FOXO4在大鼠急性酒精性肝病中的表达及作用机制

The mechanism of FOXO4 in acute alcoholic liver disease

目的 通过酒精灌胃诱导急性酒精性肝病的方法建立大鼠实验性急性酒精性肝病模型,明确TNFα在急性酒精摄入导致肠黏膜损伤的机制,进一步阐明TNFα、叉头框蛋白O4（FOXO4）、NF-κB及紧密连接蛋白之间的关系.方法 雄性Wister大鼠64只,按随机数字表法分为正常组;模型组;模型＋TNFα组;模型＋抗TNFα-IgG抗体组;模型＋wortmannin组;模型＋ IGF-1组;模型＋等量生理盐水腹腔注射组;模型＋等量生理盐水尾静脉注射组,每组8只.采用Western blotting检测小肠标本Occludin、紧密连接蛋白-1（ZO-1）、FOXO4及NF-κB,生物化学及ELISA等方法检测血清肝功能、TNFα等指标.结果 正常对照组大鼠血清的ALT、AST、TNFα表达水平最低,造模后的大鼠上述指标表达水平升高,与正常组比较差异有统计学意义（P＜0.05）;造模前给予TNFα及胰岛素样生长因子-1（IGF-1）后,表达水平进一步升高,与模型组比较差异有统计学意义（P＜0.05）.相反造模前给予抗TNFα-IgG抗体及wortmannin后,其表达水平明显低于模型组,差异有统计学意义,而二者之间差异无统计学意义（P＞0.05）.Western Blotting法检测ZO-1及NF-κB mRNA水平与蛋白质水平：正常对照组大鼠小肠组织TJ（包括ZO-1和occludin）表达水平最高,造模后的大鼠上述指标表达水平降低,与正常对照组比较差异有统计学意义（P＜0.05）.造模前给予TNFα及IGF-1后,表达水平进一步降低,与模型组比较差异有统计学意义（P＜0.05）,相反造模前给予抗TNFα-IgG抗体及wortmannin后,其表达水平明显高于模型组,差异有统计学意义,而二者之间差异无统计学意义（P＞0.05）.结论 急性酒精性肝病时血清大鼠体内增高的TNFα能降低小肠黏膜上皮细胞紧密连接蛋白ZO-1的分布及表达;血清TNFα水平影响FOXO4的活性,FOXO4通过磷酸化与去磷酸化调节NF-κB的表达,进而影响ZO-1的表达.

Objective To investigate the role of tumor necrosis factor alpha （TNFα） in a rat model of experimental acute alcoholic liver disease,and further elucidate the relationship among TNFα,forkhead box subtype O4 （FOXO4）,nuclear factor κB （NF-κB）,and zonula occludens-1 （ZO-1）.Methods Sixty four Wister （WT） rats were divided into eight groups （8 in each group）：normal group,alcohol group,alcohol ＋ TNFα group,alcohol ＋ wortmannin group,alcohol ＋ insulin-like growth factor-1 （IGF-1） group,alcohol ＋ anti-TNFo group,and two placebo groups （treated with saline）.TNFα in plasma was measured by enzyme linked immunosorbent assay （ELISA）.Western blot was used to identify the mechanisms of FOXO4 in regulating the epithelial permeability.Electron microscopy,reverse transcription polymerase chain reaction and western blot were used to examine the tightjunction proteins and NF-κB.Results Compared to control group,TNFα in alcohol group was obviously high.At the same time,TNFα could induce the phosphorylation of FOXO4.Phosphorylated FOXO4 was excluded into cytoplasm and was inactive.The inactive FOXO4 at a high level lose the ability to restrain NF-κB.Therefore,the expression of NF-κB was increased,then it down-regulated the expressions of tight junction proteins （including ZO-1 and occludin） and increased epithelial permeability.As a result,the intestinal bacteria were grown excessively,endotoxin was released into portal circulation and liver injury was deteriorated.Conclusions TNFα can up-regulate phosphorylation of FOXO4.Phosphorylated FOXO4 in the nucleus is excluded into cytoplasm and is inactive.The inactive FOXO4 lose the ability to restrain NF-κB activity,then down-regulate the expression of tight junction proteins and increase epithelial permeability.