Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide

Pan TANG; Bo LIU

doi:10.16016/j.2097-0927.202203143

您当前的位置：

首页 >

文章列表页 >

Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide

Basic Medicine | 更新时间：2026-01-09

- Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide
- Journal of Army Medical University Vol. 44, Issue 16, Pages: 1613-1620(2022)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院骨科
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81572202)
- DOI：10.16016/j.2097-0927.202203143
  CLC： R322.72;R329.25;R341
- Received：16 March 2022，
  
  Revised：2022-05-11，
  
  Published：30 August 2022
- 稿件说明：
移动端阅览
Pan TANG, Bo LIU. Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide[J]. Journal of Army Medical University, 2022, 44(16): 1613-1620.
DOI：

Pan TANG, Bo LIU. Effects of zinc finger protein A20 on senescence and apoptosis of human degenerative nucleus pulposus cells stimulated by lipopolysaccharide[J]. Journal of Army Medical University, 2022, 44(16): 1613-1620. DOI： 10.16016/j.2097-0927.202203143.

摘要

目的

探究人退变髓核细胞中锌指蛋白A20对衰老及凋亡的影响。

方法

提取并培养人退变髓核细胞，小干扰RNA(siRNA)降低A20表达，脂多糖(lipopolysaccharide

LPS)作为炎症诱导因素，设置对照组、LPS组、siRNA-A20组、siRNA-A20+LPS组。通过Western blot检测A20、P21、P16、BAX、BID、BCL2、P65、p-P65蛋白表达量。细胞衰老β-半乳糖苷酶染色试剂盒检测β-半乳糖苷酶。应用AnnexinV-PI双标法及TUNEL染色检测细胞凋亡，观察锌指蛋白A20对髓核细胞衰老及凋亡的影响。应用咖啡酸苯乙酯(caffeic acid phenethyl ester

CAPE)进行挽救实验，检测A20、P21、P16、BAX、BID、BCL2、P65、P-P65蛋白表达量，探索下调锌指蛋白A20诱导细胞衰老及凋亡可能通路。

结果

与对照组相比，LPS组P21、P16蛋白表达量无明显变化，siRNA-A20组及siRNA-A20+LPS组P21、P16蛋白表达明显增加(

0.05)，β-半乳糖苷酶染色实验显示着色阳性细胞数显著增多(

0.05)。Annexin V-PI双标法凋亡结果显示，单纯LPS刺激及单纯siRNA-A20处理组中细胞凋亡占比高于对照组(

0.05)，但低于siRNA-A20+LPS组(

0.05)，TUNEL染色结果与Annexin V-PI双标法结果一致(

0.05)。凋亡相关蛋白BAX、BID在siRNA-A20+LPS中显著增加(

0.05)，BCL2则明显降低(

0.05)，P-P65蛋白表达量在siRNA-A20+LPS组明显上升(

0.05)。与siRAN-A20+LPS组相比，应用CAPE预处理后P-P65、P21、P16、BID、BAX蛋白表达量明显下降(

0.05)，BCL2上升(

0.05)。

结论

下调锌指蛋白A20，LPS过度激活NF-κB信号通路诱导人退变髓核细胞衰老与凋亡。

Abstract

Objective

To explore the effect of zinc finger protein A20 on senescence and apoptosis in human degenerative nucleus pulposus cells.

Methods

Human degenerative nucleus pulposus cells were extracted and cultured. Small interference RNA (siRNA) decreased the expression of A20. Lipopolysaccharide (LPS) was used as an inflammatory inducing factor. Control group

LPS group

siRNA-A20 group and siRNA-A20+LPS group were set up. The protein expression levels of A20

P21

P16

BAX

BID

BCL2

P65 and P-P65 were detected by Western blotting. β-galactosidase was detected by aging β-galactosidase staining kit

and Annexin V-PI double labeling and TUNEL staining was employed to detect apoptosis and to explore the effect of zinc finger protein A20 on senescence and apoptosis of nucleus pulposus cells. Caffeic acid phenethyl ester (CAPE) was used in rescue experiment

and the protein expression levels of A20

P21

P16

BAX

BID

BCL2

P65 and P-P65 were detected to explore the possible pathway of cell senescence and apoptosis induced by down-regulation of zinc finger protein A20.

Results

Compared with the control group

there was no significant change in protein levels of P21 and P16 in LPS group

but P21 and P16 increased significantly in siRNA-A20 group and siRNA-A20+LPS group (

0.05). β-galactosidase staining test showed that the number of positive cells increased significantly in siRNA-A20 treated groups (

0.05). The proportion of apoptosis in LPS group and siRNA-A20 group was higher than that of control group (

0.05)

but lower than that of siRNA-A20+LPS group (

0.05). The result of TUNEL staining was consistent with that of AnnexinV-PI double labeling(

0.05). Apoptosis related protein BID and BAX increased (

0.05)

and BCL2 decreased in siRNA-A20+LPS group (

0.05)

while P-P65 increased significantly in siRNA-A20+LPS group(

0.05). Compared with siRNA-A20+LPS group

through the pretreatment of CAPE

protein levels of P-P65

P21

P16

BID and BAX decreased (

0.05)

and the protein levels of BCL2 increased (

0.05).

Conclusion

Through down-regulation of zinc finger protein A20

LPS induces senescence and apoptosis of human degenerative nucleus pulposus cells through NF-κB signal pathway.

关键词

Keywords

references

WANG Y J, CHE M X, XIN J G, et al . The role of IL-1β and TNF-α in intervertebral disc degeneration[J ] . Biomed Pharmacother, 2020, 131: 110660. DOI: 10.1016/j.biopha.2020.110660.

RISBUD M V, SHAPIRO I M. Role of cytokines in intervertebral disc degeneration: pain and disc content[J]. Nat Rev Rheumatol, 2014, 10(1): 44-56. DOI: 10.1038/nrrheum.2013.160.

SHEMBADE N, MA A, HARHAJ E W. Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes[J]. Science, 2010, 327(5969): 1135-1139. DOI: 10.1126/science.1182364.

BOONE D L, TURER E E, LEE E G, et al . The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses[J ] . Nat Immunol, 2004, 5(10): 1052-1060. DOI: 10.1038/ni1110.

ZHANG Y, YI W W, XIA H Q, et al . A20 regulates inflammation through autophagy mediated by NF-κB pathway in human nucleus pulposus cells and ameliorates disc degeneration in vivo [J ] . Biochem Biophys Res Commun, 2021, 549: 179-186. DOI: 10.1016/j.bbrc.2021.02.115..

CHEN J, MA Y F, YANG Z J, et al . TNFAIP 3 ameliorates the degeneration of inflammatory human nucleus pulposus cells by inhibiting mTOR signaling and promoting autophagy[J ] . Aging (Albany NY), 2020, 12(23): 24242-24254. DOI: 10.18632/aging.104160..

HERRANZ N, GIL J. Mechanisms and functions of cellular senescence[J]. J Clin Invest, 2018, 128(4): 1238-1246. DOI: 10.1172/JCI95148.

MANNARINO M, CHERIF H, LI L, et al . Toll-like receptor 2 induced senescence in intervertebral disc cells of patients with back pain can be attenuated by o-vanillin[J ] . Arthritis Res Ther, 2021, 23(1): 117. DOI: 10.1186/s13075-021-02504-z.

MA K, CHEN S, LI Z, et al . Mechanisms of endogenous repair failure during intervertebral disc degeneration[J ] . Osteoarthri Cartilage, 2019, 27(1): 41-48. DOI: 10.1016/j.joca.2018.08.021.

LI F D, SUN X F, ZHENG B, et al . Arginase Ⅱ promotes intervertebral disc degeneration through exacerbating senescence and apoptosis caused by oxidative stress and inflammation via the NF-κB pathway[J ] .Front Cell Dev Biol, 2021, 9: 737809. DOI: 10.3389/fcell.2021.737809..

彭鑫, 张聪, 王锋, 等. A20介导TNF-α炎症微环境下髓核细胞衰老的机制研究[J]. 中国脊柱脊髓杂志, 2019, 29(9): 834-840. DOI: 10.3969/j.issn.1004-406X.2019.09.09.

PENG X, ZHANG C, WANG F, et al . Mechanism research of senescence of NP cells mediated by A20 in TNF-α inflammation microenvironment[J ] . Chin J Spine Spinal Cord,2019, 29(9): 834-840. DOI: 10.3969/j.issn.1004-406X.2019.09.09.

MCARTHUR K, CHAPPAZ S, KILE B T. Apoptosis in megakaryocytes and platelets: the life and death of a lineage[J]. Blood, 2018, 131(6): 605-610. DOI: 10.1182/blood-2017-11-742684.

PENG X, WANG K, ZHANGC, et al . The mitochondrial antioxidant SS-31 attenuated lipopolysaccharide-induced apoptosis and pyroptosis of nucleus pulposus cells via scavenging mitochondrial ROS and maintaining the stability of mitochondrial dynamics[J ] . Free Radic Res, 2021, 55(11/12): 1080-1093. DOI: 10. 1080/10715762.2021.2018426.

ZHANG X B, HU Y C, CHENG P, et al . Targeted therapy for intervertebral disc degeneration: inhibiting apoptosis is a promising treatment strategy[J ] . Int J Med Sci, 2021, 18(13): 2799-2813. DOI: 10.7150/ijms.59171.

KAUFMANN T, STRASSER A, JOST P J. Fas death receptor signalling: roles of Bid and XIAP[J]. Cell Death Differ, 2012, 19(1): 42-50. DOI: 10.1038/cdd.2011.121.

ZHAO Y P, QIU C, WANG W H, et al . Cortistatin protects against intervertebral disc degeneration through targeting mitochondrial ROS-dependent NLRP3 inflammasome activation[J ] . Theranostics, 2020, 10(15): 7015-7033. DOI: 10.7150/thno.45359.

CHEN J, XIE J J, JIN M Y, et al . Sirt6 overexpression suppresses senescence and apoptosis of nucleus pulposus cells by inducing autophagy in a model of intervertebral disc degeneration[J ] . Cell Death Dis, 2018, 9(2): 56. DOI: 10.1038/s41419-017-0085-5.

张野, 夏辉强, 易威威, 等. 锌指蛋白A20对兔腰椎间盘退变影响的实验研究[J]. 中国修复重建外科杂志, 2021, 35(3): 366-374.

ZHANG Y, XIA H Q, YI W W, et al . Ex perimental study on the effect of zinc finger protein A20 on lumbar intervertebral disc degeneration in rabbits[J ] . Chin J Repar Reconstr Surg, 2021, 35(3): 366-374.

蓝海洋, 杨智杰, 夏辉强, 等. 锌指蛋白A20对内毒素刺激时人椎间盘髓核细胞炎症及退变的影响[J]. 第三军医大学学报, 2019, 41(6): 543-548. DOI: 10.16016/j.1000-5404.201810110.

LAN H Y, YANG Z J, XIA H Q, et al . Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide[J ] . J Third Mil Med Univ, 2019, 41(6): 543-548. DOI:10.16016/j.1000-5404.201810110.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Expression of miR-616 in osteosarcoma and its role in proliferation, apoptosis, migration and invasion of tumor cells

Regulation and mechanism of Gm49394 on islet-β cell apoptosis

Kallistatin inhibits apoptosis and extracellular matrix degradation of human nucleus pulposus cells by inducing SIRT1 expression

Molecular mechanism of cepharanthine synergistically acting with doxorubicin in reversing multidrug resistance of K562/ADR cells

Down-regulation of integrin-linked kinase suppresses glycolysis and proliferation and promotes apoptosis of cervical cancer cells possibly by inhibiting Wnt/β-catenin pathway

Related Author

FU Wanlei

HAO Xianglin

CAO Ya

XIA Jiying

ZHOU Xiaogang

XU Jiayi

GUO Qiaonan

LIU Dong

Related Institution

Department of Pathology， Second Affiliated Hospital， Army Medical University （Third Military Medical University）

Department of Immunology, Faculty of Pharmacy and Laboratory Medicine， Army Medical University （Third Military Medical University）

Department of Laboratory Animal Science， College of Basic Medical Sciences, Faculty of Pharmacy and Laboratory Medicine， Army Medical University （Third Military Medical University）

Department of Pharmaceutical Analysis, Faculty of Pharmacy and Laboratory Medicine， Army Medical University （Third Military Medical University）

Department of Pathology, College of Basic Medicine, Chongqing Medical University

⁰