全反式维甲酸在肝缺血再灌注损伤中的作用机制研究

发布时间：2018-09-05 20:53

【摘要】：背景：肝脏缺血再灌注(ischemia-reperfusion, IR)损伤是临床工作中常会遇见的问题,并且,随着国家取消尸肝供体移植,心死亡后器官捐献的(Donation after circulation death, DCD)肝脏已经成为当今肝移植的主要来源,而DCD供肝的缺血再灌注损伤程度将会明显影响移植肝的功能恢复,甚至会决定肝移植手术的成败,因此如何有效地减轻肝脏IR损伤程度将是DCD供肝肝移植手术成功的关键。全反式维甲酸(all-trans retinoic acid, ATRA)是临床上用来治疗急性早幼粒白血病的常用药,现在研究发现ATRA也具有减轻肝IR损伤,但是其具体作用机制还不清楚,本实验旨在阐释ATRA在肝IR损伤中的作用机制。方法：体内实验：采用70%肝IR损伤模型,分为空白组,IR组和ATRA预处理组,在相应时间点,采血后处死小鼠并取材,进行肝功能检测,观察肝脏病理并评分,并利用RT-PCR和蛋白印记等技术对相应指标进行检测；体外实验：采用过氧化氢(Hydrogen peroxide, H2O2)和脂多糖(1ipopolysaccharides, LPS)分别处理小鼠肝细胞FL83B和巨噬细胞RAW264.7,分组情况均为DMSO、 H2O2/LPS、ATRA预处理和LE540预处理组,在相应时间点,收集细胞后,再利用RT-PCR和蛋白印记等技术进行分析。结果：(1)IR组中,肝功能、肝脏组织病理评分均高于空白组且有统计学差异；(2) ATRA预处理组中,肝功能及肝脏组织病理评分均低于IR组,炎症因子TNF-α、IL-6和IL-1β mRNA等水平也有明显下降；(3)ATRA预处理组中,维甲酸受体α、Bcl-2、p-Akt、Beclin1和LC3Ⅱ/Ⅰ等蛋白水平均高于IR组,cleaved-caspase 3、 Foxol、 TLR4、NF-κB p65和p62等蛋白水平低于IR组；(4)在FL83B试验中,ATRA预处理后使细胞凋亡减少,维甲酸受体α、Bc1-2、p-Akt、 Beclin1、LC3Ⅱ/Ⅰ等蛋白水平均高于H202组,cleaved-caspase 3、Foxo1、p62等蛋白水平低于H202组,而LE540预处理后损伤明显加重；(5)在RAW264.7试验中,ATRA预处理组中,炎症因子TNF-α、IL-6和IL-1β mRNA等水平明显下降,维甲酸受体a、p-Akt、Beclinl和LC3Ⅱ/LC3Ⅰ等蛋白水平均高于LPS组,Foxo1、TLR4、NF-κB p65和p62等蛋白水平低于LPS组,而LE540预处理后,却进一步加重了炎症反应。结论：(1)70%肝IR损伤模型建立成功；(2) ATRA可以减轻肝脏R损伤：①减少细胞凋亡,②调节先天免疫反应；(3) ATRA的保护作用可能与激活维甲酸受体α有关。
[Abstract]:Background: liver ischemia-reperfusion (ischemia-reperfusion, IR) injury is a common problem in clinical work. With the cancellation of cadaveric liver donor transplantation, (Donation after circulation death, DCD) liver donated after cardiac death has become the main source of liver transplantation. However, the degree of ischemia-reperfusion injury of DCD donor liver will significantly affect the functional recovery of the transplanted liver and even determine the success or failure of liver transplantation. Therefore, how to effectively reduce the degree of liver IR injury will be the key to the success of DCD donor liver transplantation. All trans retinoic acid (all-trans retinoic acid, ATRA) is a common drug used in the treatment of acute promyelocytic leukemia. Now it has been found that ATRA can also attenuate liver IR damage, but its mechanism is not clear. The purpose of this study was to elucidate the role of ATRA in liver IR injury. Methods: in vivo experiment, 70% liver IR injury model was used and divided into two groups: control group (IR group) and ATRA pretreatment group (P < 0.05). The mice were sacrificed at the corresponding time point after blood collection, and the liver function was detected, liver pathology was observed and evaluated. RT-PCR and protein imprinting techniques were used to detect the corresponding indexes. In vitro experiments, FL83B and RAW264.7, of mouse hepatocytes were treated with hydrogen peroxide (Hydrogen peroxide, H2O2 and lipopolysaccharide (LPS) respectively as DMSO, H _ 2O _ 2 / LPSN _ (ATRA) pretreatment group and LE540 pretreatment group. Cells were collected at the corresponding time points, and then analyzed by RT-PCR and protein imprinting techniques. Results: (1) in IR group, liver function and liver histopathological score were significantly higher than those in blank group, (2) in ATRA preconditioning group, liver function and liver histopathological score were lower than those in IR group, and the levels of inflammatory factor TNF- 伪, IL-6 and IL-1 尾 mRNA were significantly decreased. (3) in the ATRA pretreatment group, the protein levels of retinoic acid receptor 伪 -Bcl-2p-Akton-Beclin1 and LC3 鈪，

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