高压氧对大鼠肠缺血-再灌注损伤致细胞凋亡的影响

目的 观察大鼠在小肠缺血-再灌注(I/R)不同时期应用高压氧(HBO)治疗对小肠黏膜细胞凋亡的影响,并探讨其作用机制.方法 采取大鼠肠系膜上动脉(SMA)钳夹缺血60 min,松钳夹再灌注60 min,建立S-D大鼠小肠I/R损伤模型,并随机(随机数字法)分成4组:I/R组、缺血前HBO治疗组或HBO预处理组(HBO-P)、缺血期HBO治疗组(HBO-I)和再灌注期HBO治疗组(HBO-R).再灌注60 min后,取回肠末端小肠标本.采用酶连免疫吸附试验法(Elisa)检测肠道组织TNF-α,用比色法检测肠道组织匀浆含量ATP,用免疫组化法检测小肠组织中半胱氨酸天门冬氨酸特异蛋白酶(Caspase-3)的表达.数据以均数±标准差(-x±s)表示,采用单因素方差分析检验,独立样本间比较采用SNK-q检验.结果 肠道组织TNF-α含量在HBO-I组最低,其次为HBO-P组(每两组比较P＜0.05),但前两组明显低于HBO-R组和I/R组(P＜0.05),HBO-R组略低于I/R组,但差异无统计学意义(P＞0.05);Caspase-3表达在HBO-I组最低,HBO-P组次之(两组比较P＜0.05);HBO-R和I/R组最高(与前两组比较P＜0.05),尽管HBO-R组略低于I/R组,但差异无统计学意义(P＞0.05);ATP含量在HBO-I组最低,HBO-P组次之(两组比较P＜0.05),HBO-R和I/R组最高(与前两组比较P＜0.05),尽管HBO-R组略低于I/R组,但差异无统计学意义(P＞0.05).结论 HBO、小肠I/R损伤和小肠黏膜上皮凋亡之间存在联系;HBO治疗可以维持黏膜上皮细胞能量代谢,减少ATP耗竭,降低肠道组织TNF-α含量,减轻I/R损伤小肠黏膜上皮凋亡;在缺血期和缺血前应用HBO治疗显示有益结果,特别是在缺血期应用HBO效果最好,再灌注期应用HBO无效.

Effects of hyperbaric oxygen on the intestinal mucosal apoptosis caused by ischemia-reperfusion injury in rats

Objective To observe the effects of hyperbaric oxygen (HBO) on the apoptosis expression of intestinal mucosa during the different periods of ischemia-reperfusion injury in order to elucidate the underlying mechanisms. Method Rats were subjected to ischemia for 60 min by clamping superior mesenteric artery, and then had reperfusion for 60 min by unclamping. Rats were randomly divided into four groups: ischemia-reperfusion group (I/R), pre-emptive HBO or HBO treatment before ischemia (HBO-P) group, HBO treatment during ischemia period (HBO-I) group, and HBO treatment during reperfusion (HBO-R) group. After reperfusion for 60 min, samples of small intestine tissue were taken from the end portion of ileum for detecting the levels of ATP by using colorimetric method and the levels of caspase-3 by using immunochemistry. The levels of TNF-α in intestinal tissue were measured by using enzyme-linked immunosorbent assay method ( ELISA). All values were expressed in Mean ± Standard Deviation (x ± s). The different groups were compared among them with SNK- q test of OneWay analysis of variance (One-Way ANOVA plus SNK). Results The levels of TNF-α in HBO-I group were significantly lower than that in HBO-P group ( P ＜ 0.05), and significantly lower in HBO-P group than those in HBO-R or I/R groups ( P ＜ 0.05), and there was no significant difference in TNF-α between HBO-R and I/R group ( P ＞ 0.05). The levels of caspase-3 were significantly lower in HBO-I group than those in HBO-P group ( P ＜ 0. 05), and also significantly lower in HBO-P group than those in I/R or HBO-R groups ( P ＜ O. 05), and no significant difference caspase-3 was found between HBO-R and I/R groups. The ATP levels were significantly lower in HBO-I group than those in HBO- P group ( P ＜ 0. 05), and also significantly lower in HBO- P group than those in I/R or HBO-R group ( P ＜ 0.05), and no significant difference in ATP level between in HBO-R and I/R group. Conclusions There was a connection between HBO and small intestinal I/R injury as well as mucosal cell apoptosis. And HBO maintained ATP and aerobic metabolism, and inhibited the genesis of TNF-α, and thus in turn prevented intestinal mucosa cell from apoptosis. The best result was obtained when HBO was administered during ischemia period, and there was no effect found when HBO was employed during reperfusion period.