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目的探讨肝缺血/再灌注损伤过程中肝和小肠细胞凋亡的发生及褪黑素的保护作用。方法 90只Wistar大鼠(190～210 g,6～7周龄)随机分为褪黑素处理组(Mel),酒精溶媒对照组(Alc)和生理盐水对照组(NS)。通过阻断门静脉左支、左肝动脉和肝总管左支60 min建立肝左叶缺血/再灌注模型,之后分别于复流后的30、60和120 min收集血清、肝脏和小肠的标本进行检测。利用自动生化分析仪检测血清中的ALT和AST,利用酶联免疫吸附法检测血清中的TNF-α,利用比色法检测肝脏及小肠组织中的MDA含量,通过免疫组织化学方法对肝脏和小肠组织的Caspase-3进行染色,利用原位末端标记技术(TUNEL)对肝脏和小肠的凋亡细胞进行染色,利用透射电镜观察组织中的凋亡小体。结果 Mel组在复流后所有时点的ALT均显著低于两个对照组(P<0.05);Mel组在复流120 min时的AST显著地低于两个对照组(P<0.05)。Mel组在复流后所有时点的TNF-α含量及线粒体中的MDA含量均显著低于两个对照组(P<0.05)。Mel组在复流后120 min的Caspase-3的积分光密度(IOD)显著地低于两个对照组(P<0.05)。另外Mel组在复流后所有时点的TUNEL染色的IOD值均显著地低于两个对照组(P<0.05)。结论肝缺血/再灌注可以升高血清中TNF-α的水平和线粒体中的MDA含量,增加肝及小肠细胞的Caspase-3的表达和凋亡的发生。褪黑素减轻肝缺血/再灌注损伤的机制可能与抑制自由基损伤线粒体,进而抑制细胞凋亡有关。
Objective To investigate the occurrence of hepatocyte and intestinal cell apoptosis and the protective effect of melatonin during hepatic ischemia / reperfusion injury. Methods 90 Wistar rats (190-210 g, 6-7 weeks old) were randomly divided into melatonin group, alcohol vehicle control group and saline control group. The left hepatic lobe ischemia / reperfusion model was established by blocking the left branch of the portal vein, the left hepatic artery and the left common hepatic duct for 60 min, then the serum, liver and small intestine specimens were collected at 30, 60 and 120 min after reperfusion respectively Detection. Serum ALT and AST were measured by automatic biochemical analyzer. The level of TNF-α in serum was detected by enzyme-linked immunosorbent assay (ELISA). The content of MDA in liver and intestine was detected by colorimetric assay. The liver and small intestine Tissue samples of Caspase-3 were stained and the apoptotic cells in the liver and small intestine were stained by TUNEL. The apoptotic bodies in the tissues were observed by transmission electron microscopy. Results The ALT of Mel group at all time points after reflow was significantly lower than that of the two control groups (P <0.05). The AST of Mel group at 120 minutes after reflow was significantly lower than that of the two control groups (P <0.05). The levels of TNF-α and mitochondrial MDA in Mel group at all time points after reperfusion were significantly lower than those in two control groups (P <0.05). The integral optical density (IOD) of Caspase-3 in Mel group at 120 min after reflow was significantly lower than that of the two control groups (P <0.05). In addition, the IOD values of TUNEL staining in all Mel groups after reperfusion were significantly lower than those in the two control groups (P <0.05). Conclusion Hepatic ischemia / reperfusion can increase the level of TNF-α in serum and the content of MDA in mitochondria, and increase the expression of Caspase-3 and apoptosis in hepatocytes and small intestine. The mechanism of melatonin in reducing hepatic ischemia / reperfusion injury may be related to the inhibition of free radical damage to mitochondria and then apoptosis.