过氧化氢对成肌细胞的氧化损伤作用研究

doi:10.11843/j.issn.0366-6964.2019.05.012

摘要/Abstract

摘要：

旨在以体外培养的小鼠成肌细胞（C2C12）为研究对象，探讨氧化损伤可能的路径。本试验分为3个处理组，每个处理组6个重复。处理组分别为：对照组（Control），以DMEM培养基培养的细胞作为空白对照，培养24 h；吡咯烷二硫代氨基甲酸酯（PDTC）组（PDTC），以每个孔1 mL的PDTC（20 μmol·L^-1）孵育细胞24 h；H₂O₂组（H₂O₂），加入0.5 mmol·L^-1的H₂O₂处理细胞24 h。检测各组细胞的存活率、ROS水平与凋亡率，采用荧光定量PCR和蛋白免疫印记法检测细胞凋亡和自噬相关基因的表达以及NF-κB信号通路相关因子的表达。结果表明，与对照组相比，PDTC可显著提高细胞的总凋亡率（P<0.05），可显著上调细胞半胱天冬蛋白酶-3（caspase-3）、caspase-6、caspase-9的mRNA表达量以及caspase-3蛋白表达水平（P<0.05），显著上调Beclin 1的mRNA表达量和微管相关蛋白1轻链3（LC3）-Ⅱ/I的值以及LC3-Ⅱ蛋白表达水平（P<0.05），显著下调核因子kappa B副族1（p50）、B型白细胞/2型淋巴细胞样蛋白（Bcl-2）、v-rel禽网状内皮增生病病毒癌基因同源物A（RelA）和环氧合酶（Cox）-2的mRNA表达量以及NF-κB蛋白表达水平（P<0.05）；H₂O₂可显著提高细胞的ROS水平和细胞总凋亡率（P<0.05），可显著上调caspase-3、caspase-6、caspase-8、caspase-9的mRNA表达量以及caspase-3蛋白表达水平（P<0.05），显著上调Beclin 1和LC3-Ⅱ/LC3-I的mRNA表达量以及LC3-Ⅱ蛋白表达水平（P<0.05），显著提高Bcl-2相关X蛋白（Bax）的mRNA表达量（P<0.05），显著下调p50、Bcl-2、RelA和Cox-1、Cox-2的mRNA表达量以及核因子kappa B（NF-κB）蛋白表达水平（P<0.05）。结果提示，H₂O₂会引起C2C12细胞的ROS升高，并且能够通过抑制NF-κB信号通路介导细胞凋亡和自噬的发生，这与NF-κB因子的特异性抑制剂PDTC的作用相类似。

Abstract:

The change of C2C12 cells exposed to H₂O₂ was investigated and the possible path of oxidative damage was explored in this study. There was 3 treatments, and 6 repeats in each treatment. C2C12 cells attached to the substrate and cultured in DMEM for 24 h were treated as control (n=6); the medium was added with PDTC (20 μmol·L^-1, PDTC treatment, n=6) for 24 h or H₂O₂ (0.5 mmol·L^-1, H₂O₂ treatment, n=6) for 24 h. The viability, ROS level and apoptosis rate of cells in different treatments were detected. The expression of genes related to cell apoptosis and autophagy and factors related to NF-κB signaling pathway were determined by real-time PCR and Western blot. The results showed that cells in PDTC treatment had significantly higher total apoptosis rate, mRNA expression of caspase-3, caspase-6, caspase-9 and the protein expression of total caspase-3, mRNA expression of Beclin1, LC3-Ⅱ/LC3-I and the protein expression level of total LC3-Ⅱ(P<0.05), and had significantly lower mRNA expression of p50, Bcl-2, RelA, Cox-2 and protein expression of total NF-κB compared to that in the control treatment(P<0.05). Moreover, cells in H₂O₂ treatment exhibited significantly higher ROS level and total apoptosis rate(P<0.05), mRNA expression of caspase-3, caspase-6, caspase-8, caspase-9 and protein expression of total caspase-3(P<0.05), mRNA expression of Beclin 1, LC3-Ⅱ/LC3-I and protein expression of total LC3-Ⅱ(P<0.05), mRNA expression of Bax(P<0.05), and had significantly lower mRNA expressions of p50, Bcl-2, RelA, Cox-1, Cox-2 and protein expression of total NF-κB(P<0.05) compared to that in the control treatment. These results indicated that H₂O₂ could promote the ROS formation, mediated the apoptosis and autophagy processes by suppressing the NF-κB signaling pathway in C2C12 cells, which was similar with the role of PDTC, the inhibitor of NF-κB factor.

中图分类号:

S852.2

陈祥兴, 李蛟龙, 邢通, 张林, 高峰. 过氧化氢对成肌细胞的氧化损伤作用研究[J]. 畜牧兽医学报, 2019, 50(5): 1016-1025.

CHEN Xiangxing, LI Jiaolong, XING Tong, ZHANG Lin, GAO Feng. Effects of H₂O₂ on Oxidative Damage of C2C12 Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(5): 1016-1025.

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