JAK/STAT通路在大鼠肠缺血再灌注所致肠损伤中的作用

Role of JAK/STAT in intestinal injury induced by intestinal ischemia/reperfusion in rats

目的:探讨Janus激酶/信号转导和转录激活子(JAK/STAT)通路在大鼠肠缺血再灌注(I/R)所致肠损伤中的作用。方法:雄性健康SD大鼠40只,体重230～255 g,随机分为5组(n=8):假手术组(sham组),仅分离肠系膜上动脉(SMA)但不夹闭;肠缺血再灌注组(I/R组),采用阻断SMA 60 min后开放120 min的方法制备肠I/R损伤模型;AG490(JAK特异性抑制剂)组,于夹闭SMA前30 min在大鼠侧腹部皮下注射8 mg.kg-1AG490,余同I/R组;雷帕霉素(STAT特异性抑制剂)组(RPM组),于夹闭SMA前30 min在大鼠侧腹部皮下注射0.4 mg.kg-1 RPM,余同I/R组;二甲基亚砜组(DMSO组),于夹闭SMA前30 min侧腹部皮下注射2μmol.kg-1DMSO,余同I/R组,DMSO系AG490和RPM的溶剂。所有大鼠均于再灌注120 min后取小肠观察肠黏膜形态学变化并行Chiu's评分,同时取肠黏膜组织检测髓过氧化物酶(MPO)和超氧化物歧化酶(SOD)活性,以及丙二醛(MDA)和乳酸(LD)含量,采集动脉血检测血清二胺氧化酶(DAO)活性及15-F2t-isoprostane、内皮素-1(ET-1)和血栓烷素B2(TXB2)浓度。TUNEL法检测肠上皮细胞凋亡情况并计算凋亡指数;采用Western blotting检测磷酸化的JAK2(p-JAK2)及STAT3(p-STAT3)的表达水平。结果:与sham组比较,其余各组的Chiu's评分和肠黏膜细胞凋亡指数均显著升高(P<0.05);而I/R组和DMSO组的LD和MDA含量、DAO活性、MPO活性、15-F2t-iso-prostane浓度、ET-1浓度及TXB2浓度均显著升高,p-JAK2及p-STAT3蛋白表达量也显著升高,SOD活性显著降低(P<0.05)。与I/R组和DMSO组比较,AG490组和RPM组的DAO活性、MPO活性、肠黏膜上皮细胞凋亡指数、ET-1浓度、p-JAK2及p-STAT3蛋白表达均显著降低,SOD活性则显著升高(P<0.05)。结论:JAK/STAT信号通路的激活参与了肠I/R所致的肠损伤的发生,其作用与促进氧化应激损伤、中性粒细胞的聚集和肠黏膜细胞凋亡有关。

AIM： To investigate the role of Janus kinase/signal transducer and activator of the transcription factor（JAK/STAT） signaling pathway in intestinal injury induced by intestinal ischemia/reperfusion（I/R） in rats.METHODS： Rat intestinal I/R injury was produced by clamping the superior mesenteric artery（SMA） for 60 min followed by reperfusion for 120 min.Forty healthy male SD rats weighing 230~255g were randomly divided into the following 5 groups（8 per group）： in sham operation group（S group）,SMA was isolated without occlusion;in intestinal I/R（I/R group）,SMA was occluded for 60 min followed by reperfusion for 120 min;in inhibitor of JAK（AG490） treatment ＋I/R group（AG490 group）,AG490 at a dose of 8 mg/kg was applied by lateral abdominal subcutaneous injection 30 min before intestinal ischemia,and the rest procedures were performed as I/R group;in inhibitor of STAT（rapamycin,RPM） treatment ＋I/R group（RPM group）,RPM 0.4 mg/kg was applied by lateral abdominal subcutaneous injection 30 min before intestinal ischemia,and the rest procedures were performed as I/R group;in DMSO（solvent of AG490 and RPM）＋I/R group（DMSO group）,DMSO at a dose of 2 μmol/kg was applied by lateral abdominal subcutaneous injection 30 min before inducing 60 min of intestinal ischemia and 120 min of reperfusion as described in group I/R.The experiment in each group was terminated after 120 min of reperfusion.The intestinal mucosa was scraped off for determining the activity of myeloperoxidase（MPO） and superoxide dismutase（SOD）,and content of malondialdehyde（MDA） and lactic acid（LD）.The damage degree of intestinal mucosa was evaluated according to Chiu＇s score under light microscope.Arterial blood samples were taken for detecting the activity of plasma diamine oxidase（DAO） and the concentrations of 15-F2t-isoprostane,endothelin（ET）-1 and thromboxane B2（TXB2）.Intestinal mucosal apoptosis was observed using TUNEL assay.The protein levels of phosphorylated JAK2（p-JAK2） and STAT3（p-STAT3） in the intestine were measured by Western blotting.RESULTS： Compared with S group,Chiu＇s score and apoptotic index calculated as the percentage of apoptotic cells were significantly increased in the other 5 groups（all P0.05）.In I/R group and DMSO group,the content of LD and MDA,the activity of DAO and MPO,plasma concentrations of 15-F2t-isoprostane,ET-1 and TXB2,and the protein levels of p-JAK2 and p-STAT3 were significantly higher while SOD activity was lower than those in S group（all P0.05）.The activity of DAO and MPO,apoptotic index,the plasma concentration of ET-1 and the protein levels of p-JAK2 and p-STAT3 were significantly reduced,and SOD activity was restored in AG490 group and RPM group as compared to I/R group and DMSO group（all P0.05）.No significant difference was detected in the content of MDA and 15-F2t-isoprostane between I/R group and DMSO group（all P0.05）.CONCLUSION： Activation of intestinal JAK/STAT pathway plays a critical role in intestinal I/R-induced injury,which is associated with the increase in oxidative stress,neutrophil accumulation and intestinal mucosal apoptosis.