MAPKs信号通路在光气吸入性肺损伤中的表达及作用

Expression and role of mitogen activated protein kinasses signaling pathway in lung injury induced by phosgene

目的探讨丝裂原活化蛋白激酶（mitogen activated protein kinases，MAPKs）的亚通路信号蛋白-细胞外信号调节蛋白激酶（extracellularsignal-regulatedkinase，ERKI／2）、P38和c-氨基末端蛋白激酶（c—JunN-terminalkinase，JNK）在大鼠光气吸入肺损伤组织中的表达及各通路在光气吸人性肺损伤中的作用。方法选取30只雄性Wistar大鼠随机分成空气对照组、光气吸入组、PD98059（ERK1／2特异性阻断剂）、SB203580（P38特异性阻断剂）和SP600125（JNK特异性阻断剂）干预组，每组6只，应用定向流动光气吸入装置，复制大鼠光气（8．33Umg）吸人性肺损伤的模型，空气对照组吸人空气，3个干预组均在光气（8．33L／mg）吸入前给予PD98059、SB203580、SP600125。分别采用免疫组化及蛋白质印迹（Westernblot）法检测肺组织MAPKs（ERK1／2、P38、JNK）蛋白及磷酸化蛋白p-MAPKs（p．ERK1／2、P—P38、p-JNK）的定性定量表达。观察肺组织病理学改变、计数支气管灌洗液（BALF）中中性粒细胞数、测定肺湿干比。结果空气对照组仅肺泡上皮细胞及气道上皮细胞见少量p-ERKl／2、p-P38、p-JNK阳性表达；光气吸人组p-ERK1／2、p-P38、p-JNK阳性细胞数明显增多，广泛分布于肺内细胞：肺泡上皮细胞、气道上皮细胞、胸膜间皮细胞、浸润炎细胞及间质纤维细胞；干预组p-ERK1／2、p-P38、p-JNK阳性细胞数减少。各组ERK、P38、JNK的表达无明显变化；光气吸人组较空气对照组p-P38、p-JNK蛋白表达增强，3个干预组的p-ERK1／2、p-P38、p-INK的蛋白表达水平均低于光气吸入组，差异均有统计学意义（／9〈0．05，P〈0．01）。与空气对照组[中性粒细胞计数：（2．05±0．75）×10^4，肺湿干比：（3．64％±0．72％）]比较，光气吸入组肺损伤程度严重，表现出肺损伤的典型病理学特征，且BALF中中性粒细胞数[（10．78±2．16）×10^4增多，肺湿干比（7．65％±0．58％）升高，SP600125干预组和SB203580干预组BALF中中性粒细胞数ISP600125组：（7．86±2．12）×10^4，SB203580组：（8．43±1．51）×10^4和肺湿干比[SP600125组：（6．10％±0．97％），SB203580组：（6．09％±1．43％）1降低，差异均有统计学意义（P〈0．05，P〈0．01）。结论光气吸人可能激活MAPK信号通路，通过p-MAPKs表达增加在光气吸入后的肺损伤中发挥作用，其中以p-P38、p-JNK活性变化为主。

Objective This study aimed to investigate the expression and role of the mitogen activated protein kinases （ERK1/2, P38, JNK） in phosgene induced lung injury in rats in vivo. Method 30 male wistar rats were randomized into the group as follows, Gas inhalation control group, Phosgene inhalation group,and the following groups of the inhibitors of MAPK, involving SP600125, PD98059 and SB203580,6 animals in each group, we copy the model of phosgene-induced lung injury, used the directional flow-inhalation device,the air control group inhaled the air,and the intervention groups were given PD98059 （intraperitoneal injection）, SB203580（hypodermic injection）, SP600125 （intravenous） respectively before the inhalation of phosgene. The locations and quantities of three subfamilies of MAPKs （ERK1/2, P38, JNK） and p-MAPKs （p-ERK1/2, p-P38, p-JNK） were analyzed by immunohistochemistry and Western Blot analysis respectively; The histopathological changes of lung tissues, the number of neutrophil cells and the W/D were examined. Result There were rare p- ERK1/2, p-P38 and p-JNK positive expression in alveolar and airway epithelial cells in control group, while the positive cells increased strikingly in phosgene inhalation groups,these cells involved in this process mainly in-cluded alveolar epithelial cells, air way epithelial cells,pleuralmesothelial cells, infiltrative inflammatory cells, interstitium fibrocytes. Af- ter the intervention of the specific inhibitor,the positive cells decreased. As Western Blot analysis show, Protein quantities of p-P38 and p- JNK were higher in phosgene inhalation groups than those in control group, and the differences were significant （P〈0.05）. Protein quanti- ties of p-ERK1/2, p-P38 and p-JNK were lower in intervention groups than phosgene inhalation group,and the differences were significant （P〈0.05, P〈0.01 ）. The lung injury in phosgene inhalation groups was more severer compared with the control group,the typical pathologi- cal characters of acute lung injury were discovered,the increase of the number of neutrophil cells and W/D. After the intervention of the specific inhibitor SP600125 and SB203580, the number of neutrophil cells and W/D reduced, and the differences were significant （P〈 0.05, P〈0.01 ）. Conclusion Phosgene inhalation may activate the MAPK signaling pathway, and the expression of the phosphorylation of MAPKs increased, especially the P38 ang JNK. The results may contribute to the lung injury induced by phosgene.