PLCE1在机械通气致肺微血管内皮细胞通透性增加中的作用机制研究

Study on the mechanism of phospholipase C epsilon-1 in mechanical ventilation-induced increased permeability of pulmonary microvascular endothelial cells

目的对比观察不同磷脂酶CE1(pathogenic role of phospholipase C epsilon-1,PLCE1)表达水平对机械通气(mechanical ventilation,MV)实验大鼠肺组织胞质型磷脂酶A2(cytoplasmic phospholipase A2,C-PLA2)表达及其代谢产物生成的影响,阐明PLCE1在MV致肺微血管内皮细胞(Pulmonary microvascular endothelial cell,PMVEC)通透性增加中的作用。方法SPF级大鼠24只,雌雄不拘,8~10周龄,体重180~200 g,随机均分为4组(n=6):①野生型大鼠+“肺保护性”MV组(WPM组);②野生型大鼠+“伤害性”MV组(WIM组);③PLCE1+/-杂合子大鼠+“肺保护性”MV组(PPM组);④PLCE1+/-杂合子大鼠+“伤害性”MV组(PIM组)。对实验大鼠实施时长为2 h的“肺保护性”MV[潮气量(tidal volume,VT)=7 mL/kg,呼气末正压(positive end expiratory pressure,PEEP)=5 cmH 2O(1 cmH_(2)O=0.098 kPa)]或“伤害性”MV(VT=20 mL/kg,PEEP=0 cmH_(2)O)MV。蛋白免疫印迹(Western-blot)和实时定量聚合酶链式反应(real-time quantitative PCR,Q-PCR)分别检测肺组织PLCE1和C-PLA2蛋白及mRNA表达水平;酶联免疫法测定肺组织花生四烯酸(arachidonic acid,AA)代谢产物前列环素(prostacyclin,PGI2)、血栓素A2(thromboxane A2,TXA2)和白三烯B4(leukotriene B4,LTB4)含量;肺通透性指数和肺湿/干重(W/D)比值评价PMVEC通透性;肺组织病理形态学评分评估肺损伤的严重程度。结果与WPM组及PPM组相比,WIM组及PIM组大鼠肺组织PLCE1和C-PLA2蛋白及mRNA表达明显上调(P<0.05);肺组织PGI2、TXA2和LTB4含量增多(P<0.05);肺通透性指数、肺W/D比值及肺组织病理形态学评分明显增高(P<0.05)。相同通气模式下,PIM组及PPM组大鼠肺组织PLCE1和C-PLA2蛋白及mRNA表达水平、肺内PGI2、TXA2和LTB4含量及肺损伤评价各项指标较WIM组及WPM组大鼠均明显降低(P<0.05)。结论下调PLCE1可以通过抑制C-PLA2活性减轻MV导致的PMVEC通透性增加,发挥肺保护作用,提示PLCE1可能是抗VILI治疗的一个潜在干预作用的靶点。

Objective To compare and observe the effects of different levels of phospholipase C epsilon-1(PLCE1)expression on the expression of cytoplasmic phospholipase A2(C-PL2)and its metabolic products in lung tissue of rats undergoing mechanical ventilation(MV),and to elucidate the role of phospholipase C epsilon-1(PLCE1)in MV-induced increased permeability of pulmonary microvascular endothelial cells(PMEC).Methods Twenty-four 8-10 week-old SPF rats(weighing 180-200 g)with an equal number of males and females were randomized equally into wild-type rats+"lung protective"or"injurious"MV group(WPM group or WIM group)and PLCE1 knock-down(PLCE1-KD)rats+"lung protective"or"injurious"MV group(PPM group or PIM group).Rats were subjected to 2 h"lung protective"tidal volume(TV)=7 mL/kg,positive end expiratory pressure(PEEP=5 cmH_(2)O)or"injurious"(TV=20 mL/kg,PEEP=0 cmH_(2)O)MV.The expressions of PLCEl and cytoplasmic phospholipase A2(C-PLA2)were examined by Western blotting and real-time quantitative PCR(QPCR).Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of arachidonic acid(AA)metabolites such as prostacyclin(PGI2),thromboxane A2(TXA2)and leukotriene B4(LTB4)in the lung tissue.The pulmonary permeability index and the lung wet/dry weight(W/D)ratio were determined to evaluate the permeability of PMEC(PMVECs).The severities of lung injury were evaluated by pathological morphology scores of lung tissues.Results In WIM and PIM groups,PLCE1 and C-PLA2 expressions at both the protein and mRNA levels were significantly upregulated,the levels of PGI2,TXA2 and LTB4 in lungs were increased,and pulmonary permeability index,lung W/D ratio,and pathological morphology scores of lung tissues were significantly increased as compared with those in WPM and PPM groups(P<0.05).Under the same MV mode,the expression levels of PLCE1 and C-PL2 at both the protein and mRNA levels in rat lung tissue,the levels of PGI2,TXA2,and LTB4 in the lungs,and various indicators for evaluating lung injury were significantly lower in PIM and PPM groups than in WIM and WPM groups(P<0.05).Conclusion Downregulation of PLCE1 can alleviate MV-induced increased PMVEC permeability by inhibiting C-PL2 activity,exerting a protective effect on the lungs,suggesting that PLCE1 may be a potential intervention target for anti-VILI therapy.