海藻糖对肝脏缺血再灌注损伤的保护作用及机制研究

目的探讨海藻糖是否对肝脏缺血再灌注损伤具有保护作用及其相关机制。方法C57BL/6J小鼠数字随机分为无缺血组、缺血再灌注组、海藻糖处理组和生理盐水对照组,缺血90 min后于再灌注的0h和6h,收集血液和肝组织,通过分离血清测丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)肝功能指标及肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-2(IL-2)炎症因子水平和肝组织病理改变研究海藻糖在肝脏缺血再灌注损伤中的作用;AML12小鼠肝细胞系构建缺糖缺氧-复糖复氧细胞模型,分为实验组和对照组,实验组根据给予的海藻糖浓度不同分为低剂量组和高剂量组,对照组无海藻糖,收集细胞,流式细胞仪检测凋亡水平以研究海藻糖对肝脏缺血再灌注损伤诱导的细胞凋亡的影响,蛋白印迹法检测Caspase-3、Cleaved Caspase-3和Bcl-2蛋白水平以研究海藻糖在肝脏缺血再灌注损伤诱导的细胞凋亡中的分子机制。结果体内动物实验显示肝脏缺血再灌注后,缺血再灌注组ALT、AST及TNF-α、IL-1β和IL-2等肝功能指标及炎症因子水平升高(P<0.05),且肝组织发生坏死;而在给予海藻糖处理后,ALT、AST、TNF-α、IL-1β和IL-2等水平较生理盐水对照组降低且肝组织坏死面积也减少(P<0.05)。体外细胞实验显示与对照组相比,实验组肝细胞凋亡水平下降;且实验组活化的促凋亡蛋白Cleaved Caspase-3水平下降、抗凋亡蛋白Bcl-2水平升高。结论在体内和体外条件下海藻糖对肝脏缺血再灌注损伤有保护作用,其机制可能是通过抑制肝脏缺血再灌注损伤诱导的炎症发生和抑制Caspase-3的活化并促进Bcl-2的表达,减轻细胞凋亡,从而保护肝脏缺血再灌注损伤。

Protective effects and mechanism of trehalose on ischemia-reperfusion injury in liver

Objective:To investigate the role of trehalose in hepatic ischemia-reperfusion injury and its underlying mechanisms.Methods C57BL/6J mice were randomly divided into no-ischemia group,ischemia-reperfusion group,trehalose-treated group and normal saline control group.After ischemia for 90 minutes,reperfusion immediately or 6h,blood and liver tissues were collected,and serum was separated.The liver function parameters of ALT,AST,the inflammatory factors of TNF-α,IL-1βand IL-2,and the pathological changes of liver were detected to study the role of trehalose during hepatic ischemia-reperfusion injury.Hypoxia-reoxygenation cell model was established by AML12 mouse hepatocyte line,and divided into experimental group and control group.The experimental group was divided into low dose group and high dose group according to the concentration of trehalose administrated.And the control group had no use of trehalose.The level of apoptosis was measured to study the effect of trehalose on apoptosis induced by hepatic ischemia-reperfusion injury with flow cytometry.Western blot was utilized for detecting the levels of Caspase-3,Cleaved Caspase-3 and Bcl-2 protein to understand the molecular mechanisms of trehalose in apoptosis during hepatic ischemia-reperfusion injury.Results In vivo animal experiments showed that liver function and such inflammatory factors as ALT,AST,TNF-α,IL-1βand IL-2 increased in ischemia-reperfusion group after hepatic ischemia-reperfusion(P<0.05),and liver tissue became necrotic.After a treatment of trehalose,the levels of ALT,AST,TNF-α,IL-1βand IL-2 were lower than those of normalsaline control group and the area of liver tissue necrosis also decreased(P<0.05).In vitro cell experiments showed that the apoptosis level of hepatocytes in the experimental group decreased compared with the control group.And the level of activated pro-apoptotic protein Cleaved Caspase-3 decreased,the level of anti-apoptotic protein Bcl-2 increased.Conclusions Trehalose has protective effects on hepatic ischemia-reperfusion injury in vivo and in vitro.The mechanism may be involved in inhibiting inflammation induced by hepatic ischemia-reperfusion injury,suppressing the activation of Caspase-3 and promoting the expression of Bcl-2,thus played a protective role by extenuation of hepatocyteapoptosis.