MitoQ对犬骨髓间充质干细胞线粒体功能及抗氧化能力的影响

doi:10.3969/j.issn.1004-1524.2022.05.07

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (5): 934-941.DOI: 10.3969/j.issn.1004-1524.2022.05.07

MitoQ对犬骨髓间充质干细胞线粒体功能及抗氧化能力的影响

钟丽君(), 邓嘉强, 古丛伟, 沈留红, 曹随忠, 余树民()

四川农业大学动物医学院,四川成都 611130

收稿日期:2021-01-18 出版日期:2022-05-25 发布日期:2022-06-06
通讯作者: 余树民
作者简介:* 余树民,E-mail: yayushumin@sicau.edu.cn
钟丽君(1995—),女,四川射洪人,硕士研究生,主要从事生殖内分泌与动物细胞工程研究。E-mail: 18728571971@163.com

Effects of MitoQ on mitochondrial function and antioxidant capacity of canine bone marrow mesenchymal stem cells

ZHONG Lijun(), DENG Jiaqiang, GU Congwei, SHEN Liuhong, CAO Suizhong, YU Shumin()

College of Veterinary Medicine,Sichuan Agricultural University,Chengdu 611130,China

Received:2021-01-18 Online:2022-05-25 Published:2022-06-06
Contact: YU Shumin

摘要/Abstract

摘要：

研究线粒体靶向抗氧化剂Mitoquinone(MitoQ)对犬骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSCs)的线粒体功能与抗氧化基因表达的作用,为进一步提高犬BMSCs的临床应用效率提供理论依据。以犬BMSCs为材料,添加MitoQ预处理48 h,采用荧光定量PCR、荧光探针等方法评估BMSCs线粒体功能以及抗氧化基因的表达水平。结果显示,随着传代次数的增加,BMSCs线粒体融合明显增强,线粒体分裂明显减弱;线粒体膜电位与ATP含量均显著下降;抗氧化基因的表达显著下调。添加MitoQ预处理后,BMSCs线粒体融合基因Mfn2的表达略有上调,且线粒体分裂基因Drp1和线粒体生物合成相关基因PGC-1α的表达显著上调;线粒体膜电位与ATP生成显著增加;抗氧化基因SOD1、SOD2、CAT和GSH-Px的表达显著上调。结果表明,MitoQ可改善犬BMSCs线粒体功能,增强BMSCs抗氧化能力。

关键词: 犬, 线粒体, 氧化应激

Abstract:

The aim of this study was to investigate the effect of Mitoquinone (MitoQ), a mitochondrial-targeted antioxidant, on the mitochondrial function and antioxidant gene expression of canine bone marrow mesenchymal stem cells (BMSCs), which may provide a theoretical basis for improving the clinical application efficiency of canine BMSCs. Canine BMSCs treated with MitoQ for 48 h were used as materials to evaluate the mitochondrial function and the expression level of antioxidant genes.The results showed that with the increase of passage times,the mitochondrial fusion of BMSCs was significantly advanced, and mitochondrial division was significantly weakened. In addition, the mitochondrial membrane potential and ATP content were significantly decreased and the expression of antioxidant genes of BMSCs was also significantly down-regulated.After MitoQ pretreatment, the expression of BMSCs mitochondrial fusion gene Mfn2 was slightly up-regulated, and the expression of mitochondrial division gene Drp1 and mitochondrial biosynthesis-related gene PGC-1α were significantly up-regulated. More over, the mitochondrial membrane potential and ATP content increased significantly and the expression of antioxidant genes SOD1, SOD2, CAT and GSH-Px was significantly up-regulated.In summary, MitoQ can improve the mitochondrial function and enhance the antioxidant capacity of canine BMSCs.

Key words: canine, mitochondria, oxidative stress

中图分类号:

S829.2

钟丽君, 邓嘉强, 古丛伟, 沈留红, 曹随忠, 余树民. MitoQ对犬骨髓间充质干细胞线粒体功能及抗氧化能力的影响[J]. 浙江农业学报, 2022, 34(5): 934-941.

ZHONG Lijun, DENG Jiaqiang, GU Congwei, SHEN Liuhong, CAO Suizhong, YU Shumin. Effects of MitoQ on mitochondrial function and antioxidant capacity of canine bone marrow mesenchymal stem cells[J]. Acta Agriculturae Zhejiangensis, 2022, 34(5): 934-941.

图/表 6

表1 引物参数

Table 1 Primer parameters

基因名称Gene ID	上游引物Forward primer (5'→3')	下游引物Reverse primer (5'→3')	长度Length/bp
SOD2	CTCAAGTTCAATGGAGGAGGTCA	CTTGGACACCGACGGATACAG	169
SOD1	CATTACAGGGCTGACTGAAGGC	TTGGACAGAGGATTAAAGTGAGGA	105
CAT	GATACCTGTGAACTGTCCTT	CATTGGCTGCTATGCTCTA	109
GSH-Px	CAACCAGTTCGGGCATCA	CGTTCACCTCGCACTTCTCA	122
Drp1	TAGAAATGCTACTGGTCCTCGTCC	CAGTTCCACACAACGCAGGC	115
Fis1	GTCTGTGGACGACCTGCTGAA	TGCGATGCCTTTACGGATG	142
Mfn2	GGCCTTGATGGGCTACAATGAC	CGCCTCCGACAACGAGAATG	179
Mfn1	CAGGGCGGTTACAGAAGAGGT	CAATACGCTTGGAGTGGGATGA	121
Opa1	GCAGAATCCTAACGCCATCATAC	ACAAATATCGTCCTCCTTCCATG	117
PGC-1α	TTCGGTCATCCCAGTCAAGC	TGTCATCAAACAGGCCATCCA	145
GAPDH	TCCCGCCAACATCAAA	TCACGCCCATCACAAAC	163

图1 体外传代对BMSCs形态的影响 A-C 分别为P1、P3和P6代BMSCs的形态(比例尺=100 μm)。

Fig.1 Effects of in vitro expansion on morphology of BMSCs A-C represented the morphology of P1, P3 and P6 BMSCs (scale bar=100 μm).

图2 BMSCs的鉴定 A-F 分别为细胞表面标志物CD105、CD90、CD31、阴性对照的免疫荧光检测结果(比例尺=200 μm);BMSCs成骨分化诱导和成脂分化诱导后的染色结果(比例尺=100 μm)。

Fig.2 Identification of BMSCs A-F represented the immunofluorescence detection results of cell surface markers CD105, CD90, CD31 and negative control (scale bar=200 μm) and the results of staining after osteogenic differentiation and adipogenic differentiation of BMSCs (scale bar=100 μm).

图3 体外传代对BMSCs抗氧化基因表达与线粒体功能的影响 A-F分别为BMSCs的SOD1、SOD2、CAT、GSH-Px;PGC-1α;Opa1、Mfn1、Mfn2、Fis1和Drp1基因的相对表达量;线粒体膜电位水平(比例尺=100 μm)和ATP含量。*和**分别表示在P<0.05和P<0.01水平上差异显著。ns,不显著。下同。

Fig.3 Effects of in vitro expansion on antioxidant gene expression and mitochondrial function of BMSCs A-F represented the relative expression of SOD1, SOD2, CAT, GSH-Px, PGC-1α, Opa1,Mfn1, Mfn2, Fis1and Drp1,mitochondrial membrane potential level(scale bar=100 μm) and ATP content of BMSCs, respectively. * and **,Significant difference at P<0.05 and P<0.01. ns, No significance. The same as below.

图4 MitoQ对BMSCs抗氧化基因与线粒体功能相关基因表达的影响

Fig.4 Effects of MitoQ on the expression of BMSCs antioxidant genes and mitochondrial dynamics-related genes

图5 MitoQ对BMSCs线粒体膜电位与ATP含量的影响 A、B分别为MitoQ处理后BMSCs的线粒体膜电位水平(比例尺=100 μm)与ATP含量。

Fig.5 Effects of MitoQ on mitochondrial membrane potential and ATP content of BMSCs A and B represented the mitochondrial membrane potential level (scale bar=100 μm) and ATP content of BMSCs after MitoQ treatment.

参考文献 21

[1]	WANG Y H, WANG D R, GUO Y C, et al. The application of bone marrow mesenchymal stem cells and biomaterials in skeletal muscle regeneration[J]. Regenerative Therapy, 2020, 15: 285-294. DOI URL
[2]	DRELA K, STANASZEK L, NOWAKOWSKI A, et al. Experimental strategies of mesenchymal stem cell propagation: adverse events and potential risk of functional changes[J]. Stem Cells International, 2019, 2019: 7012692.
[3]	ZHANG F, PENG W X, ZHANG J, et al. New strategy of bone marrow mesenchymal stem cells against oxidative stress injury via Nrf2 pathway: oxidative stress preconditioning[J]. Journal of Cellular Biochemistry, 2019, 120(12): 19902-19914. DOI URL
[4]	CHEN X J, WANG L, SONG X Y. Mitoquinone alleviates vincristine-induced neuropathic pain through inhibiting oxidative stress and apoptosis via the improvement of mitochondrial dysfunction[J]. Biomedicine & Pharmacotherapy, 2020, 125: 110003. DOI URL
[5]	PLECITÁ-HLAVATÁ L, JEŽEK J, JEŽEK P. Pro-oxidant mitochondrial matrix-targeted ubiquinone MitoQ10 Acts as anti-oxidant at retarded electron transport or proton pumping within Complex I[J]. The International Journal of Biochemistry & Cell Biology, 2009, 41(8/9): 1697-1707. DOI URL
[6]	APOSTOLOVA N, GARCIA-BOU R, HERNANDEZ-MIJARES A, et al. Mitochondrial antioxidants alleviate oxidative and nitrosative stress in a cellular model of Sepsis[J]. Pharmaceutical Research, 2011, 28(11): 2910-2919. DOI URL
[7]	ZHANG J, BAO X W, ZHANG M Y, et al. MitoQ ameliorates testis injury from oxidative attack by repairing mitochondria and promoting the Keap1-Nrf2 pathway[J]. Toxicology and Applied Pharmacology, 2019, 370: 78-92. DOI URL
[8]	TURINETTO V, VITALE E, GIACHINO C. Senescence in human mesenchymal stem cells: functional changes and implications in stem cell-based therapy[J]. International Journal of Molecular Sciences, 2016, 17(7): 1164. DOI URL
[9]	STAB B R, MARTINEZ L, GRISMALDO A, et al. Mitochondrial functional changes characterization in young and senescent human adipose derived MSCs[J]. Frontiers in Aging Neuroscience, 2016, 8: 299.
[10]	SEOK J, JUNG H S, PARK S, et al. Alteration of fatty acid oxidation by increased CPT1A on replicative senescence of placenta-derived mesenchymal stem cells[J]. Stem Cell Research & Therapy, 2020, 11(1): 1-13.
[11]	LI X, HONG Y M, HE H W, et al. FGF21 mediates mesenchymal stem cell senescence via regulation of mitochondrial dynamics[J]. Oxidative Medicine and Cellular Longevity, 2019, 2019: 4915149.
[12]	LEE J H, YOON Y M, SONG K H, et al. Melatonin suppresses senescence-derived mitochondrial dysfunction in mesenchymal stem cells via the HSPA1L-mitophagy pathway[J]. Aging Cell, 2020, 19(3): e13111.
[13]	YANG F, YAN G G, LI Y, et al. Astragalus polysaccharide attenuated iron overload-induced dysfunction of mesenchymal stem cells via suppressing mitochondrial ROS[J]. Cellular Physiology and Biochemistry, 2016, 39(4): 1369-1379. DOI URL
[14]	YOON Y M, KIM S, HAN Y S, et al. TUDCA-treated chronic kidney disease-derived hMSCs improve therapeutic efficacy in ischemic disease via PrPC[J]. Redox Biology, 2019, 22: 101144. DOI URL
[15]	LI X, ZHAN J H, HOU Y, et al. Coenzyme Q10 regulation of apoptosis and oxidative stress in H₂O₂ induced BMSC death by modulating the nrf-2/NQO-1 signaling pathway and its application in a model of spinal cord injury[J]. Oxidative Medicine and Cellular Longevity, 2019, 2019: 6493081.
[16]	ZHANG D Y, YAN B X, YU S S, et al. Coenzyme Q10 inhibits the aging of mesenchymal stem cells induced by D-galactose through Akt/mTOR signaling[J]. Oxidative Medicine and Cellular Longevity, 2015, 2015: 867293.
[17]	TAN D Q, SUDA T. Reactive oxygen species and mitochondrial homeostasis as regulators of stem cell fate and function[J]. Antioxidants & Redox Signaling, 2018, 29(2): 149-168.
[18]	ESCRIBANO-LOPEZ I, BAÑULS C, DIAZ-MORALES N, et al. The mitochondria-targeted antioxidant MitoQ modulates mitochondrial function and endoplasmic Reticulum stress in pancreatic β cells exposed to hyperglycaemia[J]. Cellular Physiology and Biochemistry, 2019, 52(2): 186-197. DOI URL
[19]	RAMSEY H, ZHANG Q, WU M X. Mitoquinone restores platelet production in irradiation-induced thrombocytopenia[J]. Platelets, 2015, 26(5): 459-466. DOI URL
[20]	KANG L, LIU S W, LI J C, et al. The mitochondria-targeted anti-oxidant MitoQ protects against intervertebral disc degeneration by ameliorating mitochondrial dysfunction and redox imbalance[J]. Cell Proliferation, 2020, 53(3): e12779.
[21]	ZHOU J, WANG H D, SHEN R M, et al. Mitochondrial-targeted antioxidant MitoQ provides neuroprotection and reduces neuronal apoptosis in experimental traumatic brain injury possibly via the Nrf2-ARE pathway[J]. American Journal of Translational Research, 2018, 10(6): 1887-1899.

MitoQ对犬骨髓间充质干细胞线粒体功能及抗氧化能力的影响

Effects of MitoQ on mitochondrial function and antioxidant capacity of canine bone marrow mesenchymal stem cells

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 6

参考文献 21

相关文章 15

编辑推荐

Metrics

本文评价

[1]	熊昕宜, 许泽玉, 何念佳, 何俊博, 陈正礼, 黄超, 刘文涛, 罗启慧. 大豆异黄酮干预肥胖大鼠肝氧化应激及炎症反应[J]. 浙江农业学报, 2022, 34(5): 942-948.
[2]	姬凯元, 邱月阳, 程敖, 蒋书东, 彭梦玲. 安徽合肥地区2018—2019年犬细小病毒遗传进化分析[J]. 浙江农业学报, 2021, 33(10): 1817-1825.
[3]	刘凯, 冯晓宇, 马恒甲, 谢楠. 钱塘江三角鲂线粒体基因组测序及其结构特征分析[J]. 浙江农业学报, 2020, 32(9): 1591-1608.
[4]	杨明霞, 连红娟, 王晓芳, 杜宜洋, 董志刚, 纪薇. 葡萄MPT基因家族鉴定与表达分析[J]. 浙江农业学报, 2020, 32(12): 2173-2185.
[5]	袁献宇, 杨龙斌, 何赞赞, 毛天骄, 何长生, 占松鹤, 孙裴, 魏建忠, 李郁. 安徽省猪伪狂犬病毒的分离鉴定及其主要毒力基因分子特征[J]. 浙江农业学报, 2020, 32(1): 43-56.
[6]	易可可, 阴文奇, 周远成, 蒋金蓁, 张白玉, 李中银, 颜其贵. 四株猪伪狂犬病毒株的分离鉴定与主要毒力基因分析[J]. 浙江农业学报, 2019, 31(9): 1429-1436.
[7]	张浩杰, 刘梅, 李春燕, 何冉, 兰景超, 罗娌, 古小彬, 谢跃, 杨光友. 犬恶丝虫核苷二磷酸激酶(NDPK)基因的原核表达及其免疫荧光定位[J]. 浙江农业学报, 2019, 31(9): 1453-1460.
[8]	尹潇潇, 李燕方, 古江, 廖艳, 谢跃, 杨光友, 古小彬. 基于线粒体ATP6基因全序列分析中国绵羊痒螨(兔亚种)的遗传多样性[J]. 浙江农业学报, 2019, 31(8): 1231-1238.
[9]	周秀敏, 杨永江, 毕英杰, 任卫合, 张丽. 基于线粒体COXⅠ基因变异探讨国内外猪种的遗传多样性及系统进化研究[J]. 浙江农业学报, 2017, 29(8): 1271-1280.
[10]	李捷, 白利鹏, 陈曦, 杨芳, 沈留红, 曹随忠, 左之才, 任志华, 马晓平, 余树民. 犬骨髓间充质干细胞的体外分离培养及鉴定[J]. 浙江农业学报, 2017, 29(5): 751-759.
[11]	樊毅, 李碧, 郭万柱, 李萍, 黄剑波, 杨凡, 姜子义, 赵军, 许思遥, 邓益超, 殷玥, 毛汐语, 吕雯婷, 徐志文, 朱玲. 伪狂犬病毒gE、gI和US9三基因缺失株的构建[J]. 浙江农业学报, 2017, 29(4): 542-547.
[12]	魏斌, 田一男, 李平, 石梅, 曹雪峰, 肖启程, 涂蕊, 但佳明, 杨亭玉, 彭广能, 钟志军. 犬瘟热病毒原液TaqMan荧光定量RT-PCR检测方法的建立[J]. 浙江农业学报, 2017, 29(11): 1819-1826.
[13]	耿文学, 唐波, 华涛, 侯继波, 张道华, 许家荣. 免疫增强剂对猪伪狂犬灭活疫苗的免疫增强作用[J]. 浙江农业学报, 2016, 28(11): 1828-1833.
[14]	李杨，高祝，荣茜，杨晓敏，张睿，李英伦*. “复方柴芩颗粒”防治鸭黄曲霉毒素B1慢性中毒的作用机理 [J]. 浙江农业学报, 2015, 27(8): 1337-.
[15]	罗厚强1，宋显章1，*，王清艳1，段龙川1，涂宜强1，付华2. 温州地区犬细小病毒病门诊的流行病学调查[J]. 浙江农业学报, 2014, 26(4): 887-.