基于腺苷酸活化蛋白激酶/Unc-51样自噬激活激酶1信号通路探讨高原低氧适应对肝缺血再灌注损伤大鼠模型的保护作用

夏鑫; 林治宇; 雷寰; 罗豫川; 陈儒德; 汪涛

doi:10.12449/JCH250725

基于腺苷酸活化蛋白激酶/Unc-51样自噬激活激酶1信号通路探讨高原低氧适应对肝缺血再灌注损伤大鼠模型的保护作用

DOI: 10.12449/JCH250725

夏鑫^{1, 2},
林治宇^{1, 2},
雷寰²,
罗豫川²,
陈儒德²,
汪涛^{1, 2, , ,}

1.
西南医科大学临床医学院肝胆外科，四川泸州 646000
2.
中国人民解放军西部战区总医院全军普外中心，成都 610000

基金项目:

中国人民解放军西部战区总医院院管课题 (2021-XZYG-A12);

四川省自然科学基金 (41C4137G)

伦理学声明：本研究方案于2024年1月15日经由中国人民解放军西部战区总医院伦理委员会审批，批号：2023ky080-1，符合实验室动物管理与使用准则。

利益冲突声明：本文不存在任何利益冲突。

作者贡献声明：夏鑫负责实验操作，整理数据，撰写文章；雷寰、罗豫川、林治宇负责指导实验和写作，分析数据；汪涛、陈儒德负责实验设计，数据分析，拟定写作思路，修改文章及定稿。

详细信息

通信作者:
汪涛， watopo@163.com （ORCID： 0000-0003-4064-8867）

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出版历程
- 收稿日期: 2024-12-01
- 录用日期: 2025-01-20
- 出版日期: 2025-07-25

Protective effect of high-altitude hypoxia acclimatization against hepatic ischemia-reperfusion injury in rats： A study based on the adenosine monophosphate-activated protein kinase/Unc-51 like autophagy activating kinase 1 signaling pathway

Xin XIA^{1, 2},
Zhiyu LIN^{1, 2},
Huan LEI²,
Yuchuan LUO²,
Rude CHEN²,
Tao WANG^{1, 2
, ,
,}

1.
Department of Hepatopancreatobiliary Surgery，School of Clinical Medicine，Southwest Medical University，Luzhou，Sichuan 646000，China
2.
Department of General Surgery，The General Hospital of Western Theater Command，Chengdu 610000，China

Research funding:

The Management Project of General Hospital of Western Theater Command of PLA (2021-XZYG-A12);

Sichuan Provincial Natural Science Foundation Project (41C4137G)

More Information

Corresponding author: WANG Tao， watopo@163.com （ORCID： 0000-0003-4064-8867）

摘要

摘要: 目的观察高原低氧适应对肝缺血再灌注损伤（HIRI）大鼠模型的保护作用，探索高原低氧适应激活自噬的作用机制。方法将56只SD雄性大鼠随机分为以下7组：平原假手术组（P-S）、平原模型组（P-M）、急性高原低氧假手术组（AHH-S）、急性高原低氧模型组（AHH-M）、高原低氧适应假手术组（HHA-S）、高原低氧适应模型组（HHA-M），以及含AMPK抑制剂复合物C（CC）的高原低氧适应模型组（HHA-M-CC），每组8只。急性高原低氧组和高原低氧适应组分别将大鼠置于海拔5 000米的低压氧舱中1周和12周；假手术组仅开腹暴露肝门静脉，未做血管夹闭处理；HHA-M-CC组术前1 h行腹腔注射20 mg/kg剂量CC，其余组注射等体积生理盐水。采用全自动生化分析仪检测肝功能指标ALT、AST、TBil水平；苏木素-伊红染色观察肝组织病理改变；透射电镜观察肝组织自噬体形成情况；RT-qPCR检测肝脏腺苷酸活化蛋白激酶（AMPK）、Unc-51样自噬激活激酶1（ULK1）mRNA表达水平；Western Blot技术检测磷酸化腺苷酸蛋白激酶（p-AMPK）、磷酸化UNC-51样激酶（p-ULK1）、Beclin-1、微管相关蛋白1轻链3Ⅱ型（LC3Ⅱ）蛋白的表达水平。计量资料多组间比较采用方差分析，进一步两两比较采用LSD-t检验。结果与AHH-M组和HHA-M-CC组比较，HHA-M组ALT、AST、TBil水平明显降低（P值均<0.05），肝组织病理损伤减弱，Suzuki评分显著降低（P值均<0.05），透射电镜下肝细胞形态结构异常程度减弱，自噬体数量明显增多，AMPK、ULK1 mRNA表达水平均明显上升（P值均<0.05），p-AMPK、p-ULK1、Beclin-1、LC3Ⅱ蛋白表达明显上调（P值均<0.05）。结论高原低氧适应可通过AMPK/ULK1信号通路相关蛋白的激活，增强肝细胞的自噬作用，进而缓解SD大鼠HIRI。
- 低氧 /
- 再灌注损伤 /
- 自噬 /
- 大鼠， Sprague-Dawley
Abstract: Objective To investigate the protective effect of high-altitude hypoxia acclimatization against hepatic ischemia-reperfusion injury （HIRI） in rats， as well as the mechanism of action of high-altitude hypoxia acclimatization in activating autophagy. Methods A total of 56 male Sprague-Dawley rats were randomly divided into plain sham-operation group （P-S group）， plain model group （P-M group）， acute high-altitude hypoxia sham-operation group （AHH-S group）， acute high-altitude hypoxia model group （AHH-M group）， high-altitude hypoxia acclimatization sham-operation group （HHA-S group）， high-altitude hypoxia acclimatization model group （HHA-M group）， and high-altitude hypoxia acclimatization model group with the adenosine monophosphate-activated protein kinase （AMPK） inhibitor compound C （HHA-M-CC group）， with 8 rats in each group. The rats in the acute high-altitude hypoxia groups and the high-altitude hypoxia acclimatization groups were placed in a low-pressure oxygen chamber at an altitude of 5 000 meters for 1 week and 12 weeks， respectively； the rats in the sham-operation groups were given laparotomy to expose the portal vein without vascular clamping； the rats in the HHA-M-CC group were given abdominal injection of 20 mg/kg CC at 1 hour before surgery， while those in the other groups were given injection of an equal volume of normal saline. An automatic biochemical analyzer was used to measure the levels of liver function parameters including alanine aminotransferase （ALT）， aspartate aminotransferase （AST）， and total bilirubin （TBil）； HE staining was used to observe liver histopathological changes； transmission electron microscopy was used to observe the formation of autophagosomes in liver tissue； RT-qPCR was used to measure the mRNA expression levels of AMPK and Unc-51 like autophagy activating kinase 1 （ULK1） in liver tissue； Western Blot was used to measure the protein expression levels of phosphorylated AMPK （p-AMPK）， phosphorylated ULK1 （p-ULK1）， Beclin-1， and microtubule-associated protein 1 light chain 3 Ⅱ （LC3Ⅱ）. An analysis of variance was used for comparison of continuous data between multiple groups， and the least significant difference t-test was sued for comparison between two groups. Results Compared with the AHH-M and HHA-M-CC groups， the HHA-M group had significantly reductions in the levels of ALT， AST， and TBil （all P<0.05）， alleviation of liver histopathological injury， a significant reduction in Suzuki score （all P<0.05）， a reduction in the degree of abnormal morphological structure of hepatocytes under transmission electron microscopy， and significant increases in the number of autophagosomes， the mRNA expression levels of AMPK and ULK1 （all P<0.05）， and the protein expression levels of p-AMPK， p-ULK1， Beclin-1， and LC3Ⅱ （all P<0.05）. Conclusion High-altitude hypoxia acclimatization can alleviate HIRI in SD rats by activating the AMPK/ULK1 signaling pathway and enhancing autophagy in hepatocytes.
- Hypoxia /
- Reperfusion Injury /
- Autophagy /
- Rats， Sprague-Dawley

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图 1 各组大鼠血清生化水平比较

Figure 1. Expression levels of serum biochemical factor in different groups of rat

下载: 全尺寸图片幻灯片

注： a，P-S组；b，AHH-S组；c，HHA-S组；d，P-M组；e，AHH-M组；f，HHA-M组；g，HHA-M-CC组。

图 2 各组大鼠肝组织的病理变化（HE染色，×400）

Figure 2. Pathological changes of the hepatic tissue of rat in each group （HE，×400）

下载: 全尺寸图片幻灯片

注： a，P-S组；b，AHH-S组；c，HHA-S组；d，P-M组；e，AHH-M组；f，HHA-M组；g，HHA-M-CC组。线粒体（红色箭头）；自噬小体（绿色箭头）。

图 3 透射电镜观察肝脏自噬体（TEM，×20 000）

Figure 3. Observing autophagosome by transmission electron microscope （TEM，×20 000）

下载: 全尺寸图片幻灯片

图 4 各组大鼠肝组织AMPK/ULK1信号通路mRNA相对表达量

Figure 4. Relative expression of AMPK/ULK1 signaling pathway mRNA in rat hepatic tissues in each group

下载: 全尺寸图片幻灯片

图 5 大鼠肝脏自噬相关Beclin-1、LC3Ⅱ和AMPK/ULK1信号通路相关蛋白表达及半定量分析

Figure 5. Expression of AMPK/ULK1 signaling pathway and autophagy related proteins in each group of rat

下载: 全尺寸图片幻灯片

表 1 引物序列

Table 1. Primer sequence

基因	上游（5'-3'）	下游（5'-3'）
β-actin	GGGAAATCGTGCGTGA‑ CATT	GCGGCAGTGGCCATC‑ TC
AMPK	ATGATGAGGTGGTGGA‑ GCAGAGG	GGTTCTCGGCTGTGCT‑ GGAATC
ULK1	TACACAGCAAGGGCAT‑ CATTCACC	CGGGCAAATCCAAAG‑ TCAGCAATC

下载: 导出CSV

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