中华危重病急救医学
中華危重病急救醫學
중화위중병급구의학
Chinese Critical Care Medicine
2015年
7期
568-573
,共6页
钟荣%肖军%于志辉%周吉%戴春光
鐘榮%肖軍%于誌輝%週吉%戴春光
종영%초군%우지휘%주길%대춘광
小窝蛋白-1,磷酸化%血红素加氧酶-1%机械通气相关性肺损伤
小窩蛋白-1,燐痠化%血紅素加氧酶-1%機械通氣相關性肺損傷
소와단백-1,린산화%혈홍소가양매-1%궤계통기상관성폐손상
Phosphorylation of caveolin -1%Heme oxygenase-1%Ventilation-induced lung injury
目的：探讨在活体动物用酪氨酸激酶抑制剂（PP2）阻断小窝蛋白酪氨酸残基14（Cav-1-Y14）磷酸化，是否会上调血红素加氧酶-1（HO-1）活性，以对抗机械通气所致的肺损伤。方法54只雄性SD大鼠按随机数字表法分为9组，每组6只。A组为正常对照组，只行气管切开；B1、B2组为保护性通气1 h、2 h组；C1、C2组为大潮气量（40 mL/kg）通气1 h、2 h组；D1、D2组为PP2预处理+大潮气量通气1 h、2 h组；E1、E2组为PP2、HO-1抑制剂锌原卟啉Ⅸ（ZnPPⅨ）预处理+大潮气量通气1 h、2 h组。各组动物在预定实验时间结束后立即放血处死并采集肺组织标本及支气管肺泡灌洗液（BALF），观察肺组织病理学改变，并计算弥漫性肺泡损伤系统评分（DAD评分），测定髓过氧化物酶（MPO）活性，计算肺湿/干质量（W/D）比值；蛋白质免疫印迹试验检测磷酸化小窝蛋白-1（P-Cav-1-Y14）、小窝蛋白-1（Cav-1）、HO-1的表达；免疫组化法检测肺组织中高迁移率族蛋白B1（HMGB1）和晚期糖基化终末产物受体（RAGE）的表达，酶联免疫吸附试验（ELISA）检测BALF中肿瘤坏死因子-α（TNF-α）水平。结果与A组比较，B组各指标均无明显改变。与B1组比较，C1组肺组织DAD评分、W/D比值、MPO活性及BALF中TNF-α浓度均显著升高〔DAD评分（分）：7.97±0.59比0.55±0.13，W/D比值：5.70±1.61比5.04±0.63，MPO（U/g）：1.82±0.14比0.77±0.26， TNF-α（ng/L）：370.10±29.61比54.38±8.18，均P＜0.05〕，且通气2 h组较1 h组损伤更为严重。D组各指标较C组明显下降；E组各指标明显高于A、B、D组，与C组比较则差异无统计学意义。C组肺组织Cav-1、P-Cav-1-Y14蛋白表达（灰度值）均显著高于B组（1 h：1.49±0.02比1.26±0.13，1.34±0.02比0.87±0.04；2 h：1.58±0.02比1.27±0.27，1.31±0.01比0.95±0.02，均P＜0.05），HO-1蛋白表达（灰度值）显著低于B组（1 h：0.59±0.02比1.10±0.01；2 h：0.49±0.01比1.20±0.02，均P＜0.05）；D组、E组Cav-1蛋白表达与C组无差异，P-Cav-1-Y14蛋白表达明显低于C组；D组HO-1蛋白表达明显高于C组，而E组HO-1蛋白表达与C组无差异。与A组比较， C组及E组肺组织HMBG1和RAGE阳性表达明显增多，但B组、D组与A组比较无差异。结论 Cav-1-Y14磷酸化是肺通气损伤的关键因素，其不仅可导致血管屏障功能的完整性降低，亦可抑制HO-1活性，从而进一步加重机械通气所致肺组织炎症损伤。
目的：探討在活體動物用酪氨痠激酶抑製劑（PP2）阻斷小窩蛋白酪氨痠殘基14（Cav-1-Y14）燐痠化，是否會上調血紅素加氧酶-1（HO-1）活性，以對抗機械通氣所緻的肺損傷。方法54隻雄性SD大鼠按隨機數字錶法分為9組，每組6隻。A組為正常對照組，隻行氣管切開；B1、B2組為保護性通氣1 h、2 h組；C1、C2組為大潮氣量（40 mL/kg）通氣1 h、2 h組；D1、D2組為PP2預處理+大潮氣量通氣1 h、2 h組；E1、E2組為PP2、HO-1抑製劑鋅原卟啉Ⅸ（ZnPPⅨ）預處理+大潮氣量通氣1 h、2 h組。各組動物在預定實驗時間結束後立即放血處死併採集肺組織標本及支氣管肺泡灌洗液（BALF），觀察肺組織病理學改變，併計算瀰漫性肺泡損傷繫統評分（DAD評分），測定髓過氧化物酶（MPO）活性，計算肺濕/榦質量（W/D）比值；蛋白質免疫印跡試驗檢測燐痠化小窩蛋白-1（P-Cav-1-Y14）、小窩蛋白-1（Cav-1）、HO-1的錶達；免疫組化法檢測肺組織中高遷移率族蛋白B1（HMGB1）和晚期糖基化終末產物受體（RAGE）的錶達，酶聯免疫吸附試驗（ELISA）檢測BALF中腫瘤壞死因子-α（TNF-α）水平。結果與A組比較，B組各指標均無明顯改變。與B1組比較，C1組肺組織DAD評分、W/D比值、MPO活性及BALF中TNF-α濃度均顯著升高〔DAD評分（分）：7.97±0.59比0.55±0.13，W/D比值：5.70±1.61比5.04±0.63，MPO（U/g）：1.82±0.14比0.77±0.26， TNF-α（ng/L）：370.10±29.61比54.38±8.18，均P＜0.05〕，且通氣2 h組較1 h組損傷更為嚴重。D組各指標較C組明顯下降；E組各指標明顯高于A、B、D組，與C組比較則差異無統計學意義。C組肺組織Cav-1、P-Cav-1-Y14蛋白錶達（灰度值）均顯著高于B組（1 h：1.49±0.02比1.26±0.13，1.34±0.02比0.87±0.04；2 h：1.58±0.02比1.27±0.27，1.31±0.01比0.95±0.02，均P＜0.05），HO-1蛋白錶達（灰度值）顯著低于B組（1 h：0.59±0.02比1.10±0.01；2 h：0.49±0.01比1.20±0.02，均P＜0.05）；D組、E組Cav-1蛋白錶達與C組無差異，P-Cav-1-Y14蛋白錶達明顯低于C組；D組HO-1蛋白錶達明顯高于C組，而E組HO-1蛋白錶達與C組無差異。與A組比較， C組及E組肺組織HMBG1和RAGE暘性錶達明顯增多，但B組、D組與A組比較無差異。結論 Cav-1-Y14燐痠化是肺通氣損傷的關鍵因素，其不僅可導緻血管屏障功能的完整性降低，亦可抑製HO-1活性，從而進一步加重機械通氣所緻肺組織炎癥損傷。
목적：탐토재활체동물용락안산격매억제제（PP2）조단소와단백락안산잔기14（Cav-1-Y14）린산화，시부회상조혈홍소가양매-1（HO-1）활성，이대항궤계통기소치적폐손상。방법54지웅성SD대서안수궤수자표법분위9조，매조6지。A조위정상대조조，지행기관절개；B1、B2조위보호성통기1 h、2 h조；C1、C2조위대조기량（40 mL/kg）통기1 h、2 h조；D1、D2조위PP2예처리+대조기량통기1 h、2 h조；E1、E2조위PP2、HO-1억제제자원계람Ⅸ（ZnPPⅨ）예처리+대조기량통기1 h、2 h조。각조동물재예정실험시간결속후립즉방혈처사병채집폐조직표본급지기관폐포관세액（BALF），관찰폐조직병이학개변，병계산미만성폐포손상계통평분（DAD평분），측정수과양화물매（MPO）활성，계산폐습/간질량（W/D）비치；단백질면역인적시험검측린산화소와단백-1（P-Cav-1-Y14）、소와단백-1（Cav-1）、HO-1적표체；면역조화법검측폐조직중고천이솔족단백B1（HMGB1）화만기당기화종말산물수체（RAGE）적표체，매련면역흡부시험（ELISA）검측BALF중종류배사인자-α（TNF-α）수평。결과여A조비교，B조각지표균무명현개변。여B1조비교，C1조폐조직DAD평분、W/D비치、MPO활성급BALF중TNF-α농도균현저승고〔DAD평분（분）：7.97±0.59비0.55±0.13，W/D비치：5.70±1.61비5.04±0.63，MPO（U/g）：1.82±0.14비0.77±0.26， TNF-α（ng/L）：370.10±29.61비54.38±8.18，균P＜0.05〕，차통기2 h조교1 h조손상경위엄중。D조각지표교C조명현하강；E조각지표명현고우A、B、D조，여C조비교칙차이무통계학의의。C조폐조직Cav-1、P-Cav-1-Y14단백표체（회도치）균현저고우B조（1 h：1.49±0.02비1.26±0.13，1.34±0.02비0.87±0.04；2 h：1.58±0.02비1.27±0.27，1.31±0.01비0.95±0.02，균P＜0.05），HO-1단백표체（회도치）현저저우B조（1 h：0.59±0.02비1.10±0.01；2 h：0.49±0.01비1.20±0.02，균P＜0.05）；D조、E조Cav-1단백표체여C조무차이，P-Cav-1-Y14단백표체명현저우C조；D조HO-1단백표체명현고우C조，이E조HO-1단백표체여C조무차이。여A조비교， C조급E조폐조직HMBG1화RAGE양성표체명현증다，단B조、D조여A조비교무차이。결론 Cav-1-Y14린산화시폐통기손상적관건인소，기불부가도치혈관병장공능적완정성강저，역가억제HO-1활성，종이진일보가중궤계통기소치폐조직염증손상。
ObjectiveTo determine whether the inhibition of caveolin-1 tyrosine residues 14 (Cav-1-Y14) phosphorylation with protein tyrosine kinase inhibitors (PP2) will upregulate heme oxygenase-1 (HO-1) activity to protect against ventilation induced lung injury in vivo of an animal model.Methods Fifty-four male Sprague-Dawley (SD) rats were randomly divided into nine groups (eachn = 6). Group A served as normal control group, in which rats did not receive ventilation but tracheotomy. Groups B1 and B2 received lung protective ventilation respectively for 1 hour or 2 hours. Groups C1 and C2 received high tidal volume (40 mL/kg) ventilation for 1 hour or 2 hours, respectively. The group D1 or D2 also received high tidal volume ventilation for 1 hour or 2 hour respectively, but they were given PP2 1 hour before high tidal volume ventilation. The groups E1 and E2 also received high tidal volume ventilation respectively for 1 hour or 2 hours, but tyrosine kinase inhibitor PP2 and HO-1 inhibitor zinc protoporphyrinⅨ(ZnPPⅨ) were given to animals 18 hours before high tidal volume ventilation. All the animals were sacrificed after ventilation, and the specimens of lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. Then the changes in pathology of lung tissue was observed, and diffuse alveolar damage scores (DAD) were calculated, myeloperoxidase (MPO) activity was measured by colorimetric analysis, lung wet/dry ratio (W/D) was estimated. The expressions of phosphorylated caveolin-1 (P-Cav-1-Y14), caveolin-1 (Cav-1) and HO-1 were determined by Western Blot. The expressions of high mobility group B1 (HMGB1) and advanced glycation end product receptor (RAGE) in lung tissues were assayed with immunohistochemistry staining. The levels of tumor necrosis factor-α(TNF-α) in BALF were measured by enzyme linked immunosorbent assay (ELISA).Results There was no significant difference in all the parameters between group A and groups B. Compared with group B1, DAD score, W/D ratio, the activity of MPO and the concentration of TNF-α in BALF in group C1 were significantly increased [DAD score:7.97±0.59 vs. 0.55±0.13, W/D ratio: 5.70±1.61 vs. 5.04±0.63, MPO (U/g): 1.82±0.14 vs. 0.77±0.26, TNF-α(ng/L): 370.10±29.61 vs. 54.38±8.18, allP< 0.05], and the injury in ventilation 2 hours group was more serious than that in ventilation 1 hour group. Compared with groups C, all the parameters in groups D were significantly decreased. The parameters in groups E were significantly higher than those in groups A, B, and D, but no significant difference was found as compared with groups C. Compared with groups B, the protein expressions of Cav-1 and P-Cav-1-Y14 (gray value) in groups C were significantly increased (1 hour: 1.49±0.02 vs. 1.26±0.13, 1.34±0.02 vs. 0.87±0.04;2 hours: 1.58±0.02 vs. 1.27±0.27, 1.31±0.01 vs. 0.95±0.02, allP< 0.05), and the expression of HO-1 protein (gray value) was significantly decreased (1 hour: 0.59±0.02 vs. 1.10±0.01, 2 hours: 0.49±0.01 vs. 1.20±0.02, both P< 0.05). No significant difference in Cav-1 protein expression between groups D as well as groups E and groups C. The protein expression of P-Cav-1-Y14 in groups D and E was significantly lower than that in groups C. The protein expression of HO-1 in groups D was significantly higher than that in groups C, but the phenomenon was not found in groups E as compared with groups C. Compared with group A, the positive expression of HMGB1 and RAGE in lung tissue in groups C and E was significantly increased, but no significant difference was found between groups B as well as groups D and group A.Conclusion Cav-1-Y14 phosphorylation is the key factor for ventilator induced lung injury, which can not only lead to a decrease in vascular barrier function, but also inhibit the activity of HO-1 enzyme, thus further aggravates inflammatory injury of the lung as induced by mechanical ventilation.
