Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide

Haiyang LAN; Zhijie YANG; Huiqiang XIA; Jie CHEN; Bo LIU

doi:10.16016/j.1000-5404.201810110

您当前的位置：

首页 >

文章列表页 >

Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide

Basic Medicine | 更新时间：2026-01-09

- Effects of zinc finger protein A20 over-expression on inflammatory response and degeneration of human intervertebral disc nucleus pulposus cells stimulated with lipopolysaccharide
- Journal of Third Military Medical University Vol. 41, Issue 6, Pages: 543-548(2019)
- 作者机构：
  
  400016 重庆，重庆医科大学附属第一医院骨科，重庆市脊柱外科中心
- 作者简介：
- 基金信息：
  
  the General Program of National Natural Science Foundation of China(81572202)
- DOI：10.16016/j.1000-5404.201810110
  CLC： R341; R392.3; R681.302
- Received：19 October 2018，
  
  Revised：2018-12-08，
  
  Published：30 March 2019
- 稿件说明：
移动端阅览
Haiyang LAN, Zhijie YANG, Huiqiang XIA, 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]. Journal of Third Military Medical University, 2019, 41(6): 543-548.
DOI：

Haiyang LAN, Zhijie YANG, Huiqiang XIA, 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]. Journal of Third Military Medical University, 2019, 41(6): 543-548. DOI： 10.16016/j.1000-5404.201810110.

摘要

目的

观察锌指蛋白A20在内毒素(lipopolysaccharide

LPS)刺激情况下，对人椎间盘髓核细胞炎症因子、核因子-kappB(NF-κB)以及细胞退变指标表达变化的影响。

方法

获取2017年6月至2018年7月本院骨科腰椎间盘退变患者术中切除的椎间盘组织，提取原代髓核细胞并进行培养，选择P1、P2代髓核细胞进行实验。采用完全随机法将同来源同代培养的髓核细胞分为对照组、单纯过表达A20基因腺病毒感染组、单纯LPS(10 μg/mL)刺激组及LPS(10 μg/mL)刺激+过表达A20基因腺病毒感染组。采用Real-time PCR测定A20 mRNA表达水平，Western blot检测A20蛋白、TNF-α、IL-1β、NF-κB结合蛋白p65、p-p65以及退变相关指标蛋白聚糖(aggrecan)、Ⅱ型胶原(collagen Ⅱ)等的表达；双抗体夹心酶联免疫吸附测定(ELISA)方法检测上清液TNF-α、IL-1β的表达。

结果

与对照组相比，单纯过表达A20基因组A20 mRNA与蛋白表达增高(

0.05)，而TNF-α、IL-1β、p-p65、蛋白聚糖、Ⅱ型胶原蛋白表达无明显变化；与对照组相比，单纯LPS刺激组IL-1β、TNF-α、p-p65蛋白表达明显升高(

0.05)，而蛋白聚糖、Ⅱ型胶原蛋白表达则明显降低(

0.05)；而与单纯LPS刺激组相比，LPS刺激+过表达A20基因组TNF-α、IL-1β和p-p65蛋白表达显著下降(

0.05)，同时蛋白聚糖与Ⅱ型胶原蛋白表达显著升高，差异有统计学意义(

0.05)。

结论

人椎间盘组织的退变与髓核细胞炎症反应的发生发展密切相关，锌指蛋白A20有效抑制髓核细胞炎症因子的产生和活化，可能在髓核细胞退变调控机制中发挥重要作用。

Abstract

Objective

To observe the effects of zinc finger protein A20 over-expression on the expression of inflammatory cytokines and nuclear factor-κB (NF-κB) in human intervertebral disc nucleus pulposus cells and on cell degeneration in response to lipopolysaccharide (LPS) stimulation.

Methods

We collected surgical specimens of intervertebral discs from patients undergoing orthopedic surgeries for lumbar intervertebral disc degeneration in our hospital between June

2017 and July

2018. The nucleus pulposus cells were isolated from the specimens and cultured

and the cells in passages 1 and 2 were divided into 4 groups

namely the control group

adenovirus-mediated A20 overexpression group

LPS (10 μg/mL) stimulation group

and LPS stimulation with A20 overexpression group. The cellular mRNA expression of A20 was examined using real-time PCR in all the 4 groups. Western blotting was used to detect the expression of A20 protein

tumor necrosis factor-α (TNF-α)

interleukin-1β (IL-1β)

NF-κB-binding proteins (p65 and phosphorylated p-p65) and the degeneration-related proteins (aggrecan and collagen Ⅱ). The levels of TNF-α and IL-1β were also detected in the supernatant of the cell cultures using ELISA.

Results

Compared with the control cells

the cells transfected with the adenoviral vector showed significantly increased mRNA and protein expression levels of A20 (

0.05) without obvious changes in the protein levels of TNF-α

IL-β

p-p65

aggrecan or collagen Ⅱ. LPS stimulation of the cells significantly increased the levels of TNF-α

IL-1β

and p-p65 proteins (

0.05) and lowered the levels of aggrecan and collagen Ⅱ proteins (

0.05). Compared with the cells with LPS stimulation alone

the A20-overexpressing cells with LPS stimulation showed obviously increased levels aggrecan and collagen Ⅱ (

0.05) and reduced levels of

TNF-α

IL-1β

and p-p65 proteins (

0.05).

Conclusion

The degeneration of human intervertebral disc tissue is closely related to inflammatory responses of the nucleus pulposus cells. The production and activation of the inflammatory cytokines in nucleus pulposus cells can be effectively inhibited by over-expression of Zinc finger protein A20

which may play an important role in the regulatory mechanism of nucleus pulposus cell degeneration.

关键词

Keywords

references

TEIXEIRA G Q, PEREIRA C L, FERREIRA J R, et al .Immunomodulation of human mesenchymal stem/stromal cells in intervertebral disc degeneration: insights from a proinflammatory/degenerative Ex vivo model[J ] . Spine, 2018, 43(12): E673-E682. DOI: 10.1097/BRS.0000000000002494.

MOLINOS M, ALMEIDA C R, CALDEIRA J, et al . Inflammation in intervertebral disc degeneration and regeneration[J ] .J R Soc Interface, 2015, 12(104): 20141 191.DOI: 10.1098/rsif.2014.1191.

DE WINTHER M P, KANTERS E, KRAAL G, et al .Nuclear factor κB singnaling in atherogenesis[J ] . Arterioscler Thromb Vasc Biol, 2005, 25(5): 904-914. DOI: 10.1161/01.ATV.0000160340.72641.87.

刘浠, 易威威, 温亚枫, 等.锌指蛋白A20及其相关炎症因子在椎间盘髓核细胞退变前后的表达变化[J].第三军医大学学报, 2016, 38(18): 2077-2081. DOI: 10.16016/j.1000-5404.201602105.

LIU X, YI W W, WEN Y F, et al .Expression of zinc finger protein A20 and related inflammatory factors in nucleus pulposus cells in degenerative intervertebral disc[J ] .J Third Mil Med Univ, 2016, 38(18): 2077-2081. DOI: 10.16016/j.1000-5404.201602105.

VO N V, HARTMAN R A, PATIL P R, et al . Molecular mechanismsof biological aging in intervertebral discs[J ] . J Orthop Res, 2016, 34(8): 1289-1306. DOI: 10.1002/jor.23195.

PARK J B, BYUN C H, PARK E Y. Rat notochordal cells undergo premature stress-induced senescence by high glucose[J]. Asian Spine J, 2015, 9(4): 495-502. DOI: 10.4184/asj.2015.9.4.495.

PARK J S, PARK J B, PARK I J, et al . Accelerated premature stress-in duced senescence of young annulus fibrosus cells of rats by high glucose-induced oxidative stress[J ] . Int Orthop, 2014, 38(6): 1311-1320. DOI: 10.1007/s00264-014-2296-z.

SUZUKI S, FUJITA N, HOSOGANE N, et al . Excessive reactive oxygen species are therapeutic targets for intervertebral disc degeneration[J ] . Arthritis Res Ther, 2015, 17: 316. DOI: 10.1186/s13075-015-0834-8.

LIPSON S J, MUIR H. Experimental intervertebral disc degeneration: morphologic and proteoglycan changes over time[J]. Arthritis Rheum, 1981, 24(1): 12-21. DOI: 10.1002/art.1780240103.

JONES P, GARDNER L, MENAGE J, et al .Intervertebral disc cells as competent phagocytes in vitro : implications for cell death in disc degeneration[J ] . Arthritis Res Ther, 2008, 10(4): R86. DOI: 10.1186/ar2466..

WANG S, LI J, TIAN J, et al . High amplitude and low frequency cyclic mechanical strain promotes degeneration of human nucleus pulposus cells via the NF-κB p65 pathway[J ] . J Cell Physiol, 2018, 233(9): 7206-7216. DOI: 10.1002/jcp.26551..

ANDERSSON G B. Epidemiological features of chronic low-backpain[J]. Lancet, 1999, 354(9178): 581-585. DOI: 10.1016/S0140-6736(99)01312-4.

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.

易威威, 温亚枫, 刘浠, 等. NF-κB抑制剂对脂多糖刺激的退变人椎间盘髓核细胞炎症因子表达的影响[J].第三军医大学学报, 2017, 39(8): 755-758. DOI: 10.16016/j.1000-5404.201610083.

YI W W, WEN Y F, LIU X, et al . Effect of nuclear factor-κB inhibitor on expression of inflammatory factors in lipopolysaccharide-stimulated degenerative human nucleus pulposus cells[J ] . J Third Mil Med Univ, 2017, 39(8): 755-758. DOI: 10.16016/j.1000-5404.201610083.

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Hypoxia-induced expression of zinc finger protein A20 inhibits apoptosis and attenuates inflammation in nucleus pulposus cells

Sirt1 is involved in intervertebral disc degeneration via regulating histone H3K18 lactylation in nucleus pulposus cells

Metformin attenuates brain damage in mouse model of lipopolysaccharide-induced sepsis

Effect of nuclear factor-κB inhibitor on expression of inflammatory factors in lipopolysaccharidestimulated degenerative human nucleus pulposus cells

YAP inhibits extracellular matrix catabolism of nucleus pulposus cells by promoting autophagy

Related Author

郭帅池

刘豪

韩正瀚

刘雯隽

刘渤

OUYANG Jian

ZHAI Yu

ZHANG Yuyao

Related Institution

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

Department of Pediatric Research Institute，Children’s Hospital of Chongqing Medical University， National Clinical Research Center for Child Health and Disorders， Ministry of Education Key Laboratory of Child Development and Disorders

Department of Dermatology， Children’s Hospital of Chongqing Medical University

Department of Ophthalmology， First Affiliated Hospital， Army Medical University （Third Military Medical University）

Department of Ophthalmology， General Hospital of Southern Theater Command

⁰