胸腺素β4对氧化应激诱导的脊髓源性神经干细胞/祖细胞损伤的作用及机制

[Effect and mechanism of thymosin beta 4 on spinal cord-derived neural stem /progenitor cell injury induced by oxidative stress].

作者信息

Li Hong-Wei, Zhang Hai-Hong

机构信息

Department of Spine Surgery, Lanzhou University Second Hospital, Lanzhou 730030, Gansu, China.

出版信息

Zhongguo Gu Shang. 2022 Aug 25;35(8):763-71. doi: 10.12200/j.issn.1003-0034.2022.08.012.

DOI:10.12200/j.issn.1003-0034.2022.08.012

PMID:35979771

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 (HO).

METHODS

NSPCs were isolated from Sprague-Dawley (SD) adult male rats, and divided into control group (untreated NSPCs cells), HO group (NSPCs cells damaged by 500 μM HO), Tβ4 -3 groups (NSPCs were treated with 1, 2.5, 5 μg/ml Tβ4 on the basis of HO 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 HO treatment]. NSPCs were transfected with lentivirus vector of myeloid differentiation factor 88(MyD88) to construct MyD88-overexpressing cell lines, which were treated with HO 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 HO injured NSPCs(<0.05). Compared with HO 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 (<0.05). After overexpression of MyD88, cell viability, SOD activity and GSH content of NSPCs decreased, LDH release and apoptosis increased significantly (<0.05), while after treatment with Tβ4, cell viability, SOD activity and GSH content increased, LDH release and apoptosis decreased (<0.05).

CONCLUSION

Tβ4 attenuates HO-induced NSPCs oxidative stress, apoptosis and inflammation in NSPCs via inhibiting TLR4 and MyD88 pathways.

摘要

目的

探讨胸腺素β4（Tβ4）在过氧化氢（HO）诱导的脊髓源性神经干细胞/祖细胞（NSPCs）氧化应激损伤中的作用及机制。

方法

从成年雄性Sprague-Dawley（SD）大鼠中分离NSPCs，分为对照组（未处理的NSPCs细胞）、HO组（用500μM HO损伤的NSPCs细胞）、Tβ4 -3组（在HO处理基础上分别用1、2.5、5μg/ml Tβ4处理的NSPCs）和TAK-242组[在HO处理基础上用5μg/ml Tβ4和Toll样受体4（TLR4）抑制剂TAK-242处理的NSPCs]。用髓样分化因子88（MyD88）慢病毒载体转染NSPCs构建MyD88过表达细胞系，并用HO和Tβ4处理。通过qRT-PCR和蛋白质免疫印迹法检测Tβ4、TLR4、MyD88的表达。用MTT法和乳酸脱氢酶（LDH）检测试剂盒检测细胞活力。用Fluo-3/AM探针法检测Ca2+浓度。通过流式细胞术以及Caspase-3和Caspase-9试剂盒检测NSPCs的凋亡；用相应试剂盒检测活性氧（ROS）、超氧化物歧化酶（SOD）活性和谷胱甘肽（GSH）含量。用酶联免疫吸附测定法检测白细胞介素（IL）-6和IL-1β。

结果

HO损伤的NSPCs中Tβ4表达降低（<0.05）。与HO组相比，Tβ4 -3组和TAK-242组细胞活力和Ca2+浓度显著升高，LDH释放和凋亡显著降低，ROS和促炎细胞因子产生显著减少，TLR4和MyD88蛋白表达水平显著降低（<0.05）。MyD88过表达后，NSPCs的细胞活力、SOD活性和GSH含量降低，LDH释放和凋亡显著增加（<0.05），而用Tβ4处理后，细胞活力、SOD活性和GSH含量增加，LDH释放和凋亡减少（<0.05）。