右美托咪定对小鼠肠缺血再灌注时内质网应激的影响

Effect of dexmedetomidine on endoplasmic reticulum stress during intestinal ischemia-reperfusion in mice

目的评价右美托咪定对小鼠肠缺血再灌注时内质网应激的影响。方法SPF级健康雄性C57BL/6小鼠30只,8周龄,采用随机数字表法分为3组(n=10):假手术组(S组)、肠缺血再灌注组(I/R组)和右美托咪定组(DEX组)。S组只分离不阻断肠系膜上动脉;I/R组和DEX组采用阻断肠系膜上动脉20min,再灌注24h的方法制备小鼠肠缺血再灌注损伤模型;缺血前30min,DEX组腹腔注射右美托咪定25μg/kg,S组和I/R组注射等容量生理盐水。再灌注24h时处死小鼠,取小肠组织,光镜下观察病理学结果并计算Chiu评分;采用TUNEL法检测细胞凋亡情况并计算凋亡指数(AI);电镜下观察上皮细胞超微结构;RT-PCR法检测CCAAT增强子结合蛋白同源蛋白(CHOP)、活化的转录因子4(ATF4)和X-盒结合蛋白-1(XBP1)mRNA的表达;Westernblot法检测CHOP、Bcl-2、Bax及caspase-3的表达并计算Bcl-2/Bax比值。结果与S组比较,I/R组和DEX组Chiu评分和AI升高,CHOP、ATF4和XBP-1mRNA及CHOP、Bax和caspase-3表达上调,Bcl-2表达下调,Bcl-2/Bax比值降低(P<0.05);与I/R组比较,DEX组Chiu评分和AI降低,CHOP、ATF4和XBP-1mRNA及CHOP、Bax和caspase-3表达下调,Bcl-2表达上调,Bcl-2/Bax比值升高(P<0.05)。结论右美托咪定减轻小鼠肠缺血再灌注损伤的机制可能与调节内质网应激抑制细胞凋亡有关。

Objective To evaluate the effect of dexmedetomidine on endoplasmic reticulum stress during intestinal ischemia-reperfusion(I/R)in mice. Methods Thirty SPF healthy male C57BL/6 mice, aged 8 weeks, were divided into 3 groups(n=10 each)using a random number table method: sham operation group(S group), intestinal I/R group(I/R group)and dexmedetomidine group(DEX group). The superior mesenteric artery was only isolated but not clamped in S group.The model of intestinal I/R injury was established by clamping superior mesenteric artery for 20 min followed by 24 h of reperfusion in I/R group and DEX group.Dexmedetomidine 25 μg/kg was intraperitoneally injected at 30 min before ischemia in DEX group, while the equal volume of normal saline was given instead of dexmedetomidine in S group and I/R group.Mice were sacrificed at 24 h of reperfusion, and small intestinal tissues was obtained for examination of the pathological changes and ultrastructure of intestinal epithelial cells and for determination of cell apoptosis, expression of CCAAT-enhancer binding protein homologous protein(CHOP), transcription factors(ATF4), and X-4 box binding protein 1(XBP1)mRNA(by real-time polymerase chain reaction), and expression of CHOP, Bcl-2, Bax and caspase-3 in intestinal tissues(by Western bolt). The apoptosis index(AI)and ratio of Bcl-2 to Bax were calculated.Intestinal damage was assessed and scored according to Chiu. Results Compared with S group, Chiu′s score and AI were significantly increased, the expression of CHOP, ATF4 and XBP-1 mRNA, CHOP, Bax and caspase-3 was up-regulated, and the expression of Bcl-2 was down-regulated, and Bcl-2/Bax ratio was decreased in I/R group and DEX group(P<0.05). Compared with I/R group, Chiu′s score and AI were significantly decreased, the expression of CHOP, ATF4 and XBP-1 mRNA, CHOP, Bax and caspase-3 was down-regulated, and the expression of Bcl-2 was up-regulated, and Bcl-2/Bax ratio was increased in DEX group(P<0.05). Conclusion The mechanism by which dexmedetomidine reduces intestinal I/R injury may be related to regulating endoplasmic reticulum stress and inhibiting cell apoptosis in mice.