糖原合成酶激酶-3β在肝脏热缺血再灌注损伤中的作用及其干预

目的 研究细胞内信号分子糖原合成酶激酶-3 β在肝脏缺血再灌注损伤中的作用.方法 以C57BL/6小鼠为研究对象,夹住肝脏动脉和门静脉以阻断肝脏向头侧肝叶血供90 min,随后建立不同灌注时间的小鼠热缺血再灌注损伤模型.动物实验组包括:假手术组,缺血再灌注模型组,SB216763干预组(SB216763溶于二甲基亚砜,质量分数为0.025,腹腔注射,缺血前2 h给予).通过Western blot检测肝脏组织中糖原合成酶激酶-3 β磷酸化的表达;检测血清ALT及组织病理学观察肝脏组织的损伤情况,实时定量PCR检测细胞炎症因子的基因表达.多组样本均数的两两比较采用one-way ANOVA分析(方差齐者用LSD-t检验,方差不齐者用Games-Howell法),P<0.05为差异具有统计学意义.结果 Western blot检测结果显示,在缺血再灌注的90 min缺血过程中,糖原合成酶激酶-3 β被去磷酸化而活性显著激活.抑制糖原合成酶激酶-3 β活性显著改善了肝脏的功能:ALT明显下降干预组对模型组:(2046±513)U/L对比(5809±1689)U/L,P=0.0153],肝脏的组织病理损伤明显改善,并显著抑制了肝脏的炎症反应:促进肝脏组织中抗炎细胞因子白细胞介素10(IL-10)的基因高表达,并同时抑制了促炎细胞因子IL-12、IL-1 β、肿瘤坏死因子α、IL-6的基因表达.结论 肝脏缺血再灌注损伤过程中,缺血提高了糖原合成酶激酶-3 α活性,促进了炎症反应的发生并导致了肝脏组织的损伤.因此,以糖原合成酶激酶-3 α为靶点进行干预,有可能为肝移植患者提供一种新的、有效的治疗方法来改善肝脏缺血再灌注损伤.

Abstract：

Objective To investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR). Methods C57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR. Results By western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046 ±513 U/L vs 5809 ± 1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNF α, IL-1 β and IL-6. Conclusions This study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3β may be a new therapeutic target to ameliorate liver IRI in transplant patients.

The role of glycogen synthase kinase-3 beta in the pathogensis of liver ischemia reperfusion injury

Objective To investigate the role of the key intracellular signaling molecule glycogen synthase kinase-3 beta in the mechanism of liver ischemia reperfusion (IR). Methods C57BL/6 mice were subjected to 90 min warm liver cephalad lobe ischemia, followed by various length of reperfusion. Experiment groups included sham control group, liver IRI model group and glycogen synthase kinase-3 beta inhibitor-treated group (SB216763 in DMSO, 25 g/kg, i.p, 2 hour prior to the onset of liver ischemia). The expression of glycogen synthase kinase-3 beta protein was analysed by Western blotting. The serum ALT levels were determined to reflect the function of liver. The affected liver lobes were harvested for histology analysis. The inflammatory gene expression was detected by Quantitative PCR. Results By western blot analysis, we found that ischemia itself activated glycogen synthase kinase-3 beta by a significant decrease of its phosphorylation. Glycogen synthase kinase-3 beta inhibitor SB216763-pretreatment ameliorated the liver damages significantly as compared to the controls (sALT: 2046 ±513 U/L vs 5809 ± 1689 U/L, P = 0.0153), and suppressed the gene expressions of IL-12, TNF α, IL-1 β and IL-6. Conclusions This study demonstrated that the ischemia process modulated liver innate immune activation via a GSK-3-dependent mechanism which favored the development of a pro-inflammation response and lead to liver tissue damages. GSK-3β may be a new therapeutic target to ameliorate liver IRI in transplant patients.