中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2014年
11期
815-820
,共6页
赖洁%汤展宏%胡军涛%周威%张驰%陈显峰
賴潔%湯展宏%鬍軍濤%週威%張馳%陳顯峰
뢰길%탕전굉%호군도%주위%장치%진현봉
急性肺损伤%亚低温%Toll样受体2%髓样分化因子88%核转录因子-κB%脂多糖
急性肺損傷%亞低溫%Toll樣受體2%髓樣分化因子88%覈轉錄因子-κB%脂多糖
급성폐손상%아저온%Toll양수체2%수양분화인자88%핵전록인자-κB%지다당
Acute lung injury%Hypothermia%Toll-like receptor 2%Myeloid differentiation factor 88%Nuclear factor-κBp65%Lipopolysaccharide
目的：研究亚低温对脂多糖（LPS）吸入导致的急性肺损伤（ALI）大鼠Toll样受体2/髓样分化因子88（TLR2/MyD88）通路中TLR2、MyD88、核转录因子-κBp65（NF-κBp65）及相关炎性蛋白纤溶酶原激活物抑制剂-1（PAI-1）表达的影响。方法将90只雄性SD大鼠按随机数字表法分成对照组（n＝18）、亚低温组（n＝24）、控温组（n＝24）、非控温组（n＝24）。用气管内滴入LPS 0.5 mL/kg方法建立ALI模型，对照组滴入等量生理盐水；1 h后取颈动脉血并予以物理降温，亚低温组、控温组分别控制肛温在32～34℃、36～37℃，对照组、非控温组体温不加干预。分别于制模前、制模后1 h及干预后1、6、12 h进行血气分析；分别于干预后1、6、12 h处死大鼠，光镜下观察肺组织病理改变并进行半定量评分；酶联免疫吸附试验（ELISA）测定支气管肺泡灌洗液（BALF）中PAI-1蛋白表达；反转录-聚合酶链反应（RT-PCR）检测肺组织TLR2 mRNA、MyD88 mRNA表达；蛋白质免疫印迹试验（Western Blot）检测肺组织NF-κBp65蛋白表达。结果滴入LPS后，各组氧合指数（PaO2/FiO2）明显降低，肺组织损伤加重，肺损伤评分、BALF中PAI-1、肺组织TLR2 mRNA、MyD88 mRNA、NF-κBp65蛋白表达均明显升高。给予亚低温治疗后各指标均明显改善，亚低温疗程持续6 h时效果最好，持续6～12 h可能获益。与控温组比较，亚低温组PaO2/FiO2（mmHg，1 mmHg＝0.133 kPa）于干预后1 h、6 h明显升高（1 h：402.49±38.61比324.36±28.93，6 h：349.72±98.20比284.35±13.68，均P＜0.01）；肺损伤评分（分）于干预后1、6、12 h明显降低（1 h：6.04±0.74比7.96±0.65，6 h：9.09±0.80比13.13±1.02，12 h：10.79±1.42比13.42±0.68，均P＜0.01）；BALF中PAI-1蛋白表达（ng/L）于干预后1、6、12 h明显下降（1 h：121.36±4.62比197.74±9.42，6 h：230.53±10.76比294.06±16.60，12 h：270.48±13.20比319.40±10.24，均P＜0.01）；TLR2、MyD88的mRNA表达（2-ΔΔCt）于干预后1、6和12 h均明显下降（TLR2 mRNA表达1 h：2.18±0.26比3.04±0.39，6 h：4.09±0.29比4.90±0.35，12 h：6.02±0.43比7.10±0.54；MyD88 mRNA表达1 h：2.25±0.41比3.04±0.30，6 h：5.67±0.55比7.01±0.76，12 h：7.14±0.60比8.87±0.54，均P＜0.01）；NF-κBp65蛋白表达（A值）于干预后6 h、12 h明显下降（6 h：0.31±0.08比0.53±0.12，12 h：1.05±0.17比1.76±0.35，均P＜0.01）。控温组与非控温组干预后各指标比较差异均无统计学意义。结论亚低温可通过下调TLR2/MyD88通路中TLR2 mRNA、MyD88 mRNA、NF-κBp65及相关炎性蛋白PAI-1的表达，对LPS吸入致ALI大鼠肺组织起保护性作用。
目的：研究亞低溫對脂多糖（LPS）吸入導緻的急性肺損傷（ALI）大鼠Toll樣受體2/髓樣分化因子88（TLR2/MyD88）通路中TLR2、MyD88、覈轉錄因子-κBp65（NF-κBp65）及相關炎性蛋白纖溶酶原激活物抑製劑-1（PAI-1）錶達的影響。方法將90隻雄性SD大鼠按隨機數字錶法分成對照組（n＝18）、亞低溫組（n＝24）、控溫組（n＝24）、非控溫組（n＝24）。用氣管內滴入LPS 0.5 mL/kg方法建立ALI模型，對照組滴入等量生理鹽水；1 h後取頸動脈血併予以物理降溫，亞低溫組、控溫組分彆控製肛溫在32～34℃、36～37℃，對照組、非控溫組體溫不加榦預。分彆于製模前、製模後1 h及榦預後1、6、12 h進行血氣分析；分彆于榦預後1、6、12 h處死大鼠，光鏡下觀察肺組織病理改變併進行半定量評分；酶聯免疫吸附試驗（ELISA）測定支氣管肺泡灌洗液（BALF）中PAI-1蛋白錶達；反轉錄-聚閤酶鏈反應（RT-PCR）檢測肺組織TLR2 mRNA、MyD88 mRNA錶達；蛋白質免疫印跡試驗（Western Blot）檢測肺組織NF-κBp65蛋白錶達。結果滴入LPS後，各組氧閤指數（PaO2/FiO2）明顯降低，肺組織損傷加重，肺損傷評分、BALF中PAI-1、肺組織TLR2 mRNA、MyD88 mRNA、NF-κBp65蛋白錶達均明顯升高。給予亞低溫治療後各指標均明顯改善，亞低溫療程持續6 h時效果最好，持續6～12 h可能穫益。與控溫組比較，亞低溫組PaO2/FiO2（mmHg，1 mmHg＝0.133 kPa）于榦預後1 h、6 h明顯升高（1 h：402.49±38.61比324.36±28.93，6 h：349.72±98.20比284.35±13.68，均P＜0.01）；肺損傷評分（分）于榦預後1、6、12 h明顯降低（1 h：6.04±0.74比7.96±0.65，6 h：9.09±0.80比13.13±1.02，12 h：10.79±1.42比13.42±0.68，均P＜0.01）；BALF中PAI-1蛋白錶達（ng/L）于榦預後1、6、12 h明顯下降（1 h：121.36±4.62比197.74±9.42，6 h：230.53±10.76比294.06±16.60，12 h：270.48±13.20比319.40±10.24，均P＜0.01）；TLR2、MyD88的mRNA錶達（2-ΔΔCt）于榦預後1、6和12 h均明顯下降（TLR2 mRNA錶達1 h：2.18±0.26比3.04±0.39，6 h：4.09±0.29比4.90±0.35，12 h：6.02±0.43比7.10±0.54；MyD88 mRNA錶達1 h：2.25±0.41比3.04±0.30，6 h：5.67±0.55比7.01±0.76，12 h：7.14±0.60比8.87±0.54，均P＜0.01）；NF-κBp65蛋白錶達（A值）于榦預後6 h、12 h明顯下降（6 h：0.31±0.08比0.53±0.12，12 h：1.05±0.17比1.76±0.35，均P＜0.01）。控溫組與非控溫組榦預後各指標比較差異均無統計學意義。結論亞低溫可通過下調TLR2/MyD88通路中TLR2 mRNA、MyD88 mRNA、NF-κBp65及相關炎性蛋白PAI-1的錶達，對LPS吸入緻ALI大鼠肺組織起保護性作用。
목적：연구아저온대지다당（LPS）흡입도치적급성폐손상（ALI）대서Toll양수체2/수양분화인자88（TLR2/MyD88）통로중TLR2、MyD88、핵전록인자-κBp65（NF-κBp65）급상관염성단백섬용매원격활물억제제-1（PAI-1）표체적영향。방법장90지웅성SD대서안수궤수자표법분성대조조（n＝18）、아저온조（n＝24）、공온조（n＝24）、비공온조（n＝24）。용기관내적입LPS 0.5 mL/kg방법건립ALI모형，대조조적입등량생리염수；1 h후취경동맥혈병여이물리강온，아저온조、공온조분별공제항온재32～34℃、36～37℃，대조조、비공온조체온불가간예。분별우제모전、제모후1 h급간예후1、6、12 h진행혈기분석；분별우간예후1、6、12 h처사대서，광경하관찰폐조직병리개변병진행반정량평분；매련면역흡부시험（ELISA）측정지기관폐포관세액（BALF）중PAI-1단백표체；반전록-취합매련반응（RT-PCR）검측폐조직TLR2 mRNA、MyD88 mRNA표체；단백질면역인적시험（Western Blot）검측폐조직NF-κBp65단백표체。결과적입LPS후，각조양합지수（PaO2/FiO2）명현강저，폐조직손상가중，폐손상평분、BALF중PAI-1、폐조직TLR2 mRNA、MyD88 mRNA、NF-κBp65단백표체균명현승고。급여아저온치료후각지표균명현개선，아저온료정지속6 h시효과최호，지속6～12 h가능획익。여공온조비교，아저온조PaO2/FiO2（mmHg，1 mmHg＝0.133 kPa）우간예후1 h、6 h명현승고（1 h：402.49±38.61비324.36±28.93，6 h：349.72±98.20비284.35±13.68，균P＜0.01）；폐손상평분（분）우간예후1、6、12 h명현강저（1 h：6.04±0.74비7.96±0.65，6 h：9.09±0.80비13.13±1.02，12 h：10.79±1.42비13.42±0.68，균P＜0.01）；BALF중PAI-1단백표체（ng/L）우간예후1、6、12 h명현하강（1 h：121.36±4.62비197.74±9.42，6 h：230.53±10.76비294.06±16.60，12 h：270.48±13.20비319.40±10.24，균P＜0.01）；TLR2、MyD88적mRNA표체（2-ΔΔCt）우간예후1、6화12 h균명현하강（TLR2 mRNA표체1 h：2.18±0.26비3.04±0.39，6 h：4.09±0.29비4.90±0.35，12 h：6.02±0.43비7.10±0.54；MyD88 mRNA표체1 h：2.25±0.41비3.04±0.30，6 h：5.67±0.55비7.01±0.76，12 h：7.14±0.60비8.87±0.54，균P＜0.01）；NF-κBp65단백표체（A치）우간예후6 h、12 h명현하강（6 h：0.31±0.08비0.53±0.12，12 h：1.05±0.17비1.76±0.35，균P＜0.01）。공온조여비공온조간예후각지표비교차이균무통계학의의。결론아저온가통과하조TLR2/MyD88통로중TLR2 mRNA、MyD88 mRNA、NF-κBp65급상관염성단백PAI-1적표체，대LPS흡입치ALI대서폐조직기보호성작용。
Objective To investigate the effect of hypothermia on the expression Toll-like receptor 2 (TLR2),myeloid differentiation factor 88(MyD88),nuclear factor-κBp65(NF-κBp65),plasminogen activator inhibitor-1(PAI-1)in the TLR2/MyD88 pathway in rats with acute lung injury(ALI)induced by lipopolysaccharide (LPS)inhalation. Methods Ninety male Sprague-Dawley(SD)rats were randomly divided into control group (n=18),hypothermia group(n=24),temperature controlled group(n=24),and temperature-uncontrolled group(n=24). The ALI model was reproduced by 0.5 mL/kg LPS intratracheal instillation,while only normal saline was instilled intratracheally for control group. Arterial blood was collected and physical cooling was started 1 hour after instillation. The body temperature was lowered to 32-34 ℃in hypothermia group and 36-37 ℃in temperature controlled group,and no intervention was used for temperature-uncontrolled group and control group. The arterial blood gas was determined in all the groups before and 1 hour after instillation of saline or LPS and 1,6, 12 hours after intervention. Rats were sacrificed respectively at 1,6 and 12 hours after temperature control therapy, the morphological changes in lung tissue were observed under light microscope. The protein expression of PAI-1 in bronchoalveolar lavage fluid(BALF)was determined by enzyme linked immunosorbent assay(ELISA). TLR2 mRNA and MyD88 mRNA transcriptional level were determined by reverse transcription-polymeras chain reaction (RT-PCR). NF-κBp65 protein level was determined by Western Blot. Results After instillation of LPS,the oxygenation index(PaO2/FiO2)of each group was decreased obviously,the damage of lung tissues was aggravating,the lung injury score was increased significantly,PAI-1 protein in BALF and the expressions of TLR2 mRNA,MyD88 mRNA, NF-κBp65 protein in lung tissues were increased obviously. Each index was improved by therapeutic Hypothermia, the effect of which was best in using a cooling period in the 1-6 hours,while might be benefit at 6-12 hours. Compared with temperature controlled group,PaO2/FiO2(mmHg,1 mmHg=0.133 kPa)at 1 hour and 6 hours of hypothermia group was improved(1 hour:402.49±38.61 vs. 324.36±28.93,6 hours:349.72±98.20 vs. 284.35±13.68, both P<0.01),the lung injury score at 1,6 and 12 hours were significantly decreased(1 hour:6.04±0.74 vs. 7.96±0.65,6 hours:9.09±0.80 vs. 13.13±1.02,12 hours:10.79±1.42 vs. 13.42±0.68,all P<0.01),the PAI-1 protein(ng/L)in BALF at 1,6 and 12 hours were significantly decreased(1 hour:121.36±4.62 vs. 197.74±9.42, 6 hours:230.53±10.76 vs. 294.06±16.60,12 hours:270.48±13.20 vs. 319.40±10.24,all P<0.01),TLR2 mRNA and MyD88 mRNA expressions(2-ΔΔCt)in the lung tissues at 1,6 and 12 hours were significantly decreased (TLR2 mRNA 1 hour:2.18±0.26 vs. 3.04±0.39,6 hours:4.09±0.29 vs. 4.90±0.35,12 hours:6.02±0.43 vs. 7.10±0.54;MyD88 mRNA 1 hour:2.25±0.41 vs. 3.04±0.30,6 hours:5.67±0.55 vs. 7.01±0.76,12 hours:7.14±0.60 vs. 8.87±0.54,all P<0.01),NF-κBp65 protein expression(A value)at 6 hours and 12 hours was significantly decreased(6 hours:0.31±0.08 vs. 0.53±0.12,12 hours:1.05±0.17 vs. 1.76±0.35,both P<0.01). There was no difference in each index between temperature controlled group and temperature-uncontrolled group. Conclusion Hypothermia can down-regulate the expression of TLR2 mRNA,MyD88 mRNA,NF-κBp65 protein and PAI-1 in the TLR2/MyD88 pathway to protect lung tissue of rats with ALI induced by LPS inhalation from injury.
