自噬在缺血后处理减轻小鼠肠缺血再灌注损伤中的作用

Role of autophagy in ischemia postconditioning-induced attenuation of intestinal ischemia-reperfusion injury in mice

目的评价自噬在缺血后处理减轻小鼠肠缺血再灌注损伤中的作用。方法SPF级健康成年雄性C57BL/6J小鼠32只,9~12周龄,体重25~29 g,采用随机数字表法分为4组(n=8):假手术组(S组)、肠缺血再灌注组(IIR组)、缺血后处理组(IPO组)和缺血后处理+自噬抑制剂3-甲基腺嘌呤组(IPO+3-MA组)。采用夹闭肠系膜上动脉根部45 min恢复灌注2 h的方法制备小鼠肠缺血再灌注损伤模型,IPO组于恢复灌注前3 min给予3个循环灌注30 s,缺血30 s的处理。于再灌注2 h时采集股动脉血样,测定血清二胺氧化酶(DAO)、D-乳酸及肠型脂肪酸结合蛋白(I-FABP)的浓度,随后处死小鼠取小肠组织,光镜下观察病理学结果并行Chiu评分,计算肠组织含水量;Western blot法检测自噬相关蛋白Beclin-1和p62的表达。结果与S组比较,IIR组和IPO组Chiu评分升高,血清DAO、D-乳酸和I-FABP的浓度及肠组织含水量升高,肠组织LC3Ⅱ/LC3Ⅰ比值升高,Beclin-1表达上调,p62表达下调(P<0.05);与IIR组比较,IPO组Chiu评分降低,血清DAO、D-乳酸、I-FABP浓度和肠组织含水量降低,LC3Ⅱ/LC3Ⅰ比值升高,Beclin-1表达上调,p62表达下调(P<0.05);与IPO组比较,IPO+3-MA组Chiu评分升高,血清DAO、D-乳酸、I-FABP浓度和肠组织含水量升高,LC3Ⅱ/LC3Ⅰ比值降低,Beclin-1表达下调,p-62表达上调(P<0.05)。结论自噬参与了缺血后处理减轻小鼠肠缺血再灌注损伤的过程。

Objective To evaluate the role of autophagy in ischemia postconditioning(IPO)-induced attenuation of intestinal ischemia-reperfusion(I/R)injury in mice.Methods Thirty-two SPF healthy adult male C57BL/6J mice,aged 9-12 weeks,weighing 25-29 g,were divided into 4 groups(n=8 each)using a random number table method:sham operation group(group S),intestinal I/R group(group IIR),group IPO and IPO plus 3-methyladenine(3-MA)group(group IPO+3-MA).The model of intestinal I/R was established by occlusion of superior mesenteric artery for 45 min followed by 2-h reperfusion in anesthetized animals.The mice underwent 3 cycles of 30-s reperfusion followed by 30-s ischemia before restoration of reperfusion in group IPO.Blood samples from the femoral artery were collected at 2 h of reperfusion for determination of concentrations of serum diamine oxidase(DAO),D-lactate(D-LA)and intestinal fatty acid binding protein(I-FABP).The animals were then sacrificed and intestinal tissues were removed for microscopic examination of the pathologic changes which were scored according to Chiu and for determination of the expression of autophagy-related proteins Beclin-1 and p62(by Western blot).The water content of intestinal tissues was calculated.Results Compared with group S,the Chiu′s score,concentrations of serum DAO,D-LA and I-FABP,water content of intestinal tissues,and LC3Ⅱ/LC3Ⅰratio were significantly increased,Beclin-1 expression was up-regulated,and p62 expression was down-regulated in IIR,IPO and IPO+3-MA groups(P<0.05).Compared with group IIR,the Chiu′s score,concentrations of serum DAO,D-LA and I-FABP,water content of intestinal tissues,and LC3Ⅱ/LC3Ⅰratio were significantly decreased,Beclin-1 expression was up-regulated,and p62 expression was down-regulated in group IPO(P<0.05).Compared with group IPO,the Chiu′s score,concentrations of serum DAO,D-LA and I-FABP,and water content of intestinal tissues were significantly increased,LC3Ⅱ/LC3Ⅰratio was decreased,Beclin-1 expression was down-regulated,and p62 expression was up-regulated in group IPO+3-MA(P<0.05).Conclusion Autophagy is involved in ischemia postconditioning-induced attenuation of intestinal I/R injury in mice.