Mitochondrial-targeting antioxidant MitoQ modulates angiogenesis and promotes functional recovery after spinal cord injury
Mitochondrial-targeting antioxidant MitoQ modulates angiogenesis and promotes functional recovery after spinal cord injury
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线粒体靶向抗氧化剂MitoQ调节脊髓损伤后血管生成和促进功能恢复
DOI：
10.1016/j.brainres.2022.147902
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发表时间：
2022-04-11
期刊：
影响因子：
2.9
通讯作者：
Zheng, Xianyou
中科院分区：
文献类型：
--
作者：
Huang, Tengli;Shen, Junjie;Zheng, Xianyou
背景：在创伤性脊髓损伤(SCI)中，继发性损伤，包括细胞死亡、线粒体功能障碍和血管损伤，被认为是脊髓损伤后功能恢复受损的重要原因。损伤后血管新生被认为是治疗脊髓损伤的一种潜在策略。新生血管可能在神经再生中起关键作用，提示血管生成在神经再生中的重要性。最近的研究揭示了活性氧自由基(ROS)与血管生成之间的串扰。线粒体作为细胞内ROS的主要来源，在血管生成过程中起着至关重要的作用。方法：在T10夹压动物模型上建立脊髓损伤模型。然后，分别于术后第0、1、2天腹腔注射MitoQ(5 mg/kg/d)。采用Basso Mouse Scale(BMS)评分和足迹分析(T型步分析)评价脊髓损伤后功能恢复情况。免疫荧光法和免疫荧光法(LEL-FITC/CD31/Iba-1/神经细丝)检测血管生成、小胶质细胞活化和神经再生。RT-qPCR(VEGFR-1、VEGFR-2、VEGFA)检测损伤脊髓组织中血管生成相关因子的表达。用ATP生成法和Western-blotting法(MFN-1和DRP-1)检测损伤脊髓线粒体功能。以BV2细胞作为体外细胞模型。经TBHP或TBHP-MitoQ处理后，用ELISA法和免疫荧光法检测BV2细胞VEGFA的分泌水平。建立人脐静脉内皮细胞与BV2细胞共培养体系。在共培养体系中对人脐静脉内皮细胞进行管状形成试验和免疫荧光试验(CD31)，以评价其促进血管生成的作用。用三磷酸腺苷(ATP)生成试验检测BV2细胞线粒体功能。结果：体外培养的BV2细胞中，MitoQ可促进VEGFA的分泌，这一作用经ELISA法和免疫荧光法证实。在BV2细胞和人脐静脉内皮细胞共培养体系中，通过管形成和免疫荧光分析(CD31)来评价MitoQ处理的BV2细胞促进血管生成的作用。在经TBHP处理的BV2细胞中，MitoQ抑制细胞和线粒体来源的ROS。经MitoQ处理的BV2细胞的ATP产量增加。为了在体内验证MitoQ的作用，成功地建立了T10夹压动物模型。BMS测试和步态分析显示，MitoQ显著促进功能恢复。通过神经丝免疫荧光分析鉴定神经再生的促进作用。免疫荧光和荧光检测(LEL-FITC/CD31/IBA-1)和RT-qPCR(VEGFR-1、VEGFR-2和VEGFA)显示，MitoQ可促进脊髓损伤后损伤部位血管生成，抑制巨噬细胞/小胶质细胞活化。线粒体特异性抗氧化剂MitoQ通过促进脊髓损伤后血管生成，改善线粒体功能，促进脊髓损伤后线粒体功能的恢复和组织保护。
Background: In traumatic spinal cord injury (SCI), secondary injuries, including cellular death, mitochondrial dysfunction, and vascular injury, have been considered as important causes of impaired functional recovery after SCI. Postinjury angiogenesis has been considered to be a potential strategy for SCI treatment. New-born vessels may play a key role in nerve regeneration, which indicates the importance of angiogenesis in nerve regeneration. Recent studies have revealed the crosstalk between reactive oxygen species (ROS) and angiogenesis. As the main source of cellular ROS, mitochondria have been proven to be essential to the angiogenesis process. Methods: SCI was established in a T10 clip-compression animal model. Then, the animals received an intraperitoneal injection of MitoQ (5 mg/kg/d) on Days 0, 1, and 2 after surgery. The Basso Mouse Scale (BMS) score and footprint analysis (CatWalk analysis) were performed to evaluate functional recovery after SCI. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1/Neurofilament) were performed to evaluate angiogenesis, microglia activation and neural regeneration. RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) was performed to evaluate angiogenesis-related factor in injured spinal cord. ATP production assay and western-blotting assay (Mfn-1 and Drp-1) were performed to evaluate mitochondrial function in the injured spinal cord. BV2 cells were used as in vitro cell model. After receiving TBHP or TBHP-MitoQ treatment, ELISA and immunofluorescence assays were used to evaluate the level of VEGFA secretion from BV2 cells. A coculture system of HUVECs and BV2 cells was established. Tube formation assays and immunofluorescence assays (CD31) were performed on HUVECs in a coculture system to evaluate angiogenesis promotion. ATP production assays were performed to evaluate mitochondrial function in BV2 cells. MitoSOX Red and DCFH-DA staining were performed to evaluate mitochondrial and cellular ROS.Results: In vitro MitoQ promoted the secretion of VEGFA from BV2 cells, which was verified through ELISA and immunofluorescence assays. The angiogenic promotion of MitoQ-treated BV2 cells was evaluated by tube formation and immunofluorescence assays (CD31) in a coculture system of BV2 cells and HUVECs. MitoQ inhibited cellular and mitochondrial-derived ROS in TBHP-treated BV2 cells. ATP production was increased in MitoQtreated BV2 cells. To verify MitoQ's effect in vivo, a T10 clip-compression animal model was established successfully. MitoQ significantly promoted functional recovery, as shown by the BMS assay and gait analysis. The promotion of neural regeneration was identified through immunofluorescence assay of neurofilament. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1) and RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) indicated that MitoQ could promote angiogenesis and inhibit macrophage/microglia activation in lesion-site after SCI. Enhanced ATP production and increased Mfn-1 with decreased Drp-1 protein expression showed MitoQ could promote mitochondrial function in SCI.Conclusion: The mitochondrial-specific antioxidant MitoQ promotes functional recovery and tissue preservation through the enhancement of angiogenesis with the modification of mitochondrial function after SCI.