| 胸腺肽β4对氧化应激诱导的脊髓源性神经干/祖细胞损伤的作用及机制 |
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投稿时间：2022-01-20
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| 作者 | Author | 单位 | Address | E-Mail |
| 李宏维 |
LI Hong-wei |
兰州大学第二医院脊柱外科, 甘肃 兰州 730030 |
Department of Spine Surgery, Lanzhou University Second Hospital, Lanzhou 730030, Gansu, China |
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| 张海鸿 |
ZHANG Hai-hong |
兰州大学第二医院脊柱外科, 甘肃 兰州 730030 |
Department of Spine Surgery, Lanzhou University Second Hospital, Lanzhou 730030, Gansu, China |
haihongzhlz@163.com |
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| 期刊信息：《中国骨伤》2022年，第35卷，第8期，第763-771页 |
| DOI：10.12200/j.issn.1003-0034.2022.08.012 |
| 基金项目:国家自然科学基金（编号：31960175） |
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中文摘要:
目的：探讨胸腺肽β4（thymosin beta 4，Tβ4）在过氧化氢（hydrogen peroxide，H2O2）诱导的脊髓源性神经干/祖细胞（neural stem/progenitor cells，NSPCs）氧化应激损伤中的作用及机制。
方法：分离Sprague-Dawley （SD）成年雄性大鼠中的原代NSPCs，将其分为对照组（未处理的NSPCs细胞），H2O2处理组（500 μM H2O2损伤的NSPCs细胞），Tβ4处理3组（H2O2处理组基础上分别用1、2.5、5 μg/ml Tβ4处理的NSPCs细胞），TAK-242处理组[H2O2处理组基础上用Tβ4（5 μg/ml）和Toll样受体4（Toll-like receptors 4，TLR4）抑制剂TAK-242处理的NSPCs细胞]。采用髓样分化因子88（myeloid differentiation factor 88，MyD88）过表达慢病毒感染NSPCs细胞，构建MyD88过表达细胞系，并经H2O2和Tβ4处理。qRT-PCR和Western blot检测Tβ4，TLR4，MyD88的表达水平；MTT法和乳酸脱氢酶（lactate dehydrogenase，LDH）试剂盒检测细胞活力；采用Fluo-3/AM探针法检测细胞内Ca2+水平；流式细胞术和半胱氨酸天冬氨酸蛋白酶-3（Caspase-3）和Caspase-9试剂盒检测细胞凋亡水平；相应试剂盒分别检测活性氧（reactive oxygen species，ROS）含量，超氧化物歧化酶（superoxi dedismu-tase，SOD）活性和谷胱甘肽（glutathione，GSH）含量。酶联免疫吸附测定法检测白细胞介素（interleukin，IL）-6和IL-1β的含量。
结果：H2O2损伤的NSPCs中Tβ4的表达降低（P<0.05）。与H2O2处理组相比，Tβ4处理3组和TAK-242处理组NSPCs的细胞活力、Ca2+浓度显著增加（P<0.05），LDH释放量、细胞凋亡显著减少（P<0.05），ROS和促炎细胞因子的生成显著减少（P<0.05），TLR4和MyD88蛋白表达水平显著降低（P<0.05）。MyD88过表达后NSPCs细胞活力，SOD活性和GSH含量降低，LDH释放量、细胞凋亡显著增加（P<0.05）；而MyD88过表达后NSPCs经Tβ4处理后，细胞活力、SOD活性和GSH含量升高，LDH释放量以及细胞凋亡降低（P<0.05）。
结论：Tβ4通过抑制TLR4，MyD88途径减轻H2O2诱导的NSPCs氧化应激、凋亡和炎症等损伤。 |
| 【关键词】胸腺肽β4 神经干细胞 祖细胞 氧化应激损伤 髓样分化因子88 |
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| Effect and mechanism of thymosin beta 4 on spinal cord-derived neural stem/progenitor cell injury induced by oxidative stress |
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ABSTRACT
Objective: To investigate the role and mechanism of thymosin beta 4 (Tβ4) in oxidative stress injury of spinal cord-derived neural stem/progenitor cells (NSPCs) induced by hydrogen peroxide (H2O2).
Methods: NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), H2O2 group (NSPCs cells damaged by 500 μM H2O2), Tβ4 -3 groups (NSPCs were treated with 1, 2.5, 5 μg/ml Tβ4 on the basis of H2O2 treatment) and TAK-242 group[NSPCs were treated with 5 μg/ml Tβ4 and Toll-like receptor 4(TLR4) inhibitor TAK-242 on the basis of H2O2 treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with H2O2 and Tβ4. The expression of Tβ4, TLR4, MyD88 were detected by qRT-PCR and Western blot. Cell viability was detected by MTT assay and lactate dehydrogenase(LDH) assay kit. Ca2+ concentration was detected by Fluo-3/AM probe method. The apoptosis of NSPCs was detected by flow cytometry and Caspase-3 and Caspase-9 kits;reactive oxygen species (ROS), superoxi dedismu-tase dismutase(SOD) activity and glutathione (GSH) content were detected by corresponding kits. Interleukin(IL)-6 and IL-1β were detected by enzyme-linked immunosorbent assay.
Results: The expression of Tβ4 was decreased in H2O2 injured NSPCs(P<0.05). Compared with H2O2 group, the cell viability and Ca2+ concentration was significantly increased, release of LDH and apoptosis were significantly decreased, production of ROS and pro-inflammatory cytokines were significantly decreased, and the expression levels of TLR4 and MyD88 protein were significantly decreased in Tβ4-3 groups and TAK-242 group (P<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (P<0.05), while after treatment with Tβ4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (P<0.05).
Conclusion: Tβ4 attenuates H2O2-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways. |
| KEY WORDS Thymosin β4 Neural stem cells Progenitor cells Oxidative stress injury Myeloid differentiation factor 88 |
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| 引用本文，请按以下格式著录参考文献： |
| 中文格式: | 李宏维,张海鸿.胸腺肽β4对氧化应激诱导的脊髓源性神经干/祖细胞损伤的作用及机制[J].中国骨伤,2022,35(8):763~771 |
| 英文格式: | LI Hong-wei,ZHANG Hai-hong.Effect and mechanism of thymosin beta 4 on spinal cord-derived neural stem/progenitor cell injury induced by oxidative stress[J].zhongguo gu shang / China J Orthop Trauma ,2022,35(8):763~771 |
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