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目的研究不同浓度的异氟醚预处理对大鼠肠缺血再灌注损伤的影响,并初步探讨其作用机制。方法成年雄性SD大鼠60只,按照随机分层区组设计分为5组(n=12):肠缺血再灌注组(缺血再灌注损伤组):暴露腹腔后夹闭肠系膜上动脉1h后开放再灌注2 h;0.25肺泡最低有效浓度、0.5肺泡最低有效浓度、1.0肺泡最低有效浓度异氟醚预处理30 min组(即0.25M组、0.5M组和1.0M组):在暴露腹腔前预吸入相应浓度的异氟醚30 min,再行肠缺血再灌注;假手术组:仅暴露腹腔,不行异氟醚预处理及肠系膜上动脉夹闭。颈动脉插管监测平均动脉压。在再灌注2 h时采血检测血浆超氧化物歧化酶活力、丙二醛和肿瘤坏死因子α含量;取小肠组织行组织切片HE染色,在光镜下观察其结构病理变化程度及改良Chiu’s评分,并用免疫组化染色法和Western blot方法检测肠组织Caspase-3活化表达情况。结果与缺血再灌注损伤组比,0.5M组、1.0M组的病理评分值显著性减低(P<0.01),而0.25M组无显著差异(P>0.05)。在再灌注期,缺血再灌注损伤组的平均动脉压随时间延长呈明显下降趋势,显著低于假手术组(P<0.05),而异氟醚预处理能明显减缓平均动脉压下降(P<0.01)。0.5M组、1.0M组较缺血再灌注损伤组的超氧化物歧化酶活力显著性增高,而丙二醛水平减低(P<0.01)。与缺血再灌注损伤组相比,1.0M组、0.25M组的肿瘤坏死因子α含量无显著性差异(P>0.05),而0.5M组较缺血再灌注损伤组显著性降低(P<0.01)。0.5M组、1.0M组的caspase-3蛋白表达量较0.25M组和缺血再灌注损伤组明显减少(P<0.05)。结论 0.5M与1.0M异氟醚预处理均能相似地减轻缺血再灌注后的肠黏膜结构的病理改变,并可减缓再灌注期的低血压,其保护效应可能与提高超氧化物歧化酶活性、减少丙二醛生成、下调肿瘤坏死因子α生成的炎症反应级联效应以及抑制细胞凋亡有关。
Objective To study the effects of different concentrations of isoflurane preconditioning on intestinal ischemia-reperfusion injury in rats and its possible mechanism. Methods Sixty adult male Sprague Dawley rats were randomly divided into 5 groups (n = 12): intestinal ischemia-reperfusion group (ischemia-reperfusion injury group): the superior mesenteric artery was occluded 1 h after exposure to the abdominal cavity After 2 hours of reperfusion, the lowest effective concentration of alveolar 0.25, the lowest effective concentration of 0.5 alveolar, the lowest effective concentration of alveolar 1.0 of isoflurane 30 min (ie 0.25M, 0.5M and 1.0M) Pre-inhalation of the corresponding concentration of isoflurane 30 min, reperfusion intestinal ischemia reperfusion; sham-operated group: only exposed peritoneal cavity, isoflurane preconditioning and superior mesenteric artery clamping. Carotid artery cannulation to monitor mean arterial pressure. Blood samples were collected at 2 h after reperfusion for determination of plasma superoxide dismutase (SOD), malondialdehyde (MDA) and tumor necrosis factor alpha (TNF-α) content. Tissue sections of small intestine tissue were stained with hematoxylin and eosin and histopathological changes and modified Chiu’s score were observed under light microscope. The expression of Caspase-3 in intestinal tissue was detected by immunohistochemistry and Western blot. Results Compared with ischemia reperfusion injury group, pathological score of 0.5M group and 1.0M group decreased significantly (P <0.01), but no significant difference was found in 0.25M group (P> 0.05). During reperfusion, the mean arterial pressure of ischemic reperfusion injury group decreased significantly with time, significantly lower than that of sham operation group (P <0.05), while isoflurane preconditioning significantly reduced the mean arterial pressure (P <0.01). The activities of superoxide dismutase (SOD) in the 0.5M and 1.0M groups were significantly higher than those in the ischemia-reperfusion group (P <0.01). Compared with ischemia reperfusion injury group, the content of tumor necrosis factor alpha in 1.0M group and 0.25M group had no significant difference (P> 0.05), while 0.5M group was significantly lower than ischemia reperfusion injury group (P < 0.01). Compared with 0.25M group and ischemia reperfusion injury group, the expression of caspase-3 protein in 0.5M group and 1.0M group decreased significantly (P <0.05). Conclusions 0.5M and 1.0M isoflurane preconditioning can both reduce the pathological changes of intestinal mucosa after ischemia-reperfusion and reduce the hypotension during reperfusion. The protective effect may be related to the increase of superoxide dismutase Activity, reducing the formation of malondialdehyde, down-regulating the cascade of inflammatory reaction induced by tumor necrosis factor-α, and inhibiting cell apoptosis.