糖原合成酶激酶-3β在D-GalN/LPS诱导的小鼠急性肝衰竭中的作用

Role of glycogen synthase kinase 3β in the pathogenesis of D-GalN/LPS-induced acute liver injury in mice

目的:研究细胞信号分子糖原合成酶激酶-3β(glycogen synthase kinase-3β,GSK-3β)在D-氨基半乳糖/脂多糖联合注射诱导小鼠重型肝炎肝衰竭中的作用.方法:以C57BL/6小鼠为研究对象,腹腔注射D-氨基半乳糖/脂多糖建立小鼠重型肝炎肝衰竭模型.动物实验分组:对照组,重型肝炎肝衰竭模型组,SB216763干预组(建模前2h腹腔注射),SB216763治疗组(建模后2h腹腔注射).Western blot检测肝脏组织GSK-3β磷酸化水平,检测血清转氨酶(alanine aminotransferase,ALT)、天门冬酸氨基转移酶(aspartate aminotransferase,AST)评价肝脏功能,观察肝脏组织病理变化评价肝脏损伤情况,实时荧光定量PCR法检测肝脏细胞炎症因子基因表达,并检测凋亡相关蛋白Caspase 3的活性表达.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有统计学意义.结果:Western blot结果显示,GSK-3β在急性肝衰竭过程中磷酸化水平先降低(活性升高)后再次升高;抑制GSK-3β活性,无论是干预还是治疗都改善肝脏功能(血清ALT、AST水平明显下降,肝组织病理损伤明显改善),并且抑制炎症反应[抑制促炎因子肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、白介素6(Interleukin-6,IL-6)、IL-1β表达,促进抗炎因子IL-10高表达],并可降低凋亡相关蛋白Caspase 3表达.结论:在D-氨基半乳糖/脂多糖诱导小鼠急性肝衰竭过程中GSK-3β被激活,抑制GSK-3β活性通过降低炎症反应和肝细胞凋亡从而改善肝损伤.因此,对信号分子GSK-3β活性进行干预有可能为重型肝炎肝衰竭的治疗提供一个新的靶点.

AIM：To study the role of glycogen synthase kinase-3β（GSK-3β） in the pathogenesis of acute liver failure（ALF） induced by injection of D-galactosamine/lipopolysaccharide（D-GalN/LPS） in mice.METHODS：ALF was induced in C57BL/6 mice by intraperitoneal injection of D-GalN/LPS.Animal experimental groups included control group,ALF model group,SB216763 pretreatment group（SB216763 in DMSO,i.p,two hours before the induction of ALF） and SB216763 treatment group（SB216763 in DMSO,i.p,two hours after the induction of ALF）.Phosphorylation level of GSK-3β was analyzed by Western blot.Serum levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） were measured to assess the liver function.HE staining was conducted to analyze histological injury.Inflammatory gene expression was detected by quantitative real-time PCR.The expression of apoptosis-related protein Caspase 3 was detected by Western blot.One-way ANOVA was used for pair-wise comparison of means of multiple samples（homogeneity of variance with LSD-t test,unequal variances with Games-Howell method）.RESULTS：The phosphorylation level of GSK-3β decreased initially and then increased in the progression of ALF.Inhibition of GSK-3β,either by pretreatment or treatment with SB216763,could improve liver function（serum ALT and AST levels decreased significantly,and there was obvious improvement in liver tissue injury）,suppress inflammatory responses（inhibition of expression of pro-inflammatory cytokines,such as TNF-α,IL-6 and IL-1β,and promotion of expression of anti-inflammatory cytokine IL-10）,and reduced the expression of apoptosis-related protein Caspase 3.CONCLUSION：GSK-3β is activated in D-GalN/LPS-induced ALF in mice,and inhibition of GSK-3β activity can improve liver injury by reducing inflammation and hepatocyte apoptosis,GSK-3β may be a new target for the treatment of ALF.