Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells

Zhengli WANG; Yafei ZI; Yangdong LIU; Qining FU; Xuehu WANG

doi:10.16016/j.2097-0927.202112106

您当前的位置：

首页 >

文章列表页 >

Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells

Basic Medicine | 更新时间：2026-01-09

- Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells
- Journal of Army Medical University Vol. 44, Issue 12, Pages: 1221-1228(2022)
- 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：
- 基金信息：
- DOI：10.16016/j.2097-0927.202112106
  CLC： R322.123;R329.25;R363.21
- Received：13 December 2021，
  
  Revised：2022-01-28，
  
  Published：30 June 2022
- 稿件说明：
移动端阅览
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：

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.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Role of SIRT1/UCP2 signaling pathway in resveratrol-induced inhibition of oxidative injury in vascular endothelial cells

Role and mechanism of SIRT3 in oxidative stress and mitochondrial biogenesis in alveolar type II cells

p300 promotes hepatic lipid accumulation in dyslipidemia by regulating SREBP-1c acetylation

Protein phosphatase 2A promotes mitophagy to alleviate fructose-induced mitochondrial oxidative damage in M2-type macrophages

Regulation and mechanism of Gm49394 on islet-β cell apoptosis

Related Author

周曦

易龙

金鑫

陈明亮

陈春烨

王丽

高燕翔

施琳颖

Related Institution

Department of Human Anatomy， Anhui Medical University

Laboratory of Stem Cell and Tissue Engineering， Chongqing Medical University

Institute of Field Surgery， Daping Hospital， Army Medical University

Department of Geriatrics and Special Service Medicine， First Affiliated Hospital（Southwest Hospital）， Army Medical University （Third Military Medical University）

达州市中西医结合医院药学科

⁰