Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway

MA Rong; DU Xiaoye; CUI Jingang; XU Jing; CHEN Yu

doi:10.3969/j.issn.1006-2157.2023.06.011

您当前的位置：

首页 >

文章列表页 >

Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway

Experimental Studies | 更新时间：2023-07-05

- Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway
- Journal of Beijing University of Traditional Chinese Medicine Vol. 46, Issue 6, Pages: 811-819(2023)
- 作者机构：
  
  1.上海中医药大学附属岳阳中西医结合医院　上海　200437
  2.上海市中医药研究院中西医结合临床研究所
- 作者简介：
  
  Prof. CHEN Yu, Ph.D., Doctoral Supervisor. Yueyang Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai University of Traditional Chinese Medicine, No. 110, Ganhe Road, Hongkou District, Shanghai 200437. E-mail: chenyu@shutcm.edu.cn
- 基金信息：
  
  National Natural Science Foundation of China(81903887);Outstanding Academic Leader of Shanghai Municipality(81673790)
- DOI：10.3969/j.issn.1006-2157.2023.06.011
  CLC： R285.5
- Received：21 October 2022，
  
  Published Online：26 May 2023，
  
  Published：30 June 2023
- 稿件说明：
移动端阅览
MA Rong, DU Xiaoye, CUI Jingang, et al. Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(6): 811-819.
DOI：

MA Rong, DU Xiaoye, CUI Jingang, et al. Icariin protects cones by inhibiting endoplasmic reticulum-mitochondria interaction through the PERK/CHOP pathway[J]. Journal of beijing university of traditional chinese medicine, 2023, 46(6): 811-819. DOI： 10.3969/j.issn.1006-2157.2023.06.011.

摘要

目的

探讨在细胞内质网应激状态下，淫羊藿苷保护光感受器细胞的作用机制。

方法

以毒胡萝卜素(0.2 μmol/L)诱导小鼠视锥细胞(661W细胞)内质网应激模型，采用阿尔玛蓝染色法检测细胞活性，蛋白免疫印迹法检测未折叠蛋白效应相关蛋白——磷酸化蛋白激酶样内质网激酶(p-PERK)、葡萄糖调节蛋白78(GRP78)、CCAAT增强子结合蛋白同源蛋白(CHOP)表达水平，荧光成像和钙比例法检测Ca

2＋

含量，荧光染色法评价线粒体功能(线粒体通透性转换孔和膜电位)。以体外分子对接技术分析淫羊藿苷与内质网钙－ATP酶的结合及相互作用。

结果

毒胡萝卜素可诱导661W细胞死亡；淫羊藿苷(2.22～20.00 nmol/L)能够抑制毒胡萝卜素引起的661W细胞死亡(

0.01)。毒胡萝卜素促进661W细胞PERK磷酸化(

0.01)、上调转录因子CHOP的蛋白表达(

0.01)；淫羊藿苷(2.0 μmol/L)能够抑制模型组细胞CHOP蛋白表达(

0.01)，有抑制PERK(Thr980)磷酸化的趋势。毒胡萝卜素作用于661W细胞的10 min内，引起胞质内钙离子含量升高；淫羊藿苷预处理(1.0 μmol/L)能够抑制胞内钙离子含量的升高。毒胡萝卜素诱导661W细胞线粒体通透性转换孔的开放，进而引起线粒体膜电位降低；淫羊藿苷(2.0 μmol/L)能够抑制毒胡萝卜素诱导的线粒体通透性转换孔开放和线粒体膜电位降低。体外分子对接分析显示，淫羊藿苷能够与内质网钙－ATP酶2b亚型的核苷酸结合结构域及磷酸化结构域稳定结合，结合能为－8.776 kcal/mol，且存在多个氢键结合。

结论

淫羊藿苷可部分拮抗毒胡萝卜素对内质网钙－ATP酶的抑制作用，维持细胞内Ca

2＋

稳态，保护线粒体功能，进而发挥视锥细胞保护作用。

Abstract

Objective

To elucidate the mechanism of action of icariin to protect photoreceptor cells suffering from endoplasmic reticulum stress.

Methods

Thapsigargin(0.2 μmol/L)

was used to induce endoplasmic reticulum stress in the mouse cone cell line 661W. AlamarBlue staining was used to detect cell viability. The levels of proteins related to the unfolded protein response p-PERK

GRP78

CHOP were analyzed by Western blotting. The intracellular Ca

level was analyzed by fluorescence imaging and the F340/F380 ratio method. Permeability transition pore protein and the membrane potential in mitochondria were analyzed by fluorescence staining. The binding and interaction between icariin and SERCA2b were analyzed

in vitro

by molecular docking.

Results

Thapsigargin induced 661W cell death. Icariin significantly inhibited 661W cell death caused by thapsigargin in the concentration of 2.22-20.00 nmol/L (

0.01). Thapsigargin induced the unfolded protein responses in 661W cells

significantly promoting the phosphorylation of PERK (Thr980) (

0.01) and increasing the protein levels of transcription factor CHOP (

0.01). Icariin (2.0 μmol/L) significantly downregulated the expression of CHOP protein (

0.01) and had the trend of inhibitation of phosphorylation of PERK(Thr980). Within 10 minutes after thapsigargin acting on 661 W cells

the calcium ions in the cytosol increased. Icariin pretreatment (1.0 μmol/L) inhibited an increase of intracellular calcium content. Thapsigargin induced the opening of mitochondrial permeability transition pores in 661W cells

which in turn caused a decrease in mitochondrial membrane potential. Icariin (2.0 μmol/L) inhibited thapsigargin-induced mitochondrial permeability transition pore opening and mitochondrial membrane potential reduction.

In vitro

molecular docking analysis showed that icariin stably bound to the nucleotide-binding and phosphorylation domains of SERCA2b

with a binding energy of -8.776 kcal/mol and multiple hydrogen bonds.

Conclusion

Icariin partially antagonized the effect on the binding of thapsigargin and SERCA

which maintained Ca

homeostasis in cones

contributing to mitochondrial health. This may be one mechanism of action of icariin in cone cell protection.

关键词

Keywords

references

KROEGER H , CHIANG WC , FELDEN J , et al . ER stress and unfolded protein response in ocular health and disease [J]. FEBS J , 2019 , 286 ( 2 ): 399 － 412 .

MCLAUGHLIN T , MEDINA A , PERKINS J , et al . Cellular stress signaling and the unfolded protein response in retinal degeneration: mechanisms and therapeutic implications [J/OL]. Mol Neurodegener , 2022 , 17 ( 1 ): 25 [ 2022－09－02 ]. https：//molecularneurodegeneration.biomedcentral.com/articles/10.1186/s13024－022－00528－w https：//molecularneurodegeneration.biomedcentral.com/articles/10.1186/s13024－022－00528－w .

马荣，吴晗晗，郭红，等 . 淫羊藿对光损伤小鼠视网膜光感受器细胞的保护作用 [J]. 上海中医药杂志， 2020 , 54 ( 2 ): 91 － 96 .

SEHGAL P , SZALAI P , OLESEN C , et al . Inhibition of the sarco/endoplasmic reticulum (ER) Ca 2＋－ATPase by thapsigargin analogs induces cell death via ER Ca 2＋ depletion and the unfolded protein response [J]. J Biol Chem , 2017 , 292 ( 48 ): 19656 － 19673 .

HESS K , DE SILVA T , GRISSO P , et al . Evaluation of Cone－ and Rod-Mediated Parameters in Dark Adaptation Testing as Outcome Measures in Age-Related Macular Degeneration [J]. Ophthalmol Retina , 2022 , 6 ( 12 )： 1173 － 1184 .

RODRIGUEZ JD , LANE K , HOLLANDER DA , et al . Cone photoreceptor macular function and recovery after photostress in early non-exudative age-related macular degeneration [J]. Clin Ophthalmol , 2018 , 12 : 1325 － 1335 .

CONWAY BR . Color vision, cones, and color-coding in the cortex [J]. Neuroscientist , 2009 , 15 ( 3 ): 274 － 290 .

KRIZAJ D . Serca isoform expression in the mammalian retina [J]. Exp Eye Res , 2005 , 81 ( 6 ): 690 － 699 .

SCHWARZ DS , BLOWER MD . The endoplasmic reticulum: structure, function and response to cellular signaling [J]. Cell Mol Life Sci , 2016 , 73 ( 1 ): 79 － 94 .

READ A , SCHRÖDER M . The Unfolded Protein Response: An Overview [J/OL]. Biology (Basel) , 2021 , 10 ( 5 ): 384 [ 2022－09－02 ]. https：//www.mdpi.com/2079－7737/10/5/384 https：//www.mdpi.com/2079－7737/10/5/384 .

YANG LP , WU LM , GUO XJ , et al . Activation of endoplasmic reticulum stress in degenerating photoreceptors of the rd1 mouse [J]. Invest Ophthalmol Vis Sci , 2007 , 48 ( 11 ): 5191 － 5198 .

JING GJ , WANG JJ , ZHANG SX . ER stress and apoptosis: a new mechanism for retinal cell death [J/OL]. Exp Diabetes Res , 2012 , 2012 : 589589 [ 2022－09－02 ]. https：//www.hindawi.com/journals/jdr/2012/589589 https：//www.hindawi.com/journals/jdr/2012/589589 .

FAN B , SUN YJ , LIU SY , et al . Neuroprotective Strategy in Retinal Degeneration: Suppressing ER Stress-Induced Cell Death via Inhibition of the mTOR Signal [J/OL]. Int J Mol Sci , 2017 , 18 ( 1 ): 201 [ 2022－09－02 ] https：//www.mdpi.com/1422－0067/18/1/201 https：//www.mdpi.com/1422－0067/18/1/201 .

NAKANISHI T , SHIMAZAWA M , SUGITANI S , et al . Role of endoplasmic reticulum stress in light-induced photoreceptor degeneration in mice [J]. J Neurochem , 2013 , 125 ( 1 ): 111 － 124 .

YANG LP , WU LM , GUO XJ , et al . Endoplasmic reticulum stress is activated in light-induced retinal degeneration [J]. J Neurosci Res , 2008 , 86 ( 4 ): 910 － 919 .

CLAPHAM DE . Calcium signaling [J]. Cell , 2007 , 131 ( 6 ): 1047 － 1058 .

WEI T , SCHUBERT T , PAQUET-DURAND F , et al . Light-driven calcium signals in mouse cone photoreceptors [J]. J Neurosci , 2012 ， 32 ( 20 )： 6981 － 6994 .

KULKARNI M , TRIFUNOVIC＇ D , SCHUBERT T , et al . Calcium dynamics change in degenerating cone photoreceptors [J]. Hum Mol Genet , 2016 , 25 ( 17 ): 3729 － 3740 .

TOYOSHIMA C , MIZUTANI T . Crystal structure of the calcium pump with a bound ATP analogue [J]. Nature , 2004 , 430 ( 6999 ): 529 － 535 .

INOUE M , SAKUTA N , WATANABE S , et al . Structural Basis of Sarco/Endoplasmic Reticulum Ca 2＋－ATPase 2b Regulation via Transmembrane Helix Interplay [J]. Cell Rep , 2019 , 27 ( 4 ): 1221 － 1230 .

WINTHER AM , LIU H , SONNTAG Y , et al . Critical roles of hydrophobicity and orientation of side chains for inactivation of sarcoplasmic reticulum Ca 2＋－ATPase with thapsigargin and thapsigargin analogs [J]. J Biol Chem , 2010 , 285 ( 37 ): 28883 － 28892 .

WONG-RILEY MT . Energy metabolism of the visual system [J]. Eye Brain , 2010 , 2 : 99 － 116 .

NARAYAN DS , CHIDLOW G , WOOD JP , et al . Glucose metabolism in mammalian photoreceptor inner and outer segments [J]. Clin Exp Ophthalmol , 2017 , 45 ( 7 ): 730 － 741 .

AOYAMA-ISHIWATARI S , HIRABAYASHI Y . Endoplasmic Reticulum-Mitochondria Contact Sites-Emerging Intracellular Signaling Hubs [J/OL]. Front Cell Dev Biol , 2021 , 9 : 653828 [ 2022－09－02 ]. https：//www.frontiersin.org/articles/10.3389/fcell.2021.653828/full https：//www.frontiersin.org/articles/10.3389/fcell.2021.653828/full .

ROWLAND AA , VOELTZ GK . Endoplasmic reticulum-mitochondria contacts: function of the junction [J]. Nat Rev Mol Cell Biol , 2012 , 13 ( 10 ): 607 － 625 .

RAKHVINDER KKS . The mechanism of glucose-induced RCAN1 expression in β-cells. Degree of Masters [D/OL]. South Australia : Flinders University School of Medicine , 2012 [ 2022－09－22 ]. https：//flex.flinders.edu.au/file/ce258574－c310－4a41－bee3－31146dad189a/1/Thesis－Kashmir_Singh－2012.pdf .

GOMEZ-SUAGA P , PAILLUSSON S , MILLER CCJ . ER-mitochondria signaling regulates autophagy [J]. Autophagy , 2017 , 13 ( 7 ): 1250 － 1251 .

STANSFIELD WE , RANEK M , PENDSE A , et al . The Pathophysiology of Cardiac Hypertrophy and Heart Failure [M]// WILLIS MS , HOMEISTER JW , STONE JR . Cellular and Molecular Pathobiology of Cardiovascular Disease . NewYork : Academic Press , 2014 : 51 － 78 .

NEGINSKAYA MA , PAVLOV EV , SHEU SS . Electrophysiological properties of the mitochondrial permeability transition pores: Channel diversity and disease implication [J/OL]. Biochim Biophys Acta Bioenerg , 2021 , 1862 ( 3 ): 148357 [ 2022－09－02 ]． https：//www.sciencedirect.com/science/article/pii/S0005272820302073？via%3Dihub https：//www.sciencedirect.com/science/article/pii/S0005272820302073？via%3Dihub .

KORGE P , WEISS JN . Thapsigargin directly induces the mitochondrial permeability transition [J]. Eur J Biochem , 1999 , 265 ( 1 ): 273 － 280 .

Views

159

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

The mechanism of modified Xiangsha Liujunzi Decoction in regulating apoA-I and improving endoplasmic reticulum stress in hyperlipidemic mice

Ziyin Mingmu Formula improve retinitis pigmentosa through the GRP78/IRE1/ATF6 pathway in vitro study

The effects of Shenqi Wan on renal damage, GRP78, and autophagy-related factors in Zucker diabetic fatty rats

Effect of icariin on osteogenic differentiation of rat adipose-derived stem cells in vitro

Related Author

JIA Lianqun

CAI Haoran

LIU Yu

WANG Jie

SONG Nan

SUI Guoyuan

ZHANG Qi

PENG Qinghua

Related Institution

College of Integrative Medicine, Liaoning University of Traditional Chinese Medicine

Key Laboratory of Visceral Phenomena Theory and Application, Traditional Chinese Medicine Innovation Engineering Technology Center, Liaoning University of Traditional Chinese Medicine, Ministry of Education

Hunan University of Chinese Medicine

The First Affiliated Hospital of Hunan University of Chinese Medicine

School of Traditional Chinese Medicine, Beijing University of Chinese Medicine

AI问答

Postal code：100029
Tel：(010)53911857 Email：jbutcm@bucm.edu.cn
Technical support is provided by Beijing Founder electronics co., LTD 京ICP备09064830号-19 京公网安备11010802024621
It is recommended to read the content of this site in Chrome&IE9+. Please switch to extreme mode in browser 360.
Cookies We use cookies to help provide and enhance our service and tailor content. By continuing, you agree to the use of cookies.

⁰