摘要
目的研究内质网应激在D-氨基半乳糖／脂多糖联合注射诱导小鼠急性肝衰竭致病机制中的作用。方法以C57BL／6小鼠为研究对象，腹腔注射D-氨基半乳糖／脂多糖建立小鼠急性肝衰竭模型。动物分为对照组、急性肝衰竭模型组、4-丁酸苯酯干预组（建模前6h腹腔注射）。Westemblot方法检测肝组织内质网应激标志物蛋白表达水平，检测血清ALT、AST水平评价肝脏功能，观察肝脏组织病理学变化评价肝脏损伤情况，实时荧光定量PCR检测肝细胞炎症因子如肿瘤坏死因子（TNF）α、白细胞介素（IL）-6和IL-1β基因表达情况，凋亡细胞原位末端转移酶标记试验法检测肝细胞凋亡情况。多组间均数比较采用单因素方差分析，两样本均数比较采用成组设计t检验；对于小鼠的生存分析，应用两两比较的Logrank检验。结果Westernblot方法结果显示，内质网应激蛋白标志物葡萄糖调节蛋白78和葡萄糖调节蛋白94随着急性肝衰竭的进展表达水平逐渐升高；非折叠蛋白反应蛋白1型内质网跨膜蛋白激酶1α、X盒结合蛋白1、活化转录因子6在急性肝衰竭早期被激活，而内质网应激诱导的凋亡蛋白CCAAT／增强子结合蛋白同源蛋白和caspasel2在急性肝衰竭晚期表达升高。4-丁酸苯酯抑制内质网应激可显著改善肝炎症[ALT：（1227．7±301．1）u／L与（727．5±412．5）u／L，P〈0．05；AST：（1682．9±580．2）u／L与（744．9±267．5）U／L，P〈0．051，并且抑制炎症因子的表达（TNFα：1．55±0．47与0．63±0．20，P〈0．05；IL-1β：7．39±2．03与4．51±1．44，P〈0．05；IL-6：0．43±0．14与0．18±0．03，P〈0．05）；凋亡细胞原位末端转移酶标记试验和Westemblot方法研究结果表明，4-丁酸苯酯干预内质网应激可显著抑制肝细胞凋亡，并降低了剪切型caspase3表达。结论内质网应激在D-氨基半乳糖／脂多糖诱导小鼠急性
Objective To study the role of endoplasmic reticulum stress （ERS） in acute liver failure （ALF） using a mouse model ofD-Galaetosamine/lipopolysaccharide （D-GaiN/LPS）-induced ALE Me.otis The ALF model was established by administering intraperitoneal （i.p.） injections of D-GaIN （700 mg/kg） and LPS （10 μg/kg） to six C57BL/6 mice. Three of the modeled mice were also administered 4-henylbutyrate （4-PBA; 100 mg/kg i.p.） at 6 hours before the onset of ALF and served as the intervention group. Non-modaled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of EILS-related proteins in fiver tissue. Liver function was assessed by measuring levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis, qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosisrelated protein Caspase-3. The extent of apoptosis in liver tissue was assessed by TUNEL assay. Results The ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALE The ERS effector proteins XBP-1, ATF-6 and IREla involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALE Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the pro- inflammatory cytokines TNFa, IL-6 and IL-1β, and reduced the extent of hepatocyte apoptosis. Conclusion ERS is activated in the mouse model of D-GaIN/LPS-induced ALF. Inhibition o
出处
《中华肝脏病杂志》
CAS
CSCD
北大核心
2014年第5期364-368,共5页
Chinese Journal of Hepatology
基金
国家“十二五”科技重大专项（2012ZX10002004-006、2012ZX10004904-003-001、2013zX10002002-006-001）
国家自然科学基金（81270532）,北京市优秀人才培养资助项目（2011D003034000022）I首都特色临床应用研究（Z121107001012167）,2012年度留学回国人员择优资助项目
关键词
肝功能衰竭
急性
脂多糖类
炎症
内质网应激
Liver failure, acute
Lipopolysaeeharide
Inflammation
Endoplasmie refioxlum slx^ss
