巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点

王正力; 訾亚飞; 刘杨东; 傅麒宁; 王学虎

doi:10.16016/j.2097-0927.202112106

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巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点

基础医学 | 更新时间：2026-01-09

- 巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点
- Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells
- 陆军军医大学学报 2022年44卷第12期页码：1221-1228
- 作者机构：
  
  1.400016 重庆，重庆医科大学附属第一医院血管外科
  2.518111 广东深圳，深圳大学附属华南医院血管外科
- 作者简介：
  
  [王学虎，E-mail: 184038696@qq.comWANG Xuehu, E-mail: 184038696@qq.com
- 基金信息：
  
  作者贡献声明王正力负责内容构思和实验方法设计、实验数据分析和撰写初稿；訾亚飞负责内容整理，参与实验研究及数据采集，参与文章内容撰写指导及提供实验技术材料支持；刘杨东参与酝酿和设计实验，核对实验进展及指导实验进行，并给予研究经费支持；傅麒宁参与选题方向确定及指导实验方案的设计；王学虎负责实验工作支持，全程参与论文内容修改指导并审核全文
- DOI：10.16016/j.2097-0927.202112106
  中图分类号： R322.123;R329.25;R363.21
- 收稿：2021-12-13，
  
  修回：2022-01-28，
  
  纸质出版：2022-06-30
- 稿件说明：
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王正力, 訾亚飞, 刘杨东, 等. 巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点[J]. 陆军军医大学学报, 2022,44(12):1221-1228.

Zhengli WANG, Yafei ZI, Yangdong LIU, et al. Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells[J]. Journal of Army Medical University, 2022, 44(12): 1221-1228.
王正力, 訾亚飞, 刘杨东, 等. 巨自噬和分子伴侣介导自噬在内皮细胞氧化应激损伤中的特点[J]. 陆军军医大学学报, 2022,44(12):1221-1228. DOI： 10.16016/j.2097-0927.202112106.

Zhengli WANG, Yafei ZI, Yangdong LIU, et al. Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells[J]. Journal of Army Medical University, 2022, 44(12): 1221-1228. DOI： 10.16016/j.2097-0927.202112106.

摘要

目的

探究巨自噬和分子伴侣介导自噬(chaperone-mediated autophagy，CMA)这两种自噬途径在人脐静脉内皮细胞(human umbilical vein endothelial cells，HUVECs)氧化应激损伤中特点。

方法

通过不同浓度(0、20、50、100、200 μg/mL)氧化低密度脂蛋白(oxidation low lipoprotein，OX-LDL)作用HUVECs后产生的活性氧(reactive oxygen species，ROS)水平筛选诱导HUVECs氧化应激的适宜浓度。设立空白对照组(不做任何处理)、单纯抑制巨自噬组(10 mmol/L 3-MA)、氧化应激组(50 μg/mL OX-LDL)和抑制巨自噬-氧化应激组(10 mmol/L 3-MA+50 μg/mL OX-LDL)，检测各组细胞增殖标志蛋白PCNA、凋亡标志蛋白Caspase-3、巨自噬标志蛋白Beclin-1、分子伴侣介导自噬两种标志蛋白Hsc70和Lamp-2蛋白表达，评估不同处理下HUVECs增殖、凋亡及两种自噬的变化。其中氧化应激组和抑制巨自噬-氧化应激组处理不同时间(1、3、6、12、48 h)后，采用CCK-8检测细胞增殖抑制率，Annexin-V/PI双染检测凋亡，Western blot检测两种自噬途径蛋白Beclin-1、Hsc70、Lamp-2表达，评估巨自噬抑制前后氧化应激下HUVECs的增殖、凋亡及两种自噬的时间调控特点。

结果

OX-LDL诱导氧化应激下ROS浓度呈递增关系，选择50 μg/mL为适宜浓度进行后续实验。与空白对照组比较，氧化应激组HUVECs Caspase-3、Beclin-1、Hsc70、Lamp-2上调，PCNA下调(

0.05)，同时随处理时间延长，48 h内细胞增殖抑制率、全期凋亡率及Lamp-2递增，Hsc70在6 h后递增，12 h内Beclin-1水平递增(

0.05)；与氧化应激组比较，抑制巨自噬-氧化应激组Caspase-3、Hsc70、Lamp-2上调，PCNA下调(

0.05)，随处理时间延长，同时段Hsc70、La

mp-2上调，且Hsc70在3 h后递增(

0.05)。

结论

巨自噬和分子伴侣介导自噬在内皮细胞氧化应激过程中先后激活，巨自噬抑制后提高分子伴侣介导自噬水平，并提前激活时间，减轻氧化应激对内皮细胞的损伤。

Abstract

Objective

To explore the characteristics of macroautophagy and molecular chaperone-mediated autophagy (CMA) in the oxidative stress injury of human umbilical vein endothelial cells (HUVECs).

Methods

After HUVECs were treated with oxidized low density lipoprotein (OX-LDL) at different concentrations(0、20、50、100、200 μg/mL)

the production of reactive oxygen species (ROS) was measured to screen the optimal concentration to induce HUVECs oxidative stress. The cells were divided into 4 groups: control group (without any treatment)

macroautophagy inhibition group (treated with 10 mmol/L 3-MA

specific inhibitor of macroautophagy)

oxidative stress group (treated with 50 μg/mL OX-LDL)

and macroautophagy inhibition-oxidative stress group (treated with 10 mmol/L 3-MA+50 μg/mL OX-LDL). The expression levels of proliferation marker protein PCNA

apoptosis marker Caspase-3

macroautophagy marker Beclin-1

and CMA markers Hsc70 and Lamp-2 were detected in each group with Western blotting. The proliferation

apoptosis

2 kinds of autophagy under different treatments were evaluated respectively. After the cells of the oxidative stress group and macroautophagy inhibition-oxidative stress group were treated for different duration (1

12 and 48 h)

the proliferation and apoptosis of HUVECs were tested by CCK-8 assay and Annexin-V/PI double staining

respectively. Western blotting was adopted to determine the protein levels of Beclin-1

Hsc70 and Lamp-2. Finally

the time regulation characteristics of the 2 autophagy pathways before and after macroautophagy inhibition were analyzed.

Results

Under OX-LDL induced oxidative stress

the ROS level was elevated with the increase of OX-LDL concentration

and

50 μg/mL was finally selected as the appropriate concentration for subsequent experiments. As compared with the control group

the levels of Caspase-3

Beclin-1

Hsc70 and Lamp-2 were up-regulated

while that of PCNA was down-regulated in the oxidative stress group (

0.05). With the prolongation of treatment time

proliferation inhibition rate

overall apoptotic rate and Lamp-2 level were increased within 48 h

Hsc70 level was elevated after 6 h

and that of Beclin-1 was improved within 12 h in the oxidative stress group (

0.05). In the macroautophagy inhibition-oxidative stress group

the expression levels of Caspase-3

Hsc70 and Lamp-2 were also up-regulated

that of PCNA down-regulated (

0.05)

those of Hsc70 and Lamp-2 were increased at the same time and that of Hsc70 was in an elevation after 3 h of treatment (

0.05).

Conclusion

Macroautophagy and CMA are activated successively during the process of oxidative stress

and CMA can be enhanced after macroautophagy inhibition. The early activation contributes to attenuate oxidative stress-induced damage to endothelial cells.

关键词

Keywords

references

靖永胜, 王小平, 刘芳, 等. 动静脉内瘘功能障碍病理及发病机制的研究进展[J]. 中华肾脏病杂志, 2017, 33(4): 318-320. DOI: 10.3760/cma.j.issn.1001-7097.2017.04.015.

JING Y S, WANG X P, LIU F, et al . Research progress on dysfunction and pathogenesis of arteriovenous fistula[J ] . Chin J Nephrol, 2017, 33(4): 318-320. DOI: 10.3760/cma.j.issn.1001-7097.2017.04.015.

OWENS C D, WAKE N, KIM J M, et al . Endothelial function predicts positive arterial-venous fistula remodeling in subjects with stage Ⅳ and Ⅴ chronic kidney disease[J ] . J Vasc Access, 2010, 11(4): 329-334. DOI: 10.5301/jva.2010.5833.

EGOROVA A D, VAN DER HEIDEN K, POELMANN R E, et al . Primary Cilia as biomechanical sensors in regulating endothelial function[J ] . Differentiation, 2012, 83(2): S56-S61. DOI: 10.1016/j.diff.2011.11.007.

DUNN S, VOHRA R S, MURPHY J E, et al . Thelectin-like oxidized low-density-lipoprotein receptor: a pro-inflammatory factor in vascular disease[J ] . Biochem J, 2008, 409(2): 349-355. DOI: 10.1042/BJ20071196.

FURLONG K, HWANG S. Autophagy and noroviruses[J]. Viruses, 2019, 11(3): 244. DOI: 10.3390/v11030244.

MIALET-PEREZ J, VINDIS C. Autophagy in health and disease: focus on the cardiovascular system[J]. Essays Biochem, 2017, 61(6): 721-732. DOI: 10.1042/EBC20170022.

蒋琦炜, 张德宇, 石烟祝, 等. 细胞自噬与肿瘤发生发展[J]. 军事医学, 2021, 45(3): 234-241.

JIANG Q W, ZHANG D Y, SHI Y Z, et al . Autophagy and tumor development and progression[J ] . Mil Med Sci, 2021, 45(3): 234-241.

DANCOURT J, MELIA T J. Lipidation of the autophagy proteins LC3 and GABARAP is a membrane-curvature dependent process[J]. Autophagy, 2014, 10(8): 1470-1471. DOI: 10.4161/auto.29468.

MIZUSHIMA N, YOSHIMORI T, LEVINE B. Methods in mammalian autophagy research[J]. Cell, 2010, 140(3): 313-326. DOI: 10.1016/j.cell.2010.01.028.

李钰. 分子伴侣介导自噬[J]. 临床医药文献电子杂志, 2017, 4(A3): 20364-20365.

LI Y. Molecular chaperone mediated autophagy[J]. J Clin Med Lit, 2017, 4(A3): 20364-20365.

ARIAS E, CUERVO A M. Chaperone-mediated autophagy in protein quality control[J]. Curr Opin Cell Biol, 2011, 23(2): 184-189. DOI: 10.1016/j.ceb.2010.10.009.

杨雄. 兔自体动静脉瘘术后内膜增生中的自噬现象[D]. 南宁: 广西医科大学, 2019.

YANG X. Autophagy in endometrial hyperplasia after auto genous arteriovenous fistula surgery in rabbits[D]. Nanning: Guangxi Medical University, 2019.

孔淑君, 陈卫东. 慢性肾脏病氧化应激的研究进展[J]. 国际泌尿系统杂志, 2020, 40(4): 759-762.

KONG S J, CHEN W D. Research progress of oxidative stress in chronic kidney disease[J]. Int J Urol Nephrol, 2020, 40(4): 759-762.

KIHARA A, KABEYA Y, OHSUMI Y, et al . Beclin-phosphatidylinositol 3-kinase complex functions at the trans-Golgi network[J ] . EMBO Rep, 2001, 2(4): 330-335. DOI: 10.1093/embo-reports/kve061.

和晓莹, 秦智琦, 段琦, 等. 氧化低密度脂蛋白激活PYHIN1/Caspase 1炎症复合体信号通路增强主动脉内皮细胞黏附的机制研究[J]. 中国分子心脏病学杂志, 2021, 21(5): 4248-4252. DOI: 10.16563/j.cnki.1671-6272.2021.10.021.

HE X Y, QIN Z Q, DUAN Q, et al . Mechanism of oxidized low density lipoprotein promotes PYHIN1/caspase 1 inflammatory complex signaling pathway and enhances aortic endothelial cell adhesion[J ] .Mol Cardiol China, 2021, 21(5): 4248-4252. DOI: 10.16563/j.cnki.1671-6272.2021.10.021.

ZABIRNYK O, LIU W, KHALIL S, et al . Oxidized low-density lipoproteins upregulate proline oxidase to initiate ROS-dependent autophagy[J ] . Carcinogenesis, 2010, 31(3): 446-454. DOI: 10.1093/carcin/bgp299.

ZHANG Y L, CAO Y J, ZHANG X, et al . The autophagy-lysosome pathway: a novel mechanism involved in the processing of oxidized LDL in human vascular endothelial cells[J ] . Biochem Biophys Res Commun, 2010, 394(2): 377-382. DOI: 10.1016/j.bbrc.2010.03.026.

朱琪, 林芳. 自噬的分子标志物[J]. 药学学报, 2016, 51(1): 33-38. DOI: 10.16438/j.0513-4870.2015-0634.

ZHU Q, LIN F. Molecular markers of autophagy[J]. Acta Pharm Sin, 2016, 51(1): 33-38. DOI: 10.16438/j.0513-4870.2015-0634.

NAZIO F, BORDI M, CIANFANELLI V, et al . Autophagy and cancer stem cells: molecular mechanisms and therapeutic applications[J ] . Cell Death Differ, 2019, 26(4): 690-702. DOI: 10.1038/s41418-019-0292-y.

王海杰, 谭玉珍. 细胞自噬的形态学特征和功能意义[J]. 解剖学报, 2009, 40(5): 844-849.

WANG H J, TAN Y Z. Morphological features and functional implications of autophagy[J]. Acta Anat Sin, 2009, 40(5): 844-849.

李彩丽, 辛英, 魏虎来, 等. 巨自噬与分子伴侣介导的自噬在饥饿诱导的Raji细胞中相继顺序被激活[J]. 中国药理学通报, 2019, 35(7): 958-961. DOI: 10.3969/j.issn.1001-1978.2019.07.014.

LI C L, XIN Y, WEI H L, et al . The sequential switch from macroautophagy to chaperone-mediated autophagy in Raji cells induced by starvation[J ] . Chin Pharmacol Bull, 2019, 35(7): 958-961. DOI: 10.3969/j.issn.1001-1978.2019.07.014.

曾欣程. 二甲双胍诱导分子伴侣介导自噬的功能研究[D]. 杭州: 浙江大学, 2018.

ZENG X C. Study of metformin induced chaperone mediated autophagy[D]. Hangzhou: Zhejiang University, 2018.

孙乐平. 分子伴侣介导自噬在甲基苯丙胺诱导的α-突触核蛋白相关神经毒性中的作用[D]. 广州: 南方医科大学, 2019.

SUN L P. The role of chaperone-mediated autophagy in neurotoxicity induced by alpha-synuclein after METH exposure[D]. Guangzhou: Southern Medical University, 2019.

赵彬. 通过激活和抑制EPCs的CMA探讨CMA和大自噬相互关系的实验研究[D]. 苏州: 苏州大学, 2012.

ZHAO B. Research interaction of CMA and macroautophagy through inhibition and activation CMA in EPCs[D]. Suzhou: Soochow University, 2012.

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