摘要：
目的 探讨在活体机械通气损伤模型抑制c-Abl激酶是否可以减少桩蛋白酪氨酸残基位点Y31和Y118(Pxn Y31、Pxn Y118)磷酸化,从而阻断其下游效应分子血管内皮-钙黏蛋白(VE-cad)及Rho/Rho激酶活化所致的血管屏障功能紊乱.方法 90只健康雄性SD大鼠按随机数字表法分为9组,每组10只.假手术(Sham)组仅进行气管切开;保护性通气1 h、2 h组(PVT 1h、2h组)潮气量(VT)为6 mL/kg,呼气末正压(PEEP)为5 cmH2O(1 cmH2O=0.098 kPa);大VT通气1 h、2 h组(HVT 1h、2h组)VT为30 mL/kg,PEEP为0;p42/44丝裂素活化蛋白激酶(p42/44MAPK)抑制剂UO126和c-Abl激酶抑制剂AG957预处理1 h、2 h组分别于大VT通气前1 h腹腔注射UO1261 mg/kg或灌胃AG95710 mg/kg.各组于预定实验时间结束后处死大鼠,采集肺组织标本及支气管肺泡灌洗液(BALF),用伊文思蓝(EB)实验检测肺血管渗透性,用酶联免疫吸附试验(ELISA)检测BALF中肿瘤坏死因子-α(TNF-α)水平;镜下观察肺组织病理学改变,并计算弥漫性肺泡损伤系统(DAD)评分,计算肺湿/干重(W/D)比值;用比色法测定肺组织髓过氧化物酶(MPO)活性,用蛋白质免疫印迹试验(Western Blot)检测肺组织c-Abl Y245、Pxn Y31、Pxn Y118、VE-cad Y658、p42/44MAPK Y202/Y204、肌球蛋白轻链(MLC)及肌球蛋白磷酸酯酶目标亚基Y696(MYPT Y696)的磷酸化情况.结果 ①Sham组与PVT组肺组织无明显病理学改变,且两组间各指标均无明显差异;HVT组肺组织损伤严重,且DAD评分、肺W/D比值、EB渗出量、MPO活性和BALF中TNF-α 水平较Sham组及PVT组明显升高;给予AG957或UO126预处理后,上述指标均较HVT组明显降低.②HVT组肺组织各蛋白磷酸化水平较Sham组和PVT组明显增加,以2 h更明显;给予AG957预处理后2 h,肺组织蛋白磷酸化水平均较HVT组明显降低〔p-c-Abl Y245(灰度值):0.29±0.04比0.42±0.04,p-Pxn Y31(灰度值):0.51±0.03比0.70±0.05,p-Pxn Y118(灰度值):0.65±0.04比0.91±0.04,p-VE-cad Y658(灰度值):0.77±0.07比1.32±0.07,p-p42/44MAPK Y202/Y204(灰度值):0.38±0.06比0.61±0.03,p-MLC(灰度值):0.37±0.04比0.77±0.05,p-MYPT Y696(灰度值):0.54±0.05比0.87±0.06,均P<0.05〕;给予UO126预处理后2 h,肺组织p-VE-cad Y658表达较HVT组明显降低(灰度值:0.74±0.04比1.32±0.07),且p42/44MAPK及其下游效应分子MLC、MYPT的磷酸化水平亦明显降低〔p-p42/44MAPK Y202/Y204(灰度值):0.38±0.07比0.61±0.03,p-MLC(灰度值):0.37±0.04比0.77±0.05,p-MYPT Y696(灰度值):0.55±0.05比0.87±0.06,均P<0.05〕.结论 抑制c-Abl激酶可阻断Pxn Y31、Pxn Y118磷酸化,稳定黏附连接处的VE-cad,并有可能通过阻断Pxn-鸟嘌呤核苷酸交换因子H1(GEF-H1)-p42/44MAPK信号小体的形成而抑制Rho信号链的活化、MLC的磷酸化及继发的肺血管屏障渗透性增加.%Objective To determine whether the inhibition of paxillin tyrosine residues 31 and tyrosine residues 118 (Pxn Y31 and Pxn Y118) phosphorylation via inhibition of c-Abl kinase will effectively block its downstream effector molecules vessel endothelium-cadherin (VE-cad), and whether Rho/Rho kinase activation which will induce the vascular barrier dysfunction. Methods Ninety healthy male Sprague-Dawley (SD) rats were randomly divided into nine groups (each n =10). Only tracheotomy was undergone in the sham group. Groups of protective ventilation were set at a volume tidal (VT) of 6 mL/kg, a positive end-expiratory pressure (PEEP) of 5 cmH2O (1 cmH2O =0.098 kPa) for 1 hour or 2 hours (namely group PVT 1 h and group PVT 2 h), respectively. Groups of high VT were put on mechanical ventilation (MV) at high VT 30 mL/kg, PEEP 0 for 1 hour or 2 hours (namely group HVT 1 h and group HVT 2 h), respectively. Groups UO126 and AG957 pretreatment were set on MV at HVT for 1 hour or 2 hour respectively, but they were given p42/44 mitogen-activated protein kinase (p42/44MAPK) inhibitor UO1261 mg/kg by intraperitoneal injection or c-Abl kinase inhibitor AG95710 mL/kg by intragastric injection 1 hour before HVT ventilation. All the animals were sacrificed after experiments and specimens of lung tissues and bronchoalveolar lavage fluid (BALF) were harvested. Pulmonary vascular permeability was measured by Evans blue (EB). The levels of tumor necrosis factor-α(TNF-α) in BALF were measured by enzyme linked immunosorbent assay (ELISA). Then the change of lung tissue pathology was observed with light microscope, diffuse alveolar damage system (DAD) score and lung wet/dry ratio (W/D) were estimated. The myeloperoxidase (MPO) activity was measured by colorimetric analysis, phosphorylations of c-Abl Y245, Pxn Y31, Pxn Y118, VE-cad Y658, p42/44MAPK Y202/Y204, myosin light chain (MLC) and myosin-associated phosphatasetype Y696 (MYPT Y696) were determined by Western Blot. Results ① There were no obvious pathological changes in the lung tissue in the sham group and PVT 1 h or 2 h group, and also there were no significant differences in all the parameters between above groups. However, the injury in lung tissue was severe in the HVT groups. In addition, DAD score, lung W/D ratio, EB content, the activity of MPO, and TNF-α in BALF in HVT groups were significantly higher than those in sham group and PVT groups. After pretreatment with AG957 or UO126, all the parameters were significantly decreased as compared with those of groups HVT. ② The levels of phosphorylation of the proteins in lung tissue in HVT groups were increased as compared with those of group sham and groups PVT, especially at 2 hours of MV. However, compared with groups HVT, the level of p-VE-cad Y658 in lung tissue decreased significantly in group AG957 and group UO126 at 2 hours after HVT. However, the levels of all phosphorylated proteins at 2 hours were significantly lowered in the AG957 group compared with those of the HVT group [p-c-Abl Y245 (gray value): 0.29±0.04 vs. 0.42±0.04, p-Pxn Y31 (gray value): 0.51±0.03 vs. 0.70±0.05, p-Pxn Y118 (gray value):0.65±0.04 vs. 0.91±0.04, p-VE-cad Y658 (gray value): 0.77±0.07 vs. 1.32±0.07, p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.06 vs. 0.61±0.03, p-MLC (gray value): 0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value):0.54±0.05 vs. 0.87±0.06, all P < 0.05]. After pretreatment with UO126, the phosphorylation level of VE-cad in lung tissue at 2 hours was significantly lower than that of HVT group (gray value: 0.74±0.04 vs. 1.32±0.07), and the phosphorylation levels of p42/44MAPK and its downstream effector molecules MLC and MYPT Y696 were also significantly decreased [p-p42/44MAPK Y202/Y204 (gray value): 0.38±0.07 vs. 0.61±0.03, p-MLC (gray value):0.37±0.04 vs. 0.77±0.05, p-MYPT Y696 (gray value): 0.55±0.05 vs. 0.87±0.06, all P < 0.05]. Conclusions Pxn Y31 and Pxn Y118 phosphorylation could be blocked by inhibition of c-Abl kinase, which could strengthen VE-cad at attachment junction and might block formation of Pxn-guanine nucleotide-exchange factor H1 (GEF-H1)-p44/42MAPK signalosome which induce activation local Rho signaling, lead to activation of MLC phosphorylation, actomyosin contraction, and increase endothelial permeability.
