MAPKs通路在高氧肺损伤发病机制中的作用

Role of MAP kinases signal pathway in hyperoxia-induced lung injury

目的:观察丝裂原活化蛋白激酶(Mitogen activated protein kinases,MAPKs)信号通路细胞外信号调节激酶(Extracellular signal-regulated kinase,ERK)、C-jun氨其未端激酶(C-Jun N-terminal kinase,JNK)、p38MAPKs在高氧肺损伤肺组织的分布以及表达,应用信号途径抑制剂,研究MAPKs信号在高氧诱导肺损伤发生发展中的作用。方法:幼年Wistar大鼠随机分为如下各组(n=6):空气对照组(A)、高氧暴露3、7、14 d组(O3、O7、O14组)、高氧7 d+ERK抑制剂PD98059(O7+PD)组、高氧7 d+p38抑制剂SB203580(O7+SB)组、高氧7 d+JNK抑制剂SP600125(O7+SP)组及相应空气+抑制剂组。其中,PD98059 0.3 mg/kg及SB2035800.2 mg/kg由大鼠尾静脉注入,SP600125 15 mg/kg经腹腔注射。光镜下观察肺组织形态学改变并进行肺损伤病理评分;测定肺湿/干重比(Wet/drgratio,W/D)、肺泡灌洗液(Bronchoalvedar lavage fluid,BALF)蛋白含量及肺通透系数;免疫组化检测磷酸化MAPKs在肺组织的分布,Western blot检测MAPKs蛋白含量变化。结果:与空气暴露组比较,高氧暴露各组肺组织可见不同程度损伤,O7及O14组肺W/D、BALF蛋白含量、肺通透系数明显增加(P<0.05);而抑制剂组中O7+SB、O7+SP组肺泡结构较O7组有明显改善,肺损伤病理学评分降低,同时W/D、总蛋白、肺通透系数较也明显下降(P<0.05);MAPKs阳性细胞在高氧组表达明显增多,分布广泛;各相应抑制剂组中其阳性细胞显著减少;Western blot结果显示,高氧组ERK、p38、JNK蛋白含量明显高于空气组,在高氧各组中以O7组ERK及O14组p38、JNK表达最强,各信号通路抑制剂均能有效抑制相应MAPKs蛋白的表达。结论:高浓度氧可激活肺组织ERK、p38、JNK信号途径;p38及JNK可能促进了高氧肺损伤的发生发展。

Objective：To explore distributions and expressions of ERK,JNK,p38 MAP kinase in lung tissues and roles of MAP kinases on hyperoxia-induced lung injury with signal pathway inhibitors. Methods：Sixty Wistar rats aged 3 weeks old were randomized to 10 groups with 6 rats in each：room-air group（A）,hyperoxia exposure 3,7,14 days groups（O3,O7,O14）,hyperoxia 7 d＋ PD98059 group （O7＋PD）、hyperoxia 7 d ＋SB203580 group（O7＋SB）,hyperoxia 7 d＋ SP600125 group（O7＋SP） and room-air ＋inhibitors groups.Rats had received i.v injection of PD98059（0.3 mg/kg）and SB203580（0.2 mg/kg） and injected with SP600125（15 mg/kg） intraperitoneally.Lung pathology examined by light microscopy,lung wet/dry ratio（W/D） and protein level in BALF and permeability coefficient were evaluated.The location and quantity of MAP kinases protein were detected by immunohistochemistry and Western blot analysis respectively.Results：Lung injury in hyperoxia exposure groups were developed as compared with room-air group,W/D and protein level in BALF and permeability coefficient in O7 and O14 groups were increased than those in control group（P0.05）,lung tissue pathology in O7＋SB and O7＋SP groups were improved than that in O7 group,as evidenced by decreased pathology score and decreased W/D,total protein in BALF and permeability coefficient（P0.05）.The positive cells remarkably increased in hyperoxia groups andthe positive cells labeled in many types cells.MAP kinases positive cells in inhibitors groups decreased significantly.Protein quantity of ERK,p38,JNK in hyperoxia exposure groups were remarkablely higher than those in room-air group,protein expression of ERK in O7 group and p38 in O14 group and JNK in O14 group were strongest in all hyperoxia groups.Signal inhibitors could inhibit corresponding MAP kinases protein expression.Conclusion：High concentration of oxygen activated ERK,p38,JNK signaling pathway in lung tissue,p38 and JNK may exert promotion role on hyperoxia induced lung injury development.