臭氧氧化预处理对大鼠肝脏缺血再灌注细胞凋亡的影响

Effects of Ozone Oxidative Preconditioning on Apoptosis Induced by Hepatic Ischemia Reperfusion Injury in Rats

关键词:  臭氧氧化预处理  大鼠  肝脏缺血再灌注  细胞凋亡

Key words:  O₃ oxidative preconditioning  rats  hepatic ischemia reperfusion  cell apoptosis

摘要:

目的 观察O₃氧化预处理对大鼠肝脏缺血再灌注后细胞凋亡的影响，探讨O₃对肝脏的保护作用及其可能的作用机制。方法 将18只SD大鼠（♂，6~8周龄，250~280 g）随机分为3组：O₃预处理组（OP+IR组）、单纯缺血再灌注组（IR组）和假手术组（S组），每组6只。O₃预处理组每天同一时间点行腹腔注射O₃/O₂混合气体（O₃浓度为50 μg·mL^-1，1 mg·kg^-1·d^-1），连续注射5 d。S组和IR组则注射相等容积的O₂。缺血45 min后恢复灌注3 h建立70%肝脏缺血再灌注模型（S组仅开腹不阻断肝血流），取左肝叶组织，HE染色观察肝组织形态学变化，TUNEL法测定肝细胞凋亡指数（AI），免疫组化法测定凋亡相关基因Bcl-2、Bax蛋白表达量的变化，并计算Bcl-2/Bax比值。结果 与S组比较，OP+IR组与IR组的肝组织电镜观察皆有损伤，OP+IR组的损伤程度较IR组减轻；与S组相比，OP+IR组和IR组的肝细胞凋亡指数增加，Bcl-2蛋白表达下降，Bax蛋白表达增加，Bcl-2/Bax比值下降（P均<0.05）；与IR组相比，OP+IR组的肝细胞凋亡指数降低，Bcl-2蛋白表达增加，Bax蛋白减少，Bcl-2/Bax比值增加（P均<0.05）。结论 O₃氧化预处理可以显著减轻大鼠肝脏缺血再灌注后肝细胞的凋亡，其作用机制可能与升高Bcl-2蛋白表达、降低Bax蛋白表达从而上调Bcl-2/Bax比例有关。

Abstract:

OBJECTIVE To observe the effect of ozone oxidative preconditioning on apoptosis induced by hepatic ischemia reperfusion injury in rats, and explore the protective effect of ozone on the liver and possible mechanism. METHODS Eighteen SD rats (♂, 6-8 weeks old, 250-280 g) were randomly divided into three groups: ozone oxidative preconditioning group(OP+IR group), pure ischemia reperfusion group (IR group) and sham operation group (S group), six rats in each group. OP+IR group were received intraperitoneal injection of O₃/O₂ gas mixture (O₃ concentration of 50 μg·mL^-1, 1 mg·kg^-1·d^-1) at the same time every day for 5 d; S group and IR group were received equal volume of O₂. To set up 70% of hepatic ischemia reperfusion model, OP+IR group and IR group were subjected to 45 min ischemia, then follow by 3 h perfusion, and S group was only subjected to laparotomy. The liver tissue morphology was observed, liver cell apoptosis index (AI), Bcl-2 protein expression and Bax protein expression were determined, the Bcl-2/Bax ratio was computed. RESULTS Compared with S group, the liver tissue ultrastructure of OP+IR group and IR group was damaged; but compared with IR group, the damage of OP+IR group was lighter. Compared with S group, the hepatocyte apoptosis index of OP+IR group and IR group were increased, the Bcl-2 protein expression was decreased, Bax protein expression was increased, and the Bcl-2/Bax ratio was decreased(P<0.05). Compared with IR group, the hepatocyte apoptosis index of OP+IR group was reduced, the Bcl-2 protein expression was increased, Bax protein was decreased, and the Bcl-2/Bax ratio was increased(P<0.05). CONCLUSION O₃ oxidative preconditioning may reduce apoptosis induced by hepatic ischemia reperfusion injury in rats, and it may be related to the rise of the Bcl-2 protein, the decrease of the Bax protein and the rise in the proportion of the Bcl-2/Bax.