Page 30 - 南京医科大学学报自然科学版

南京医科大学学报（自然科学版） 第43卷第6期
·772 · Journal of Nanjing Medical University（Natural Sciences） 2023年6月

·基础研究·

Drp1缺失通过ABCB10激活线粒体未折叠蛋白反应

毋柯蓉，郭 兴 *
南京医科大学基础医学院神经生物学系，江苏 南京 211166

［摘 要］ 目的：在小鼠胚胎成纤维细胞（mouse embryonic fibroblast，MEF）中探讨动力相关蛋白 1（dynamin related protein 1，
Drp1）基因缺失激活线粒体未折叠蛋白反应（mitochondrial unfolded protein reaction，mtUPR）的分子机制。方法：采用不同浓度
（0、2.5、5.0、10.0mmol/L）3⁃硝基丙酸（3⁃nitropropionic acid，3⁃NP）处理Drp1敲除或敲低的MEF细胞、Drp1抑制剂Mdivi⁃1或选择
性阻断Drp1与下游蛋白相互作用的小分子多肽P110处理的MEF细胞以及相应对照，随后进行Western blot检测CCAAT/增强
子结合蛋白同源蛋白（CCAAT/enhancer⁃binding protein homologous protein，CHOP）、ATP 结合盒 B 亚家族成员 10（ATP binding
cassette subfamily B member 10，ABCB10）、Lon肽酶1（Lon peptidase 1，LONP1）以及热休克蛋白60（heat shock protein 60，Hsp60）
的表达。RT⁃qPCR 检测 Drp1 敲低或 Mdivi⁃1 处理后 MEF 细胞的 ABCB10 的 mRNA 水平。同时敲低 Drp1 和 ABCB10，Western
blot检测CHOP蛋白的表达，试剂盒检测培养液中乳酸脱氢酶（lactate dehydrogenase，LDH）含量，流式细胞术检测线粒体活性氧和
线粒体膜电位水平。结果：3⁃NP处理后，Drp1敲除或敲低的MEF细胞以及Mdivi⁃1或P110处理的MEF细胞中CHOP表达呈现倍
数上调。Drp1敲除或敲低的MEF细胞以及Mdivi⁃1或P110处理的MEF细胞中ABCB10蛋白表达上调，mtUPR效应蛋白LONP1和
Hsp60表达上调。Drp1敲低的MEF细胞和Mdivi⁃1处理的MEF细胞中ABCB10 mRNA水平上调。同时敲低Drp1和ABCB10后，
与Drp1敲低组相比，CHOP表达下调，LDH含量降低，线粒体活性氧水平降低，线粒体膜电位水平增加。结论：在MEF细胞中，
Drp1表达下调可引起ABCB10表达量增加，导致mtUPR关键蛋白CHOP、LONP1和Hsp60蛋白表达上调，进而激活mtUPR。
［关键词］ Drp1；ABCB10；线粒体未折叠蛋白反应
［中图分类号］ R393 ［文献标志码］ A ［文章编号］ 1007⁃4368（2023）06⁃772⁃08
doi：10.7655/NYDXBNS20230604

Drp1 deficiency activates ABCB10⁃mediated mitochondrial unfolded protein response

WU Kerong，GUO Xing *
Department of Neurobiology，School of Basic Medical Sciences，Nanjing Medical University，Nanjing 211166，China

［Abstract］ Objective：The current study aims to investigate the molecular mechanism of mitochondrial unfolded protein reaction
（mtUPR）induced by down ⁃ regulation of dynamin related protein 1（Drp1）expression in mouse embryonic fibroblast（MEF）cells.
Methods：The expression levels of CCAAT/enhancer⁃binding protein homologous protein（CHOP），ATP binding cassette subfamily B
member 10（ABCB10），Lon peptidase 1（LONP1）and heat shock protein 60（Hsp60）were detected by Western blot in Drp1 KO/KD
cells，MEF cells treated with Drp1 inhibitors Mdivi1 or P110 and control cells after administration of 3⁃nitropropionic acid（3⁃NP）at
different concentrations（0，2.5，5.0，10.0 mol/L）. RT⁃qPCR was used to detect ABCB10 mRNA levels in Drp1 KD or Mdivi⁃1 treated
MEF cells. After Drp1/ABCB10 double knocking down，the expression levels of CHOP protein was detected by Western blot，lactate
dehydrogenase（LDH）release assay kit was used to detect the content of LDH in culture medium，and the levels of mitochondrial
reactive oxygen and membrane potential were detected by flow cytometry. Results：The expression levels of CHOP were upregulated in
Drp1 KO/KD cells or MEF cells treated with Drp1 inhibitors Mdivi⁃1 or P110. The expression levels of ABCB10 and mtUPR related
proteins LONP1 and Hsp60 were upregulated in Drp1 KO/KD cells or MEF cells treated with Drp1 inhibitors Mdivi ⁃ 1 or P110.
Compared with control groups，the mRNA levels of ABCB10 were upregulated in Drp1 KD or Mdivi⁃1 treated MEF cells. After both
Drp1 and ABCB10 knocking down，the expression levels CHOP was down ⁃ regulated，LDH content was decreased，mitochondrial
reactive oxygen level was decreased，and mitochondrial membrane potential level was increased. Conclusion：Deficiency of Drp1
activates the mtUPR through ABCB10，which causes the upregulation of the mtUPR proteins CHOP，LONP1 and Hsp60.
［Key words］ Drp1；ABCB10；mitochondrial unfolded protein response
［J Nanjing Med Univ，2023，43（06）：772⁃779］
［基金项目］ 国家自然科学基金（81971189）
∗
通信作者（Corresponding author），E⁃mail：guox@njmu.edu.cn