Luteolin inhibits glioblastoma by regulating ROS levels via the NFE2L2/x-CT/GPX4 signalling axis

doi:10.11958/20250608

Abstract

Abstract:

Objective To investigate the role of luteolin (Lut) in regulating reactive oxygen species (ROS) levels through nuclear factor erythroid 2-related factor 2 (NFE2L2)/cystine glutamate antitransporter (x-CT)/glutathione peroxidase 4 (GPX4) signaling axis to inhibit the viability of glioblastoma and promote apoptosis. Methods U87 MG and U251 cells were cultured in vitro. The CCK-8 assay was used to detect cell survival rates after 48 hours of treatment with different concentrations (0, 6.25, 12.5, 25, 50 and 100 μmol/L) of Lut. According to whether cells were treated with Lut, cells were divided into the U87 control group, the U87 Lut group, the U251 control group and the U251 Lut group. The half-maximal inhibitory concentration (IC₅₀) at 48 hours was used as the unified treatment concentration for subsequent experiments. The apoptosis level of cells was detected by flow cytometry double staining method. Changes of reactive oxygen species (ROS) levels in cells were detected by the DCFH-DA method. Molecular docking was conducted using AutoDock software to verify the proteins related to the Lut and oxidative stress pathway. Real-time fluorescence quantitative reverse transcription (RT-qPCR) was used to detect the mRNA levels of NFE2L2 and GPX4. The expression levels of NFE2L2, x-CT and GPX4 proteins were detected by Western blot assay. Results After U87 MG and U251 cells were treated with Lut for 48 hours, the cell viability was significantly inhibited, and with the increase of Lut concentration, the cell viability decreased (P<0.05). Compared with the U87 control group and the U251 control group respectively, the apoptosis rate of cells increased in the U87 Lut group and the U251 Lut group, the green fluorescence intensity was enhanced, and the intracellular ROS level was upregulated (P<0.05). Results of molecular docking showed that Lut was tightly bound to NFE2L2, x-CT and GPX4. The results of RT-qPCR and Western blot assay showed that compared with the U87 control group and the U251 control group respectively, the protein and mRNA levels of NFE2L2 and GPX4 in cells of the U87 Lut group and the U251 Lut group, as well as the expression level of x-CT protein, decreased (P<0.05). Conclusion Lut regulates ROS levels through the NFE2L2/x-CT/GPX4 signaling axis to inhibit the viability of glioblastoma and promote cell apoptosis.

Key words: glioblastoma, Luteolin, reactive oxygen species, NF-E2-related factor 2, phospholipid hydroperoxide glutathione peroxidase, apoptosis, GPX4

CLC Number:

R730.264

Figures/Tables 7

References 26

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基因名称	引物序列（5'→3'）	产物大小/bp
NFE2L2	上游：TCAGCGACGGAAAGAGTATGA	174
NFE2L2	下游：CCACTGGTTTCTGACTGGATGT	174
GPX4	上游：CCGATACGCTGAGTGTGGTTTG	199
GPX4	下游：CTTGCCCTTGGGTTGGATCTTC	199
GAPDH	上游：ACACCCACTCCTCCACCTTTG	112
GAPDH	下游：TCCACCACCCTGTTGCTGTAG	112

基因名称	引物序列（5'→3'）	产物大小/bp
NFE2L2	上游：TCAGCGACGGAAAGAGTATGA	174
NFE2L2	下游：CCACTGGTTTCTGACTGGATGT	174
GPX4	上游：CCGATACGCTGAGTGTGGTTTG	199
GPX4	下游：CTTGCCCTTGGGTTGGATCTTC	199
GAPDH	上游：ACACCCACTCCTCCACCTTTG	112
GAPDH	下游：TCCACCACCCTGTTGCTGTAG	112

组别	凋亡率/%	ROS	NFE2L2 mRNA	GPX4 mRNA
U87 Control组	3.02±0.62	66.06±4.57	1.00±0.02	1.00±0.02
U87 Lut组	13.27±1.88	113.89±7.46	0.55±0.01	0.58±0.01
t	7.325^＊	7.734^＊＊	29.270^＊＊	33.720^＊＊
U251 Control组	3.21±1.14	51.34±15.37	1.00±0.00	1.00±0.04
U251 Lut组	12.80±0.29	102.82±11.98	0.80±0.01	0.53±0.02
t	11.530^＊＊	3.736^＊	24.360^＊＊	16.790^＊＊

组别	凋亡率/%	ROS	NFE2L2 mRNA	GPX4 mRNA
U87 Control组	3.02±0.62	66.06±4.57	1.00±0.02	1.00±0.02
U87 Lut组	13.27±1.88	113.89±7.46	0.55±0.01	0.58±0.01
t	7.325^＊	7.734^＊＊	29.270^＊＊	33.720^＊＊
U251 Control组	3.21±1.14	51.34±15.37	1.00±0.00	1.00±0.04
U251 Lut组	12.80±0.29	102.82±11.98	0.80±0.01	0.53±0.02
t	11.530^＊＊	3.736^＊	24.360^＊＊	16.790^＊＊

组别	NFE2L2	GPX4	x-CT
U87 Control组	1.00±0.00	1.00±0.00	1.00±0.00
U87 Lut组	0.65±0.15^a	0.45±0.01^a	0.57±0.16^a
t	3.247^＊	60.430^＊＊	3.866^＊
U251 Control组	1.00±0.00	1.00±0.00	1.00±0.00
U251 Lut组	0.75±0.12^a	0.43±0.18^a	0.78±0.03^a
t	3.061^＊	4.506^＊	10.080^＊＊