Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca<sup>2+</sup> level through GRP75

Penghui HUANG; Li LI; Jian CUI

doi:10.16016/j.2097-0927.202403004

您当前的位置：

首页 >

文章列表页 >

Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca²⁺ level through GRP75

Basic Medicine | 更新时间：2026-01-09

- Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca²⁺ level through GRP75
- Journal of Army Medical University Vol. 46, Issue 18, Pages: 2081-2091(2024)
- 作者机构：
  
  400038 重庆，陆军军医大学(第三军医大学)第一附属医院疼痛科
- 作者简介：
- 基金信息：
  
  the General Project of Natural Science Foundation of Chongqing(CSTB2023NSCQ-MSX0683)
- DOI：10.16016/j.2097-0927.202403004
  CLC： R322.85;R363.21;R441.1
- Received：02 March 2024，
  
  Revised：2024-03-28，
  
  Published：30 September 2024
- 稿件说明：
移动端阅览
Penghui HUANG, Li LI, Jian CUI. Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca²⁺ level through GRP75[J]. Journal of Army Medical University, 2024, 46(18): 2081-2091.
DOI：

Penghui HUANG, Li LI, Jian CUI. Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca²⁺ level through GRP75[J]. Journal of Army Medical University, 2024, 46(18): 2081-2091. DOI： 10.16016/j.2097-0927.202403004.

摘要

目的

探讨受损神经来源的线粒体DNA(mitochondrial DNA，mtDNA)对人脑星形胶质母细胞瘤细胞(human glioblastoma cell line U-87MG，U87)内GRP75蛋白表达、线粒体Ca

水平、细胞凋亡的影响，及其诱导小鼠痛觉敏化的机制。

方法

采用坐骨神经慢性缩窄(chronic constriction injury，CCI)建立小鼠神经病理性疼痛(neuropathic pain，NeP)模型，采用随机数字表法将20只C57BL/6雄性小鼠(6~8周龄，体质量20~30 g)分为4组：假手术组、CCI-7天组、CCI-14天组、CCI-21天组，行为学检测痛觉敏化情况，实时荧光定量PCR法(quantitative Real-time PCR

qPCR)检测小鼠脊髓中mtDNA含量变化。从H

处理的人神经母细胞瘤细胞(human neuroblastoma cell line SH-SY5Y，SH-SY5Y)提取mtDNA。采用浓度为0~1 ng/μL的mtDNA处理U87细胞。CCK-8检测细胞活力变化，根据结果选择适宜的实验浓度。将U87细胞分为5组：对照组、mtDNA组、mtDNA+ GRP75抑制剂(MKT-077 1 μg/mL)组、mtDNA+钙螯合剂(BAPTA-AM 10 μmol/L)组和mtDNA+ MKT-077+ BAPTA-AM组，mtDNA处理前2 h分别予以MKT-077、BAPTA-AM预处理。Western blot检测GRP75蛋白(glucose-regulated protein 75，GRP75)的表达，内质网-线粒体共定位染色观察内质网-线粒体连接；钙离子荧光探针检测线粒体Ca

水平；活性氧(reactive oxygen species，ROS)及线粒体膜电位检测评估线粒体氧化应激功能；PI荧光标记检测U87细胞凋亡率。

结果

与假手术组相比，CCI-21天组小鼠脊髓中用于检测mtDNA含量的细胞色素c氧化酶I(cytochrome c oxidase I，CO1)和NADH脱氢酶亚基1(nicotinamide adenine dinucleotide dehydrogenase 1，ND1)均升高，CO1：1.01±0.20

2.22±0.26 (

0.05)，ND1：1.00±0.12

1.79±0.07 (

0.05)。CCK-8结果显示：mtDNA(1 ng/μL)可抑制U87细胞活力(

0.01)；0.2 ng/μL mtDNA可上调GRP75蛋白的表达(

0.05)，促进内质网-线粒体偶联，增加线粒体Ca

水平(109.4±62.6

540.3±150.3，

0.05)，并诱使线粒体释放ROS增多(

0.05)。MKT-077或/和BAPTA-AM处理后，线粒体Ca

量减少，线粒体膜电位改善，PI检测显示U87细胞凋亡率降低[mtDNA组

mtDNA+MKT-077组：(18.39±2.09)

(13.22±1.42)，

0.05；mtDNA组

mtDNA+BAPTA-AM组：(18.39±2.09)

(12.09±1.53)，

0.05；mtDNA组

mtDNA+ MKT-077+ BAPTA-AM组：(18.39±2.09)

(11.65±2.09)，

0.05

]

。

结论

受损神经来源的mtDNA可上调GRP75蛋白的表达、干扰线粒体Ca

水平、加剧线粒体氧化应激，以诱导U87细胞凋亡，这可能是一种参与NeP形成的新机制。

Abstract

Objective

To investigate the effects of damaged nerve-derived mitochondrial DNA (mtDNA) on GRP75 protein expression

mitochondrial Ca

level and apoptosis in human glioblastoma cell line U-87MG (U87) cells

and its mechanism of inducing pain sensitization in mice.

Methods

Chronic constriction injury (CCI) of the sciatic nerve was used to establish a mouse model of neuropathic pain (NeP). A total of 20 male C57BL/6 mice (6~8 weeks old

weighing 20~30 g) were randomly divided into sham group

and CCI-7

-14 and CCI-21 d groups. Paw withdrawal mechanical threshold (PWMT) was measured

and the changes in mtDNA content in the spinal cord were detected with quantitative real-time PCR (qPCR). After mtDNA was extracted from human neuroblastoma cells (SH-SY5Y) treated with H

U87 cells were treated with mtDNA at a dose of 0~1 ng/μL. CCK-8 assay was utilized to detect cell viability

and the appropriate concentration was selected according to the results. Then U87 cells were divided into: control group

mtDNA group

mtDNA+GRP75 inhibitor (MKT-077 1 μg/mL) group

mtDNA+calcium chelator (BAPTA-AM 10 μmol/L) group

and mtDNA+MKT-077+BAPTA-AM group. MKT-077 and BAPTA-AM were pre-treated in 2 h before mtDNA treatment

respectively. Western blotting was employed to assess the expression of glucose-regulated protein 75 (GRP75) protein

and endoplasmic reticulum-mitochondrial colocalization staining was conducted to observe the endoplasmic reticulum-mitochondrial junction. Calcium ion fluorescent probe was used to measure mitochondrial Ca

levels. Th

e oxidative stress and function of mitochondria were evaluated by reactive oxygen species (ROS) and mitochondrial membrane potential. PI fluorescent labeling was employed to examine apoptotic rate of U87 cells.

Results

Compared with the sham group

the cytochrome C oxidase I (CO1) and nicotinamide adenine dinucleotide dehydrogenase 1 (ND1)

which were utilized to measure mtDNA content in the spinal cord of mice in the CCI-21 group

were increased (1.01±0.20

2.22±0.26

0.05

1.00±0.12

1.79±0.07

0.05). CCK-8 assay showed that mtDNA (1 ng/μL) inhibited U87 cell viability (

0.01). mtDNA (0.2 ng/μL) up-regulated the expression of GRP75 protein (

0.05)

promoted endoplasmic reticulum-mitochondrial coupling

and consequently increased mitochondrial Ca

level (109.4±62.6

540.3±150.3

0.05) and production of ROS (

0.05). After MKT-077 or/and BAPTA-AM treatment

the level of mitochondrial Ca

was decreased

the mitochondrial membrane potential was improved

and the apoptotic rate of U87 cells was reduced by PI assays (mtDNA

mtDNA+MKT-077: 18.39±2.09

13.22±1.42

0.05; mtDNA

mtDNA+BAPTA-AM: 18.39±2.09

12.09±1.53

0.05; mtDNA

mtDNA+MKT-077+BAPTA-AM: 18.39±2.09

11.65±2.09

0.05).

Conclusion

Damaged nerve-derived mtDNA can up-regulate the expression of GRP75

interfere with mitochondrial Ca

level and exacerbate mitochondrial oxidative stress to induce apoptosis in U87 cells

which may be a novel mechanism involved in NeP formation.

关键词

Keywords

references

MOISSET X, BOUHASSIRA D, ATTAL N. French guidelines for neuropathic pain: an update andcommentary[J]. Rev Neurol, 2021, 177(7): 834-837. DOI: 10.1016/j.neurol.2021.07.004.

HERCULANO-HOUZEL S. The glia/neuron ratio: how it varies uniformly acrossbrain structures and species and what that means for brain physiology and evolution[J]. Glia, 2014, 62(9): 1377-1391. DOI: 10.1002/glia.22683.

TSUDA M, KOHRO Y, YANO T, et al . JAK-STAT3 pathway regulates spinal astrocyte proliferation and neuropathic pain maintenance in rats[J ] . Brain, 2011, 134(Pt 4): 1127-1139. DOI: 10.1093/brain/awr025.

ARENDT-NIELSEN L, KLITGAARD H, HANSEN S N. Bridging the translational gap: adenosine as a modulator of neuropathic pain in preclinical models andhumans[J]. Scand J Pain, 2024, 24(1): 1-7. DOI: 10.1515/sjpain-2023-0048.

LIU C C, GAO Y J, LUO H, et al . Interferon alpha inhibits spinal cord synaptic and nociceptive transmission via neuronal-glial interactions[J ] . Sci Rep, 2016, 6: 34356. DOI: 10.1038/srep34356.

CHENG T, XU Z L, MA X Q. The role of astrocytes in neuropathicpain[J]. Front Mol Neurosci, 2022, 15: 1007889. DOI: 10.3389/fnmol.2022.1007889.

SHARMA A, SCHAEFER S T, SAE-LEE C, et al . Elevated serum mitochondrial DNA in females and lack of altered platelet mitochondrial methylation in patients with Parkinson's disease[J ] . Int J Neurosci, 2021, 131(3): 279-282. DOI: 10.1080/00207454.2020.1738433.

GONG T, LIU L, JIANG W, et al . DAMP-sensing receptors in sterile inflammation and inflammatory diseases[J ] . Nat Rev Immunol, 2020, 20(2): 95-112. DOI: 10.1038/s41577-019-0215-7.

LIN M M, LIU N, QIN Z H, et al . Mitochondrial-derived damage-associated molecular patterns amplify neuroinflammation in neurodegenerative diseases[J ] . Acta Pharmacol Sin, 2022, 43(10): 2439-2447. DOI: 10.1038/s41401-022-00879-6.

YU C H, DAVIDSON S, HARAPAS C R, et al . TDP-43 triggers mitochondrial DNA release via mPTP to activate cGAS/STING in ALS[J ] . Cell, 2020, 183(3): 636-649. e18. DOI: 10.1016/j.cell.2020.09.020..

ZHAO Y X, LIU B H, XU L, et al . ROS-induced mtDNA release: the emerging messenger for communication between neurons and innate immune cells during neurodegenerative disorder progression[J ] . Antioxidants, 2021, 10(12): 1917. DOI: 10.3390/antiox10121917.

SLITER D A, MARTINEZ J, HAO L, et al . Parkin and PINK1 mitigate STING-induced inflammation[J ] . Nature, 2018, 561(7722): 258-262. DOI: 10.1038/s41586-018-0448-9.

YU S H, FU J Y, WANG J, et al . The influence of mitochondrial-DNA-driven inflammation pathways on macrophage polarization: a new perspective for targeted immunometabolic therapy in cerebral ischemia-reperfusion injury[J ] . Int J Mol Sci, 2021, 23(1): 135. DOI: 10.3390/ijms23010135.

KONG L Q, LI W Y, CHANG E, et al . mtDNA-STING axis mediates microglial polarization via IRF3/NF-κB signaling after ischemic stroke[J ] . Front Immunol, 2022, 13: 860977. DOI: 10.3389/fimmu.2022.860977..

PARK J H, LO E H, HAYAKAWA K. Endoplasmic reticulum interaction supports energy production and redox homeostasis in mitochondria released from astrocytes[J]. Transl Stroke Res, 2021, 12(6): 1045-1054. DOI: 10.1007/s12975-021-00892-7.

GAO P, YANG W X, SUN L. Mitochondria-associated endoplasmic reticulum membranes (MAMs) and their prospective roles in kidney disease[J]. Oxid Med Cell Longev, 2020, 2020: 3120539. DOI: 10.1155/2020/3120539.

GÖBEL J, ENGELHARDT E, PELZER P, et al . Mitochondria-endoplasmic reticulum contacts in reactive astrocytes promote vascular remodeling[J ] . Cell Metab, 2020, 31(4): 791-808. e8. DOI: 10.1016/j.cmet.2020.03.005.

LI Y, LI H Y, SHAO J, et al . GRP75 modulates endoplasmic reticulum-mitochondria coupling and accelerates Ca 2+ -dependent endothelial cell apoptosis in diabetic retinopathy[J ] . Biomolecules, 2022, 12(12): 1778. DOI: 10.3390/biom12121778..

BENNETT G J, XIE Y K. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man[J]. Pain, 1988, 33(1): 87-107. DOI: 10.1016/0304-3959(88)90209-6.

CHAPLAN S R, BACH F W, POGREL J W, et al . Quantitative assessment of tactile allodynia in the rat paw[J ] . J Neurosci Methods, 1994, 53(1): 55-63. DOI: 10.1016/0165-0270(94)90144-9.

LIU Y, SHEN Q Z, LI H Y, et al . Cell-free mitochondrial DNA increases granulosa cell apoptosis and reduces aged oocyte blastocyst development in the mouse[J ] . Reprod Toxicol, 2020, 98: 278-285. DOI: 10.1016/j.reprotox.2020.10.012.

ZHANG W F, LI G C, LUO R J, et al . Cytosolic escape of mitochondrial DNA triggers cGAS-STING-NLRP3 axis-dependent nucleus pulposus cell pyroptosis[J ] . Exp Mol Med, 2022, 54(2): 129-142. DOI: 10.1038/s12276-022-00729-9.

VAN HECKE O, AUSTIN S K, KHAN R A, et al . Neuropathic pain in the general population: a systematic review of epidemiological studies[J ] . Pain, 2014, 155(4): 654-662. DOI: 10.1016/j.pain.2013.11.013.

WANG M J, JING X Y, WANG Y Z, et al . Exercise, spinal microglia and neuropathic pain: potential molecular mechanisms[J ] . Neurochem Res, 2024, 49(1): 29-37. DOI: 10.1007/s11064-023-04025-4.

RILEY J S, TAIT S W. Mitochondrial DNA in inflammation andimmunity[J]. EMBO Rep, 2020, 21(4): e49799. DOI: 10.15252/embr.201949799.

MU Y, MEI Y, CHEN Y H, et al . Perisciatic nerve dexmedetomidine alleviates spinal oxidative stress and improves peripheral mitochondrial dynamic equilibrium in a neuropathic pain mouse model in an AMPK-dependent manner[J ] . Dis Markers, 2022, 2022: 6889676. DOI: 10.1155/2022/6889676.

TODKAR K, CHIKHI L, DESJARDINS V, et al . Selective packaging of mitochondrial proteins into extracellular vesicles prevents the release of mitochondrial DAMPs[J ] . Nat Commun, 2021, 12(1): 1971. DOI: 10.1038/s41467-021-21984-w.

GAMBARDELLA S, LIMANAQI F, FERESE R, et al . Ccf-mtDNA as a potential link between the brain and immune system in neuro-immunological disorders[J ] . Front Immunol, 2019, 10: 1064. DOI: 10.3389/fimmu.2019.01064.

YAMASHITA K, KINOSHITA M, MIYAMOTO K, et al . Cerebrospinal fluid mitochondrial DNA in neuromyelitis optica spectrum disorder[J ] . J Neuroinflamm, 2018, 15(1): 125. DOI: 10.1186/s12974-018-1162-0.

XU Z Z, XIE W L, FENG Y Q, et al . Positive interaction between GPER and β-alanine in the dorsal root ganglion uncovers potential mechanisms: mediating continuous neuronal sensitization and neuroinflammation responses in neuropathic pain[J ] . J Neuroinflamm, 2022, 19(1): 164. DOI: 10.1186/s12974-022-02524-9.

XIAN H X, WATARI K, SANCHEZ-LOPEZ E, et al . Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling[J ] . Immunity, 2022, 55(8): 1370-1385. e8. DOI: 10.1016/j.immuni.2022.06.0 07.

LINNERBAUER M, WHEELER M A, QUINTANA F J. Astrocyte crosstalk in CNS inflammation[J]. Neuron, 2020, 108(4): 608-622. DOI: 10.1016/j.neuron.2020.08.012.

PHAM J H, STANKOWSKA D L. Mitochondria-associated endoplasmic reticulum membranes (MAMs) and their role in glaucomatous retinal ganglion cell degeneration-a mini review[J]. Front Neurosci, 2023, 17: 1198343. DOI: 10.3389/fnins.2023.1198343.

WANG N, WANG C, ZHAO H Y, et al . The MAMs structure and its role in cell death[J ] . Cells, 2021, 10(3): 657. DOI: 10.3390/cells10030657.

LEWIS A, HAYASHI T, SU T P, et al . Bcl-2 family in inter-organelle modulation of calcium signaling; roles in bioenergetics and cell survival[J ] . J Bioenerg Biomembr, 2014, 46(1): 1-15. DOI: 10.1007/s10863-013-9527-7.

YOUSUF M S, MAGUIRE A D, SIMMEN T, et al. Endoplasmic reticulum-mitochondria interplay in chronic pain: The calcium connection[J]. Mol Pain, 2020, 16: 1744806920946889. DOI: 10.1177/1744806920946889.

Views

259

下载量

CSCD

Alert me when the article has been cited

提交

Tools

Publicity Resources

Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway

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

Related Author

Li LI

Penghui HUANG

LI Li

HUANG Penghui

CUI Jian

FU Wanlei

HAO Xianglin

CAO Ya

Related Institution

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

Department of Pathology， Second Affiliated Hospital， 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 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）

⁰

Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca2+ level through GRP75

DOI：10.16016/j.2097-0927.202403004

摘要

Abstract

关键词

Keywords

references

Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca²⁺ level through GRP75