CAJ | 학술논문

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.
목적 통과주정관위유도급성주정성간병적방법건립대서실험성급성주정성간병모형,명학TNFα재급성주정섭입도치장점막손상적궤제,진일보천명TNFα、차두광단백O4(FOXO4)、NF-κB급긴밀련접단백지간적관계.방법 웅성Wister대서64지,안수궤수자표법분위정상조;모형조;모형+TNFα조;모형+항TNFα-IgG항체조;모형+wortmannin조;모형+ IGF-1조;모형+등량생리염수복강주사조;모형+등량생리염수미정맥주사조,매조8지.채용Western blotting검측소장표본Occludin、긴밀련접단백-1(ZO-1)、FOXO4급NF-κB,생물화학급ELISA등방법검측혈청간공능、TNFα등지표.결과 정상대조조대서혈청적ALT、AST、TNFα표체수평최저,조모후적대서상술지표표체수평승고,여정상조비교차이유통계학의의(P＜0.05);조모전급여TNFα급이도소양생장인자-1(IGF-1)후,표체수평진일보승고,여모형조비교차이유통계학의의(P＜0.05).상반조모전급여항TNFα-IgG항체급wortmannin후,기표체수평명현저우모형조,차이유통계학의의,이이자지간차이무통계학의의(P＞0.05).Western Blotting법검측ZO-1급NF-κB mRNA수평여단백질수평:정상대조조대서소장조직TJ(포괄ZO-1화occludin)표체수평최고,조모후적대서상술지표표체수평강저,여정상대조조비교차이유통계학의의(P＜0.05).조모전급여TNFα급IGF-1후,표체수평진일보강저,여모형조비교차이유통계학의의(P＜0.05),상반조모전급여항TNFα-IgG항체급wortmannin후,기표체수평명현고우모형조,차이유통계학의의,이이자지간차이무통계학의의(P＞0.05).결론 급성주정성간병시혈청대서체내증고적TNFα능강저소장점막상피세포긴밀련접단백ZO-1적분포급표체;혈청TNFα수평영향FOXO4적활성,FOXO4통과린산화여거린산화조절NF-κB적표체,진이영향ZO-1적표체.
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.