外源性神经肽CART对大鼠海马神经元氧化应激的抵抗作用及机制研究

doi:10.11843/j.issn.0366-6964.2022.04.023

摘要/Abstract

摘要： 旨在研究外源性神经肽可卡因-苯丙胺调节转录因子(cocaine-and amphetamine-regulated transcript,CART)抵抗海马神经元氧化应激的效果及机制。本研究取初生24 h内大鼠海马进行神经元的原代培养，经纯度检测后，用200 μmol·mL^-1过氧化氢诱导以构建氧化应激模型。在此基础上，分析CART对神经元活率、ATP含量、线粒体膜电位(mitochondrial membrane potential,MMP)和凋亡通路相关基因以及trkB基因表达的影响，并对trkB基因与凋亡相关蛋白和基因的表达进行相关性分析。结果显示，与对照组相比，H₂O₂组中的神经元活率及ATP含量极显著降低(P<0.01)，MMP值显著降低(P<0.05)；与H₂O₂组相比，添加不同浓度的CART使海马神经元活率、ATP含量和MMP值显著增加(P<0.05)。与对照组相比，H₂O₂组中bax、caspase-3的mRNA水平极显著增加(P<0.01)，bcl-2(P<0.01)、trkB(P<0.05)的mRNA水平分别极显著和显著降低；与H₂O₂组相比，CART+H₂O₂组中bcl-2的mRNA水平极显著升高(P<0.01)，而trkB的mRNA水平显著升高(P<0.05)，同时bax的mRNA水平显著降低(P<0.05)，而caspase-3的mRNA水平则极显著降低(P<0.01)。Western blot分析结果显示，与对照组相比，H₂O₂组中bax、caspase-3的蛋白质水平显著升高(p<0.05)，bcl-2、trkB的蛋白质水平显著降低(P<0.05)；与H₂O₂组相比，CART+H₂O₂组中bcl-2、trkB的蛋白质水平显著升高(P<0.05)，而bax、caspase-3的蛋白质水平显著降低(P<0.05)。相关性分析结果显示，trkB基因与bax、caspase-3基因的表达呈显著负相关，而与bcl-2基因的表达呈显著正相关。结果提示，CART在增加氧化应激海马神经元中trkB基因表达水平的同时，可通过削弱凋亡信号通路抵抗海马神经元氧化应激。

关键词: 神经肽, 海马神经元, 氧化应激, BDNF/trkB, 凋亡信号通路

Abstract: The study aimed at investigating the effect and mechanism of exogenous neuropeptide cocaine-and amphetamine-regulated transcription factor (CART) against oxidative stress in hippocampal neurons. The primary culture of hippocampal neurons was carried out in rats within 24 hours of birth. Following the purity test, 200 μmol·mL^-1 hydrogen peroxide were used to induce oxidative stress. On this basis, the effects of CART on the neuronal viability, ATP content, mitochondrial membrane potential (MMP)and the expression levels of apoptosis-related genes and trkB gene were investigated. Also the correlation between trkB gene and apoptosis-related genes was analyzed. The results showed that compared with the control group, the neuron viability and ATP content in H₂O₂ group were extremely significantly decreased(P<0.01), and the MMP was significantly decreased (P<0.05); CART with different concentrations significantly increased the neuronal viability, ATP content and MMP(P<0.05). Compared with the control group, the mRNA levels of bax and caspase-3 were extremely significantly increased in H₂O₂ group(P<0.01), and the mRNA levels of bcl-2 were extremely significantly decreased (P<0.01) and trkB were significantly decreased (P<0.05) in H₂O₂ group. Compaced with the H₂O₂ group, the mRNA levels of bcl-2 (P<0.01) were extremely significantly increased and trkB (P<0.05) were significantly increased in CART + H₂O₂ group. While the mRNA levels of bax were significantly decreased (P<0.05) and the mRNA levels of caspase-3 was extremely significantly decreased (P<0.01) in CART+H₂O₂ group compared with H₂O₂ group. The results of Western blot showed that the protein levels of bax and caspase-3 in H₂O₂ group were significantly higher than those in control group (P<0.05), and the protein levels of bcl-2 and trkB were significantly lower than those in control group (P<0.05). The protein levels of bcl-2 and trkB were significantly increased in CART+H₂O₂ group (P<0.05), while the protein levels of bax and caspase-3 were significantly decreased (P<0.05) compared with H₂O₂ group. The correlation analysis showed that, trkB gene was negatively correlated with the expression level of bax and caspase-3 genes, while positively correlated with the expression level of bcl-2 gene. The results indicate that CART can resist oxidative stress in hippocampal neurons by inhibiting apoptotic signaling pathway while increasing the expression level of trkB gene.

Key words: neuropeptide, hippocampal neurons, oxidative stress, BDNF/trkB, apoptosis signaling pathway

中图分类号:

Q539.2

杨成迎, 林海烂, 黄子晴, 王乃秀, 汪锴, 甘玲. 外源性神经肽CART对大鼠海马神经元氧化应激的抵抗作用及机制研究[J]. 畜牧兽医学报, 2022, 53(4): 1231-1240.

YANG Chengying, LIN Hailan, HUANG Ziqing, WANG Naixiu, WANG Kai, GAN Ling. Effects and Mechanisms of Exogenous Neuropeptide CART on Oxidative Stress in Hippocampal Neurons[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1231-1240.

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