氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化

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氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化

神经科学 | 更新时间：2026-01-09

- 氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化
- Oxidative stress triggers neuronal injury and mouse pain sensitization by up-regulating TDP-43 to activate mtDNA-cGAS/STING pathway
- 陆军军医大学学报 2024年46卷第18期页码：2036-2045
- 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：
  
  [崔剑, E-mail: cqjian211@163.comCUI Jian, E-mail: cqjian211@163.com
- 基金信息：
  
  重庆市自然科学基金面上项目(CSTB2023NSCQ-MS×0683)
- DOI：10.16016/j.2097-0927.202403103
  中图分类号： R322.85;R363.21;R441.1
- 收稿：2024-03-27，
  
  修回：2024-04-05，
  
  纸质出版：2024-09-30
- 稿件说明：
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李丽, 黄鹏辉, 崔剑. 氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化[J]. 陆军军医大学学报, 2024,46(18):2036-2045.

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.
李丽, 黄鹏辉, 崔剑. 氧化应激通过TDP-43激活mtDNA-cGAS/STING通路诱发神经元损伤和小鼠痛觉敏化[J]. 陆军军医大学学报, 2024,46(18):2036-2045. DOI： 10.16016/j.2097-0927.202403103.

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

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