Heliox Protects SH-SY5Y Cells from Oxygen-Glucose Deprivation/Reperfusion-Induced Ferroptosis

Background: Heliox shows protective effects against acute focal ischemia-reperfusion injury in the brain. However, further research is needed to unveil the intricate molecular mechanisms involved. Determining how heliox affects ferroptosis caused by oxygen-glucose deprivation/reoxygenation (OGD/R) in SH-SY5Y cells as well as the underlying mechanism was the goal of the current work. Methods: With the use of 2 ${{}^{\prime}}$ ,7 ${{}^{\prime}}$ -Dichlorodihydrofluorescein diacetate (DCFH-DA), JC-1, and methyl thiazolyl tetrazolium, we assessed the survival, reactive oxygen species (ROS), and mitochondrial membrane potential in SH-SY5Y cells after they had been exposed to OGD/R and heliox. The expression of molecules associated with ferroptosis and the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway was analyzed using quantitative polymerase chain reaction (PCR) and immunoblotting, while malondialdehyde (MDA), oxidized glutathione disulfide (GSSG), ferrous ion (Fe ${}^{2+}$ ), and reduced glutathione (GSH) levels were evaluated using biochemical kits. Results: OGD/R treatment reduced the GSH to GSSG ratio; the potential of the mitochondrial membrane; the expression of the proteins GSH, SLC7A11, and glutathione peroxidase 4 (GPX4); and the ability of SH-SY5Y cells to survive. In contrast, OGD/R treatment increased the expression of cyclooxygenase-2 (COX2), ACSL4, and ferritin heavy chain 1 (FTH1) proteins, the production of MDA and GSSG, and the levels of ROS and Fe ${}^{2+}$ . However, heliox effectively mitigated all these OGD/R-induced effects. Furthermore, in OGD/R-treated SH-SY5Y cells, heliox administration stimulated the PI3K/AKT pathway while suppressing the nuclear factor- $\kappa{}$ B (NF- $\kappa{}$ B) pathway. When MK-2206, an AKT inhibitor, was applied concurrently to the cells, these outcomes were reversed. Conclusions: Heliox prevents OGD/R from causing ferroptosis in SH-SY5Y cells by activating the PI3K/AKT pathway. This suggests a promising therapeutic potential for heliox use in the management of ischemia/reperfusion injury.

Keywords

heliox

oxygen-glucose deprivation/reoxygenation

ferroptosis

PI3K/AKT pathway

Funding

3502Z20224ZD1056/Xiamen medical and health guiding project

Figures

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Fig. 1.

OGD/R prevents SH-SY5Y cells from surviving. 4-h OGD exposure was applied to SH-SY5Y cells, which were followed by treatment with oxygen-glucose for 0, 3, 6, 12, or 24 h. Cell survival was then evaluated using the MTT assay (A); the DCFH-DA dye was used to gauge ROS generation (B); the relative mean fluorescence intensity (MFI) of the DCFH-DA probe was quantified (C); the MMP was determined using JC-1 dye and flow cytometry (D); and the MFI of JC-1 fluorescence was quantified (E). OGD denotes oxygen-glucose deprivation, and R represents reoxygenation. IR, ischemia/reperfusion; OGD, oxygen-glucose deprivation; OGD/R, oxygen-glucose deprivation/reoxygenation; MTT, methyl thiazolyl tetrazolium; DCFH-DA, 2 ${{}^{\prime}}$ ,7 ${{}^{\prime}}$ -Dichlorodihydrofluorescein diacetate; ROS, reactive oxygen species; DCF, 2 ${{}^{\prime}}$ ,7 ${{}^{\prime}}$ -Dichlorodihydrofluorescein diacetate.

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J. Integr. Neurosci. Print ISSN 0219-6352 Electronic ISSN 1757-448X