AMPK/mTOR信号通路介导的自噬在模拟高海拔视网膜病变中的作用

Role of AMPK/mTOR signaling-mediated autophagy in simulated high-altitude retinopathy

摘要

目的 探究 AMP 活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）信号通路介导的自噬在模拟高海拔视网膜病变（HAR）中的作用及其潜在分子机制。方法 选取 6~8 周龄雄性 C 57BL/6 小鼠。模型组置于低压氧舱连续暴露 24 h，模拟海拔 5000 m 环境，对照组在常压、常氧环境下饲养。采用苏木精-伊红染色对小鼠视网膜组织进行病理学改变分析；采用二氢乙啶探针检测小鼠视网膜组织中活性氧含量；采用免疫荧光检测小鼠视网膜组织中 8-OHdG表达水平；采用 Western blotting 检测小鼠视网膜组织中凋亡相关蛋白 Bcl-2、Bax、活化型半胱氨酸蛋白酶-3，AMPK、mTOR及其磷酸化形式，以及 LC3B、P62的表达水平，免疫荧光进一步验证 LC3B 和 P62 表达情况。结果 与对照组相比，模型组视网膜组织病理损伤显著；二氢乙啶探针发现模型组视网膜组织活性氧表达增加，免疫荧光显示 8-OHdG 表达显著上调。Western blotting 检测结果显示模型组中 Bax 及 C-半胱氨酸蛋白酶-3，蛋白表达显著升高，揭示细胞凋亡增强；模型组磷酸化 AMPK 表达降低，磷酸化mTOR 表达升高，LC3B 表达下降而 P62 表达升高，提示自噬流受阻。结论 AMPK/mTOR信号通路介导的自噬失衡可能是 HAR 发生发展的重要分子机制。

Abstract

Objective To investigate the function of autophagy regulated by the AMP-activated protein kinase （AMPK）/mammalian target of rapamycin（mTOR）signaling pathway in simulated high-altitude retinopathy （HAR）and to explore its potential mechanisms．Methods Male C57BL/6 mice aged 6~8 weeks were employed．Mice in the model group were exposed to a hypobaric hypoxia chamber for 24 hours to simulate an altitude of approximately 5000 m，and those in the control group were maintained under normal conditions．Histopathological changes in retinal tissues were assessed using hematoxylin-eosin staining．Reactive oxygen species levels were detected by dihydroethidium fluorescence staining．The expression of 8-OHdG in retinal tissue was examined by immunofluorescence．Western blotting analysis was performed to evaluate apoptosis- related proteins，including Bcl-2，Bax，and cleaved caspase-3（C-caspase 3），as well as key components of the AMPK/mTOR signaling pathway，including AMPK，mTOR and their phosphorylated forms．In addition，the expression of autophagy-related proteins LC3B and P62 was assessed by Western blotting and further con-firmed by immunofluorescence．Results Compared with the control group，mice exposed to simulated high- altitude conditions exhibited marked pathological alterations in retinal tissues．Dihydroethidium fluorescence staining revealed a significant increase in reactive oxygen species levels in the model group．Immunofluores- cence analysis showed a marked elevation of 8-OHdG expression．Western blotting results demonstrated sig- nificantly increased expression of Bax and C-caspase 3，indicating enhanced apoptotic activity．Phosphoryla- tion of AMPK was reduced，whereas phosphorylation of mTOR was increased in the model group．Moreover， LC3B expression was decreased and P62 expression was accumulated，suggesting a disturbance of autophagic flux．Conclusion Dysregulation of AMPK/mTOR-mediated autophagy may represent an important molecular mechanism underlying HAR．

关键词

高海拔视网膜病变 / AMPK/mTOR信号通路 / 自噬 / 氧化应激 / 细胞凋亡

Key words

high-altitude retinopathy / AMPK/mTOR signaling pathway / autophagy / oxidative stress / apoptosis

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基金资助

朗视界•沐光明中青年眼科科研项目(BCF-KH-YK-20240906-03)

兰州大学第二医院萃英学子科研培育计划资助项目(CYXZPT2025-04，CYXZ2023-09)

甘肃省科技计划资助项目(24JRRA1105)