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目的观察O_3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O_3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:O_3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。O_3预处理组每天同一时间点行腹腔注射O_3/O_2混合气体(O_3浓度为50μg·m L~(-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O_2。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 O_3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。
Objective To observe the effect of O_3 preconditioning on apoptosis in rat liver after ischemia-reperfusion and to explore the protective effect of O_3 on the liver and its possible mechanism. Methods 18 SD rats (♂, 6-8 weeks old, 250-280 g) were randomly divided into three groups: O_3 pretreatment group (OP + IR group), ischemia reperfusion group Surgery group (S group), 6 rats in each group. O_3 preconditioning group was injected intraperitoneally with O_3 / O_2 gas mixture (50μg · m L -1, 1 mg · kg -1 · d -1) at the same time every day, 5 d. S group and IR group were injected with equal volume of O_2. Forty-five minutes after ischemia, the rats were reperfused for 3 hours to establish a model of hepatic ischemia-reperfusion injury (group S, only open abdomen did not block the blood flow of the liver). Left hepatic lobe tissue was taken and the morphological changes of liver were observed by HE staining. TUNEL assay The apoptosis index (AI) of hepatocytes and the expression of Bcl-2 and Bax proteins were detected by immunohistochemistry, and the ratio of Bcl-2 / Bax was calculated. Results Compared with S group, OP + IR group and IR group were both injured by electron microscopy, and the damage in OP + IR group was less than that in IR group. Compared with S group, the hepatocytes in OP + IR group and IR group The apoptotic index increased, the expression of Bcl-2 decreased, the expression of Bax increased and the ratio of Bcl-2 / Bax decreased (P <0.05). Compared with IR group, -2 protein expression increased, Bax protein decreased, Bcl-2 / Bax ratio increased (P <0.05). Conclusion Oxidative preconditioning can significantly reduce hepatocyte apoptosis after hepatic ischemia-reperfusion in rats. The mechanism may be related to increasing Bcl-2 protein expression, decreasing Bax protein expression and up-regulating Bcl-2 / Bax ratio.