异丙酚对肝脏缺血再灌注损伤时的保护作用

异丙酚对肝脏缺血再灌注损伤时的保护作用

异丙酚对肝脏缺血再灌注损伤时的保护作用

安世芝 王月兰 郭鸿雁

中文摘要

【目的】缺血再灌注损伤是一重要的临床问题，可以涉及到临床各科。休克、感染、肝创伤及肝移植所引起的肝功能衰竭、肝切除后肝脏的再生及肝功能的恢复等都与肝缺血再灌注损伤（HIRI）有关。大量的研究证明，氧自由基产生增加、细胞内钙超载、细胞因子的参与、Kupffer细胞（Kupffer cell,KC）激活及中性粒细胞（polymorphonuclear leukocyte,PMN）的聚集等参与肝脏缺血再灌注的损伤，但其发生的机制和途径尚不清楚。

本研究旨在通过建立大鼠部分肝叶缺血再灌注模型，从分子生物学水平到电子显微镜的超微结构，从细胞浆炎症因子到细胞核转录因子DNA结合活性等方面系统地探讨肝脏缺血再灌注损伤的机理，并观察异丙酚对其影响，为防治围术期肝脏缺血再灌注损伤、寻找合理的麻醉药物，提供理论依据和新的切入点。

【方法】采用Kobayashi法建立Waster大鼠部分肝叶缺血再灌注模型，并将72只动物随机分为4组，正常对照组、缺血再灌注组、异丙酚4 mg组和异丙酚6 mg组，并分别于肝门阻断30min再灌注30min（T1）、阻断60min再灌注60min（T2）、阻断60min再灌注120min（T3）3个时点取颈静脉血和肝组织；采用双抗体夹心酶联免疫吸附法（ELISA）检测肿瘤坏死因子-a（TNF- a）和白细胞介素-10（IL-10）的活性与含量的变化；采用凝胶电泳迁移率法（EMSA）测定T2时点核转录因子-kB（NF- kB ）的DNA结合活性；投射电镜观察T2时点肝细胞超微结构以及细胞凋亡和线粒体能荷(energy charge)的改变。

【结果】1. 血清中的细胞因子含量变化 B组大鼠于肝门阻断后，随缺血再灌注时间的延长，血清TNF-a、IL-10水平水平显著升高，（P＜0.05），但于T2时点达高峰；C、D组与B组同一时点比较TNF-a水平有显著性降低（P＜0.05），IL-10水平显著升高(P＜0.05)；但C、D组之间各时点比较无显著性差异(P＞0.05)。

2. NF－KB的DNA结合活性（NF-KB-binding activity OR affinity） 肝门阻 作者单位：250001 济南市妇幼保健院麻醉科，山东大学硕士在读，电话13153037423（安世芝）；武汉市，华中科技大学同济医学院协和医院麻醉科（王月兰）；山东大学2003级硕士（郭鸿雁）

异丙酚对肝脏缺血再灌注损伤时的保护作用

断后，B组NF- kB结合活性显著增强， C、D组NF- kB结合活性无显著增加，

3. 细胞超微结构改变与凋亡 电镜显示再灌注后，B组中肝细胞核浓缩、核

膜肿胀、染色质边居，线粒体、内质网肿胀明显增加，与B组相比，C、D组核膜、微粒体肿胀染色质边居均明显轻于B组。

【结论】综合以上结果可得出以下结论：

在肝脏缺血再灌注中，在缺血缺氧的作用下，细胞因子释放增加，NF－kB的DNA结合活性增强，肝细胞微粒体肿胀、细胞凋亡。这些因素又相互协同相互作用，并随缺血再灌注时间的延长而增强，于I/R3小时时达高峰。在这个过程中，异丙酚对肝脏缺血再灌注损伤起一定的保护作用，如：1、异丙酚在肝脏缺血再灌注损伤过程中，可降低TNF-a而升高IL-10的血清含量，起到对肝脏缺血再灌注损伤的保护作用。2、异丙酚通过抑制肝脏NF- kB的结合活性,对缺血再灌注肝脏产生保护作用.3、 异丙酚可以减少再灌注后的肝脏细胞凋亡和线粒体肿胀，其抗氧化作用可能是其减少再灌注后的肝脏细胞凋亡和线粒体肿胀的机制．但这些保护作用并不是单一的作用于某一环节，而是多位点、多途径、交互作用的结果。

【关键词】异丙酚；缺血再灌注；肝脏；细胞因子；超微结构； NF- kB

异丙酚对肝脏缺血再灌注损伤时的保护作用

Effect of propofol on hepatic ischemia reperfusion in rats

An Shi-zhi,Wang Yue-lan,Guo Hong-yan.Department of Anesthesiology,Jinan women and children hospital,Master of Shandong University,Jinan 250001,Shandong,China

Abstract

[Objective] Hepatic injury caused by ischemia and reperfusion(HIRI)is a very important problem in clinical practice.It is related to the exclusion of liver transplantation and hepatic failure,which caused by shock, infection, trauma of liver and liver transplantation. HIRI effects revival of liver and recovery of hepatic function after hepatectomy.A large number of researches testified that ,some factor participate in the HIRI,such as increase of free radical intracellular, calcium overload, cytokines, kupffer cell (KC) activation and polymorphonuclear leukocyte(PMN)aggregation and so on,but the mechanism and its pathway are not clear.

This study aimed to observe the mechanism of I/R and the effects of propofol in rat in vivo from molecularbiology to morphology ，from cytokines to NF- kB activation.Which proved the theory foundation for reducing perioperative HIRI and finding out rational anesthetic.

[Methods] First of all to build the model of I/R for rat according to the method of Kobayashi,and then seventy and two healthy Wistar rats were randomly divided into the nomal control group(A),ischemia and reperfusion group(B),I/R+propofol 4mg.h-1.100g-1 (C)and 6mg.h-1.100g-1(D)group.Blood plasma and partial hepatic issue were obtained at 30min-ischemain and 30min-reperfusion(T1),at 60min-ischemain and 60min(T2)、at 60min-ischemain and 120min-reperfusion(T3). Blood plasma levels of TNF- a and IL-10 were measured with ELISA.The NF-kB activation to special DNA sequence were measured with EMSA.The ultramicrostructure ,cell apoptosis and mitochondria energy charge were observed by electron microscope.

[Results]1.Blood plasma TNF-a and IL-10 levels of T1、 T2、 T3 significantly increased compared with group A(P＜0.05)in group B , but reached peek at 3

异丙酚对肝脏缺血再灌注损伤时的保护作用

hours-ischemia and reperfution .Campared with group B TNF-a decreased and IL-10increased significantly(P ＜0.05) in group C and group D.There is no significant difference between group C and group D(P＞0.05).

2. Hepatic NF-kB activation was induced in a time-dependent manner after

I/R.Propofol suppressed NF-kB activation.

3. Electron microscopic examination revealed that in group Bthere was a

significant increase in apoptotic hepatocytes and sinusoidal endothelial cell(SEC)after ischemia-reperfusion （P＜0.05）.Most of apoptotic cell were SEC and only a small number of hepatocytes. Mitochondria swelled obviously.Both SEC and hepatocytes apoptosis and mitochondria swell were much slighter in group C、D than in group B.

[Conclusions]In this study ,we can observed that cytokines and NF- kB activation increased,hepatocytes apoptosis and ultromicrostructure swelled during HIRI.These factors affect together.Propofol has some protective effect on HIRI in rats,such as 1. Propofol has protective effects on HIRI by inhibiting TNF-a and increasing IL-10 after hepatic ischemia reperfusion in rats.2. Hepatic is -chemia-reperfusion injury is related to the binding activation of NF- kB to special DNA sequence,propofol protects against hepatic ischemia-reperfusion by suppressing NF- kB activation.3.Propofol inhibits not only SEC and hepatocytes apoptosis but also mitochondria swell after ischemia-reperfusion through its antioxidant effect.

[Key words] Propofol ; ischemia-reperfusion ; liver ; cytokine ; ultromicrostructure ; NF- kB

异丙酚对肝脏缺血再灌注损伤时的保护作用

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