Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism

Fan Mingyu; Bian Feng; Zhou Shaohuai; Wang Xin; Wang Ping; Li Jing; Huang Yafen; Li Jiaqiong

doi:10.13406/j.cnki.cyxb.003282

您当前的位置：

首页 >

文章列表页 >

Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism

Basic research | 更新时间：2025-10-21

- Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism
- Journal of Chongqing Medical University Vol. 48, Issue 7, Pages: 777-783(2023)
- 作者机构：
  
  武汉市第三医院骨一科，武汉 430060
- 作者简介：
- 基金信息：
- DOI：10.13406/j.cnki.cyxb.003282
  CLC： R274
- Received：01 December 2022，
  
  Revised：2023-06-02，
  
  Accepted：23 June 2023，
  
  Published：28 July 2023
- 稿件说明：
移动端阅览
范明宇,卞峰,周少怀,等.穿心莲内酯对TBHP诱导的椎间盘髓核细胞凋亡的作用及机制研究[J].重庆医科大学学报,2023,48(7):777-783.

Fan Mingyu,Bian Feng,Zhou Shaohuai,et al.Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism[J].Journal of Chongqing Medical University,2023,48(7):777-783.
范明宇,卞峰,周少怀,等.穿心莲内酯对TBHP诱导的椎间盘髓核细胞凋亡的作用及机制研究[J].重庆医科大学学报,2023,48(7):777-783. DOI： 10.13406/j.cnki.cyxb.003282.

Fan Mingyu,Bian Feng,Zhou Shaohuai,et al.Effect of andrographolide on TBHP-induced apoptosis of intervertebral disc nucleus pulposus cells and its mechanism[J].Journal of Chongqing Medical University,2023,48(7):777-783. DOI： 10.13406/j.cnki.cyxb.003282.

摘要

目的

探讨穿心莲内酯（andrographolide，ANDRO）对叔丁基过氧化氢（tert-butyl hydroperoxide，TBHP）诱导的椎间盘髓核细胞（nucleus pulposus cells，NPC）凋亡的作用及其机制。

方法

用TBHP诱导NPC建立细胞模型。将NPC分为5组：对照组、模型组（100 μmol/L TBHP）、ANDRO低剂量组（100 μmol/L TBHP+9 μmol/L ANDRO）、ANDRO中剂量组（100 μmol/L TBHP+18 μmol/L ANDRO）和ANDRO高剂量组（100 μmol/L TBHP+36 μmol/L ANDRO）。流式细胞术检测细胞凋亡和活性氧（reactive oxygen species，ROS）水平，ELISA试剂盒检测氧化应激相关指标超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-PX）和过氧化氢酶（catalase，CAT）水平，JC-1探针法检测线粒体膜电位，Western blot检测动力相关蛋白1（dynamin-related protein 1，Drp1）、p-Drp1和线粒体融合蛋白2（mitofusin 2，Mfn2）的表达水平。

结果

与对照组相比，模型组NPC凋亡率、ROS水平和线粒体膜电位下降细胞比例明显升高（均

=0.000），SOD、GSH-PX和CAT水平明显降低（均

=0.000），p-Drp1/Drp1水平明显上调（

=0.000），Mfn2蛋白表达明显下调（

=0.000）。与模型组相比，经ANDRO高剂量干预后，NPC凋亡率［（41.37±0.84）% vs.（26.36±1.33）%］、ROS水平［（42.62±0.71）% vs.（22.50±0.37）%］和线粒体膜电位下降细胞比例［（43.85±0.71）% vs.（24.96±1.04）%］明显降低（均

=0.000），SOD［（288.16±14.59） ng/mL vs.（658.87±13.22） ng/mL］、GSH-PX［（6.66±0.37） ng/mL vs.（15.66±0.56） ng/mL］和CAT［（40.89±1.04） pg/mL

vs.（87.91±1.56） pg/mL］水平明显升高（均

=0.000），p-Drp1/Drp1水平（0.96±0.05

vs. 0.63±0.01）明显下调（

=0.001、0.000），Mfn2蛋白表达（0.39±0.01

vs. 0.64±0.02）明显上调（均

=0.000）。

结论

ANDRO具有抑制TBHP诱导的NPC细胞凋亡的作用，其机制可能与调节线粒体功能，缓解氧化应激有关。

Abstract

Objective

To investigate the effect and mechanism of andrographolide（ANDRO） on intervertebral disc nucleus pulposus cell（NPC） apoptosis induced by tert-butyl hydroperoxide（TBHP）.

Methods

The cell model of NPCs induced by TBHP was established. NPCs were divided into five groups： control group，model group（100 μmol/L TBHP），low-dose ANDRO group（100 μmol/L TBHP+9 μmol/L ANDRO），middle-dose ANDRO group（100 μmol/L TBHP+18 μmol/L ANDRO），and high-dose ANDRO group（100 μmol/L TBHP+36 μmol/L ANDRO）. Cell apoptosis and reactive oxygen species（ROS） levels were determined by flow cytometry. The levels of oxidative stress related indicators，such as superoxide dismutase（SOD），glutathione peroxidase（GSH-PX），and catalase（CAT），were determined by an enzyme-linked immunosorbent assay kit. Mitochondrial membrane potential was measured by a JC-1 probe. Western blot was used to determine the expression levels of dynamin-related protein 1（Drp1），phosphorylated Drp1（p-Drp1），and mitofusin 2（Mfn2）.

Results

Compared with the control group，the model group had significantly increased apoptosis rate of NPCs，ROS level，and proportion of cells with decreased mitochondrial membrane potential（all

=0.000），significantly decreased SOD，GSH-PX，and CAT levels（all

=0.000），a significantly up-regulated p-Drp1/Drp1 level（

=0.000），and significantly down-regulated protein expression of Mfn2（

=0.000）. Compared with the model group，the high-dose ANDRO group had significantly decreased apoptosis rate of NPCs［（41.37±0.84）% vs.（26.36±1.33）%］，ROS level［（42.62±0.71）% vs.（22.50±0.37）%］，and proportion of cells with decreased mitochondrial membrane potential［（43.85±0.71）% vs.（24.96±1.04）%］（all

=0.000），significantly increased SOD［（288.16±14.59） ng/mL vs.（658.87±13.22） ng/mL］，GSH-PX［（6.66±0.37） ng/mL vs. （15.66±0.56） ng/mL］，and CAT［（40.89±1.04） pg/mL

vs. （87.91±1.56） pg/mL］ levels（all

=0.000），a significantly down-regulated p-Drp1/Drp1 level（0.96±0.05

vs. 0.63±0.01）（

=0.001，0.000），and significantly up-regulated protein expression of Mfn2（0.39±0.01 vs. 0.64±0.02）（all

=0.000）.

Conclusion

ANDRO can inhibit TBHP-induced NPC apoptosis，and the mechanism may be related to the regulation of mitochondrial function and the relief of oxidative stress.

关键词

Keywords

references

Wang D ， He X ， Wang D ， et al . Quercetin suppresses apoptosis and attenuates intervertebral disc degeneration via the SIRT1-autophagy pathway [J ] . Front Cell Dev Biol ， 2020 ， 8 ： 613006 .

Zhang JW ， Zhang JY ， Zhang YL ， et al . Mesenchymal stem cells-derived exosomes ameliorate intervertebral disc degeneration through inhibiting pyroptosis [J ] . J Cell Mol Med ， 2020 ， 24 （ 20 ）： 11742 - 11754 .

Zhao YP ， Qiu C ， Wang WH ， et al . Cortistatin protects against intervertebral disc degeneration through targeting mitochondrial ROS-dependent NLRP3 inflammasome activation [J ] . Theranostics ， 2020 ， 10 （ 15 ）： 7015 - 7033 .

朱　超，阮狄克 . 椎间盘退变机制研究进展 [J ] . 中国骨与关节杂志， 2022 ， 11 （ 9 ）： 700 - 706 .

Zhu C ， Ruan DK . Research advances on intervertebral disc degeneration mechanism [J ] . Chin J Bone Jt ， 2022 ， 11 （ 9 ）： 700 - 706 .

Kang L ， Liu SW ， Li JC ， et al . The mitochondria-targeted anti-oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance [J ] . Cell Prolif ， 2020 ， 53 （ 3 ）： e12779 .

Burgos RA ， Alarcón P ， Quiroga J ， et al . Andrographolide，an anti-inflammatory multitarget drug：all roads lead to cellular metabolism [J ] . Molecules ， 2020 ， 26 （ 1 ）： 5 .

Geng J ， Liu W ， Gao J ， et al . Andrographolide alleviates Parkinsonism in MPTP-PD mice via targeting mitochondrial fission mediated by dynamin-related protein 1 [J ] . Br J Pharmacol ， 2019 ， 176 （ 23 ）： 4574 - 4591 .

Chen DH ， Xia DD ， Pan ZY ， et al . Metformin protects against apoptosis and senescence in nucleus pulposus cells and ameliorates disc degeneration in vivo [J ] . Cell Death Dis ， 2016 ， 7 （ 10 ）： e2441 .

Liu JW ， Jiang TM ， He MW ， et al . Andrographolide prevents human nucleus pulposus cells against degeneration by inhibiting the NF-κB pathway [J ] . J Cell Physiol ， 2019 ， 234 （ 6 ）： 9631 - 9639 .

Chen Y ， Wu YQ ， Shi HX ， et al . Melatonin ameliorates intervertebral disc degeneration via the potential mechanisms of mitophagy induction and apoptosis inhibition [J ] . J Cell Mol Med ， 2019 ， 23 （ 3 ）： 2136 - 2148 .

Chen Z ， Tang WJ ， Zhou YH ， et al . Andrographolide inhibits non-small cell lung cancer cell proliferation through the activation of the mitochondrial apoptosis pathway and by reprogramming host glucose metabolism [J ] . Ann Transl Med ， 2021 ， 9 （ 22 ）： 1701 .

Rider SM ， Mizuno S ， Kang JD . Molecular mechanisms of intervertebral disc degeneration [J ] . Spine Surg Relat Res ， 2019 ， 3 （ 1 ）： 1 - 11 .

Song Y ， Li S ， Geng W ， et al . Sirtuin 3-dependent mitochondrial redox homeostasis protects against AGEs-induced intervertebral disc degeneration [J ] . Redox Biol ， 2018 ， 19 ： 339 - 353 .

Lin JL ， Zhuge JR ， Zheng XQ ， et al . Urolithin A-induced mitophagy suppresses apoptosis and attenuates intervertebral disc degeneration via the AMPK signaling pathway [J ] . Free Radic Biol Med ， 2020 ， 150 ： 109 - 119 .

Yu HC ， Hou GW ， Cao JK ， et al . Mangiferin alleviates mitochondrial ROS in nucleus pulposus cells and protects against intervertebral disc degeneration via suppression of NF-κB signaling pathway [J ] . Oxid Med Cell Longev ， 2021 ， 2021 ： 6632786 .

Feng CC ， Yang MH ， Lan MH ， et al . ROS：crucial intermediators in the pathogenesis of intervertebral disc degeneration [J ] . Oxid Med Cell Longev ， 2017 ， 2017 ： 5601593 .

Li B ， Jiang TM ， Liu H ， et al . Andrographolide protects chondrocytes from oxidative stress injury by activation of the Keap1-Nrf2-Are signaling pathway [J ] . J Cell Physiol ， 2018 ， 234 （ 1 ）： 561 - 571 .

Kim B ， Park J ， Chang KT ， et al . Peroxiredoxin 5 prevents amyloid-beta oligomer-induced neuronal cell death by inhibiting ERK-Drp1-mediated mitochondrial fragmentation [J ] . Free Radic Biol Med ， 2016 ， 90 ： 184 - 194 .

Qin X ， Zhao Y ， Gong J ， et al . Berberine protects glomerular podocytes via inhibiting Drp1-mediated mitochondrial fission and dysfunction [J ] . Theranostics ， 2019 ， 9 （ 6 ）： 1698 - 1713 .

Hu L ， Ding MG ， Tang DS ， et al . Targeting mitochondrial dynamics by regulating Mfn2 for therapeutic intervention in diabetic cardiomyopathy [J ] . Theranostics ， 2019 ， 9 （ 13 ）： 3687 - 3706 .

Views

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Has-miR-204-5p mediates intervertebral disc degeneration and associated pain symptoms: evidence from Mendelian randomization and bioinformatics

Establishment of a goat model of lumbar static and dynamic instability for the research on intervertebral disc degeneration

Effect of Glycyrrhiza uralensis Fisch. endophytes combined with cisplatin on proliferation and apoptosis of A549 cells

Milk fat globule-EGF factor Ⅷ attenuates apoptosis and permeability damage of the intestinal mucosa in rats with obstructive jaundice

Effect of remifentanil on the intervertebral disc of rats with intervertebral disc degeneration and its mechanism

Related Author

Zhao Runhan

Zhang Jun

Tang Dagang

Li Ningdao

Luo Xiaoji

Zhou Zhi

Tian Ziyang

Wang Zhenwei

Related Institution

Department of Orthopedics,The First Affiliated Hospital of Chongqing Medical University

Department of Orthopedics,Chongqing Hospital of Traditional Chinese Medicine

Department of Orthopedic Surgery，Renji Hospital，Shanghai Jiao Tong University School of Medicine, Renji Hospital，Shanghai Jiao Tong University School of Medicine

Department of Laboratory Medicine, Renji Hospital，Shanghai Jiao Tong University School of Medicine

College of Pharmacy，Gansu University of Chinese Medicine

⁰