상동검측.대수거진행중복측량설계방차분석、석인설계방차분석、단인소방차분석화t검험. 결과 (1)체외실험1.여세균조화세균+ 1.2 mmol/L iCORM-2조비교,세균+1.2 mmol/L CORM-2조다수시상점세균증식활성명현수억,균락명현수량감소(F치분별위1170.80、217.52,P치균소우0.01)；여세균조화세균+ 1.6 mmol/L iCORM-2조비교,세균+1.6 mmol/L CORM-2조다수시상점세균증식활성역명현수억,균락수량역명현감소(F치분별위7948.34、14 432.85,P치균소우0.01).(2)체외실험2.qRT-PCR검측결과현시:여세균조비교,세균+0.8 mmol/L CORM-2조fliA기인표체하조,dnaK、marA화waaQ기인표체상조(t치분별위30.28、-165.54、-168.88、-187.28,P치균소우0.01).(3)재체연구.세균조화세균+ 1.2 mmol/L iCORM-2조、세균+1.6 mmol/L iCORM-2조소서출현제여정신위미、호흡급촉등증상,세균+ 1.2 mmol/L CORM-2조、세균+1.6 mmol/L CORM-2조소서상술증상경미혹불명현.주사후6h,세균조、세균+1.2 mmol/L iCORM-2조소서혈청TNF-α、IL-6수평분별위(647.3±3.8) pg/mL、(3.44±0.22) ng/mL이급(639.3±0.8)pg/mL、(2.47±0.32)ng/mL,명현고우세균+ 1.2 mmol/L CORM-2조[(124.6±10.7) pg/mL、(1.03 ±0.16)ng/mL,t치위15.22～84.03,P치균소우0.01].여세균조화세균+1.6 mmol/L iCORM-2조비교,주사후6、12 h세균+1.6 mmol/L CORM-2조소서혈청TNF-α、IL-6수평역균명현강저(t치위19.27～245.34,P치균소우0.01).주사후12 h,세균+1.2 mmol/L CORM-2조소서간、폐조직중MPO활성명현저우세균조、세균+ 1.2 mmol/L iCORM-2조,세균+1.6 mmol/L CORM-2조소서간、폐조직중MPO활성역명현저우세균조、세균+ 1.6 mmol/L iCORM-2조(t치분별위17.36～18.92、2.35 ～3.61,P치균소우0.01).결론 CORM-2능구명현억제대장간균적활력화독력,기억제작용가능여기조절대장간균부분중요적파기인(fliA、dnaK、marA화waaQ)유관.
Objective To 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 ofE.coliwas 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-cα 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-αt 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.
