阿托伐他汀预处理对小鼠肠缺血再灌注损伤的影响及其与PI3K/Akt信号通路的关系

Effect of atorvastatin preconditioning on intestinal ischemia-reperfusion injury in mice and the relationship with PI3K/Akt signaling pathway

目的探讨阿托伐他汀预处理对小鼠肠缺血再灌注损伤的影响及其与1-磷脂酰肌醇3-激酶/丝氨酸-苏氨酸蛋白激酶(PI3K/Akt)信号通路的关系。方法健康雄性C57BL/6小鼠24只,6~8周龄,体重18~22 g,采用随机数字表法分为4组(n=6):假手术组(S组)、缺血再灌注组(I/R组)、阿托伐他汀预处理组(A组)和阿托伐他汀+PI3K抑制剂LY294002组(AL组)。A组和AL组阿托伐他汀10 mg/kg连续灌胃3 d,AL组于阿托伐他汀末次灌胃前30 min腹腔注射LY2940020.3 mg/kg。随后采用夹闭肠系膜上动脉45 min再灌注2 h的方法,建立小鼠肠缺血再灌注损伤模型,S组只分离肠系膜上动脉不夹闭。于再灌注2 h后处死小鼠,取小肠组织,HE染色后光镜下观察病理学结果,测定肠组织Chiu评分和湿重/干重比值,Western blot法检测肠组织PI3K、磷酸化Akt(p-Akt)、自噬蛋白Beclin-1、微管相关蛋白1轻链3Ⅰ(LC3Ⅰ)和微管相关蛋白1轻链3Ⅱ(LC3Ⅱ)的表达,计算LC3Ⅱ/LC3Ⅰ比值。结果与S组比较,I/R组、A组和AL组肠组织Chiu评分和湿重/干重比值升高,PI3K和p-Akt下调,Beclin-1表达上调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05);与I/R组比较,A组肠组织Chiu评分和湿重/干重比值降低,PI3K和p-Akt表达上调,Beclin-1表达下调,LC3Ⅱ/LC3Ⅰ比值降低(P<0.05)。与A组比较,AL组肠组织Chiu评分和湿重/干重比值升高,PI3K和p-Akt表达下调,Beclin-1表达上调,LC3Ⅱ/LC3Ⅰ比值升高(P<0.05)。结论阿托伐他汀预处理可减轻小鼠肠缺血再灌注损伤,其机制与激活PI3K/Akt信号通路,抑制自噬水平有关。

Objective To investigate the effect of atorvastatin preconditioning on intestinal ischemia-reperfusion(I/R)injury in mice and the relationship with phosphatidylinositol 3-kinase(PI3K)/serine-threonine kinase(Akt)signaling pathway.Methods Twenty-four healthy male C57BL/6 mice,aged 6-8 weeks,weighing 18-22 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(S group),I/R group,atorvastatin preconditioning group(A group),atorvastatin plus PI3K inhibitor LY294002 group(AL group).Atorvastatin 10 mg/kg was given by intragastric gavage for 3 consecutive days in A and AL groups,and in addition LY2940020.3 mg/kg was intraperitoneally injected at 30 min before the last administration of atorvastatin in AL group.Intestinal I/R was produced by occlusion of superior mesenteric artery(SMA)for 45 min followed by 2 h reperfusion in anesthetized mice.The superior mesenteric artery was only isolated but not clamped in S group.The mice were sacrificed at the end of reperfusion,and small intestinal tissues were taken for determination of the pathological changes with a light microscope after HE staining and for determination of wet to dry weight ratio(W/D ratio)and expression of PI3K,phosphorylated Akt(p-Akt),autophagy-related proteins Beclin-1,microtubule-associated protein 1 light chain 3Ⅰ(LC3Ⅰ)and LC3Ⅱ.The intestinal damage was assessed and scored according to Chiu.The ratio of LC3Ⅱexpression to LC3Ⅰexpression(LC3Ⅱ/LC3Ⅰ)was calculated.Results Compared with S group,Chiu′s scores and W/D ratio were significantly increased,the expression of PI3K and p-Akt was down-regulated,the expression of Beclin-1 was up-regulated,and LC3Ⅱ/LC3Ⅰratio was increased in I/R,A and AL groups(P<0.05).Compared with I/R group,Chiu′s scores and W/D ratio were significantly decreased,the expression of PI3K and p-Akt was up-regulated,the expression of Beclin-1 was down-regulated,and LC3Ⅱ/LC3Ⅰratio was decreased in A group(P<0.05).Compared with A group,Chiu′s scores and W/D ratio were significantly increased,the expression of PI3K and p-Akt was down-regulated,the expression of Beclin-1 was up-regulated,and LC3Ⅱ/LC3Ⅰratio was increased in AL group(P<0.05).Conclusion Atorvastatin preconditioning can mitigate intestinal I/R injury in mice,and the mechanism is related to activating PI3K/Akt signaling pathway and inhibiting the level of autophagy.