Ren Feng, Yang Bingzhang, Zhang Xiangying, Wen Tao, Wang Xinxin, Yin Jiming, Piao Zhengfu, Zheng Sujun, Zhang Jing, Chen Yu, Chen Dexi, Duan Zhongping
Institute of hepatology, Beijing You'an hospital, Capital Medical University, beijing 100069, China.
Zhonghua Gan Zang Bing Za Zhi. 2014 May;22(5):364-8. doi: 10.3760/cma.j.issn.1007-3418.2014.05.009.
To study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF.
The ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver 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 apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay.
The ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a 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 ALF. 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 proinflammatory cytokines TNFa, IL-6 and IL-1 β, and reduced the extent of hepatocyte apoptosis.
ERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.
利用D-半乳糖胺/脂多糖（D-GalN/LPS）诱导的急性肝衰竭（ALF）小鼠模型，研究内质网应激（ERS）在急性肝衰竭中的作用。
通过对6只C57BL/6小鼠腹腔注射D-Ga1N（700mg/kg）和LPS（10μg/kg）建立ALF模型。3只建模小鼠在ALF发病前6小时还腹腔注射4-苯基丁酸盐（4-PBA；100mg/kg），作为干预组。未建模小鼠作为对照组。通过蛋白质免疫印迹法和定量逆转录聚合酶链反应（qRT-PCR）分析所有小鼠肝脏组织中ERS相关蛋白的表达水平。通过检测血清中丙氨酸转氨酶（ALT）和天冬氨酸转氨酶（AST）水平评估肝功能。通过苏木精-伊红染色和组织学分析评估肝组织损伤程度。qRT-PCR还用于检测炎症相关基因表达的差异，蛋白质免疫印迹法也用于检测凋亡相关蛋白Caspase-3表达的差异。通过TUNEL检测评估肝组织凋亡程度。
ERS标志物葡萄糖调节蛋白78（GRP78）和葡萄糖调节蛋白94（GRP94）在基因和蛋白水平上的表达均随ALF进展而增加。参与未折叠蛋白反应的ERS效应蛋白X盒结合蛋白1（XBP-1）、活化转录因子6（ATF-6）和肌醇需求酶1α（IRE 1α）在ALF早期被激活，而ERS诱导的凋亡调节因子Caspase-12和C/EBP同源蛋白（CHOP）在ALF晚期被激活。4-PBA干预抑制ERS可保护肝组织和肝功能免受损伤，血清ALT和AST水平显著降低以及组织学改变程度明显减轻证明了这一点。此外，抑制ERS可抑制促炎细胞因子肿瘤坏死因子α（TNFα）、白细胞介素6（IL-6）和白细胞介素1β（IL-1β）的表达，并减少肝细胞凋亡程度。
在D-GalN/LPS诱导的ALF小鼠模型中ERS被激活。抑制ERS可能对肝损伤具有保护作用，其作用机制可能涉及减少炎症和凋亡因子及/或信号传导。因此，抑制ERS可能代表一种治疗ALF的新方法。
