SH-SY5Y cells (IM-H227) which had been tested and validated for mycoplasma (See Supplementary Material) were purchased from Xiamen IMMOCELL Biotechnology Co., Ltd. (Xiamen, China) and cultured in 45% Dulbecco’s modified eagle’s medium (DMEM; 11965092, GIBCO, New York, NY, USA) and 45% Ham’s F12 medium (11765054, GIBCO) with 10% fetal bovine serum (FBS; 10099141C, GIBCO) in an incubator at 37 °C and with 5% CO${}_2$.

When the SH-SY5Y cell density reached 70–80%, cells were washed three times using phosphate buffered solution (PBS) to completely remove the medium and then cultured in 50% glucose-free DMEM (11966025, GIBCO) and 50% Ham’s F12 medium in an incubator at 37 °C for 4 h with 95% N${}_2$ and 5% CO${}_2$. The cells were then cultured in 45% DMEM containing glucose and 45% Ham’s F12 medium with 10% FBS in an incubator containing 95% atmosphere and 5% CO${}_2$ for 3, 6, 12, and 24 h, or in an incubator containing 80% helium and 20% oxygen for 12 and 24 h. Alternatively, cells were cultured for 24 h in 45% DMEM containing glucose and 45% Ham’s F12 medium with 10% FBS and 5 µM MK-2206 2HCl (HY-10358, MedChemExpress, Monmouth Junction, NJ, USA) in an incubator containing 80% helium and 20% oxygen.

Medium (100 µL) containing 1 $\times$ 10${}^4$ SH-SY5Y cells was cultured in a 96-well plate. After OGD/R and heliox treatment, 10 µL 5% methyl thiazolyl tetrazolium (MTT) was added to each well and the plate was incubated for 3 h at 37 °C. The medium was then carefully discarded and 100 µL dimethyl sulfoxide (DMSO, catalog number: 0219605580, MP Biomedicals, Santa Ana, CA, USA) was added to each well. The plate was incubated on a shaker at 70–80 rpm for 10 min. At 490 nm, an optical density (OD) measurement was made using a microplate reader (model: Varioskan LUX, Thermo Fisher Scientific, Waltham, MA, USA).

SH-SY5Y cells were lysed on ice in radioimmunoprecipitation assay (RIPA, catalog number: E-BC-R327, Elabscience, Wuhan, Hubei, China) buffer for 20 min. Centrifugation at 15,000 $\times$g for 10 min was then used to separate the supernatant. The malondialdehyde (MDA) assay kit (S0131S, Beyotime Biotechnology, Shanghai, China), reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG) assay kit (S0053, Beyotime Biotechnology), and cell iron content assay kit (BC5315, Solarbio Life Sciences, Beijing, China) were used to quantify the concentrations of MDA, GSH, GSSG, and ferrous ion (Fe${}^{2+}$), respectively, in the supernatant following the manufacturer’s instructions. The relative levels of MDA, GSH, GSSG, and Fe${}^{2+}$ were calculated by comparing the OD value with the standard curve and normalizing according to the cell number.

Following OGD/R or heliox treatment, reactive oxygen species (ROS) levels in SH-SY5Y cells were assessed using the 10 µM 2${}^{\prime }$,7${}^{\prime }$-Dichlorodihydrofluorescein diacetate (DCFH-DA, catalog number: 50101ES01, Yeason, Shanghai, China) fluorescence probe for 20 min at 37 °C in an incubator. The fluorescence signals from SH-SY5Y cells were measured at 560 nm using flow cytometry.

After treatment under different conditions, the JC-1 probe (C2006, Beyotime Biotechnology) was used to determine mitochondrial membrane potential (MMP) in SH-SY5Y cells for 20 min at 37 °C in an incubator. PBS was used to wash the cells twice and flow cytometry was used to measure the fluorescence signals from SH-SY5Y cells.

The RNA, separated from SH-SY5Y cells using TRIzol reagent (Invitrogen, Waltham, MA, USA), was used for cDNA synthesis using a reverse transcription kit (R323-01, Vazyme, Nanjing, Jiangsu, China). The SYBR Master Mix (Q411-02, Vazyme) was used to carry out the quantitative polymerase chain reaction (qPCR). Each sample was analyzed in triplicate on an Applied Biosystems${}^{\text{TM}}$ real-time fluorescent quantitative PCR system (model: 7500 Fast, Thermo Fisher Scientific). The following steps were used for the reaction: step I, 95 °C for 30 sec; step II, 40 cycles of 95 °C for 10 sec and 60 °C for 30 sec; and step III, 95 °C for 15 sec, 60 °C for 60 sec, and 95 °C for 15 sec. Data were analyzed using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method. Primer sequences are shown in Table 1.

Following treatment under different conditions, SH-SY5Y cells were lysed by incubation on ice for 20 min in RIPA buffer containing phosphatase inhibitor. A bicinchoninic acid (BCA) kit (PA115-02; TIANGEN Biotechnology, Beijing, China) was used to extract proteins and measure their concentration. Equal protein amounts were loaded into the wells of a 10% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel. Electrophoresis was carried out at 80 V for 0.5 h and at 120 V for 1 h. At 350 mA for 3 h, the isolated proteins were deposited onto polyvinylidene fluoride membranes. Five percent non-fat milk was used to inhibit the membranes for 1 h. After being exposed to primary antibodies overnight at 4 °C, the membranes were incubated with secondary antibody for 1 h at 25 °C. An enhanced chemiluminescence kit (WP20005, Thermo Fisher Scientific) was used to detect the protein bands, which were imaged using a Bio-Rad ChemiDoc MP imaging system (catalog number: 12003154, Bio-Rad, Hercules, CA, USA). All antibodies used are listed in Table 2.

The creation of bar charts and statistical analysis were performed using GraphPad Prism (version 8.0, GraphPad Software, Inc., San Diego, CA, USA). Student’s t-test (unpaired) and one-way analysis of variance (ANOVA) were used to compare parametric data between two groups and among multiple groups, respectively. Western blots were scanned and the signal intensity was quantified using ImageJ (version 2, National Institutes of Health, Bethesda, MD, USA). Statistical significance was determined at the following probability levels: *p 0.05, **p 0.01, ***p 0.001, and ****p 0.0001.

To examine the effect of heliox on IR injury, oxygen-glucose deprivation (OGD) was initially applied for 4 h to SH-SY5Y cells which were followed by incubation with oxygen and glucose for 0, 3, 6, 12, or 24 h. Using the MTT assay, a significant reduction in the SH-SY5Y cells’ survival was observed following treatment with oxygen and glucose for 3, 6, 12, and 24 h (Fig. 1A). DCFH-DA staining and flow cytometry were performed to investigate ROS production after OGD/R treatment. The peak shifted to the right (Fig. 1B), indicating the mean fluorescence intensity (MFI) of DCFH-DA increased after reoxygenation for 3, 6, 12, and 24 h. As shown in Fig. 1C, ROS levels in SH-SY5Y cells treated with OGD/R also increased significantly. The JC-1 probe was used to investigate whether OGD/R treatment altered the MMP. As shown in Fig. 1D, OGD/R treatment of SH-SY5Y cells resulted in a noticeable increase in the proportion of cells displaying green fluorescence. The green/red ratio increased significantly with longer treatment times (Fig. 1E). OGD/R therefore decreased the survival and MMP of SH-SY5Y cells, and increased the production of ROS.

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.

High ROS production occurs when cells undergo ferroptosis [16]. The above results indicate that OGD/R induces a large amount of ROS production in SH-SY5Y cells, suggesting that it may inhibit cell survival by inducing ferroptosis. In addition, heliox has been shown to protect the brain from acute focal IR injury [17]. To investigate the molecular mechanism by which heliox protects the brain, we therefore studied its role in ferroptosis. OGD/R-treated SH-SY5Y cells were cultured with helium and oxygen for 0, 12, or 24 h. Untreated cells acted as the controls. The ischemia reperfusion/helium-oxygen mixture (IR/HOM) 0 h group were cells that were cultured for 4 h under OGD and then for 6 h in 5% CO${}_2$ and 95% air. The levels of ferroptosis markers [18], including COX2, ACSL4, FTH1, SLC7A11, GPX4, MDA, GSSG, and GSH, were all evaluated. OGD/R treatment of SH-SY5Y cells resulted in elevated levels of COX2, ACSL4, FTH1, MDA, GSSG, and Fe${}^{2+}$; decreased levels of SLC7A11, GPX4, and GSH; and a lower GSH to GSSG (GSH/GSSG) ratio (Fig. 2A–H). Administration of heliox attenuated these OGD/R-induced effects. Moreover, the viability of OGD/R-treated cells increased significantly with heliox treatment (Fig. 2I). Furthermore, heliox treatment attenuated the increased generation of ROS observed in OGD/R-treated SH-SY5Y cells (Fig. 2J–K). JC-1 staining indicated that heliox treatment also significantly reversed the decrease in mitochondrial potential caused by OGD/R (Fig. 2L–M). In summary, heliox treatment reversed OGD/R-induced ferroptosis in SH-SY5Y cells.

Fig. 2.

Heliox protects OGD/R-treated SH-SY5Y cells from ferroptosis. OGD was applied to SH-SY5Y cells for 4 h, followed by heliox-glucose therapy for 0, 12, or 24 h. Assessments were then made of the mRNA (A) and protein levels (B,C) for COX2, ACSL4, FTH1, SLC7A11, and GPX4 using qPCR and immunoblotting, respectively. Commercial kits were used to measure MDA, GSH, GSSG, and Fe${}^{2+}$ levels, and the GSH to GSSG ratio (D–H). In order to gauge cell vitality, the MTT test was performed (I). DCFH-DA staining was used to detect ROS generation (J–K). MMP was assessed using the JC-1 probe and analyzed by flow cytometry (L–M). IR/HOM, ischemia reperfusion/helium-oxygen mixture; MDA, malondialdehyde; GSH, glutathione; GSSG, oxidized glutathione disulfide; Fe${}^{2+}$, ferrous ion. *p 0.05, **p 0.01, ***p 0.001, and ****p 0.0001.

The PI3K/AKT pathway is known to play a crucial role in regulating cell proliferation [19]. Experiments were therefore conducted to evaluate the expression of proteins involved in the PI3K/AKT pathway following heliox treatment. Heliox treatment markedly upregulated the expression levels of phosphorylated PI3K and phosphorylated AKT in OGD/R-treated SH-SY5Y cells (Fig. 3A,B). Moreover, the mRNA levels of PI3K and AKT decreased after OGD/R exposure, but increased after heliox treatment (Fig. 3C). The levels of p50 and phosphorylated p65 were markedly increased in OGD/R-treated SH-SY5Y cells, but decreased significantly following heliox treatment (Fig. 3A,B). These findings suggest that heliox treatment effectively suppresses the nuclear factor-$\kappa$B (NF-$\kappa$B) pathway. Thus, heliox activates the PI3K/AKT pathway and inhibits the NF-$\kappa$B pathway in OGD-treated SH-SY5Y cells.

Fig. 3.

Heliox therapy stimulates the PI3K/AKT pathway while impairing the NF-κB pathway in OGD-treated SH-SY5Y cells. OGD was applied to SH-SY5Y cells for 4 h, and then heliox-glucose was applied for 0, 12, or 24 h. The protein levels for P-PI3K, PI3K, P-AKT, AKT, P-p65, p65, p50, and GAPDH were then measured using immunoblotting (A,B), while the mRNA levels for PI3K and AKT were quantified using qPCR (C). ns, not significant. **p 0.01, ***p 0.001.

After heliox administration for 24 h, we introduced the AKT inhibitor MK-2206 to OGD/R-treated SH-SY5Y cells to test if heliox exerts its protective effect via activation of the PI3K/AKT pathway. MK-2206 treatment significantly reduced the protein levels of phosphorylated AKT, SLC7A11, and GPX4 in heliox-treated SH-SY5Y cells (Fig. 4A,B). The decrease in MDA, GSSG, and Fe${}^{2+}$ levels in heliox-treated cells was reversed by MK-2206, while the increased GSH level and GSH to GSSG ratio was attenuated by MK-2206 (Fig. 4C–H). In heliox-treated SH-SY5Y cells, MK-2206 also decreased the viability and the MMP and increased ROS production (Fig. 4I–L). Heliox prevented ferroptosis in SH-SY5Y cells by stimulating the PI3K/AKT pathway.

Fig. 4.

Heliox protects SH-SY5Y cells from ferroptosis via triggering the PI3K/AKT pathway. After oxygen-glucose deprivation for 4 h, SH-SY5Y cells were treated with heliox-glucose and MK-2206 for 0 or 24 h. The following assessments were then made: protein levels for P-PI3K, PI3K, P-AKT, AKT, SLC7A11, and GPX4 using immunoblotting (A,B); levels of MDA, GSH, GSSG, and Fe${}^{2+}$, and the GSH to GSSG (GSH/GSSG) ratio using commercial kits (C–G); cell viability using the MTT assay (H); ROS levels using the DCFH-DA probe (I,J); and MMP using the JC-1 probe and flow cytometry (K–L). ns, not significant. *p 0.05, **p 0.01, ***p 0.001, and ****p 0.0001.