AGK2 预处理通过调节 MFN2-PERK 轴和铁死亡信号通路来防止硫代乙酰胺诱导的急性肝衰竭。

AGK2 pre-treatment protects against thioacetamide-induced acute liver failure via regulating the MFN2-PERK axis and ferroptosis signaling pathway.

机构信息

Department of Infectious Diseases, Renmin Hospital of Wuhan University, Wuhan 430060, China.

出版信息

Hepatobiliary Pancreat Dis Int. 2024 Feb;23(1):43-51. doi: 10.1016/j.hbpd.2023.03.003. Epub 2023 Mar 16.

DOI:10.1016/j.hbpd.2023.03.003

PMID:36966125

Abstract

BACKGROUND

Acute liver failure (ALF) is an unpredictable and life-threatening critical illness. The pathological characteristic of ALF is massive necrosis of hepatocytes and lots of inflammatory cells infiltration which may lead to multiple organ failure.

METHODS

Animals were divided into 3 groups, normal, thioacetamide (TAA, ALF model) and TAA + AGK2. Cultured L02 cells were divided into 5 groups, normal, TAA, TAA + mitofusin 2 (MFN2)-siRNA, TAA + AGK2, and TAA + AGK2 + MFN2-siRNA groups. The liver histology was evaluated with hematoxylin and eosin staining, inositol-requiring enzyme 1 (IRE1), activating transcription factor 6β (ATF6β), protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylated-PERK (p-PERK). C/EBP homologous protein (CHOP), reactive oxygen species (ROS), MFN2 and glutathione peroxidase 4 (GPX4) were measured with Western blotting, and cell viability and liver chemistry were also measured. Mitochondria-associated endoplasmic reticulum membranes (MAMs) were measured by immunofluorescence.

RESULTS

The liver tissue in the ALF group had massive inflammatory cell infiltration and hepatocytes necrosis, which were reduced by AGK2 pre-treatment. In comparison to the normal group, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe in the TAA-induced ALF model group were significantly increased, which were decreased by AGK2 pre-treatment. The levels of MFN2 and GPX4 were decreased in TAA-induced mice compared with the normal group, which were enhanced by AGK2 pre-treatment. Compared with the TAA-induced L02 cell, apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe were further increased and levels of MFN2 and GPX4 were decreased in the MFN2-siRNA group. AGK2 pre-treatment decreased the apoptosis rate and levels of IRE1, ATF6β, p-PERK, CHOP, ROS and Fe and enhanced the protein expression of MFN2 and GPX4 in MFN2-siRNA treated L02 cell. Immunofluorescence observation showed that level of MAMs was promoted in the AGK2 pre-treatment group when compared with the TAA-induced group in both mice and L02 cells.

CONCLUSIONS

The data suggested that AGK2 pre-treatment had hepatoprotective role in TAA-induced ALF via upregulating the expression of MFN2 and then inhibiting PERK and ferroptosis pathway in ALF.

摘要

背景

急性肝衰竭（ALF）是一种不可预测且危及生命的危急重症。ALF 的病理特征是大量肝细胞坏死和大量炎性细胞浸润，可能导致多器官衰竭。

方法

动物分为 3 组，正常组、硫代乙酰胺（TAA，ALF 模型）组和 TAA+AGK2 组。培养的 L02 细胞分为 5 组，正常组、TAA 组、TAA+MFN2-siRNA 组、TAA+AGK2 组和 TAA+AGK2+MFN2-siRNA 组。用苏木精和伊红染色、肌醇需求酶 1（IRE1）、激活转录因子 6β（ATF6β）、蛋白激酶 R（PKR）样内质网激酶（PERK）和磷酸化-PERK（p-PERK）评估肝组织学。用 Western blot 法测定 C/EBP 同源蛋白（CHOP）、活性氧（ROS）、MFN2 和谷胱甘肽过氧化物酶 4（GPX4），测定细胞活力和肝功能。通过免疫荧光法测定线粒体相关内质网膜（MAMs）。

结果

ALF 组肝组织有大量炎性细胞浸润和肝细胞坏死，AGK2 预处理可减轻。与正常组相比，TAA 诱导的 ALF 模型组细胞凋亡率及 IRE1、ATF6β、p-PERK、CHOP、ROS 和 Fe 水平显著升高，AGK2 预处理后降低。与正常组相比，TAA 诱导的小鼠 MFN2 和 GPX4 水平降低，AGK2 预处理后升高。与 TAA 诱导的 L02 细胞相比，MFN2-siRNA 组细胞凋亡率及 IRE1、ATF6β、p-PERK、CHOP、ROS 和 Fe 水平进一步升高，MFN2 和 GPX4 水平降低。AGK2 预处理降低了 MFN2-siRNA 处理的 L02 细胞的细胞凋亡率及 IRE1、ATF6β、p-PERK、CHOP、ROS 和 Fe 水平，并增强了 MFN2 和 GPX4 的蛋白表达。免疫荧光观察显示，与 TAA 诱导组相比，AGK2 预处理组在小鼠和 L02 细胞中 MAMs 水平升高。