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

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

Neuroscience | 更新时间：2026-01-09

- Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway
- Journal of Army Medical University Vol. 46, Issue 18, Pages: 2036-2045(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-MS×0683)
- DOI：10.16016/j.2097-0927.202403103
  CLC： R322.85;R363.21;R441.1
- Received：27 March 2024，
  
  Revised：2024-04-05，
  
  Published：30 September 2024
- 稿件说明：
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Li LI, Penghui HUANG, Jian CUI. Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway[J]. Journal of Army Medical University, 2024, 46(18): 2036-2045.
DOI：

Li LI, Penghui HUANG, Jian CUI. Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway[J]. Journal of Army Medical University, 2024, 46(18): 2036-2045. DOI： 10.16016/j.2097-0927.202403103.

摘要

目的

2

探索TAR DNA结合蛋白43(transactive response DNA binding protein 43，TDP-43)在氧化应激诱导的小鼠神经元(neuro-2a，N2a)细胞损伤及小鼠痛觉敏化中的作用及机制。

方法

2

① 为评估最佳诱导浓度，不同浓度的H

2

O

2

处理N2a细胞分为4组：对照组、200 μmol/L H

2

O

2

组、400 μmol/L H

2

O

2

组和800 μmol/L H

2

O

2

组。②为评估最佳诱导时间，400 μmol/L H

2

O

2

处理N2a细胞分为4组：对照组、6 h H

2

O

2

组、12 h H

2

O

2

组和24 h H

2

O

2

组。③为验证线粒体DNA(mitochondria DNA，mtDNA)释放途径，使用环孢素(cyclosporin，CsA)抑制线粒体通透性转换孔(mitochondrial permeability transition pore，mPTP)分为3组：对照组、24 h H

2

O

2

组和24 h H

2

O

2

+CsA组。④为验证TDP-43介导的细胞损伤机制，siRNA抑制TDP-43后分为3组：对照组、24 h H

2

O

2

组、24 h H

2

O

2

+siTDP-43组。⑤采用CCK-8检测细胞活性，EdU检测细胞增殖，Western blot检测TDP-43、神经元标志物(neuronal nuclei，NeuN)、环状GMP-AMP合酶(cylic GMP-AMP synthase，cGAS)和干扰素基因刺激因子(stimulator of interferon，STING)表达，qPCR检测mtDNA，免疫染色观察细胞内TDP-43表达变化，Calcein AM染色评估mPTP开放。⑥为验证TDP-43在神经病理性疼痛(neuropathic pain，NP)中的作用，将24只6~8周健康SPF级雄性C57BL/6J小鼠(体质量25~30 g)使用随机数字表法分为3组：对照组、慢性压迫性损伤(chronic constriction injury，CCI)组、CCI+siTDP-43组，术前1 d和术后7、14、21 d进行鞘内注射siTDP-43；术前1 d和术后1、3、5、7、14、21 d通过von Frey纤维丝和热辐射法测定小鼠机械痛阈值和热痛阈值，免疫荧光检测术后21 d腰段(L5-L6)脊髓背角中TDP-43与NeuN的变化。

结果

2

氧化应激刺激诱导N2a中TDP-43蛋白表达增加，刺激mtDNA通过mPTP释放，上调cGAS、STING的表达，影响N2a的细胞活性(

P

<

0.05)；CsA抑制mPTP通道的开放并减少mtDNA释放(

P

<

0.05)；下调TDP-43的表达后可显著降低mtDNA的释放，抑制cGAS和STING的表达，并恢复N2a细胞的增殖能力(

P

<

0.05)。CCI术后5 d，小鼠机械痛阈值和热痛阈值出现明显下降并持续至21 d(

P

<

0.05)；CCI小鼠术后21 d脊髓背角神经元中TDP-43表达增加(

P

<

0.05)；鞘内注射siRNA抑制TDP-43后，可提高CCI小鼠的机械痛阈值和热痛阈值(

P

<

0.05)。

结论

2

氧化应激诱导神经元细胞TDP-43蛋白增加，刺激mtDNA通过mPTP释放到细胞质，激活cGAS/STING通路，导致神经元损伤并加重CCI小鼠痛觉敏化。

Abstract

Objective

2

To investigate the role and possible mechanisms of transactive response DNA binding protein 43 (TDP-43) in mediating neuronal injury induced by oxidative stress in mouse neuro-2a (N2a) cells and mouse pain sensitization.

Methods

2

① To evaluate the optimal induction concentration

N2a cells were treated with different concentrations of H

2

O

2

and the cells were divided into control group

200

400 and 800 μmol/L H

2

O

2

groups. ②To assess the optimal induction duration

N2a cells were treated with 400 μmol/L H

2

O

2

and the cells were divided into control group

and the cell groups treated for 6

12 and 24 h

respectively. ③To validate the mitochondrial DNA (mtDNA) release pathway

cyclosporin A (CsA) was used to inhibit the mitochondrial permeability transition pore (mPTP)

and the cells were divided into control group

24 h H

2

O

2

group and 24 h H

2

O

2

+CsA group. ④To validate TDP-43-mediated cellular damage

the cells were divided into control group

24 h H

2

O

2

group and 24 h H

2

O

2

+siTDP-43 group. ⑤ Cell viability was assessed using CCK-8 assay

while cell proliferation was determined using EdU assay. Western blot analysis was employed to examine the expression levels of TDP-43

neuronal nuclei (NeuN)

cyclic GMP-AMP synthase (cGAS)

and stimulator of interferon genes (STING). qPCR was utilized to measure the release of mtDNA. Immunostaining was conducted to observe intracellular expression of TDP-43

and Calcein AM staining was employed to evaluate mPTP opening status. ⑥To elucidate the role of TDP-43 in neuropathic pain (NP)

24 healthy SPF male C57BL/6J mice (6~8 weeks old

25~30 g) were randomly divided into control group

chronic cons

triction injury (CCI) group

and CCI+siTDP-43 group. In 1 d before and 7

14 and 21 d after surgery

intrathecal injections of siTDP-43 were administered. Mechanical and thermal pain thresholds of the mice were assessed using von Frey filaments and radiant heat

respectively

on 1 preoperatively and 1

3

5

7

14 and 21 d postoperatively. Immunofluorescence assay was conducted on 21 d postoperatively to examine the changes in TDP-43 and NeuN in the lumbar spinal dorsal horn (L5-L6).

Results

2

Oxidative stress induced a significant increase in TDP-43 protein level in N2a cells

prompted mtDNA release through mPTP

markedly up-regulated the expression of cGAS and STING

and consequently impacted the viability of N2a cells (

P

<

0.05). CsA treatment inhibited mPTP channel opening and thus effectively blocked mtDNA release (

P

<

0.05)

down-regulated TDP-43 and thus significantly reduced mtDNA release

suppressed the expression of cGAS and STING

and finally restored the proliferation ability of N2a cells (

P

<

0.05). The mechanical and thermal pain thresholds exhibited a significant decrease since 5 d after CCI

which then persisted until 21 d (

P

<

0.05). The expression of TDP-43 in spinal cord dorsal horn neurons was increased in the mice in 21 d after CCI (

P

<

0.05)

and intrathecal injection of siRNA inhibited TDP-43 expression and effectively increased the mechanical and thermal pain thresholds in the CCI mice (

P

<

0.05).

Conclusion

2

Oxidative stress induces an up-regulation in TDP-43 protein in neurons

which stimulates the release of mtDNA into the cytoplasm through mPTP

and subsequently activates the cGAS/STING pathway

and finally

results in neuronal injury and pain sensitization in CCI mice.

关键词

Keywords

references

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Related Institution

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

Laboratory of Stem Cell and Tissue Engineering， Chongqing Medical University

Department of Human Anatomy， Anhui Medical University

Institute of Field Surgery， Daping Hospital， Army Medical University

Department of Geriatrics and Special Service Medicine， First Affiliated Hospital（Southwest Hospital）， Army Medical University （Third Military Medical University）

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