小窝蛋白-1/血红素加氧酶-1信号链轴对机械通气所致肺损伤调控效应的研究

Research on the effect of protection against ventilator-induced lung injury via regulation of caveolin-1/heme oxygenase-1 signaling

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。

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.