中国危重病急救医学
中國危重病急救醫學
중국위중병급구의학
CHINESE CRITICAL CARE MEDICINE
2009年
4期
215-218,前插2
,共5页
白建文%邓伟吾%章剑%许淑敏%张斗霞
白建文%鄧偉吾%章劍%許淑敏%張鬥霞
백건문%산위오%장검%허숙민%장두하
肌球蛋白轻链激酶抑制剂%肌球蛋白轻链激酶%急性肺损伤
肌毬蛋白輕鏈激酶抑製劑%肌毬蛋白輕鏈激酶%急性肺損傷
기구단백경련격매억제제%기구단백경련격매%급성폐손상
myosin light-chain kinase inhibitor%myosin light-chain kinase%acute lung injury
目的 探讨肌球蛋白轻链激酶(MLCK)抑制剂ML-7对细菌脂多糖(LPS)诱导的人肺动脉内皮细胞(HPAEC)和急性肺损伤(ALI)的影响.方法 体外培养HPAEC,待4～6代予ML-7(10 μmol/L)孵育60 min后,再给予LPS刺激60 min,四甲基偶氮唑盐(MTT)比色法检测HPAEC活性;荧光显微镜下观察磷酸化肌球蛋白轻链激酶(p-MLCK)免疫反应细胞.20只雌性BALB/c小鼠随机分组,LPS组鼻内注入LPS(1 μg/g);ML-7组在LPS滴鼻前进行ML-7干预.观察小鼠肺湿/于重(W/D)比值、支气管肺泡灌洗液(BALF)蛋白含量、肺组织髓过氧化物酶(MPO)活性和病理学改变;用免疫组化法检测肺组织MLCK和CD11b阳性免疫反应细胞,逆转录-聚合酶链反应(RT-PCR)检测肺组织MLCK mRNA表达,蛋白质免疫印迹法(Western blotting)检测肺组织MLCK蛋白表达.结果 与LPS组比较,HPAEC在ML-7孵育下吸光度(A)值增加(P<0.01),p-MLCK免疫反应细胞明显减少(P<0.05).肺W/D比值、肺组织MPO活性、BALF蛋白含量均明显降低(P<0.05或P<0.01).病理结果显示,LPS组小鼠肺部炎症反应明显,以中性粒细胞浸润为主,肺泡充血、水肿;ML-7组肺部炎症及充血明显改善.免疫组化显示位于内皮的MLCK免疫反应细胞和位于炎性细胞的CD11b在ML-7组均较LPS组明显减少.ML-7组肺组织MLCK的mRNA和蛋白表达均较LPS组降低(P均<0.05).结论 MLCK特异性抑制剂ML-7能提高LPS诱导的HPAEC生长活性,降低p-MLCK表达;减轻LPS诱导的中性粒细胞在肺内浸润、肺水肿及MLCK、CD11b蛋白和MLCK mRNA的表达.表明抑制MLCK活性可能通过减弱MLCK磷酸化而达到稳定血管屏障功能,防治ALl的作用.
目的 探討肌毬蛋白輕鏈激酶(MLCK)抑製劑ML-7對細菌脂多糖(LPS)誘導的人肺動脈內皮細胞(HPAEC)和急性肺損傷(ALI)的影響.方法 體外培養HPAEC,待4～6代予ML-7(10 μmol/L)孵育60 min後,再給予LPS刺激60 min,四甲基偶氮唑鹽(MTT)比色法檢測HPAEC活性;熒光顯微鏡下觀察燐痠化肌毬蛋白輕鏈激酶(p-MLCK)免疫反應細胞.20隻雌性BALB/c小鼠隨機分組,LPS組鼻內註入LPS(1 μg/g);ML-7組在LPS滴鼻前進行ML-7榦預.觀察小鼠肺濕/于重(W/D)比值、支氣管肺泡灌洗液(BALF)蛋白含量、肺組織髓過氧化物酶(MPO)活性和病理學改變;用免疫組化法檢測肺組織MLCK和CD11b暘性免疫反應細胞,逆轉錄-聚閤酶鏈反應(RT-PCR)檢測肺組織MLCK mRNA錶達,蛋白質免疫印跡法(Western blotting)檢測肺組織MLCK蛋白錶達.結果 與LPS組比較,HPAEC在ML-7孵育下吸光度(A)值增加(P<0.01),p-MLCK免疫反應細胞明顯減少(P<0.05).肺W/D比值、肺組織MPO活性、BALF蛋白含量均明顯降低(P<0.05或P<0.01).病理結果顯示,LPS組小鼠肺部炎癥反應明顯,以中性粒細胞浸潤為主,肺泡充血、水腫;ML-7組肺部炎癥及充血明顯改善.免疫組化顯示位于內皮的MLCK免疫反應細胞和位于炎性細胞的CD11b在ML-7組均較LPS組明顯減少.ML-7組肺組織MLCK的mRNA和蛋白錶達均較LPS組降低(P均<0.05).結論 MLCK特異性抑製劑ML-7能提高LPS誘導的HPAEC生長活性,降低p-MLCK錶達;減輕LPS誘導的中性粒細胞在肺內浸潤、肺水腫及MLCK、CD11b蛋白和MLCK mRNA的錶達.錶明抑製MLCK活性可能通過減弱MLCK燐痠化而達到穩定血管屏障功能,防治ALl的作用.
목적 탐토기구단백경련격매(MLCK)억제제ML-7대세균지다당(LPS)유도적인폐동맥내피세포(HPAEC)화급성폐손상(ALI)적영향.방법 체외배양HPAEC,대4～6대여ML-7(10 μmol/L)부육60 min후,재급여LPS자격60 min,사갑기우담서염(MTT)비색법검측HPAEC활성;형광현미경하관찰린산화기구단백경련격매(p-MLCK)면역반응세포.20지자성BALB/c소서수궤분조,LPS조비내주입LPS(1 μg/g);ML-7조재LPS적비전진행ML-7간예.관찰소서폐습/우중(W/D)비치、지기관폐포관세액(BALF)단백함량、폐조직수과양화물매(MPO)활성화병이학개변;용면역조화법검측폐조직MLCK화CD11b양성면역반응세포,역전록-취합매련반응(RT-PCR)검측폐조직MLCK mRNA표체,단백질면역인적법(Western blotting)검측폐조직MLCK단백표체.결과 여LPS조비교,HPAEC재ML-7부육하흡광도(A)치증가(P<0.01),p-MLCK면역반응세포명현감소(P<0.05).폐W/D비치、폐조직MPO활성、BALF단백함량균명현강저(P<0.05혹P<0.01).병리결과현시,LPS조소서폐부염증반응명현,이중성립세포침윤위주,폐포충혈、수종;ML-7조폐부염증급충혈명현개선.면역조화현시위우내피적MLCK면역반응세포화위우염성세포적CD11b재ML-7조균교LPS조명현감소.ML-7조폐조직MLCK적mRNA화단백표체균교LPS조강저(P균<0.05).결론 MLCK특이성억제제ML-7능제고LPS유도적HPAEC생장활성,강저p-MLCK표체;감경LPS유도적중성립세포재폐내침윤、폐수종급MLCK、CD11b단백화MLCK mRNA적표체.표명억제MLCK활성가능통과감약MLCK린산화이체도은정혈관병장공능,방치ALl적작용.
Objective To investigate the influence of inhibitor of myosin light-chain kinase (MLCK) on the human pulmonary arterial endothelial cell (HPAEC) challenged with lipopolysaccharide (LPS) and LPS induced of acute lung injury (ALI) in mice. Methods HPAECs were cultured in ECM medium and its passages 4-6 were used. After treatment with inhibitor of MLCK (ML-7) for 60 minutes, the HPAECs were incubated in LPS for another 60 minutes, and then cell viability was measured by the methyl thiazolyl tetrazolium (MTT) assay. Immunofluorescence microscope was used to detect phosphorylated-MLCK (p-MLCK) immunoreactive cells. Twenty female BALB/c mice were randomly divided into two groups. The mice of LPS group were exposed to LPS (1 μg/g) through nasal instillation, and the mice of ML-7 group were pretreated with ML-7 before intranasal instillation of LPS. Wet/dry weight (W/D) ratio of lung, bronchoalveolar lavage fluid (BALF) protein content, myeloperoxidase (MPO) activity and histopathological changes of lung tissue were observed. Immunohistochemistry assays were used to determine the status of MLCK and CD11b immunoreactive cells in lung tissue, and expression of MLCK mRNA in lung tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR). Expression of MLCK protein in lungs was assayed by Western blotting. Results Compared with LPS group, increased absorbance (A) value of HPAEC was found in ML-7 group (P＜0. 01). Immunoreactive cells of p-MLCK were more reduced in the ML-7 group (P＜0. 05), and W/D ratio of lung, MPO activity and BALF protein content of lung tissue were decresead in ML-7 group (P＜0. 05 or P＜0. 01). Histological examination showed that an extensive lung inflammation was seen in mice of LPS group, with an accumulation of a large number of neuyrophils, marked pulmonary edema and hemorrhage, but the inflammation and parenchymal hemorrhage was significantly alleviated in ML-7 group. Both MLCK immunoreactive cells located in endothelium and CD11b in infiltrated inflammatory cells were decresead in ML-7 group compared with those in LPS group. Compared with LPS group, MLCK mRNA and protein expressions (A) in ML-7 group were significantly decreased (both P＜0. 05). Conclusion ML-7, an MLCK inhibitor, enhances activity of HPAEC induced by LPS and reduces expression of p-MLCK. It also reduces the LPS-induced infiltration of neutrophils in lung tissues, pulmonary edema and expression of MLCK and CD11b protein and MLCK mRNA in lung tissues, demonstrating that inhibition of activation of MLCK, leading to an abatement of phosphorylation of myosin light chain or MLCK, resulting in stabilization of vascular barrier function. The results suggest that MLCK has a crucial role in the pathogenesis of ALI.
