褪黑素对过氧化氢诱导人神经母细胞瘤SH-SY5Y细胞氧化应激的改善作用及其机制

doi:10.13481/j.1671-587X.20220210

Abstract

Abstract: Objective

To investigate the protective effect of melatonin on the hydrogen peroxide（H₂O₂）-induced oxidative stress injury of human neuroblastoma SH-SY5Y cells， and to explore its mechanism.

Methods

The SH-SY5Y cells were cultured in vitro and divided into control group，H₂O₂ group， melatonin groups， and N-acetyl-2-benzyltryptamine（Luzindole） group. The medium containing 200 μmol·L^－1 H₂O₂ was added in H₂O₂ group；different concentrations （1， 5 and 10 μmol·L^－1） of melatonin and medium containing 200 μmol·L^－1 H₂O₂ were added in melatonin groups； 50 μmol·L^－1 Luzindole， 10 μmol·L^－1 melatonin and the medium containing 200 μmol·L^－1 H₂O₂ were added in Luzindole group. The survival rates of human neuroblastoma SH-SY5Y cells were measured by CCK-8 method， the apoptotic rates of SH-SY5Y cells in various groups were detected by flow cytometry， and DCFH-DA fluorescence probe was used to detect the levels of reactive oxygen species（ROS） in the cells in various groups. The expression levels of microtubule-associated protein light chain 3-Ⅱ（LC3-Ⅱ）protein in the cells in various groups were detected by Western blotting method， and the fluorescence intensities of autophagic vesicles in various groups were observed by fluorescence microscope.

Results

Compared with control group， the survival rate of SH-SY5Y cells in H₂O₂ group was decreased （P<0.01），the ROS level and the apoptotic rate were increased （P<0.01）. Compared with H₂O₂ group， the survival rates of SH-SY5Y cells in different concentrations of melatonin groups were increased（P<0.05），the ROS levels and the apoptotic rates were significantly decreased （P<0.01），the LC3-Ⅱ protein expression levels were significantly increased （P<0.01）， and the fluorescence intensity of autophagic vesicles in 10 μmol·L^－1 melatonin group was increased（P<0.05）. Compared with 10 μmol·L^－１ melatonin group， the survival rates of SH-SY5Y cells in 1 μmol·L^－１ melatonin group and Luzindole group were significantly decreased（P<0.05）；the ROS levels and the apoptotic rates in the SH-SY5Y cells in 1 and 5 μmol·L^－1 melatonin groups and Luzindole group were significantly increased（P<0.05），and the LC3-Ⅱ protein expression levels in the SH-SY5Y cells were decreased（P<0.05 or （P<0.01）， and the fluorescence intensities of autophagic vessicles in the SH-SY5Y cells in 1 μmol·L^－1 melatonin group and Luzindole group were significantly decreased （P<0.05）.

Conclusion

Melatonin can inhibit the H₂O₂-induced oxidative stress injury of SH-SY5Y cells and has a neuroprotective effect，and its mechanism may be related to reducing the ROS levels and enhancing the autophagy of cells.

Key words: SH-SY5Y, Melatonin, Oxidative stress, Reactive oxygen species, Autophagy

CLC Number:

R741

Yang ZHOU,Xuguang MI,Wenxing PU,Wentao WANG,Meng JING,Fankai MENG. Ameliorative effect of melatonin on oxidative stress of human neuroblastoma SHSY5Y cells induced by hydrogen peroxide and its mechanism[J].Journal of Jilin University(Medicine Edition), 2022, 48(2): 340-347.

Figures/Tables 8

Tab. 1

Fig. 1

Tab. 2

Fig. 2

Tab. 3

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Tab. 4

References 25

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Group	Survival rate
Control	100.00±2.85
H₂O₂	54.64±5.85^*
Melatonin
1 μmol·L^－1	61.32±4.28^△#
5 μmol·L^－1	65.93±1.10^△
10 μmol·L^－1	67.72±2.29^△
Luzindole	57.72±2.48^△#

Group	ROS level
Control	1.80±0.54
H₂O₂	75.37±5.87^*
Melatonin
1 μmol·L^－1	66.19±0.68^△△#
5 μmol·L^－1	57.50±0.71^△△#
10 μmol·L^－1	52.50±2.17^△△
Luzindole	62.33±2.20^△##

Group	Apoptotic rate
Control	2.84±0.69
H₂O₂	50.85±1.69^*
Melatonin
1 μmol·L^－１	47.13±1.29^△#
5 μmol·L^－１	46.34±1.31^△#
10 μmol·L^－１	32.52±1.66^△△
Luzindole	43.02±2.40^△△#

Group	Fluorescence intensity
Control	1.00±0.18
H₂O₂	1.67±0.27^*
Melatonin
1 μmol·L^－1	1.91±0.24^*#
5 μmol·L^－1	2.46±0.51^*
10 μmol·L^－1	2.69±0.43^△
Luzindole	1.89±0.21^#

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Ameliorative effect of melatonin on oxidative stress of human neuroblastoma SHSY5Y cells induced by hydrogen peroxide and its mechanism

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