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受损神经来源的mtDNA通过GRP75增加线粒体内Ca2+水平诱导U87细胞凋亡和小鼠痛觉敏化
Damaged nerve-derived mtDNA induces neural cell apoptosis and mouse pain sensitization by increasing mitochondrial Ca2+ level through GRP75
- 陆军军医大学学报 2024年46卷第18期 页码：2081-2091
- 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-MSX0683)
DOI：10.16016/j.2097-0927.202403004
中图分类号： R322.85;R363.21;R441.1收稿：2024-03-02，
修回：2024-03-28，
纸质出版：2024-09-30
- 稿件说明：
移动端阅览
黄鹏辉, 李丽, 崔剑. 受损神经来源的mtDNA通过GRP75增加线粒体内Ca2+水平诱导U87细胞凋亡和小鼠痛觉敏化[J]. 陆军军医大学学报, 2024,46(18):2081-2091.
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.
黄鹏辉, 李丽, 崔剑. 受损神经来源的mtDNA通过GRP75增加线粒体内Ca2+水平诱导U87细胞凋亡和小鼠痛觉敏化[J]. 陆军军医大学学报, 2024,46(18):2081-2091. DOI： 10.16016/j.2097-0927.202403004.
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
2
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
2+
水平、细胞凋亡的影响，及其诱导小鼠痛觉敏化的机制。
方法
2
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
2
O
2
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
2+
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
2
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
vs
2.22±0.26 (
P
<
0.05)，ND1：1.00±0.12
vs
1.79±0.07 (
P
<
0.05)。CCK-8结果显示：mtDNA(1 ng/μL)可抑制U87细胞活力(
P
<
0.01)；0.2 ng/μL mtDNA可上调GRP75蛋白的表达(
P
<
0.05)，促进内质网-线粒体偶联，增加线粒体Ca
2+
水平(109.4±62.6
vs
540.3±150.3，
P
<
0.05)，并诱使线粒体释放ROS增多(
P
<
0.05)。MKT-077或/和BAPTA-AM处理后，线粒体Ca
2+
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
vs
mtDNA+MKT-077组：(18.39±2.09)
vs
(13.22±1.42)，
P
<
0.05；mtDNA组
vs
mtDNA+BAPTA-AM组：(18.39±2.09)
vs
(12.09±1.53)，
P
<
0.05；mtDNA组
vs
mtDNA+ MKT-077+ BAPTA-AM组：(18.39±2.09)
vs
(11.65±2.09)，
P
<
0.05
]
。
结论
2
受损神经来源的mtDNA可上调GRP75蛋白的表达、干扰线粒体Ca
2+
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
2
To investigate the effects of damaged nerve-derived mitochondrial DNA (mtDNA) on GRP75 protein expression
mitochondrial Ca
2+
level and apoptosis in human glioblastoma cell line U-87MG (U87) cells
and its mechanism of inducing pain sensitization in mice.
Methods
2
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
2
O
2
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
2+
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
2
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
vs
2.22±0.26
P
<
0.05
1.00±0.12
vs
1.79±0.07
P
<
0.05). CCK-8 assay showed that mtDNA (1 ng/μL) inhibited U87 cell viability (
P
<
0.01). mtDNA (0.2 ng/μL) up-regulated the expression of GRP75 protein (
P
<
0.05)
promoted endoplasmic reticulum-mitochondrial coupling
and consequently increased mitochondrial Ca
2+
level (109.4±62.6
vs
540.3±150.3
P
<
0.05) and production of ROS (
P
<
0.05). After MKT-077 or/and BAPTA-AM treatment
the level of mitochondrial Ca
2+
was decreased
the mitochondrial membrane potential was improved
and the apoptotic rate of U87 cells was reduced by PI assays (mtDNA
vs
mtDNA+MKT-077: 18.39±2.09
vs
13.22±1.42
P
<
0.05; mtDNA
vs
mtDNA+BAPTA-AM: 18.39±2.09
vs
12.09±1.53
P
<
0.05; mtDNA
vs
mtDNA+MKT-077+BAPTA-AM: 18.39±2.09
vs
11.65±2.09
P
<
0.05).
Conclusion
2
Damaged nerve-derived mtDNA can up-regulate the expression of GRP75
interfere with mitochondrial Ca
2+
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
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